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MAGNETIC GLYCO-NANOPARTICLES: A NEW TOOL
TO DETECT BACTERIA, CANCER AND ATHEROSCLEROSIS

presented by

Kheireddine El-Boubbou

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MAGNETIC GLYCO-NANOPARTICLES: A NEW TOOL TO DETECT
BACTERIA, CANCER AND ATHEROSCLEROSIS

Kheireddine El—Boubbou

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Chemistry

2010

ABSTRACT

MAGNETIC GLYCO-NANOPARTICLES: A NEW TOOL TO DETECT
BACTERIA, CANCER AND ATHEROSCLEROSIS

By

Kheireddine El—Boubbou

Rapid and sensitive detection of pathogenic bacteria, cancer, and
atherosclerotic plaques is critical for the prevention of such diseases and
bioterrorism. To address these issues, we developed a magnetic iron oxide
glyco—nanoparticle (MGNP)-based system as novel “detecting and imaging”
vehicle with unique properties. In fact, there is an urgent need for an effective
method for microbial decontamination and rapid pathogen detection without time
consuming cell culturing. We proved that MGNPs can be utilized not only for fast
pathogen detection, but also for strain differentiation and efficient pathogen
decontamination. Using MGNPs, we were able to detect the presence of the
bacterium Escherichia coli (E. coli) within five minutes as well as remove up to
88% of the microbe. Moreover, three different E. coli strains were easily
identified using two MGNPs highlighting its potentials in bio-sensing. These
results gave us great confidence to apply MGNPs for cancer detection. The
development of simple and effective techniques to identify reliable detection
methdds and to delineate the fine characteristics of cancer cells can have great
potential impacts on cancer diagnosis and treatment. We demonstrated the
utilization of MGNP nanocomposites not only to detect and differentiate cancer

cells but also to quantitatively profile their carbohydrate bindings by magnetic

resonance imaging (MRI). Using an array of MGNPs, a range of cells including
closely related isogenic tumor cells, cells with different metastatic potential and
malignant vs normal cells were readily distinguished based on their respective
“MRI signatures”. As the interactions between glyco-conjugates and endogenous
Iectins present on cancer cell surface are crucial for cancer development and
metastasis, the ability to characterize and unlock the glyco—code of individual cell
lines can facilitate both the understanding of the roles of carbohydrates as well
as the expansion of diagnostic and therapeutic tools for cancer. Building on the
success of bacterium and cancer detection, we moved on to examine the utility of
MGNPs for in vivo atherosclerotic detection. Despite the significant progress in
cardiology, there remain large unmet needs to detect atherosclerotic plaques.
One of the major causes of such dramatic event is “inflammation” which occurs
during early onset of the disease leading to over-expression of cell-adhesion
receptors. Our proposed work is based on the knowledge that hyaluronic acid
(HA) is upregulated in atherosclerotic lesions and its principal cell-adhesion
receptor, CD44 is involved in several atherogenic processes. Thus, we
engineered hyaluronic-coated magnetic nanoparticles (HA-MGNPs) to non-
invasively image atherosclerotic plaques via MRI. Today’s nanotechnologies are
enabling better detection and diagnosis systems with great therapeutic
potentials. Tomorrow is likely to bring a full understanding of the “cell-NP bio-
conversation” where major problems relating to detection will be solved

translating insights from the “nano-world” into clinical practice.

DEDICATION

I would like to dedicate this thesis to my father, Hassan Wafic El-Boubou
for his endless support, to my mother, Bochra Saad EI-Masri for her prayers, love
and faith in me and to my lovely girl, Najwa Aown, who stood by me through
difficult times. They all taught me how to believe in myself, keep my dreams alive

and achieve success which requires belief, vision, hard work, determination, and

dedication.

iv

ACKNOWLEDGEMENTS

Lots of people, professors, students, and family contributed to my
achievements and set me up to be able to reach my future goals. Without them, I
could not accomplish so much today.

First of all, I would like to express deep gratitude, passionate appreciation
and many thanks to my advisor, Prof. Xuefei Huang. He was that “person in life”
that left a big impact on me and gave me reasons to fill my life with specific
purposes. He taught me the “real” meaning of science. His patience, inspiration
and wisdom impressed me. He changed me to a more mature, independent and
planned person. The time I spent with him was more than a journey to PhD. It’s
a journey into exploring new horizons in life, seeing things in a different
perspective, thinking more into the details and asking to learn more to be able to
give more. It was a unique experience that is well appreciated and greatly
admired. Indeed, the people we admire tend to be those who “we would most like
to be like”. And when you admire someone, you can borrow a little bit of their life
blueprint to help formulate your own. So thanks Dr. Huang for shaping my life.

I would also like to express my sincere gratitude to Prof. Cyndee Gruden
in the Department of Civil Engineering at the University of Toledo. Thanks for her
help, support and encouragement. I am grateful for her willingness to discuss
various research efforts with me at any time and providing invaluable advice. She
did not only open her lab for me, but also motivated me and taught me a lot

about E. coli bacteria, culturing and microbial sensing.

Special thanks to Prof. David Zhu in the Department of Radiology and
Psychology at MSU for the support, encouragement, fruitful arguments and
instructive ideas. Thanks for your confidence in me and so many valuable
discussions on our experiments. He taught me all the details about MRI and
helped me understand its concepts. Working with him was a pleasant experience.
Without him we will not be able to perform in vitro and in vivo studies using MRI.
Thanks for helping and reserving the time slots for the scanner. It’s greatly
appreciated.

Special thanks to our former postdoc, Dr. Lijun Huang, for his help and
excellent hands in the lab. He endured my enthusiasm in chemistry and
motivates me. One can learn a lot by just watching him. Thanks also for Dr.
Balasubramanian Srinivasan (Bala) for the sound arguments we had and great
help. I would also like to thank Dr. Zhen Wang (Jason) for teaching me the basics
of sugar chemistry and special synthetic techniques when I first joined the lab.

Thanks to Dr. Chrysoula Vassilio for her help in cancer cells. She was
always there at any time whenever we asked for assistance. She taught me the
cell culture techniques and valuable knowledge on molecular biology.

Thanks for Prof. Xudong Fan and Prof. Terry Biogini for teaching me how
to operate Transmission Electron Microscope (TEM).

Thanks for Mr. Guang for helping me in growing and counting E. coli
bacteria, and Mr. Vivek for his excellent hands in the preparation of sugar

building blocks.

vi

Moreover, I would like to acknowledge the many outstanding people I met
during my graduate studies, and all former and present lab members who
provided encouragement and help in one way or another. In no particular order
those people are: Dr. Mohamad El-Dakdouki, Dr. Medha Kamat, Dr. Adeline
Miermont, Dr. Bin Sun, Dr. Gopinath Tiruchinapally, Dr. Youlin Zeng, Bo Yang,
Gilbert Wasonga, Luyuan Zhou, Nardos Teumelsan, Xiaowei Lu, Yuqing Jing,
Philip Bentley, Steven DuLaney, and Hovlg Kouyoumdjian. Although we all face
certain anxieties and pressures, good moments and notable times always
outweigh and persist. l have learnt a lot from discussions with them either on
group meetings or at casual time. Thanks guys.

Thanks for Dr. Steven Sucheck for his assistance and guidance in the
courses I took with him.

Thanks for Dr. Viranga Tillekeratne, Dr. Richard Hudson, and Dr. James
Slama for their inspiring discussions on synthetic organic mechanisms in
“Organic Journal Clubs” we had every Thursday. lt was an outstanding
experience that makes your mechanistic way of thinking much stronger.

Special Thanks for my committee members: Dr. Gregory Baker, Dr. Gary
Blanchard and Dr. John McCracken.

Last but not least, I would like to thank the people who will never forget
you no matter what happened. The only people that do not understand any
chemistry but are, chemically, the most connected to you. Thanks for their never-
ending love, care, and support (financially and spiritually) at any time. Many and

deep thanks to mom, dad, sisters and brothers.

vii

TABLE OF CONTENTS

LIST OF FIGURES ............................................................................................ xi

LIST OF ABBREVIATIONS ............................................................................ xvii
CHAPTER 1 Gcho—Nanoparticles: Translating Insights from the

“Sugar-Code” into Diagnostics and Therapeutics .................. 1

1.1. Introduction ................................................................................... 1

1.2. The “Glyco” Perspective ............................................................... 3

1.3. The “Nano” Perspective ................................................................ 4

1.4. Glyco-nanotechnology .................................................................. 5

1.5. Nano—biosensors .......................................................................... 6

1.6. Magnetic Iron Oxide Nanoparticles (MNPs) ................................. 8

1.6.a. Magnetic Properties ......................................................... 10

1.6.b. Synthesis of MNPs ......................................................... 13

1.6.c. Mechanistic Insights ........................................................ 19

1.6.d. Colloidal Stability ............................................................ 20

1.6.e. Characterization of MNPs ............................................... 22

1.6.f. Applications of MNPs ...................................................... 26

1.7. Glyco-nanoparticles for in vitro and in vivo Detection ................. 29

1.7a. Magnetic Iron Oxide Glyco-nanoparticles ........................ 29

1.7.b. Gold Glyco—nanoparticles ................................................ 32

1.7.0. Gcho—Quantum Dots ...................................................... 5O

1.7.d. Glyco-Carbon Nanotubes ................................................ 52

1.7.e. Glyco-Micelles and Liposomes ........................................ 53

1.8. References ................................................................................. 61

CHAPTER 2 Magnetic Glyco—Nanoparticles: A Unique Tool for

2.1.
2.2.
2.3.

2.4.
2.5.
2.6.
2.7.

Rapid Pathogen Detection, Decontamination and

Strain Differentiation ................................................................ 79
Pathogen Detection ................................................................... 79
Syntheses of MGNPs ................................................................. 80
Characterization of MGNPs ....................................................... 82
2.3.a. Transmission Electron Microscopy (TEM) ...................... 82
2.3.b. X-ray Diffraction (XRD) ................................................... 83
2.3.c. Fourier Transform Infrared Spectroscopy (FT-IR) ............ 83
2.3.d. Thermogravimetric analysis (TGA) ................................. 84
2.3.e. High-Resolution Magic Angle Spinning (HR-MAS)

1H NMR ............................................................................ 85
Lectin Binding Assay: MGNP/Con A Interactions ....................... 86
E. coli Detection using MGNPs ................................................. 89
Bacterial Differentiation ............................................................. 91

Lipoic Carbohydrate Conjugates: Controlling Nonspecific
Adsorption at Biointerfaces for Biosensor and Biomedical
Application ................................................................................. 93

viii

2.8.

2.9.

Experimental Section ................................................................. 98

2.8.3. Materials and Instrumentation .......................................... 98
2.8.b. E. coli Detection using MGNPs ..................................... 100
2.8.c. Evaluation of Capture Efficiency Variation ..................... 101
2.8.d. HR-MAS ‘H NMR Analyses ........................................... 101
2.8.e QCM Measurement ......................................................... 102
2.8.e. Syntheses of MGNPs .................................................... 103
1. Synthesis of sugar building blocks (Mannose
and Galactose) ......................................................... 103
2. Synthesis of AIkyne—Siloxane (AS) Linker for
Click Chemistry ......................................................... 108
3. Synthesis of magnetic Fe3O4 NPs and subsequent
Immobilization .......................................................... 109
2.8.f. Synthesis of Lipoic Carbohydrate Derivatives ................ 111
1. Synthesis of Lipoic Amine .......................................... 1 1 1
2. Synthesis of Lipoic Galactose (LGal) ......................... 113
3. Synthesis of Lipoic Mannose (LMan) ......................... 117
4. Synthesis of Lipoic Fucose (LFuc) ............................. 119
5. Synthesis of Lipoic Glucose (LGlc) ............................ 121
6. Synthesis of Lipoic Sialic (LSia) ................................. 123
7. Synthesis of Lipoic N-acetyl glucosamine (LGINAc) 125
References ............................................................................... 127

CHAPTER 3 Magnetic Glyco—Nanoparticles: A Tool to Detect, Differentiate,

3.1.
3.2.
3.3.
3.4.
3.5.
3.6.
3.7.
3.8.

3.9.

3.10.

3.11.

3.12.

3.13.

and Unlock the Gcho-Codes of Cancer via MRI .................. 132
Cancer Facts ........................................................................... 132
Cancer Detection and Diagnosis ............................................. 132
Glyco-conjugates as Cancer Biomarkers ................................ 134
Nanoparticles for Cancer Detection ......................................... 136
Synthesis and Characterization of MGNPs ............................. 137
Validation of MGNP Binding Specificities ................................ 139
Monitoring MGNP Binding by MRI ........................................... 142
Mammalian Cells Selectively Bound with MGNPs as

Detected by MRI ...................................................................... 145
Biological Implications of Carbohydrate-receptors

on Cancer Cells ........................................................................ 146
Establishment of MR Responses as Molecular Signatures for

Full Differentiation of All Ten Cell Lines via LDA Analysis ....... 148
Detection of Cancer Cells vs Normal Cells based on the MR
Signature ................................................................................. 1 50
Differentiation of Closely Related lsogenic Cancer Cells

including Metastatic Cancer Cells ............................................ 151
Cellular Uptake and Bio-compatibility of MGNPs .................... 152

ix

3.14. Cytotoxicity of MGNPs ............................................................. 154

3.15. Anti-adhesive Properties of MGNPs ........................................ 154
3.16. Experimental Section .............................................................. 157
3.16.a. Cells and Culture Conditions ....................................... 157
3.16.b. MRI and Relaxivity Measurement ................................ 158
3.16.c. Detection of Cancer Cells using MGNPs ..................... 158
3.16.d. Prussian Blue Staining ............................................... 159
3.16.e. Cell Viability ................................................................ 159
3.16.f. Anti-adhesive Assay ................................................... 160
3.16.9. Preparation of MGNPs ............................................... 161

1. Syntheses of Carboxylic Acid-functionalized Sugars 161
2. Synthesis of Alkynyl-GIcNAc (7) for Click Chemistry 168

3. Synthesis of Azido Siloxane Derivative (8) .............. 169

4. Synthesis of MGNPs (2-6) ........................................ 170

3.16.b. Characterization of MGNPs ........................................ 172

3.17. References .............................................................................. 175

CHAPTER 4 Hyaluronan Magnetic Gcho—Nanoparticles (HA-MGNPs)

for Detection and Molecular Imaging of Atherosclerosis 186

4.1 . Introduction .............................................................................. 186
4.2. Biological Significance of Hyaluronan and CD44 ................... 188
4.3. Atherosclerotic Plaque Detection using HA-MGNPs ............... 191
4.3.a. Plaque Imaging via MRI ................................................ 192
4.3.b. Nanoparticles as Imaging Agents ................................. 193
4.4. Synthesis of HA-MGNPs ........................................................ 196
4.5. HA-MGNPs as Nanoprobes for Diagnosis and Imaging
of Injured Rabbit ..................................................................... 199
4.6. Current Studies and Future Insights ....................................... 203
4.7. Conclusion ............................................................................... 207
4.8. Experimental Section ................................................................ 208
4.9. References ............................................................................. 214
Appendices Supplementary Materials ....................................................... 222
A1 NMR spectra for Chapter 2 ...................................................... 222
A2 NMR spectra for Chapter 3 ...................................................... 243

LIST OF FIGURES
Images in this dissertation are presented in color

Figure 1-1. Magnetic iron oxide nanocrystal with corresponding magnetic
domains and spinel crystal structure .......................................................... 9

Figure 1-2. Different types of magnetic materials and their spin responses in the
presence or absence of an external magnetic field (H) ................................. 11

Figure 1-3. Disorganization of spin close to the surface of a nanoparticle due to
lack of crystallinity organization, a phenomenon known as spin canting ........... 12

Figure 1-4. Synthesis of highly crystalline monodispersed nanocrystals via
thermal decomposition of metal-oleate complex; TEM images of 9 nm iron oxide
nanocrystals ....................................................................................... 17

Figure 1-5. Synthesis of highly crystalline monodispersed nanocrystals via
thermal decomposition of Fe(acac)3 in benzyl ether; TEM images of 10-12 nm
Fe304 nanoparticles .............................................................................. 18

Figure 1-6.72-weighted MR image (a) preinjection and (b) after 1-hour injection
of PVLA-coated SPIONs; (c) pre- and (d) post-injection of pyrroridone-coated
SPIONs. Significant signal drop (darkening) of liver compared to control
pyrroridone-coated SPIONs .................................................................... 31

Figure 1-7. Seed-mediated growth of gold nanoparticles ............................. 37

Figure 1-8. Various strategies for studying specific carbohydrate interactions that
mimic sugar presentation on the cell surface, thus representing important roles in
cell-cell adhesion and recognition ............................................................ 40

Figure 1-9. Inhibition of HIV binding to DC-SIGN using oligomannoside-
AuNP ................................................................................................ 43

Figure 1-10. Selective binding of mannose-AuNPs to the wild-type E. coli strain
ORN178. The mutant ORN208 showed no binding ...................................... 45

Figure 1-11.Schematic presentation of Pk-AuNPs for detection, separation and
inhibition of Shiga-Iike toxin .................................................................... 46

Figure 1-12. Specific effect of lacto-AuNPs on the metastatic potential of B16F 10
‘ to induce lung tumoral foci upon intravenous (iv) inoculation in C57/Bl6 mice. A
positive control group was injected with a single dose of B16F10 melonoma cells
(105 cells/animal). Other groups were treated with single doses of B16F10

xi

melanoma cells (105 cells/animal), preincubated for 5 min at 37 °C with 90 uM of
the control gluco—GNPs or with 90 (M of Iacto-GNPs. After three weeks, the
animals were sacrificed, and lungs were evaluated under a microscope for tumor
foci analysis. Inspection of lungs inoculated with B16F10 cells pretreated with
Iacto-AuNPs showed a strong protective effect against lung metastasis (~ 70%
inhibition) in contrast to those obtained from animals primed with B16F 10 cells or

B16F 10 cells pretreated with gluco-GNPs ................................................. 49
Figure 1-13. Construction of glycovirus. Growth of glycocluster amphiphile Gly8
or Gly5 through GNP to glycovirus and its aggregates....................................54
Figure 2-1. Syntheses of MGNPs ............................................................ 81
Figure 2-2. TEM image of MGNPs ........................................................... 82
Figure 2-3. XRD of MGNPs .................................................................... 83
Figure 2- 4. FT-IR spectra of bare Fe3O4 NP (brown), NP 1 (blue) and MGNP 3
(black) ................................................................................................ 84
Figure 2-5. TGA curves for NP 1 (blue), Man-MGNP 3 (pink) and Gal-MGNP 4
(green) ............................................................................................... 85
Figure 2-6. a) 1H-NMR spectrum of Gal-MGNP 4 acquired in solution with a
conventional 5 mm probe; and b) HR-MAS 1H NMR .of the same sample ......... 86
Figure 2-7. Schematic representation of MGNP-Con A interactions ................ 87

Figure 2-8. Fluorescent emission spectra of the supernatants of fluorescein-
labeled Con A solutions: (a) after incubation with MGNPs or NP 1 and
subsequent magnet mediated separation; (b) after incubation with NP 1 and
various amounts of MGNP 3 followed by magnetic
separation ....................................................................................... ..88

Figure 2-9. Schematic demonstration of E. coli detection by MGNPs .............. 90

Figure 2-10. Representative fluorescence microscopic images of captured E.
coli. The concentration of bacteria (cells/mL) incubated with MGNP 3 is indicated
on each image ..................................................................................... 91

Figure 2-11. TEM images of MGNP 3/E. coli complexes at different
magnifications. Arrows indicate where MGNP 3 binds to the pill, which were
faintly stained in the pictures. A drop of 1% phosphotungustic acid was used to
negatively stain MGNP/E. coli aggregates ................................................. 91

Figure 2-12. E. coli strain differentiation by MGNPs 3 and 4 ......................... 93

xii

Figure 2-13. Synthesis of Lipoic Carbohydrate Conjugates ........................... 96

Figure 2-14. A) Blocking effect of various lipoic acid carbohydrate derivatives. (a)
LGal, (b) LSia, (c) LGlc, (d) LGINAc, (e) LMan, (f) LFuc; B) Real-time frequency
responses to the addition of 2X104 Hela cells onto (a) the mixture of lipoic
galactose and BSA modified Au quartz and (b) PEG-thiol modified Au
quartz ................................................................................................ 98

Figure 2-15. Reagents and conditions: a) 3—chloro-1-propanol, BF3.Et20, DCM;
b) NaNg, DMF, 80 °C; c) succinic anhydride, H2/Pd-C, THF, 3h; d) NaOMe,
MeOH .............................................................................................. 103
Figure 2-16. Synthesis of Azido-mannose for Click Chemistry ..................... 105

Figure 2-17. Reagents and conditions: a) 3-chloro-1-propanol, BF3.Et20, DCM;
b) NaN3, DMF, 80 °C; c) succinic anhydride, H2/Pd-C, THF, 3h; d) NaOMe,

MeOH .............................................................................................. 106
Figure 2-18. Reagents and conditions: a) succinic anhydride, DMAP, DCM, 6h;
b) APTES, EDC, DCM, 12h ................................................................... 108
Figure 2-19. Reagents and conditions: a) EDC, Et3N, DCM, overnight; b) TFA,
DCM, 1 h .......................................................................................... 111
Figure 2-20. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA,
DMF, 3hrs; b) NaOMe, MeOH, 1h .......................................................... 113
Figure 2-21. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA,
DMF, 3hrs; b) NaOMe, MeOH, 1h .......................................................... 117
Figure 2-22. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA,
DMF, 3hrs; b) NaOMe, MeOH, 1h .......................................................... 119
Figure 2-23. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA,
DMF, 3hrs; b) NaOMe, MeOH, 1h .......................................................... 121
Figure 2-24. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA,
DMF, 3hrs; b) NaOMe, MeOH, 1h, then 0.2 mL water ................................ 123
Figure 2-25. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA,
DMF, 3hrs; b) NaOMe, MeOH, 1h .......................................................... 125
Figure 3-1. Syntheses of MGNPs .......................................................... 138

Figure 3-2. a) Benzoyl chloride, pyridine, CHzClz, 0 °C to r.t.,2 hrs; b) 3-
bromopropyne, K2003, acetone, reflux for 8 hrs; c) 6% MeOH in HCI, reflux for 2

xiii

hrs; d) N-acetyl glucosamine, 0.1 M acetate buffer (pH=6.5) I DMF (3:1), 50 °C,
24 hrs .............................................................................................. 139

Figure 3-3. Fluorescent emission intensities of supernatents of FITC-labeled a)
Con A, b) WGA, c) BS-I and d) TPA (100 ug/mL) upon incubation with various
MGNPs 2-6 or control NP 1 (1 mg/mL) followed by magnet mediated separation
(Aexcltation = 494 nm) .............................................................................. 141

Figure 3-4. a) Incubation of Man-MGNP 2 (shown as pink balls) with Con A, a
tetrameric mannose selective lectin (shown as blue rectangles) resulted in the
formation of aggregates, leading to shorter T2 relaxation time and consequently a
darkened MRI image. b) T2 weighted MRI images of Man-MGNP 2 (20 ug/mL)
and NP 1 (20 ug/mL) upon incubation with increasing concentrations of Con A. 0)
T2 changed linearly upon incubation of Man-MGNP 2 with increasing
concentrations of Con A, while incubation with NP 1 devoid of carbohydrates did
not lead to any change ......................................................................... 143

Figure 3-5. a) Percentage changes of T2 relaxation time (% AT2) obtained upon
incubating MGNPs 2-6 or the control NP 1 (20 pg/mL) with ten cell lines (105
cells/mL). The AT2 was calculated by dividing the T2 differences between
MGNP and MGNP/cancer cell by the corresponding highest AT2 from each
MGNP category. b) % AT2 obtained upon incubating MGNPs 2-6 or the control
NP 1 (20 uglmL) with ten cell lines (106 cells/mL). The above data represent the
averages of 8 individual measurements with the error bars showing standard
deviations ......................................................................................... 147

Figure 3-6. LDA plots for the first three LDs of AT2 patterns obtained with the
MGNP array upon binding with the ten cell lines (105 cells/mL). Full differentiation
of the ten cell lines was achieved ........................................................... 149

Figure 3-7. % AT2 obtained upon incubating MGNPs 2-6 or the control NP 1 (20
ug/mL) with a) breast cancer MCF-7/Adr-res vs normal breast cell 18485; b)
TA3ST vs TA3HA cell lines; and c) B16F1 vs B16F10 (105 cells/mL). Significant
differences in binding with MGNPs were observed differentiating these cell
fines ................................................................................................ 151

Figure 3-8. Prussian blue staining images of 20 ug/mL of a) Man-MGNP 2; b)
Gal—MGNP 3; c) Fuc—MGNP 4; d) GlcNAc-MGNP 6; and e) control NP 1
incubated with B16F 10 cancer cells after unbound particles were removed by
washing and f) B16F 10 cells. The images clearly indicated the high intracellular
uptake of Man-MGNP 2, Gal—MGNP 3 and Fuc-MGNP 4. No Prussian blue
stains were visible with the non-binding GlcNAc-MGNP 6 and the control NP 1
proving the selectivity in binding ............................................................ 153

xiv

Figure 3-9. As an example MTT assay of A498 cells incubated with Fuc—MGNP 4
showed no cytotoxicity after one week incubation, while Adriamycin killed most of
the cells ........................................................................................... 154

Figure 3-10. The adhesion of mouse melanoma B16F 10 (~ 5 x 104 cells/mL) to
the surface was significantly reduced by incubation with Gal-MGNP 3 (blue line),
while the control NP 1 (green line) had no effect on cell adhesion as compared to
cells without treatment with any NPs (red line). Error bars indicate standard
deviations (triplicate readings) ............................................................... 155

Figure 3-11. a) 33% HBr in AcOH, CHZCIZ, 2 hrs; b) 3-bromo-1—propanol, MS 4A,
Ang03, CH2CI2, 18 hrs; c) NaNg, DMF, 80 °C, 18 hrs; d) succinic anhydride,
H2/Pd-C, THF, 3 hrs; e) NaOMeIMeOH ................................................... 161

Figure 3-12. a) 3-chloro-1-propanol, NIS, TfOH, 3A MS, CH2CI2:CH3CN, -40 °C
to r.t. 3 hrs; b) NaN3, DMF, 80 °C, 12 hrs; c) succinic anhydride, H2/Pd-C, THF, 3
hrs; d) NaOMe/MeOH, 2hrs .................................................................. 164

Figure 3-13. a) Benzoyl chloride, pyridine, CH2CI2, 0 °C to r.t.,2 hrs; b) 3-
bromopropyne, K2C03, acetone, reflux for 8 hrs; 0) 6% MeOH in HCI, reflux for 2
hrs; d) N-acetyl glucosamine, 0.1 M acetate buffer (pH=6.5) / DMF (3:1), 50 °C,
24 hrs .............................................................................................. 168

Figure 3-14. a) Succinic anhydride, DMAP, CHzClz, 12 hrs; b) APTES, EDC,
CH2CI2, 8 hrs ..................................................................................... 169

Figure 3-15. TEM images of MGNP shows that NP core is ~ 5 — 10 nm ........ 172
Figure 3-16. FT-IR spectra of a-) Azido-NP b-) GlcNAc-MGNP 6 ................. 173
Figure 3-17. a) TGA curves for TEOS-NP 1 (light blue), Man-MGNP 2 (dark

blue), Gal-MGNP 3 (red), Fuc-MGNP 4 (violet), and Sia-MGNP 5 (green). b) TGA
curves for TEOS-NP 1 (blue), Azide-NP 9 (red), and GlcNAc-MGNP 10

(green) ............................................................................................. 1 74
Figure 4-1. a) Structure of hyaluronic acid (HA) ....................................... 188
Figure 4-2.Crystal structure of CD44 complexed with HA ........................... 190
Figure 4-3. Synthesis of HA-MGNP ....................................................... 197

Figure 4-4. TGA for HA—MGNP showed that ~ 90% of the particle weight was
from dextran and HA coating. Sample was heated at a constant heating rate of
20 °Clmin from 50 to 1000 °C under nitrogen .......................................... 198

XV

Figure 4-5. a) TEM of HA-MGNP (scale bar is 20 nm) and b) HR-MAS 1H-NMR
of HA-MGNP ..................................................................................... 199

Figure 4—6. Binding of HA-MGNP with rabbit artery tissues after removal of
unbound particles. a) healthy non-atherosclerotic tissue surface; b)
atherosclerotic tissue surface; c) cross section of the atherosclerotic tissue; and
d) MRI image of the atherosclerotic tissue ................................................ 200

Figure 4-7. MRI images of rabbit artery. a) in vivo image before HA-MGNP
injection; b) in vivo image after HA-MGNP injection; and 0) ex vivo image
following HA-MGNP injection and artery removal with the arrow showing
corresponding in vivo slice location ......................................................... 202

Figure 4-8. MRI images of rabbit artery. 3) in vivo image before Feridex injection;
b) in vivo image after Feridex (1.5 mg Fe/kg) injection; 0) in vivo image before
HA-MGNP injection; d) in vivo image after HA-MGNP (1.5 mg Fe/kg)
injection ............................................................................................ 203

xvi

LIST OF ABBREVIATIONS
Magnetic glyco-nanoparticles (MGNPs)
Escherichia coli (E. coli)
Magnetic resonance imaging (MRI)
Hyaluronan or hyaluronic acid (HA)
Hyaluronan-coated magnetic nanoparticles (HA-MGNPs)
Nanoparticles (NPs)
Enzyme-linked immunosorbent assay (ELISA)
Polymerase chain reaction (PCR)
Quartz crystal microbalance (QCM)
Surface plasmon resonance (SPR)
Dynamic light scattering (DLS)
Magnetic iron oxide nanoparticles (MNPs)
Polyethylene glycol (PEG)
Polyvinylpyrrolidone (PVP)
Polyglutamic acid (PGA)
Polyvinyl alcohol (PVA)
Van der Waals (vdW)
Transmission Electron Microscopy (TEM)
Thermogravimetric Analysis (TGA)
Fourier Transform Infrared (FT-IR)
High-Resolution Magic Angle Spinning (HR-MAS)

Nuclear magnetic resonance (NMR)

xvii

Photochemically activated phosphate perfluorophenylazides (PF PAs)
Colony-fonning unit (cfu)

Hyaluronic-dopamine (HA-DN)

Polyvinylbenzyl- O-B-D-galactose-D-gIuconamide (PVLA)
Superparamagnetic iron oxide nanoparticles (SPIONs)
sialyl Lewisx (sLex)

Gold nanoparticles (AuNPs)

Auric acid (HAuCl4)

Cetyltrimethyl ammonium bromide (CTAB)

Gold nanosphere (GNS)

Gold nanorod (GNR)

Tetraoctylammonium bromide (TOAB)
Ethylene-diamine tetraacetic acid (EDTA)
Self-assembled monolayers (SAMs)

Atomic force microscopy (AF M)

Isothermal titration calorimetry (ITC)

Mannose (Man)

Glucose (Glc)

Galactose (Gal)

Lactose (Lacto)

Maltose (Malto)

N—acetylglucosamine (GlcNAc)

Galactosamine (GaIN)

xviii

Sialic acid (Sia)

Fucose (F uc)

Cellobiose (Cel)

Concanavalin A (Con A)

Wheat Germ Agglutinin (VVGA)

Bandeiraea Simplicifolia isolectin (BS-l)
Tetragonolobus Purpureas Agglutinin (TPA)
Fluorescein isothiocyanate (F ITC)
Macromolecular aggregation factor (MAF)
Shiga toxins (Sts)

Cholera toxin (CT)

Dextran (Dex)

Reversible addition-fragmentation chain transfer (RAFT)
Quantum dots (QDs)

Lymphatic vessel endothelial receptor 1 (LWE-1)
Lymphatic endothelial cells (LEC)
Single-walled carbon nanotube (SWNT)
poly(lactide-co-glycolide) (PLGA)

Ehrlich ascites tumor (EAT)

Intravenous (IV)

Chitosan (CS)

Human corneal epithelial (HCE)

Mitomycin C (MMC)

xix

Doxorubicin (DOX)

Tetraethoxysilane (TEOS)

Aminopropyltriethoxysilane (APTES)

Alkyne-siloxane (AS)

Double deionized water (DDVV)
1-[3—(Dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride (EDC)

Benzotriazole—1-yI-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate
(BOP)

2-(1 H-benzotriazole-1-yl)-1 ,1 ,3,3-tetra-methyluronium hexafluorophosphate
(H BTU)

1-Hydroxybenzotriazole (HOBT)
Diisopropylethylamine (DIPEA)
Dimethylfonnamide (DMF)
Dichloromethane (DCM)

Computed tomography (CT)

Optical coherence tomography (OCT)
Ultrasound (US)

Positron emission tomography (PET)
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
Time of echo (TE)

Region of interest (ROI)

Extracellular matrices (ECM)

Smooth muscle cells (SMCs)

HA-binding domain (HABD)

XX

CHAPTER 1

Glyco-Nanoparticles: Translating Insights from the
“Sugar-Code” into Diagnostics and Therapeutics

1.1. Introduction

Rapid and sensitive detection of infectious pathogenic agents, cancer, and
atherosclerosis is critical for the prevention of such diseases and bioterrorism.
Although biosensor technology have been extensively studied1 and carefully
translated to “point of care” devices at the clinical level, nano-biosensorsz' 3 are
actually in the early stages of development and their major potentials remain to
be exploited. As the understanding of nano-fabrication, the control of nano-
synthesis and the “cell-nanoparticle bio-conversation” become more advanced,
major problems relating to biological detection will be solved.

Glyco-nanotechnolgy is gaining more eminence as the next great frontier
of modern science.4' 5 Recently, nano—based glyco-sensors have attracted a
great deal of attention owing to their carbohydrate functionality, small sizes,
polyvalency, biocompatibility, simplicity, as well as beneficial optical, electronic
and magnetic properties. Knowing that the surfaces’ of cells, microorganisms,
pathogens, and viruses have either carbohydrates or carbohydrate receptors, it is
of tremendous importance to use this natural phenomenon for sensing, detection
and identification purposes. Thus, glycoscience is a very instructive example of
how one common topic of interest stimulates both chemistry and biology to
collectively open novel scientific frontiers?"8 In spite of this great potential, nano-

based glyco—sensing, diagnosis and therapeutics are yet to be fully explored.

In fact, there is a tremendous need for developing biosensors for selective
detection of biomolecules which can be more sensitive, less time- and labor-
consuming. Herein, a novel magnetic carbohydrate-based nano-sensing system
for the detection of pathogens, cancer and atherosclerotic plaques was
developed and evaluated. Our system incorporates the use of sugar-coated
magnetic iron oxide nanocomposites as innovative “detecting and imaging"
agents with unique properties. This work will be divided into three parts, which

will be discussed in three separate chapters. Briefly, we explored the following:

1) atho en detection: synthesis of magnetic glyco-nanoparticles (MGNPs) not
only for fast Escherichia coli (E. coli) detection and enumeration, but also for

strain differentiation and efficient pathogen decontamination.

2) cancer detection: fabrication of monosaccharide-functionalized MGNP array

 

for sensitive detection and differentiation of nine types of cancer cells using

magnetic resonance imaging (MRI).

3) atherosclerotic detection: engineering of highly colloidal, monodispersed and
superparamagnetic hyaluronan-coated magnetic nanoparticles (HA-MGNPs) as
targeted MRI contrast agents for the detection and imaging of atherosclerotic

plaque in vivo.

1.2. The “Glyco” Perspective

Signal transduction and appropriate communication between two cells are
chief factors for proper cell growth. Indeed, the "bio-conversation" between two
cells involves a messenger from the sender (called a ligand) and a site on the
cell surface (called a receptor) receiving the signal. When the signal is received,
it is passed along within the cell. Carbohydrates are one of the most common
cell-surface ligands that direct the initiation of many medicinally important
physiological processes where they intervene in a wide variety of events,
including inflammatory and immunological responses, tumor metastasis, cell-cell
signaling, apoptosis, adhesion, bacterial and viral recognition, and
anticoagulation.9'12

Glyco-recognition is generally a multivalent process. Carbohydrates bind
Iectins (carbohydrate-binding proteins) in a cooperative manner improving the
typically weak affinity of monomeric sugar ligands to their Iectins.13 Accordingly,
the recognition of glycosyl residues on the cell surface requires the clustering of
surface receptors, a phenomenon referred to as the “cluster-glycoside effect".“
Indeed, this cluster or polyvalency effect is proved to be responsible for
enhancing the binding affinities by several orders of magnitude.

All these characteristics are particularly attractive as they offer an
alternative non-routine identification of biological targets. Routine discrimination
of bio-analytes is primarily performed using antibodies. However, antibody
based-sensors are commonly prone to protein denaturation. Moreover,

antibodies are specific for one particular target and are of the “key-lock” identity,

possessing minimal cross-reactivity for other biomarkers.15 Moreover, extensive

3

 

prior knowledge on specific target is required, which can be very time-
consuming. Carbohydrates do not denature nor lose activity. They are smaller
than antibodies which allow higher ligand-immobilized densities, better uniformity
and conjugation on the nano-surfaces. In response to all these observations,
extensive work has been conducted for conjugating of multivalent carbohydrates
to the scaffolds of polymers,16 liposomes,17 dendrimers,18 beads,19 and recently
nanomaterials.20 The ability to architect different sugars on nano-surfaces in a
controlled polyvalent fashion can lead to better understanding of the
carbohydrate-recognition events that promises to have diagnostic and
therapeutic potentials and may uncover the important “sugar code” in cellular

interactions.

1.3. The “Nano” Perspective

Nanomaterials are promising platforms for carbohydrate display and have
recently attracted much attention where extensive efforts have been devoted to
methodological studies toward their synthesis and their surface modifications.”24
In fact, advances in nanoresearch have led to the development of novel
nanoparticles where size, geometry and surface functionality can be controlled at
the nanoscale.2527 Diverse metal and semiconductor core materials have been
used to construct nanoparticles (NPs) with important physical and electronic
properties ranging from high optical extinctions (gold),28 stable photoemission
(quantum dots),29 superparamagnetism (iron oxide),30 and surface-enhanced

Raman effect (silver and gold)“ 32 The constructed NPs, because of their small

nanometer sizes, possess novel electronic, optical and structural properties

 

rendering them excellent vehiclesfor biological applications at the cellular and
molecular level. When coupled to affinity ligands, such NPs can function as
biological mediators. However, to be suited for biomedical applications, several
features should be fine-tuned including synthesis, magnetic, electronic
properties, stability, characterization and targeting specificity (which will be
discussed in details in the upcoming sections).

With all the advances in this field, NPs still present a challenging research
area since little is known about their behavior and interaction with
microorganisms, particularly at the cellular and molecular level. The question that
remains to be answered is: How do microbial and mammalian cells respond in
the presence of surface-modified nanoparticles? It will be ~ 10 years from now
to fully understand the exact mechanisms of how NPs network with the cells and
biological entities in vitro as well as in vivo. Size, shape and surface charge are
among the most prominent factors that can influence this special “bio-
conversation”. Other features, such as surface modification (hydrophilicity and
hydrophobicity), density, molecular weight, and crystallinity direct their targeting,
sensing and electronic properties, which significantly affects the interactions with

biological environments.

1.4. Glyco-nanotechnology

Extensive work has been done using carbohydrate-functionalized
polymers to detect Iectins,33 virus?’4 and bacteria.16 Moreover, carbohydrate-
microarrays have been employed to study carbohydrate-carbohydrate

interactions, investigate the carbohydrate-binding specificities of bacteria, detect

 

pathogens, and screen anti-adhesive therapeutics.” “‘39 Seeberger et al.
reported the use of carbohydrate microarray to detect strain-specific differences
in binding bacteria in complex biological systems.36 Glyco-nanotechnology have
emerged as promising machinery to prepare, study and evaluate saccharide-
coated nanoparticles to better investigate carbohydrate-binding recognition
events at both the cellular and molecular level.” 4‘ Glyco-nanoparticles combine
the properties of nanometer-scale objects with the unique architecture of the
polyvalent-carbohydrate display, greatly enhancing the weak affinities of
individual ligands to their binding partners. Although numerous reports of
nanoparticles functionalized with carbohydrates as stabilizing agents have been
reported,“47 few reviews have focused on NPs immobilized with biologically-
relevant carbohydrates that have shown to play key role in the recognition
processes affecting the fate of the nanoparticles and its specificity to certain
receptors. Critical to the performance of glyco-nanomaterials is the proper
display of carbohydrate ligands, taking into consideration the coupling chemistry,

the type and length of the spacer linkage, and the ligand density.

1.5. Nana-biosensors

Important progress has been reported using various nano-biosensors to

52-55 56-59

detect DNA,48 proteins,” 5° viruses,“’1 pathogens, cancer cells, and
atherosclerotic plaques.” 6‘ Of the various substances employed for biosensing
applications, glyco-sensors62 have become a major target because of the

significant molecular characteristics of carbohydrates in living systems.

Generally, biosensors used for detection employ a substrate comprising a
ligand that binds to a receptor producing a detectable signal. In nanosensors,
the substrate is usually a nanocomposite made of any of iron oxide (magnetic),
gold and silver (cptical), silica, carbon nanotubes, or quantum dots.63 The most
common ligands employed are antibodies,‘54 proteins or peptides,55
oligonucleotides,49 or carbohydrates66 to name few. The functionalized-
nanocomposites, thereafter, may be used for the recognition of specific targeted
receptor, e.g., to detect specific enzymes, tumor cells, signaling molecules,
infected sites and proteins. Current available methods for detection of pathogenic
bacteria and disease biomarkers include optical, colorimetric and fluorescent
assays, enzyme-linked immunosorbent assay (ELISA), polymerase chain
reaction (PCR) approaches, quartz crystal microbalance (QCM) analyses,
surface plasmon resonance (SPR), dynamic light scattering (DLS), and
electrochemical methods." 67 Despite these wide choices of platforms, important
challenges still remain in minimizing sensor size, reducing detection time,
eliminating target-labeling and developing simple and inexpensive fabrication
protocols. The urgent necessity of biological detection will expectantly lead to
more economic, simple to use, and versatile sensors that will have a great impact
on biomedical applications.68 Recently, on the diagnostic front, biocompatible
magnetic iron oxide nanoparticles (MNPs) attracted much attention as they can
be used not only as carriers in immunoassay,49 biosensing,69 and bioseparation70
but also as drug delivery vehicles59 and MRI contrast agentsse. Moreover,

MGNPs were also shown to be ideally suited for these purposes where their

 

biological applications highlight the important roles they play in glycoscience and
biomedicine.5

To extend the scope of biomedical research on carbohydrate—mediated
molecular recognition and anti-infection responses, the studies of carbohydrate-
based nano-systems will be reviewed. The use of magnetic glyco-nanomaterials
in bio-separation, bacterium detection, cell interactions, diagnosis, imaging and in
vivo applications will be discussed. A special section on the preparation,
synthetic insights, and functionalization of iron oxide NPs will be thoroughly
discussed as those were the nanocomposites used in our study. Iron oxide NPs
offer grand advantages due to their ability to respond to magnetic fields making

them suitable for molecular imaging, biological and medical applications.

1.6. Magnetic Iron Oxide Nanoparticles (MNPs)

MNPs have the potential to revolutionize chemical sectors, biotechnology
industries, and the medical fields. Of the various magnetic nanocrystals studied,
magnetite (Fe304) and maghemite (y-Fe203) nanomaterlals protected with a

71-74

polymeric coating, proved to be superb nanoprobes for both in vitro and in

ViVOsg' 64, 65, 75, 76

applications. Due to their intrinsic magnetic properties, iron oxide
NPs have been the subject of intense investigation as diagnostic and therapeutic
agents. Some of the chief properties of iron oxide nanoparticles are: a)
biocompatibility; b) superparamagnetism; c) imagebility (via MRI); d) uniformity
(control over size and shape); e) functionality (surface modification), and f)

colloidal stability. To better understand the behavior of iron oxide nanoparticles, it

is of immense significance to understand their properties in more details.

 

MNPs consist mainly of two central parts: a) a magnetic core having one
or more magnetic Fe304lFe203 crystals, and b) a polymer coating associated
with the core. The crystalline structures of magnetite have the general formula
Fe2(3*’03.Fe(2+)O. Each iron oxide crystal is made of magnetic domains and has
inverse spinel structure with oxygen ions forming a close—packed face-centered
cubic lattice and Fe cations occupying interstitial tetrahedral and octahedral sites
(Figure 1-1). In magnetite, magnetization arises from electron hopping between

ferrous (Fey) and ferric (Fe3*) ions that coexist at the octahedral sites.

‘5.

‘5.

 

 

\‘\ C. D.
-- 4 __ 1:13 “-333. .0 0.0 ..
ate; ., \;::-; x )0.o.o.o.c
r.~~‘~'/ '3'" .4.-N? . i
..-.iv-r' 1).} s - ‘ .
~ ...:43 ;~.::§\./ii o'o'o'o'o'
is; , \~..--*...\ My . . . . .
....r” K‘, f; w.
“New B '.'.' C
(A) (B) (C)
0 Oxygen Magnetic domains 0 Iron oxrde crystals
9 Ferrous ion (Fe2”) 0 Coating
o Ferric ion (Fe3*)

 

 

 

Figure 1-1. Mag/netic iron oxide nanocrystal with corresponding magnetic domains and spinel
crystal structure. 7

The “polymer coating” is a natural, or synthetic polymer that functions to
keep the metal oxides dispersed and colloidal. If they are used for in vitro
applications, good chemical stability is necessary and adequate. For in vivo
applications, besides having the proper constituents, good control over size,
stability, biocompatibility, and biodegradability are required. Thus, a brief

discussion of some of the basic concepts of magnetism and synthetic pathways

is needed to better support the use of MNPs and understand their function in

driving certain processes.

1.6.a. Magnetic Properties

Iron oxide nanoparticles have unique magnetic properties that allow them
to move in high magnetic field gradient making them useful in areas of bio-
separation, detection, imaging and hence medicine.

In fact, all materials are magnetic to a certain extent, with their response
depending on their atomic structure and temperature. Most materials display
small magnetism, even in the presence of an applied magnetic field (H). These
are classified as either diamagnetic or paramagnetic (Figure 1-2). However,
some materials exhibit ordered magnetic states and are magnetic even without a
field applied, and these are classified as ferromagnetic, ferrimagnetic, and
antiferromagnetic (depending on the nature of the coupling interaction between
the spins within the material). The classification of a material's magnetic
properties is based on its magnetic susceptibility (x), which is defined by the ratio
of induced magnetization (M) l (H).

In diamagnetic materials, the magnetic moment is anti-parallel to H
resulting in very small and negative susceptibilities. Diamagnetic materials do not
retain magnetic properties when the external field is removed. Paramagnetic
materials, on the other hand, have their magnetic moments aligned parallel to H
and higher susceptibilities. While in ferri- and ferromagnetic materials, magnetic
moments also align parallel to H, coupling interactions between the electrons of

the material result in ordered magnetic domains and large spontaneous

10

 

magnetization. Superparamagnetism is exhibited by small ferromagnetic or

ferrimagnetic nanoparticles.

 

 

H = o _l"___. H = o _'1_.
0000 @999 cede 9663
0000 case Qa¢¢ 9699
0000 @696 (bdbdb? 666-8
0000 ©©®© game 43-99-43

Diamagnetic materials Paramagnetic materials

H = 0 H = 0 H =.O

eeee~ 9969 9669.

66-90 O 6 © 9 9'6 69

6690 9 © 9 © '6 69 9-

6-9-69 © 6 63-9 96 99

Ferromagnetic Ferrlmagnetic Antiferromagnetlc

 

 

Figure 1-2. Different types of magnetic materials and their spin responses in the presence or
absence of an external magnetic field (H).78

When the size of the NPs is below a critical value (~15 nm), the individual
nanoparticles behave as a single magnetic domain exhibiting superparamagnetic
behavior. Multiple domains in one particle, therefore, maintain one large
magnetic moment. At low temperature, nanoparticles self-organize in solution
where all magnetic spins align. At adequately high temperatures (i.e. block
temperature TB), thermal energy is sufficient to induce free rotation of the
particles resulting in a loss of net magnetization in the absence of an external
magnetic field. Application of a magnetic field can cause the magnetic dipoles to
reorient. Thus, superparamagnetic nanoparticles respond rapidly to an applied
magnetic field, but exhibit negligible residual magnetism away from the magnetic
field. Superparamagnetism is essential for the synthesis of stable colloidal

nanoparticles, as the non-alignment of spin limits the inter-particle attraction that

11

would othenivise occur. These features make superparamagnetic nanoparticles
attractive for a broad range in medicine.

In brief, the main advantages of using small iron oxide nanoparticles (~15
nm) are their superparamagnetism, higher effective surface areas (more surface,
more area for reactions), lower sedimentation rates (high stability) and improved
cellular and tissue diffusion. The small particle size, however, has some
consequences. As particle sizes decrease, surface area/volume ratios increase
which can dramatically changes the magnetic properties. Typically, the saturation
magnetization (M) values of nanoparticles (corresponding to the complete
alignment of all individual moments), decrease with NP size due to disordered
crystal structure resulting from high surface curvature. The magnetic spins of
atoms close to the surface are less organized than for those in the bulk near the

core, a phenomenon known as “spin canting” (Figure 1-3).

  
 

l “ ' " Canted spin
Aligned spin "‘" .'

Figure 1-3. Disorganization of spin close to the surface of a nanoparticle due to lack of
crystallinity organization, a phenomenon known as spin canting.79

All the above discussed phenomena arise from finite size and surface
effects that dominate the magnetic behavior of iron oxide NPs. Thus, detailed

insights into the most-widely used synthetic methods for NPs along with factors

12

affecting their size, stability, surface charge and shape are extremely important

and will be discussed.

1.6.b. Synthesis of MNPs

Particular attention should be paid to the synthetic methods of iron oxide
nanoparticles as they can significantly affect the size, shape, magnetic properties
and hence the fate of nanocomposites in practical applications. Numerous
chemical methods can be used to synthesize such particles: co-precipitation,
thermal decomposition, microemulsions, sol-gel syntheses, hydrothermal
reactions, and spray pyrolysis to name few. All those techniques were excellently
reviewed.” 23 The following central features dictate the success of any of these
methods to be commercialized. The synthesis of monodispersed (i.e. uniform-
size variation <5%) nanocomposites is the first main chemical challenge where
the properties of NPs depend strongly on their dimensions. The second key
factor consists of defining experimental conditions, leading to stable and colloidal
suspensions of magnetic nanoparticles of suitable size. The third critical point is
to select a reproducible process that can be industrialized. Most commonly, iron
oxide nanoparticles are prepared by co-precipitation of ferrous and ferric salts
solution in a basic media stabilized using biocompatible surfactant“: 80' 81 Target
biomolecules can then be attached by covalent or electrostatic coupling to the
protected nanoparticle. Alternatively, high-temperature thermal decomposition
(> 200°C) of organo-metallic precursors in the presence of surfactants has been
successfully employed to produce iron nanocrystals with marked improvements

in size and shape control, size distribution and crystallinity.” 82'“ Herein, I will

13

focus on those two most extensively studied methods that were successfully

used in clinical applications and were explored in our lab.

1) Co-precipitation Method

The co-precipitation technique is probably the simplest, easily scalable
and most efficient chemical pathway to obtain magnetic nanoparticles with a
good control in size, shape and uniformity. Iron oxides, either Fe304 or y—Fe203
are usually prepared via the Massart method85 by aging appropriate
stoichiometric ratios of Fe2+ and Fe3+ salts in an aqueous basic media (usually
NH4OH or NaOH). Usually hydrolysis of ferric ion (solvated cations) will occur

with the formation of oxo—ligands.
Fe2+ + 2Fe3” + 8OH‘ ——> FegO4 + 4HzO

The magnetic nanoparticles thus produced are spherical, homogenous in
size and shape, hydrophilic and negatively charged in an alkaline medium. Their
yield, size, stability and polydispersity depend on many factors: the addition of
the base, pH value, nature and concentration of the counterions, and Few/Fe2+
ratio. According to the thermodynamics of this reaction, complete precipitation of
Fe304 in the presence of a base should be expected at a pH (~10-14), with a
stoichiometric ratio of (Fey/Fe2+ 2:1) under nitrogen. It was observed that purging
nitrogen not only protects against oxidation of the magnetite to maghemite but
also reduces the particle size. An increase of the mixing rate also tends to
decrease the particle size. Synthesis under vigorous stirring results in the

formation of small particles by reducing their tendency to agglomerate. In the

14

 

same way, a decrease of the size and polydispersity is noted when the base is
added to the salt mixture as compared to the opposite process. Moreover, the
size is strongly dependent upon the acidity and the ionic strength of the
precipitation medium. The higher the pH and ionic strength, the smaller the
particle size and size distribution will be. These parameters determine the
chemical composition of the crystal surface and consequently the electrostatic
surface charge of the particles. The shape variation is also related to the
variation of the electrostatic surface density of the nanoparticles.

In an alkaline medium of pH>8, polarizing or highly charged cations or
anions, such as ammonium or alkaline may give rise to flocculation (the process
by which NPs clump together). The addition of chelating organic anions (i.e.
carboxylate ions such as acrylic or oleic acid) or surface complexing polymers
(e.g. dextran, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP),
polyglutamic acid (PGA), or polyvinyl alcohol (PVA)) during the formation of
magnetite can help control the distribution, size and stability of the
nanoparticles.” According to the molar ratio between the organic ion and the iron
salts, the chelation of these organic ions on the iron oxide surface can either
prevent nucleation and then lead to larger particles or inhibit the growth of the
crystal nuclei, leading to small nanoparticles. Ideally, the coating does not only
protect and stabilize the core but also makes the nanoparticles biocompatible
and make this method especially appropriate for in vivo applications.” 87

Magnetite nanoparticles can also be stabilized with silica, especially

aminosilanes, to form well~dispersed magnetic silica nanospheres.”91 An

15

 

advantage of silica coating is the established surface chemistry for silica surface
modification. The surface hydroxyl groups can be chemically modified to afford
different bio-conjugation groups, such as amines and carboxylates.92 The size of
the particles can be controlled by changing the silica/iron oxide ratio.88 Surface
functionalization of MNPs is extremely important not only for stability purposes,
but also to increase the targeting—proficiency. Finding means to control the size

while maintaining stable suspensions is the subject of intense study.

2) Thermal Decomposition Method

Several researchers report the use of elevated reaction temperatures and
suggest its significance in optimal crystal formation and hence higher
magnetization. Different studies show that enhanced uniformity with better size
distributions occur at high temperatures. The investigations support the theory of
nucleation and growth of the particles that will be discussed in the upcoming
section.

Among the many available methods, thermal decomposition of
organometallic compounds (iron-oleate complex) in high-boiling-point nonpolar
organic solvents (benzyl ether) has proven to be an attractive route for the
synthesis of highly monodispersed nanocrystals with high yield, uniformity, good
crystallinity and reproducibility. Hyeon et al. first synthesized superparamagnetic
maghemite y-Fe203 nanocrystals via a high-temperature aging of iron—oleic metal
complex using iron pentacarbonyl (Fe(CO)5) in the presence of oleic acid at
1 00 °C (cited by more than 530 publications).83 In 2004, instead of using the toxic

and expensive Fe(CO)5, they reported the elegant ultra-Iarge-scale synthesis of

16

monodispersed nanocrystals via thermal decomposition of metal-oleate

precursors in high boiling solvent (Figure 14).”

Metal Chloride Metal -oleate Thermal decomposition i;
+ Na-oleate complex high boiling point solvent

 

 

 

Figure 1-4. Synthesis of highly crystalline monodispersed nanocrystals via thermal
decomposition of metal-oleate complex; TEM images of 9 nm iron oxide nanocrystals. 3

Later, Sun et al. reported the synthesis of highly monodispersed magnetite
Fe304 nanoparticles (cited by more than 450 publications) from a high-
temperature (200-300 °C) 1,2-hexadecanediol solution of iron(lll) acetylacetonate
(Fe(acac)3) in the presence of oleic acid and oleylamine (Figure 1-5).27 Metal
ferrites (MFe204, where M=Co, Fe, Mn, etc.) were also synthesized in the form of
monodisperse cubic and spherical nanoparticles by the seed-mediated growth

process using Fe(acac)3 and M(acac)2 as reactants.82

3) Ligand Exchange
Despite its success, high-temperature therrnolysis typically produces
nanocrystals with hydrophobic surfaces and hence only dispersible in nonpolar

organic solvents. The resulting insolubility in water greatly limits their biological

l7

 

applications. It is therefore of great importance to find an effective approach to
tune their solubility and make them fully dispersed in aqueous media. One of the
most popular methods is “ligand-exchange”,94 where the hydrophobic chains on

the particle surface are replaced by molecules containing polar groups.

0
M(acaC)2 "I" Fe(acac)3 ROH SOIVent, 200 C
RCOOH then, refluxing
RNH2

 

of Fe(acac)3 in benzyl ether; TEM images of 10-12 nm Fe3O4 nanoparticles.82

At room temperature, the exchange ratio is typically low, leading to
irreversible desorption of the new surfactants from the particle surface
destabilizing the system and resulting in subsequent aggregation. Moreover, the
efficacy of exchange is usually not that high (hydrophobic surface ligands are not
fully exchanged). As a result the nanoparticles will not be fully dispersed in water
and precipitation might occur. Indeed, surface modification by ligand-exchange is
usually more complicated due to the dynamic nature of the outer layer coating
and the different stabilization factors that will be discussed later. That’s why
researchers tend to use amphiphilic65 or tri-block polymers29 or further stabilize

the polymer layer through cross-linking,95 making it well-suited for subsequent

18

chemical modification. Here, it is worth mentioning that we explored a robust

ligand-exchange method that will be discussed in Chapter 4.

1.6.c. Mechanistic Insights

Although there are numerous reports on the synthesis of iron
nanocomposites, little on the understanding of the synthesis mechanism and
control of uniformity are present.

Crystal growth typically follows an initial stage of either homogeneous
(without the influence of foreign particles) or heterogeneous (with the influence of
foreign particles) surface-catalyzed nucleation. The creation of a nucleus implies
the formation of an interface at the boundaries of a new phase. In a
homogeneous nucleation, a short single burst occurs when the concentration of
constituent species reaches critical supersaturation. Then, the nuclei are allowed
to grow uniformly by diffusion of solutes from the solution to their surface until the
final size is attained. To achieve monodispersity, these two stages must be
separated and nucleation should be avoided during the period of growth. For
heterogeneous nucleation, the seeded-growth technique is effective in providing
good size-controlled nanocrystals. Here, the uniformity of the initial nanocrystal
seeds determines the monodispersity and uniformity of the final product. Indeed,
mechanistic studies indicate that both heterogeneous and homogeneous
nucleation followed by growth can lead to the formation of monodispersed
nanocrystals.

As mentioned earlier, thermolysis in a high boiling-point solvent can result

in the controlled synthesis of monodisperse nanocrystals. This involves a burst of

19

homogeneous nucleation followed by a diffusion controlled growth process.
Recently, a detailed study of the shape control and formation mechanism of
monodisperse magnetic ferrite (MFezO4) produced by thermolysis has' been
investigated.96 Researchers were able to reproducibly control the shape and the
size of the nanocrystals by varying three conditions: a) concentration of
precursors, b) heating rate and c) aging time. In brief, for reaction temperatures ~
250-320 °C, the nucleation and growth dynamics dictate the size and shape
evolution of the nanocrystals. Prenucleation of MFe204 occurs at ~ 250-300 °C
but without any growth of nanocrystals. Heating between 300-320 °C, results in
homogeneous nucleation. Moreover, the shape of MFeZO4 nanocrystals can be
reproducibly controlled by prolonging the aging time at 320 °C. Practically,
mixing can be done in 2 means: Rapid addition of reactants into a preheated hot
solvent (high degree of supersaturation), or heating the reactants premixed with
the solvent gradually (careful control of the heating rate). Both ways can help
control the degree of supersaturation. The point is to accomplish a homogeneous

nucleation separated from the growth process.

1.6.d. Colloidal Stability

“Dispersion or Colloidal Stability” is one of the most impOltant factors that
dictate the fate of nanoparticles in aqueous and physiological conditions. “Bare"
iron oxide nanoparticles are not stable in water at neutral pH or in physiological
fluids, tending to aggregate and precipitate quickly. Nanoparticles are likely to
agglomerate in order to reduce their surface energy by strong magnetic dipole-

dipole attractions between particles. Thus, either steric or electrostatic

20

 

stabilization is required to ensure stable aqueous dispersions which are usually
achieved by different range of functionalities. Hence, colloidal stability is directly
correlated to polymer or surfactant coating grafted on the iron oxide core. Indeed,
stability has been a major topic of study, and there is still much to be understood
concerning the origin and nature of interparticle and intraparticle forces and how
they affect coagulation in dilute dispersions.

At the nanoscale dimensions, nanoparticles exhibit different properties
from their bulk where forces of a different nature become significant: attractive
long-range van der Waals (vdW) interactions, repulsive short-range interactions
(steric stabilization) and electrostatic forces. The overall effect of the three forces
buffers the particles against irreversible aggregation or adsorption to surfaces.

To produce stable nanoparticle dispersions, surfactants which provide
control over the forces, miscibility and steric repulsion must be employed. This
repulsion is largely entropic in nature, countering for long-range vdW attractions,
and highly dependent on the chain and its mobility. Unfortunately, metal
nanoparticles typically have facets which encourage surfactants to assemble into
closed-packed domains, resulting in low chain mobility where short-range steric
repulsion is overwhelmed by vdW attractive forces. This leads to poor control
over dispersion stability and particle aggregation. Entropic steric repulsion can
be greatly increased by modulating the nature and density of the surfactant or
polymer. Two dispersant qualities for an ideal choice of a polymer are: a)
polyvalent hydrophilic groups for adsorption to nanoparticle surface; b)

hydrophobic linkers intercepted by hydrophilic cationic or anionic groups to

21

ensure high degree of configurational freedom, which translates into effective
entropic steric repulsion in addition to adequate electrostatic repulsion regardless
of the molecular packing density on the nanoparticle surface.

The role of polymers on stability is more complicated than electrostatic or
steric stability due to many other factors such as solvent effects and
polyelectrolytes present. In brief, there is a combination of electrostatic effects as
well as effects that arise from the polymeric nature of the additive; this combined
effect is referred to as electrostatic stabilization. Moreover, as mentioned earlier
there is steric stabilization since the polymer molecules are adsorbed or
anchored on the particle surfaces. As a rule of thumb, the higher the potential at
the surface of a particle, the larger the repulsion between the particles, the more

stable the colloid is.

1.6.e. Characterization of MNPs

An important challenge in the area of nanomaterlals is the ability to
characterize the structure of the capping agents on the surface of NPs.
Identifying the size, shape, charge density, surface functionalities and getting
deeper insights into the structural properties and hence the behavior of iron oxide
nanoparticles would not have been possible without the following powerful
characterization techniques. The following techniques are used to study the
structure and organization of different organic/inorganic materials on the surface

of NPs.

22

1) Transmission Electron Microscopy (TEM)

TEM is the most common technique used to examine the size of the
crystalline core, the morphology and shape of nanoparticles. TEM is used
because of the limited image resolution in light microscopes imposed by the
wavelength of visible light. Electrons have wave-like characteristics, with a
wavelength substantially less than visible light. TEM visualize objects using thin
beam of rapidly moving electrons that encounter a specimen where the
accelerated emitted electrons can be absorbed, scattered or transmitted.
Because different regions of sample are variously transparent to electrons,
different amount of electrons with changed energies pass through these region.
This difference is responsible for the contrast. Since electrons are smaller than

atoms, TEMs are capable of resolving atomic level detail.

2) Thermogravimetric Analysis (TGA)

TGA registers the loss or gain in weight of immobilized ligands or polymers
(organic constituents) of a nanocomposite during heating. The variation in weight
as a function of temperature is recorded showing how much of the organic
substance is lost from the composite material. The change in weight of an
untreated nanoparticle would be compared to that of a coated nanoparticle,
which can then be used to calculate the average number of functionalized

molecules on the surface of the metal core.

23

3) Fourier Transform Infrared (FT-IR)

In infrared spectroscopy, radiation energy is absorbed by organic
molecules and converted into energy of molecular vibration. There are 2 types of
molecular vibrations, stretching and bending. Only those vibrations which result
in a change in the dipole moment of the molecule are observed in IR. Although
IR is useful to characterize organic molecules functionalized on nanoparticles
and gives qualitative information about the functional groups, however it remains
difficult to fully identify the structural information of the functionalized ligand. Thus,

other spectroscopic methods are necessary for accurate characterization.

4) High-Resolution Magic Angle Spinning (HR-MAS) NMR

A major hurdle in magnetic nanoparticle biofunctionalization is the ability to
elucidate the chemical structure of the anchored organic ligands on the surface
of the particles. Nuclear magnetic resonance (NMR) is a powerful tool if properly
utilized to obtain detailed structural analysis of surface ligands on nanomaterials.
However, study of ligands on magnetic materials by 1H-NMR is difficult due to
large broadening effects caused by the paramagnetic particles that produce field
inhomogeneity. However, relatively sharp NMR resonances are possible by the
use of high-resolution magic angle spinning NMR. HR-MAS NMR enables the
fine-structure-resolved characterization of complex organic molecules bound to
magnetic nanocomposites by strongly decreasing the effects of paramagnetic
disturbances.97 Compared to conventional probes, in HR-MAS it is possible to
work with considerably higher concentrations, thus avoiding the loss of structural

information.

24

5) Dynamic Light Scattering (DLS)

DLS also known as quasi-elastic light scattering, is a technique used
widely for particle size and size distribution studies. DLS measures the overall
hydrodynamic size (diameter of core spheres with their adsorbed and solvated
coating materials) of the nanoparticles in suspension or aqueous media. This is
different from TEM that measures only the core size (diameter of the core in its
dried form). DLS is based on the Brownian motion of spherical particles which
causes a Doppler shift of incident laser light. The average solution particle sizes
are calculated by monitoring the diffusion characteristics of the nanoparticles in
solution. Moreover, DLS can tell us about the dispersity and size distribution of
the particles. Measurements of the nanoparticle may be taken in growth media or
serum and tell more about the behavior of the particles in various salt
concentrations. It is worth mentioning that nanoparticles can agglomerate in
saline environments due to the reduction of the protective surface charge known

as the electric double layer that surrounds the surface.

6) Zeta Potential
Zeta potential (it) is the electric potential difference between the outer
dispersion medium and the fixed layer attached to the dispersed particle. Each
particle dispersed in a solution is surrounded by oppositely charged ions that are
strongly bound (fixed layer), and an outer diffuse cloud-like area (diffuse double
layer) with varying compositions of ions of opposite polarities. When a voltage is
applied to the solution, particles are attracted to the electrode of the opposite

polarity, accompanied by the fixed layer and part of the diffuse double layer, or

25

their internal side of what is known as the "sliding surface". Zeta potential is
considered to be the potential of this inner area. The magnitude of the zeta
potential gives an indication of the potential stability of a colloidal system. If the
particles have relatively large negative or positive zeta potentials (E, z -30 mV or
+30 mV), they will repel each other and create dispersion stability. If the particles
have low zeta potential values (§ ~ 0 mV), there is no force to prevent the
particles from agglomerating, which can lead to dispersion instability. It is worth
mentioning that among the most important factors that affect zeta potential are
pH and salt concentrations. A zeta potential value without a quoted pH or a

specified media is a meaningless number.

1.6.f. Applications of MNPs

Biomedical applications of magnetic nanoparticles can be classified
according to their application inside (in vivo) or outside (in vitro) the body. In vivo
applications includes diagnosis (detection and imaging) and therapeutics
(hyperthennia, gene and drug-delivery), while the main use of in vitro
applications is in diagnosis (detection and bio-separation).

Potential in vitro applications employ magnetic nanoparticles with
appropriate surface-functionalization by selectively binding the particles to
species of interest and placing them under a magnetic field thus separating them
from other species. The advantage of using magnetic nanoparticles over
magnetic micropalticles for such applications is that magnetic nano-suspensions
are stable against sedimentation in absence of an applied magnetic field and are

removable when magnetic field is applied. Moreover, unlike larger microbeads,

26

the small dimensions of NPs present minimal steric hindrance to reactants in
solution for accessing the active sites of the bio-target. Thus, magnetic
nanocrystals can have great impacts in bioseperation, sensing and biomedical
applications.

Areas of in vivo applications include, but are not limited to, drug and gene

I,99 and hyperthermia.100 Several reports have been

delivery,98 imaging via MR
published on the use of MNPs as nano-carriers for such applications“:101 Iron
oxide nanoparticles can serve as efficient contrast agents because they have
high magnetic moments and can therefore be used at very low concentrations.
When coated with hydrophilic polymers, magnetite can be coupled to targeted
molecules like antibodies, polypeptides, and carbohydrates that offer the promise
of targeting specific organs within the body. Iron oxide nanoparticles hold great
promise as nano-vehicles for targeting, imaging and delivering drug in one

102"“ In fact, several groups

package, thus revolutionizing medicine.
demonstrated the promising biological applications of MNPs. For example,
Weissleder et al. showed that MNPs can be used for monitoring specific
enzymes and detecting biomolecules such as viruses.” 7" 105407 Cheon et al.
reported the use of multifunctional magnetic nanocrystals to detect cancer in
vivo.108 Willner et al. demonstrated that magnetic nanoparticles can act as a
magnetoswitch to induce selective bio-electrocatalysis, detect cancer, and
amplify DNA detection.68 The above studies and other explorations verify the

potential of magnetic nanoparticles in life science, as summarized in several

authoritative reviewsz' 86 mentioned earlier.

27

As shown from the above examples and discussions, the ability to control
the size, shape, chemical composition, linker chemistry, and surface properties of
the nanocrystals is critically important because it determines the performance of
the nanomaterlals in biological applications. Specifically, Sailor et al. have
demonstrated that factors such as targeting ligand density, targeted-receptor
nature, and nanoparticle shape evidently affect the extent of tumor-targeting

efficacy.‘°3

It was found that the in vivo tumor-targeting ability in xenograft
models of human tumors of elongated nanoworm is superior to that of the
nanosphere (shape effect). Moreover, the smaller, neutral targeting ligand is
more effective in tumor-targeting and has longer blood half-life than the larger,
positively charged molecule (chemical composition), and that incorporation of a
5-kDa PEG linker improves targeting to tumor types relative to a short linker
(linker chemistry). It is also well known and proved that uptake of nanoparticles
into a wide variety of cells in vivo and in vitro is largely dictated by the size of the
particles.‘°9'111 Moreover, surface modifications (positively or negatively charged)
with polymers, peptides, proteins or carbohydrates enhance this uptake.
Positively charged NPs bind to the negatively charged cell membranes via

electrostatic interactions and are then internalized, while negatively charged NPs

are uptaken by either receptor-mediated endocytosis or diffusion.112

28

1.7. Gcho-nanoparticles for in vitro and in viva Detection

Despite the clinical success of iron oxide dextran-coated nanoparticles as
MRI contrast agents for enhancing the T2 relaxation times, glyco-nanoparticles
where the functionalized-carbohydrate plays a key role in recognition event
rather than in stabilizing phenomenon have not been extensively studied in many
biological and medical applications. Here, a number of important examples of

sugar-coated NPs used for in vitro or in vivo applications will be discussed.

1.7.a. Magnetic Iron Oxide Gcho-nanoparticles
1) In vitro Detection:

Sykova et al. showed that mannose-modified iron oxide NPs are efficient
probes for labeling living cells particularly stem cells.”3 They showed that
mannose modified-NPs crossed the cell membranes and were internalized well
by rat bone marrow stromal cells. Yan et al. used mannose-functionalized iron
oxide NPs prepared via photochemically activated phosphate
perfluorophenylazides (PFPAs) coupling chemistry and showed by TEM that
when the NPs were treated with E. coli strain ORN178, they selectively bound to
the FimH lectin on the bacteria.114 We observed similar phenomenon where we
explored the utilization of MGNPs not only to detect E. coli bacteria but also
calculate the capture efficiencies (~ 88% bacteria was removed). Here, it is worth
mentioning that lyer et al. immobilized biotinylated mannose conjugates on
magnetic beads to capture and detect E. coli and compared them to antibody-
coated beads.19 They observed that the glyco—beads were better than their

antibody-counterparts in both sensitivity and selectivity. Capture efficiencies of

29

~ 20-35% were observed with glyco-beads compared to only ~ 5-15% with
antibodies, depending on the E. coli concentration (105-107 colony-forming unit
(cfu)/mL) respectively. This is likely due to the smaller size of the glycoconjugate,
which can result in better packing density on E. coli and thus binding. This fact
was clearly demonstrated in our experiments where the exceptionally small sizes
of MGNPs enhanced both sensitivity and selectivity of biosensing to yield capture
efficiencies as high as ~ 88% (discussed In details in Chapter 2).

Moreover, Pieters et al. detected a gram-positive pathogenic
Streptococcus suis bacteria known to bind to galabiose (Gald1,4Gal) using
biotinylated sugars coated on 250 nm magnetic streptavidin-particles.115 Using a
luminescence assay that quantifies the bacterial ATP, monovalent and
tetravalent gaIabioside-functionalized particles yielded strong signals (mono >>
tetra) with a detection limit in the order of 104 bacteria/mL, whereas GlcNAc-
coated particles yielded no signals indicating the galabiose recognition specificity.
Importantly, experiments with larger magnetic particles (diameter ~10 mm) did
not enable successful bacterial detection which pinpoints the importance of the
much larger surface area of glycoparticles in detection.

Park et al. reported the fabrication of highly colloidal HA-DN (hyaluronic-
dopamine)-coated superparamagnetic iron oxide nanocrystals as target-specific
MR imaging vehicle and showed that the cellular uptake of HCT116
overexpressing CD44 was greatly enhanced compared to the low expressing
NIH3T3 by measuring relative relaxation rates.116 Mohapatra et al. reported the

synthesis of HA-Fe203 hybrid nanocomposites via electrostatic interactions of oil-

30

in-water HA nanoemulsion and F6203 nanoparticles and showed their
effectiveness in delivering encapsulated atrial natriuretic peptides to the nuclei of
A549 and HEK293 cells overexpressing CD44.117

2) In vivo Qetectjon:

Cho et al. reported the synthesis of polyvinylbenzyl- O-fi-D-galactose-D—
gluconamide (PVLA)-functionalized superparamagnetic iron oxide nanoparticles
(SPIONs) and demonstrated their utility to be targeted specifically into liver via
the ASGP-R receptor, a galactosyl binding asialoglycoprotein, expressed
predominantly on hepatocytes.118 Galactose and galactosamine are known to
accumulate selectively in the liver via ASGP-R receptor leading to hepatocyte
necrosis.119 In fact, in vitro studies showed time-dependent uptake of PVLA-
SPIONs into the cytosol and cell membrane of hepatocytes, suggesting a
receptor-mediated endocytosis.118 In vivo MR images of rat liver indicated that
the PVLA-SPIONs accumulated predominately in the liver compared with control
nontargted pyrrolidone—SPIONs (T2 signal drop 75 % vs 36 %), which suggests

their potential utility as liver-targeting MRI contrast agent (Figure 1-6).

 

(a)
Figure 1-6.72-weighted MR image (a) preinjection and (b) after 1-hour injection of PVLA-coated
SPIONs; (c) pre- and (d) post-injection of pyrroridone-coated SPIONs. Significant signal drop

(darkening) of liver compared to control pyrroridone-coated SPIONs.

Davis et al. demonstrated the use of glycan sialyl LewisX (sLe‘)

functionalized iron oxide coated-nanoparticles (NP-sLe") to specifically target

31

CD62 protein.66 The carbohydrate-binding transmembrane proteins CD62E (E-
selectin) and CD62P (P-selectin) are important in recruiting leukocytes to sites of
inflammation and are up-regulated on the activated brain endothelium in
response to injury. Specific visualization of the early-activated cerebral
endothelium using magnetic NPs, thus, provides a unique tool for the pre-
symptomatic diagnosis of brain disease and evaluation of new therapies. To this
end, selectin expression on activated endothelium in the brain was induced by
microinjection of interleukin-1B (IL-18) followed by the systematic injection of NP-
sLe" (4 mg Fe/kg) in a rat model. Direct targeted detection of endothelial markers
E/P selectin (CD62E/CD62P) in acute inflammation was observed via MRI. The
resulting sensitivity and binding selectivity of GNPs, displaying multiple copies of
the natural complex glycan ligand of selectins sLe", allowed acute detection of
the disease in mammals with beneficial implications for treatment of neurological

disease.

1.7.b. Gold Gcho—nanoparticles

Gold nanoparticles (AuNPs) exhibit several significant features:
straightfonlvard simple synthesis, high reproducibility, exceptional stability, and
excellent optical and surface plasmon enhanced properties. Such properties
make them well suited for applications in imaging, sensing, biology and medicine
and thus hold potential promise in diagnosis, therapeutics and targeting.31 As
mentioned earlier, the optical properties of metallic nanoparticles depend on their
size, shape, composition, structure, and morphology. Due to the novel properties

at the surface of gold nanomaterlals, absorption and scattering of

32

electromagnetic radiation by such nanocomposites are strongly enhanced.120
Gold nanocomposites, including nanospheres, nanorods, and nanoshells (size ~
10 to 100 nm) have large light absorption and scattering cross section in the SPR
wavelength regions.31 The magnitude of light scattering and emission by such
nano-gold materials can be orders of magnitude higher than that from strongly
fluorescing dyes.121 Basically, the light that is absorbed and emitted by metallic
nanoparticles depends on the nanoparticle diameter and its aspect ratio. The
aspect ratio is defined as the ratio of length divided by the width of a
nanocomposites (spheres have an aspect ratio of 1). Moreover, the shape and
crystallographic facets are the major factors in determining the optical and
surface properties of such particles. By changing the structure of the gold
nanocomposites from a sphere to a core-shell (silica-gold)122 to a hollow cage123
to a rod,121 the optical extinction wavelength shifts from the visible to near-
infrared region (NIR) (650-900 nm). Such structural and compositional tuning is
quite useful in potential in vivo applications where tissue absorption in NIR
window is minimal. All these important properties lead to the design of gold
nanoagents for optical detection, molecular and cell imaging, and photothermal
therapy and in vivo targeting.” 12“ For potential clinical trials, it is necessary to
understand their potential risks to human health. Although the noncytotoxicity of
gold nanoparticles in human cells has been studied in details,125'127 it is difficult to
predict how toxic nano-golds will be at certain doses for clinical endpoints.
Although the synthesis of gold nanospheres is dated back to 1857 by

Faraday128 and their use as contrast agents for biological electron microscopy

33

since the 1970s,”9 deeper insights into the fabrication of colloidal gold
nanomaterials with controlled sizes, shapes and properties emerged only
recently. Generally, reduction of metal salts, i.e. auric acid (HAuCI4), in aqueous
media by reducing agents causes Au3” to be reduced to neutral Au atoms that
gradually starts to precipitate to form nm spherical particles upon vigorous stirring.
Surfactants, such as citrate, have been utilized as surface stabilizers and/or
templates to control the synthesis. When bound to the nanoparticle surface, such
molecules can not only decrease the surface energy and control the growth and
shape of the particles, but also act as a stabilizer against aggregation. In general,
two types of AuNPs, a negatively charge citrate-stabilized gold nanosphere (GNS)
and a surface positive charge cetyltrimethyl ammonium bromide (CTAB)-
protected gold nanorod (GNR) are commonly synthesized.130

Here, the most common, successful and known techniques for the
fabrication of gold nanoparticles will be discussed. Of the simplest and most used
methods to synthesize GNSs is the one pioneered by Turkevichm' ‘32 in 1951
and refined by Frens‘33'134 in 1973. Briefly, it involves the reduction of HAuCl4 by
citrate to produce a broad size range spheres (diameters ~ 10 to 150 nm) with
low monodispersity (especially for > 30 nm particles). Another widely known
approach develOped in 1994 is the Brust's method135 and its variations that yield
~ 2-5 nm gold nanoparticles. Simply, Au ions are transferred to an organic phase
mediated by a phase transfer catalyst tetraoctylammonium bromide (T OAB),

followed by reduction with strong reducing agent, NaBH4. To prevent

aggregation, a stronger binding agent, usually a thiol ligand, is added that

34

strongly binds gold due to the soft character of both Au and S extensively studied
by the Whitesides136 group. In 1995, Gelbart et al.137 synthesized gold particles
with diameters ranging from 1.5 to 20 nm by varying the Au(lll) ion to stabilizer
thiol molar ratio. In 1996, Schmid et al.138 employed a powerful methodology,
known as seed-mediated growth (widely used to date), which gives better control
over size and shape. Briefly, gold ions are reduced by strong reducing agents to
form seeds, i.e. small particles that are then used in the next growth step.139 In
the second stage, the reducing agent is generally mild,14o reducing only the
precursor ions which are adsorbed onto the seed surface without creating any
new nucleation center. Since then, extensive studies on the mechanistic insights
and controlled-synthetic methodologies of nano-golds by Murhpy et al.129' ”"145
and EI-Sayed et al.‘2°' 12" “6449 have been conducted. In 2009, Perrault and
Chan‘5° reported a well-controlled synthesis of monodispersed gold nanospheres
(50-200 nm) with a narrow size and shape distribution using hydroquinone. In
this method, hydroquinone is used to reduce HAuCl4 in an aqueous solution that
contains gold seeds produced using the citrate method.

GNRs, on the other hand, have unique and improved surface-enhanced
Raman scattering properties and electronic properties compared to GNSs.12g
GNRs have the strongest surface plasmon band enhancement among all the
different shapes of gold and with suitable aspect ratios can absorb and scatter
strongly on the NIR region. Indeed, the use of GNRs for dual molecular imaging
and selective photothermal therapy of cancer cells using a NIR low-energy laser

have been reported.124

35

 

In general, rod-shaped metal nanoparticles can be synthesized in either

1 or in the presence of neutral or charged surfactants.152 For

hard templates15
colloidal GNR synthesis, two main approaches have been widely utilized and are
well established: the electrochemical153 and seed-mediated growth141 method. Of
the two, the seed-mediated growth method is the most popular route due to
simplicity, high quality and yield, size and shape control, and flexibility for
structural modifications.

Initially, GNRs were mainly prepared by the electrochemical method
developed by Wang et al. in the 1990s.154 Briefly, a gold metal plate anode and a
platinum plate cathode are immersed in an electrolytic solution consisting of rod-
inducing CTAB cationic surfactant as the major component and co-surfactant
TOAB as the minor. The latter surfactant induces the cylindrical shape of the gold
nanoparticle and the ratio of the two surfactants roughly determines the aspect
ratio of the nanorod. It is worth mentioning that GNSs (~ 12 nm) can be
synthesized using the above procedure either by removing the cosurfactant or by
increasing the mole ratio of the surfactant/cosurfactant above 6.

Using the seed-mediated approach, Wokaun et al.155 reported the formation
of gold colloids by adding gold nuclei to HAuCI4 growth solutions. The nuclei
were formed by reduction of HAuCl4 with phosphorus, and the growth of gold
nanorods was initiated with the addition of H202. In 2001, Murphy et al. reported
the most widely known procedure that involves, first, the preparation of small size
spherical gold nanoparticles, and second, growth of the prepared spherical

Particles in a rod-like micellar environment (Figure 1-7).“" “2' 1‘“ In this method,

36

colloidal gold nanorods were prepared by the addition of citrate-capped gold
nano-spheres to HAuCI2 growth solution obtained by the reduction of HAuCl4 with
ascorbic acid in the presence of CTAB surfactant with or without silver nitrate
(AgNOa). Briefly, three steps are required: (1) Seed: synthesis of the seed
solution by the reduction of a metal salt, i.e. HAuCl4, with strong reducing agent
such as sodium borohydride (NaBH4) in the presenCe of citrate; (2) Growth:
preparation of the growth solution using rod-like micellar template CTAB mixed
with HAuCl4 and ascorbic acid; and the final step (3) Seed-Growth: addition of
the seed solution to the growth solution in the presence143 or absence141 of Ag
ions. In the first step citrate is present as a capping agent to prevent particle
growth where the gold spheres thus produced are 3:5 nm in diameter. The

seeds then serve as nucleation sites for nanorod growth.

_ O
HAuCI4 + NaBH4 Wale 'e’

0
Gold nanospheres (GNSs)

O O
O
. .: O + metal salt + ascorbic acid CTAB _-_-___

seed Gold nanorods (GNRs)

Figure 1-7. Seed-mediated growth of gold nanoparticles.

The aspect ratio is controlled by the ratio of metal seed to metal salt, thus
restricting the size to nm regime. It was observed as the amount of seed was
decreased, the aspect ratio increased, and the longer wavelength plasmon band
gradually red-shifted and broadened. Moreover, with increasing amount of seeds,

the overall rate of particle formation increases, and hence the growth rate. In the

37

 

absence of seed, particle formation rate is very slow. It is worth mentioning that
ascorbic acid is a mild reducing agent and cannot reduce the gold salt in the
presence of the micelle without the presence of seed. Consequently, minimal
additional nucleation occurs during particle growth. Similar to iron oxide
nanoparticle synthesis, in nano-gold fabrication it is also crucial to separate the
continuous nucleation throughout the growth process in order to better control
the shape and size of the nanocomposites. The disadvantages and limitations of
this method, however, are mainly the formation of noncylindrical nanorods and a
large amount of spherical particles. Moreover, in all cases, centrifugation and
extraction are always performed to separate rods from any spheres that have
been formed.

In 2003, modifications to this method have been applied for synthesizing
nanorods with aspect ratios of 1.5 to 10 by El-Sayed.147 For synthesis of pure
gold nanorods (99%) with minimal nanospheres and aspect ratios up to 5, the
following strategies were employed: (1) replacement of the citrate-capped seed
with a stronger CTAB stabilizer in the seed formation step; (2) adjustment of Ag
ions concentration of the growth solution to control the aspect ratio (increasing
Ag ion concentration Increases the aspect ratio); (3) addition of Ag ions to the
growth solution before seed addition to facilitate rod formation and tune the
aspect ratio. To grow NRs with aspect ratios ranging from 4.6 to 10, a binary
surfactant mixture composed of benzyl-dimethylhexadecyl—ammonium chloride
(BDAC) and CTAB was used. NRs are grown in this mixture either by aging or by

addition of a growth solution suitable to shorter NRs. The yield, monodispersity,

38

size, aspect ratio and shape of gold nanorods can be controlled by many
parameters, such as seed concentration, relative concentrations of gold
precursors, ascorbic acid, salts and different surfactants, temperature, pH, purity

of additives, solvent, and the aging time.

1) In vitro Detection:

a) Studying Carbohydrate-Carbohydrate and Carbohydrate-Protein
Interactions

Gold glyco-nanoparticles have been extensively and successfully
employed by several groups for studying carbohydrate-protein and carbohydrate-
carbohydrate interactions which are the major mechanisms for cell adhesion and
recognition. Jesus de la Fuente and Soledad Penadés did elegant work in this
field where they prepared carbohydrate-functionalized gold, gold—iron, and
semiconductor nanocrystals for studying carbohydrate interactions and
investigating carbohydrate-mediated cell—cell adhesion processes. In an early
report, Penadés et al. demonstrated the use of either disaccharide lactose (lacto-)
or trisaccharide LewisX (Le"-) AuNPs as multivalent ligand carriers for studying
Cay-mediated carbohydrate interactions.156 Le" antigen or lactose was
derivatized with an alkylthiol, and AuNPs were prepared by reducing HAuCI4 with
NaBH.. in presence of the glyco-thiols. Using TEM, it was revealed that only
specific binding between Ca2+ and Le"-AuNPs result in self-aggregation, while
lacto-AuNPs did not show any clustering (Figure 1-8). Removal of the cations via
ethylene-diamine tetraacetic acid (EDTA) addition reversed the assembly,

proving the specific role of the sugar in inducing aggregation. This ability was

39

also confirmed by 'an atomic force microscopy (AFM) study of adhesion forces
between Le" antigens self-assembled on gold surfaces (2D).157 Furthermore, they
reported the quantification of the kinetics of Ca2*-mediated carbohydrate-self
interactions via SPR by using combination of self-assembled monolayers (SAMs)
of alkanothiolates on gold with carbohydrates as the substrate and AuNPs as the
analyte.158 Furthermore, thermodynamic insights using isothermal titration
calorimetry (ITC) were also investigated.159 Their results showed that Ca2+-
mediated aggregation of Lex-AuNPs is a slow process but highly exothermic,
while the heat evolved in the case of Iacto-AuNPs is very low and its thermal
equilibrium is quickly achieved. Measurements in the presence of Mg2+ and Na"
cations did not induce significant aggregation of Lex-AuNPs confirming its
selectivity for Ca”. All this work demonstrated the highly specific and chief role of
carbohydrate-carbohydrate interactions in cell-cell adhesion processes.

0
mOA/vwvvs-

Neoglycoconjugatee of Significant Oligoeacharidee

 

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/ l \ K ’3.
h K 3 r. «n
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‘1. r _j" 3,]
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1 Gold Glyconanoparticlee SAM. and Glyconanoparticlee

AFM. Adhesion Forces TEM, Oak-Mediated Aggregation SPR. Kinetic Parameters

Figure 1-8. Various strategies for studying specific carbohydrate interactions that mimic sugar
presentation on the cell surface, thus representing important roles in cell-cell adhesion and

recognition.20

40

Lin et al. used SPR to quantitatively analyze the binding affinity of the
polyvalent glyco-nanoparticles (mannose- (man), glucose- (glc), or galactose-
(gal) AuNPs) with Iectin, Concanavalin A (Con A).160 The dissociation constant K,
of man-AuNPs with Con A was determined to be 2.3 nM, representing a binding
affinity over 5 orders of magnitude higher than that of the monomeric mannose.
This work quantitatively proves the high affinity and specificity of multivalent
carbohydrate-protein interactions. Similar affinities were also observed by Wu et
al. where they get Kd values in the nM range.161 Moreover, Russell et al.
demonstrated the utilization of functionalized glyco AuNPs to detect
carbohydrate-binding Iectins using colorimetric bioassays based on recognition-
induced aggregation of the metal nanoparticles.162

For studying NP-cell interactions, Penadés et al. reported the preparation
of gold and gold—iron nanoparticles163 functionalized with maltose (malto),
glucose and lactose and evaluate their biological effects.164 Different cellular
responses were obtained for each glyco-nanoparticle type depending on the
sugar, demonstrating the importance of carbohydrate in cellular-recognition.
Incubation with a human fibroblast cell line showed that lacto-MGNP are taken
up by endocytosis without provoking apoptosis while malto-MGNP are endocyted
and promoted cell death (80 % cytotoxicity). Surprisingly gIc-MGNP had a
completely different behavior where it was not endocytosed and did not have any
effects on cell viability. This result encouraged them to test the possibility of using
glc- or Iacto- Au/Fe NPs to image an experimental C6 glioma in mice in vivo

which will be discussed later.

41

Recently, the same group prepared a small library of multivalent gold
nanoparticles functionalized with different structural mimics of the high mannose
undecasaccharide of gp120 and evaluated their effect on the inhibition of HIV
binding to DC-SIGN expressing cells via SPR (Figure 1-9).165 A major
mechanism of HIV infection implies the interaction of the virus envelop
glycoprotein gp120 with DC-SIGN receptor expressed on dendritic cells. DC-
SIGN selectively recognizes endogenous N-linked high-mannose-type
oligosaccharide structures Man9(GlcNAc)2 of the HIV envelop glycoprotein
gp120.166 A highly significant inhibition (~ 20,000-fold more effective in inhibiting
DC-SlGN/gp120 interaction) using Mand1-2Mand bearing GNPs (100% inhibition
at 120 nM) than with the corresponding monovalent disaccharide (100%
inhibition at 2.2 mM), was observed.165 Moreover, inhibition of gp120-DC-SIGN
binding was evaluated in a model using DC-SIGN-transfected Raji B cells.167
GNPs bearing the Mancr1-2Man disaccharide inhibited binding of HIV-1 to DC-
SIGN by 85%, while free mannan inhibited binding only by 60%. Moreover,
oligomannoside—functionalized AuNPs were able to inhibit the HIV DC-SIGN-
mediated trans-infection of PM-1 T cells at nM concentrations. Mand1-2Man-
AuNPs were able to inhibit trans-infection of PM-1 T cells by HIV-1 by 80%,
compared to free mannan, which inhibited binding by 50%. In brief, they showed
a successful carbohydrate-based multivalent system that can function as an anti-
adhesive barrier at an early stage of HIV-1 infection, prevent viral attachment to
DC-SlGN-expressing cells, and subsequently inhibit trans-infection of human T

lymphocytes.

42

     

V .1!
if" :3. E’s
9- ...,
>111 Ye "it.
??‘123. , .2111»! 3“?" GNPS
_ 1_ gp120 A. 1 it
‘ Add 4 x . a t
DC-SIGN}? DC-SIGN .
it
Cell i-I. Cell - -
Membrane Membran

Figure 1-9. Inhibition of HIV binding to DC-SIGN using oligomannoside-GNPs.165

In another report, Kamerling et al. grafted sugars on AuNPs to explore
carbohydrate-mediated self-recognition events in the marine sponge Microciona
prolifera and studied their effects on cell-aggregation.168 They demonstrated the
chief role carbohydrates play in the proteoglycan-like macromolecular
aggregation factor (MAF) self-aggregation, where they designed water-soluble
AuGNPs coated with synthetic sulfated disaccharide fragments related to
GlcpNAc3S(B1—3)Fucp for mimicking the g-200 self-association. It is known that
Cay-dependent self-association of MAF occurs through highly repetitive epitopes
(GIcpNAc38(B1—3)Fucp) on the g-200 glycan present on the N-glycosylated
protein, MAFp3.169 It was shown that the proteoglycan self-recognition is highly
specific and any structural changes in the disaccharide completely disrupt the
aggregation phenomenon. Examinations revealed that the stereochemistry,
hydrophobic groups on the sugar moiety, the linker type and the presence of
Ca2+ play an essential role in the specific organization of the sugar moieties.

They found that the d-anomeric product, the sulfated disaccharide and the C6

43

methyl group of L-fucose to have an irreplaceable function in the self-recognition
phenomenon.
b) Exploring Pathogen Detection

Pathogenicbacteria, viruses, and other microbes use cell-surface
carbohydrates to invade host organisms and to deliver toxins (i.e. cholera toxin
and shiga-like toxins). Protein-sugar interactions between the bacteria and the
host cell are crucial first step in the infectious process. Multivalent protein-
carbohydrate interactions generate adhesive forces where multiple copies of
sugar usually provide potential means of strengthening those binding events.
Thus instant and ultra-sensitive detection of bacteria using glyco-AuNPs, without
time-consuming procedures, such as incubation or amplification by PCR, offers
obvious clinical benefits.

At an early stages in this direction, Wu, Chen, Lin and coworkers
confirmed the concept of multivalency where they showed the specific binding of
mannose-encapsulated gold nanoparticles (man-AuNPs) to FimH adhesin of
bacterial type 1 pill in E. coli by TEM.170 That was one of the first examples to
demonstrate that carbohydrate-functionalized AuNPs can be used as efficient

labeling probe and multiligand carrier in a biological system (Figure 1-10).

44

Wild-type 5, coli selective -

binding

 

m-AuNP

Mutant
m-AuNP binding to the specific receptor of type 1 pill

Figure 1-10. Selective bindingof man-AuNPs to the wild-type E. coli strain ORN178. The mutant
ORN208 showed no binding. °

Recently, Lin et al. designed AuNPs for Shiga-like toxin detection,
separation and inhibition (Figure 1.11).171 As Escherichia coli O157:H7 is a chief
cause of foodborne illness, the development of a rapid and sensitive detection
method of Shiga toxins (Sts), which are produced by the bacterium, is
recommended. Sts are a family of AB5 bacterial toxins which specifically
recognizes cell surface glycosphingolipid Gb3 (globotriaosylceramide) through
multivalent binding of the symmetric B-subunit pentamer. The Gb3 glycolipid is
known as the globotriose (Pk) blood group antigen or CD77, which contains the
trisaccharide dGal(1-4)BGal(1-4)BGIc. Lin and coworkers engineered a
globotriose-functionalized gold nanoparticles (Pk-AuNP) that specifically captured
the recombinant Shiga-like toxin I (B-Slt) from bacterial cell lysate with > 95%

purity while maintaining its activity.171

They also demonstrated that a 20 nm
water-soluble Pk-AuNP is an antagonist for B-Slt and shows >108-fold binding
affinity enhancement over the monovalent Pk trisaccharide using SPR
competition binding assay. Moreover, they developed a Pk-AuNP-based

detection method for SIt-l by combining the technique with silver enhancement.

45

its «131%. . :

Detection Inhibition

 

 

 

Figure 1-11.Schematic presentation of Pk-AuNPs for detection, separation and inhibition of
Shiga-like toxin.171

Russell et al. developed a rapid colorimetric bioassay for the detection and
quantification of cholera toxin (CT) within 10 minutes.54 The bioassay is based on
lactose-functionalized AuNPs that upon binding to the toxin induces aggregation
and subsequent change in color upon surface plasmon absorption
measurements. The selectivity of the bioassay stems from the thiolated lactose
derivative that mimics the GM (GalB(1-3)GalNAc[Neu5Aca(2-3)]-B(1-4)GaIB(1-
4)Glc—lipid) ganglioside, the receptor to which cholera toxin binds in the small
intestine.

In another example, Perez described a quick and high-throughput
nanoparticle-based antimicrobial susceptibility assay utilizing the differential
changes in the surface plasmon band of dextran(Dex)-coated AuNP upon
clustering.172 Con A-induced clustering of Dex-AuNP yields sensitive and
reproducible changes in the nanoparticles’ absorption maximums, providing
efficient means to sense the presence of available complex carbohydrates in
bacterial suspension and hence giving the promise of identification of potential

antimicrobial agents within 3 hrs.

46

c) Investigating DNA Interactions

The study and better understanding of the binding affinity of GNPs and DNA
will hold great implications for their potential use as nonviral gene delivery
agents. Glyco-nanoparticles are attractive probes for studying the less advanced
carbohydrate-nucleic acid interactions and hence creation of DNA-binding
systems. Their large surface area, multivalent interactions upon DNA-binding and
their cationic ligands grafted onto the NP surface makes them ideal mimics for
the naturally occurring protein-nucleic acid interactions. To this end, Penades el
al. reported the interaction of GNPs functionalized with Glc or Gal and amino
ending ethylene glycol chains with a linear DNA.173 TEM, AFM and gel
electrophoresis examinations showed that mixed amino/d-Gal (1:1) nanoparticles
are highly efficient DNA-binders and condense DNA into a compact globular
shape, which is a desired property for gene transfection agents. AFM analysis
revealed the presence of free DNA (and free nanoparticles) in the case of a-Gal,
B-Glc, amino/B-Gal, and amino/B-Glc nanoparticles, pinpointing the specificity
involved in carbohydrate-DNA interactions and calls for an urgent need to better
understand the system.

Narain et al. proposed a cationic monodispersed and biocompatible
glycopolymer-stabilized gold nanoparticles as an effective gene delivery vehicle
that can enhance the cellular uptake by cell-mediated interactions, thus
promoting higher transfection efficiencies.174 The synthesis of cationic gold
nanoparticles was achieved using the photoinitiator, Irgacure-2959, where

cationic glycopolymers were first synthesized by the reversible addition-

47

fragmentation chain transfer (RAFT) process and the cationic glyconanoparticles
were then produced in one step using UV radiation. It was found that cationic
nanoparticles produced undergo receptor-mediated endocytosis due to the
glycopolymer following their vesicular escape in Hela cells, due to the net

cationic character of the nanoparticles.

2) In vivo Detection:

Penades el al. reported the utilization of lacto-AuNPs as potent inhibitors of
lung metastasis in C57/BI6 mice and evaluated their potential as anti-adhesive
tools against metastasis progression.175 They showed that short ex vivo
preincubation of tumoral 816F10 with Iacto-AuNPs is enough to substantially
inhibit lung metastasis (up to 70%) in a tumor metastasis model (Figure 1-12).

Moreover, the same group reported sugar-coated AuNPs combined with
Gd(lll) chelates as new paramagnetic probes for enhancing the relaxivity for
MRI.176 GNPs have small sizes (24 nm) and their carbohydrate-coating make
them non-toxic to cells and mice. It was shown that sugar (glc, gal or Iacto)
stereochemistry and the relative position of the sugar with respect to the Gd ion
seem to control the relaxivity values of these GNPs. Sugars may create specific
interactions with water that bring water protons in the proximity of the metal ions

favoring the water exchange with the bulk solvent.

48

B16F10 (105 cells/animal) No B16F10

Mock I..°.l|°|

6.1.3.311... O G 0 e .

 

 

 

 

 

 

“PM? I D G e e e I

Figure 1-12. Specific effect of Iacto-AuNPs on the metastatic potential of B16F 10 to induce lung
tumoral foci upon intravenous (i.v.) inoculation in CS7/Bl6 mice. A positive control group was
injected with a single dose of B16F10 melonoma cells (105 cells/animal). Other groups were
treated with single doses of B16F10 melanoma cells (105 cells/animal), preincubated for 5 min at
37 °C with 90 uM of the control gluco-GNPs or with 90 uM of lacto-GNPs. After three weeks, the
animals were sacrificed, and lungs were evaluated under a microscope for tumor foci analysis.
Inspection of lungs inoculated with B16F 10 cells pretreated with Iacto-AuNPs showed a strong
protective effect against lung metastasis (~ 70 % inhibition) in contrast to those obtained from

animals primed with B16F10 cells or B16F10 cells pretreated with gluco-GNPs.175

 

In vivo imaging of glioma (generated with GL261 tumoral cells) in mice
indicates that at the same Gd(lll) concentration gal-GNPs were able to enhance
the contrast in the tumoral zones better than lacto-GNPs or the clinically used
contrast agent, magnevist proving the specificity of sugar targeting. Indeed, it is
known that the asialoglycoprotein receptor which is expressed exclusively in
hepatic parenchymal cells binds specifically to galactosyl-terminal glycoproteins,

which might explain the difference in the enhanced relaxivity.

49

1.7. c. Gcho-Quantum Dots
1) In vitro Detection:

Less work has been done in the area of functionalizing quantum dots (QDs)
especially when it comes to in vivo applications due to the potential toxicities of
005. However, glyco-quantum dots present an advance that could help tap the
much-heralded potential of 003 in the treatment of diseases. 00s are
nanocrystals that glow when exposed to light. In comparison with organic dyes
and fluorescent proteins, 00s have unique optical and electronic properties:
size-tunable light emission, high quantum yield fluorescence, improved signal
brightness, resistance against photobleaching, and simultaneous excitation of
multiple fluorescence color.29' ‘77 QDs protected with polysaccharides were
reported in 2003 by Rosenzweig et al."'4 This group prepared CdSe-ZnS QDs
protected with carboxymethyldextran and polylysine, and they proved the high
affinity of dex-QDs toward the glucose binding protein, Con A. Along the same
lines, Chaikof et al. reported the site-specific labeling of streptavidin-QDs with
biotinylated end-tenninated functionalized glycopolymers and demonstrated their
potential in the specific carbohydrate-lectin binding phenomena using Con A and
RCA12o, a gal-binding Iectin.178 Moreover, Surolia et al. prepared sugar-QDs for
selective and sensitive detection of Iectins.179 Fang et al. have recently reported
the labeling of mice, pigs and seaurchin live sperm with CdSe/ZnS core shells
QDs functionalized with N-acetyl glucosamine or mannose.180 GlcNAc-
encapsulated QDs were concentrated at the sperm heads, while mannose-
coated QDs tended to spread over the whole sperm body, due to the different

distribution of the GIcNAc and mannose receptors on the sperm surface. Thus,
50

they proved that glyco-QDs are potential candidates that can be used as cell
labels. Moreover, Kim et al. demonstrated an approach for the rapid and simple
detection of protein glycosylation based on the energy transfer between Iectin-
conjugated AuNPs and sugar-conjugated 00.181 They also showed the potential
of their system in the high-throughput analysis of glycosylation degree, which is

critical for the development of protein therapeutics.

2) In vivo Detection:

Kim et al. reported the fabrication of water-soluble, biocompatible and
size-tunable hyaluronic acid-coated QDs (HA-QDs) where HA is coupled
electrostatically to positively charged amines grafted on the surface of ODS.182
They showed the specific labeling and uptake capability of HA-QDs to effectively
target lymphatic vessel endothelial receptor 1 (LWE-1) overexpressed on
lymphatic endothelial cells (LEC) and l-IeLa cells with low cytotoxicity. Decreased
cytotoxicity was significantly observed compared to unconjugated QD-treated
cells. Moreover, in vivo fluorescent imaging was evaluated where they were able
to selectively visualize the changes in lymphatic vessels (lymphangiogenesis) in
real-time for days.182

Seeberger et al. reported the synthesis of carbohydrate (man, gal,
galactosamine (GaIN))-capped PEGylated QDs to study specific carbohydrate-
protein interactions for in vitro applications and in vivo targeting.183 In vitro, they
showed that Gal— and GaIN-capped 00s are selectively uptaken by
hepatocellular carcinoma HepGZ cells via the ASGP-R receptor, a galactosyl

binding asialoglycoprotein, expressed predominantly on hepatocytes. The uptake

51

was partially inhibited by preincubation with poly-L-lysine galactose polymer or by
the knockdown of ASGP-R1 proving that the internalization is via receptor-
mediated endocytosis. In vivo, they demonstrated in a mice study, that Man- and
GalN-capped QDs accumulate selectively in the liver but not other parts of the
body suggesting a mannose and ASGP-R receptors on Kupffer and hepatic cells
respectively. These sugar-coated 003 became three times more concentrated in
the mice livers than the regular PEG-QDs, demonstrating their targeting abilities.
Furthermore, upon iv. injection of GaIN-capped 00s, a significant increase in
serum transaminases (ALT) was observed indicating that liver injury was
selectively mediated by GaIN-QDs. It is worth mentioning that the same group
recently presented an efficient synthesis of carbohydrate-functionalized 00s in
micro flow reactors at 160 °C.“

In brief, giving 003 an icing-like cap of specific carbohydrates makes
these nanoparticles accumulate in certain organs enhancing their effectiveness
and decreasing their cytotoxicity. Such particles have the promise of selective
targeting that could be used to deliver anti-cancer drugs to one organ, reducing

the side-effects that occur with existing cancer drugs.

1.7. d. Gcho-Carbon Nanotubes

Sun et al. reported that single-walled carbon nanotube (SWNT) serves as
an excellent scaffold for multivalent carbohydrate ligand display, with the Gal-
SWNTs exhibiting strong cell adhesion resulting in efficient capturing of
pathogenic E. coli in solution.”5 Moreover, they demonstrated that mannose or

galactose-functionalized SWNTs represent a unique displaying scaffold for

52

multivalent monosaccharide ligands that bind effectively to B. anthracis spores in
the presence of a divalent cation.186 The binding results in substantial
aggregation of the spores and corresponding colony forming units reduction
which may potentially find valuable applications in the decontamination and anti-
bioterrorism. Bertozzi and coworkers did some elegant work in functionalizing
carbon nanotubes with glycopolymers to mimic cell surface proteins and hence

study their interface with living cells."37'188

1.7. e. Gcho-Micelles and Liposomes

Aoyama et al. reported that neutral micellar glycoviruses undergo
saccharide—dependent self-aggregation and size-dependent cell endocytosis.189
They prepared calix[4]resorcarene-based macrocyclic glyco-bundle amphiphiles
having long alkyl (undecyl) chains and saccharide moieties ranging from
cellobiose (Cel), lactose or maltose. Water-soluble glycocluster micellar
amphiphilic nanoparticles (GNPs) grafted with the different saccharides were
then constructed and combined with size-controlled plasmid pCMVIuc DNA
(7040 bp) resulting in glycoviral nanoparticles. It was shown that the obtained
glycoviruses are small in size (~ 50 nm) with no net charge surface (é ~ 0 mV),
and undergo saccharide-dependent (d-Glc > B—Gal >> B—Glc) self-aggregation,
and transfect cell (Hela and HepGZ) cultures via highly size-regulated pinocytic
form of endocytosis. Alternation in stereochemistry of glycoside linkage (Mal vs
Cel) or single OH group orientation (Cel vs Lac) resulted in drastic change in the
adhesion properties of glycoclusters. It was also noted that glycovirus aggregates

(>100 nm) are incorporated only slightly in the cells. It was shown that the size

53

effects in the glycoviral gene delivery allow only monomeric viruses to work
effectively and that the activites of oligomeric and poorly active B-Gal-
functionalized nanoparticles toward hepatic HepGZ cells are ~ 100 times higher
than expected on the size basis, owing to the receptor-mediated specific pathway
involving the asialoglycoprotein receptors on the hepatic cell surfaces. In general,
they proved that neutral glyco-cluster nanoparticles are efficient in coating DNA
where a specific uncharged and highly hydrophilic carbohydrate can prevent the

glycoviruses from aggregation, rendering the system an effective gene delivery

 

 

vehicle.190
(
plasmid DNA 0
112-7.},er if it :A
1‘31», if; ‘ 1 m 06'
‘ill .l. .. ' _
Gly8 Gly5 m I
‘ Lac
-' ...... ii ee'
' ‘ .‘ 9" &52x3
glycocluster
amphiphile GNP U . c -
Mal

._. = pitch (3.4 nm)

Figure 1-13. Construction of glycovirus. Growth of glycocluster amphiphile Gly8 or Gly5 through
GNP to glycovirus and its aggregates.189

Later, the same group reported the synthesis of 15 nm TOPOQD conjugated
with glyco calyx-[4]resorcarene based sugars and compared their uptake to the
50 nm sized GNP prepared earlier in an effort to better understand the size-
effects on endocytosis.191 It was shown that TOPOQD-conjugated sugar ball is
taken up by Hela cells via endocytosis, but marks endosomes much more

efficiently than the micellar homoaggregate of the amphiphile (GNP, 5 nm) and

54

much less than the virus-like DNA-GNP conjugate (50 nm), respectively.
Knowing that all the above nanoparticles are functionalized with the same [3-
glucoside moiety not only strongly suggests that endocytosis is highly size-
dependent (50 nm>> 15 nm>> 5 nm), but also pinpoints the utmost importance of
viral-size control in designing artificial delivery vehicles.

Agrawal et al. synthesized HA-poly(ethylene glycol)-poly(lactide-co-
glycolide) (HA-PEG-PLGA) polymeric nanoparticles encapsulated with
Doxorubicin (DOX) and studied the in vivo release of the drug and the
corresponding tissue distribution.192 After intravenous (IV) injection in Ehrlich
ascites tumor (EAT)-bearing mice, it was found compared to the control
monomethoxy(polyethylene glycol) (MPEG)-PLGA nanoparticles, that HA-PEG-
PLGA showed a higher concentration of DOX in the tumor (4-fold higher) with
nonspecific uptake mainly in the liver. Moreover, HA-PEG-PLGAs were able to
deliver higher amount of DOX in the inhibition studies, and were able to sustain
the release for up to 15 days. The nontoxic HA—PEG-PLGAs not only were
effective in targeting EAT tumor but also showed long tumor retention times with
rapid clearance from normal tissues rendering them good targeted-drug delivery
candidates.

Park et al. enclosed the synthesis of nano-sized and self-assembled HA-
paclitaxel conjugate micelles and studied their promising potential as tumor-
specific therapeutic agents for targeted delivery of paclitaxel to cells
overexpressing HA receptors.193 Apoptosis-inducing effect of HA-paclitaxel

micelles on HCT-116 cells, overexpressing CD44, displayed apparent evidence

55

of cell death quantitatively assessed as increase in G2/M phase cell population
(2-fold higher than Taxol and 4-fold higher than the control). In general, HA-
paclitaxel nano-micelles exhibited greater cytotoxicity to CD44 overexpressing
cells than for the HA receptor deficient cells, suggesting the proficiency of
targeting using HA. Previously, the same group also demonstarted that HA
nanogels encapsulating small interfering RNA (siRNA) could be delivered to
HGT-116 cells in a HA receptor-mediated, target-specific manner.

Alonso et al. demonstrated that both chitosan (CS) nanoparticles and
liposome-CS nanoparticle complexes interact with and penetrate the corneal and
conjunctival epithelia.194 The same group then reported an efficient novel ocular
targeted gene delivery nano-vehicle composed of HA-CS nanoparticles prepared
by ionotropic gelification, entrapped with pDNA (pEGFP or pB-gal) and studied
their transfection abilities in proliferating ocular cells.195 It was shown that the HA-
CS nanoparticles were nontoxic, were able to provide high transfection levels (up
to 15% of cells transfected) in human corneal epithelial (HCE), and were proved
to be internalized by hyaluronan CD44-receptor mediated endocytosis. Blocking
of the receptor recognition sites of HA led to a decrease in the tumor-targeting
selectivity. This gives evidence of the potential of HA-CS nanoparticles for
targeting and intracellular delivery of genes to the cornea and conjunctiva.

Szoka et al. used HA-targeted Iiposomes (HALs) entrapped with DOX as
tumor-targeting drug vehicles to treat CD44-expressing tumors.17 They elegantly
demonstrated the selective delivery of DOX drug to B16F10 melanoma

cancerous cells, expressing high levels of CD44. B16F 10 specifically bind and

56

internalize HALs rapidly in a temperature and concentration-dependent manner
(uptake proportional to HA loading 0-3 mol % range), whereas cells expressing
low levels of CD44 do not. HAL binding to Bt6F10 was inhibited by HA and by an
anti-CD44 monoclonal antibody. Moreover, for periods up to 24 h, they observed
a remarkable 8.2 and 4.4-fold increase in potency of the HAL-delivered DOX
compared with free DOX in both transient and continuous exposure conditions,
respectively. They also showed significantly enhanced cytotoxicity (almost 1-fold
better) by HAL-DOX to CD44-overexpressing cells and much less toxicity to low-
CD44 expressing cells compared to the free drug. All this proves the promise of
targeting CD44 by HA-coated Iiposomes and the potential of using them as
chemotherapeutic agents for drug delivery in vivo.

In 2004, Margalit et al. demonstrated the use of targeted-covalently linked-
HA nano-liposomes (tHA-LIP) encapsulated with mitomycin C (MMC) as
anticancer nanocarriers delivering drugs to tumor-bearing mice models in vivo.196
In vitro, they observed 100-fold increase in potency of MMC encapsulated tHA-
LIP in cells overexpressing but not in cells underexpressing, hyaluronan
receptors. In vivo, in 3 mouse tumor models (C-26 solid tumors, B16F 10.9 and
D122 lung metastasis) tHA-LIP were long-circulating, 7-fold and 70-fold longer
than drug-loaded nontargetd liposome (nt—LIP) and free MMC, respectively.
When delivered in B16F10.9 tumor-bearing C57BU6 mice via tHA-LIP, MMC
accumulation in the tumor was 30-fold higher than the free drug, and 4-fold
higher than nt-LIP proving selectively of targeting. tHA-LIP-mediated MMC

accumulation in tumor-bearing lungs was 20% of injected dose, compared to 0.6%

57

and 4% with free drug and nt-LIP, respectively while tumor-free lungs showed
low accumulation, irrespective of drug formulation. Moreover, to evaluate tHA-LIP
effects on therapeutic responses, tumor response to treatment, tumor size,
metastatic burden and survival were tested and found superior in animals
receiving MMC-loaded tHA-LIP. Along the same lines, the same group
demonstrated the use of DOX encapsulated tHA-LIP as tumor-targeted vehicles
in both syngeneic and human xenograft mice.197 Mediated by the long-circulating
tHA—LIP, DOX accumulation in tumor-bearing lungs was 30-, 6.7-, and 3.5-fold
higher than free DOX, nt-LIP, and liposomal doxorubicin (Doxil), respectively. In
brief, DOX-encapsulating tHA-LIP generated significant improvements in
pharrnacokinetics, biodistribution, loading efficacy and cytotoxicity in four
different mouse species, in tumors generated in different organs, and in tumors
originating from different cell lines- all overexpressing HA receptors. All these
successful and promising results shows that tHA-LIPs emerge as a valid targeted
and specific cargo for tumor chemotherapy, with distinct advantages:
cryoprotection, good drug encapsulation, sustained release, long circulation and
high affinity binding to the tumor, allowing the liposomes to act as sustained-
release drug vehicles.

Very recently, Paulson et al. developed 0022 sialic acid-decorated
liposomal nanoparticles loaded with doxorubicin for in vivo targeting of B
lymphoma cells.198 A current clinical target for B cell lymphoma is CDZZ, a B cell
specific member of the sialic acid binding Ig-like lectin (siglec) family that

recognizes 02-6-Iinked sialylated glycans as ligands. In contrast to the approved

58

Doxil, which passively delivers DOX to solid tumors via leaky blood vessels, it
was demonstrated that DOX-loaded sialic acid-decorated liposomal
nanoparticles are actively targeted to and endocytosed by 022 on B cells, and
significantly extend life in a murine xenograft model of human B cell lymphoma.
Moreover, they bind and kill malignant B cells from peripheral blood samples
obtained from patients with hairy cell leukemia, marginal zone lymphoma, and
chronic lymphocytic leukemia. The results demonstrate the potential for using a
carbohydrate recognition-based approach for efficiently targeting B cells in vivo
that can offer improved treatment options for patients with B cell malignancies.

In conclusion, as shown above, carbohydrates displayed on nano-systems
are useful in many areas of biomedical application such as pathogen detection,
inhibition of metastatic diseases, gene and drug delivery and cellular imaging.
Although recent advances have demonstrated the use of carbohydrate-
functionalized nanoprobes for diagnosis, sensing, imaging and therapy, new
methods and strategies to produce targeted probes based on the “sugar code”
are certainly needed. To achieve these goals, the specificity of carbohydrate
ligands needs to be improved and the characteristics and bio-functionalization of
nanostructures should be better controlled to meet the demands of specific
biological targets. Indeed, surface engineering of nanomaterials is critical to
translate molecular recognition events into reliable biological readouts. A balance
between specific receptor expression levels, proper ligand presentation, efficient
ligand coupling, physiological barriers’ consideration, and tumor distribution is

crucial for a full successful biological system. Nonetheless, many obstacles for in

59

vivo intracellular targeting still exits: 1) suscessful application of targeted
nanoparticles, 2) preserving the physiochemical properties of the nanoparticles 3)
engineering of sizes comparable to target, 4) avoiding the reticulo-endothelial
system 5) internalization into tumoral cells vs normal cells with high efficiency.
New and better treatments are still to be explored, but there are substantial gains
to be made by applying the nano-systems already developed. The main
problems are lack of effective methods for non-surgical prevention or early
detection and diagnosis. The next decade should explicit the accumulation of
enormous amounts of data on nanotechnology and nano-biofunCtionalization to
make it to clinical practice. Nevertheless, functionalized nanoprobes are destined
to play a major role in biomedical applications for many years to come. There is

plenty of room at the bottom.

60

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CHAPTER 2
Magnetic Gcho-Nanoparticles: A Unique Tool for Rapid Pathogen
Detection, Decontamination and Strain Differentiation

2.1. Pathogen Detection

Pathogenic bacteria continue to present a significant threat to public
health. Consequently, accurate detection and enumeration of bacteria, such as
E. coli, can help quantify the risks of contamination and reduce potential
infections.1 Traditional methods that require amplification or enrichment of the
target bacteria tend to be laborious and time-consuming. Moreover, antibodies or
Iectins functionalized on magnetic particles are not superlative for detection
because of the challenges entailed in immobilizing biomacromolecules and the
low-capture efficiencies attained, typically in the 10-30% range.2 Commercial and
synthesized magnetic beads have also been used, but these detection systems
have reduced sensitivity, less capture efficiency and limited detection range due
to a wide bead and magnetic domain size distribution.3'4 Indeed, it is
advantageous to use magnetic nanoparticles for detection. The large active
surface area offered by such nanoparticles is necessary for effective sugar
presentation. The high surface/volume ratio offers more contact surface area for
attaching carbohydrates and hence for capturing pathogens.5 The sizes of
nanoparticles are typically about two orders of magnitude smaller than a
bacterium, which allows the attachment of multiple NPs onto a bacterial cell

rendering easy magnet-mediated separation.5'6 Moreover, the small

79

 

nanoparticles have faster kinetics in solution as compared to their micrometer-
sized counterparts, which can result in fast detection.

Herein, we demonstrate the potential of sugar-coated MGNPs for fast
bacterial detection and removal, which provides an attractive avenue for
diagnostic applications. In fact, the diversity and broad knowledge pertaining to
the surface-displayed carbohydrates could aid the design of indicative tests for
accurate detection of bacteria through interactions with multivalent carbohydrate
ligands.7 We hypothesized that, due to this polyvalency, MGNPs displaying
monosaccharides can be attached to E. coli, thereby producing a notably
enhanced recognition and improving the capture efficiency. Indeed, one
challenging aspect of studying carbohydrate-protein interactions is the low affinity
(~ mM range) of oligosaccharides to their receptors leading to weak signal
responses. Enhancing these responses underscores the need to generate simple
monosaccharide ligands in a multivalent decoration providing insight into relevant
protein-carbohydrate binding events. The implementation of this concept is

crucial for minimizing and eliminating potential infections.

2.2. Syntheses of MGNPs

Our strategy to construct the artificially engineered MGNPs features: 1) a
triethoxy silane anchor that can bind to the metal oxide nanoparticles; 2) a linker
with the desired functional group allowing biomolecular attachment; 3) simple
and efficient methods for covalently attaching requisite oligosaccharides to the
spacers. Our journey commenced with the preparation of silica-coated form of

Fe304 nanoparticles (NP 1) (Figure 2-1a). In an effort to control the devised

80

___-—"”

 

nanocomposites to better suit the biorecognition purposes, we used PVP as the
surfactant and tetraethoxysilane (T EOS) as the stable coated silica shell.8 Silica-
coated magnetite NP 1 was then caged with alkyne-siloxane (AS) linker or
aminopropyltriethoxysilane (APTES)":10 to yield alkyne- or amine-terminated NPs
respectively, onto which monosaccharides were subsequently affixed (Figure 2-
1a). Siloxanes provide common intermediates for a wide range of chemical

modifications, particularly for biological applications.11

 

 

 

 

AS 0
H ..
1 O A/\ \ON /\/\Si/ dig" Iklron )1“
1 v 0 H < _,\oxrde/
F63+IF62 '1' 1)NH3. H20 lag—‘37" Iron ,1 ..
_’ \oxide/l' AIkyne-NP
(2:1) 2) PVP HO—"\../ APTES
3) TEOS' Silica coated (EtO)3Si\/\,NH2 ‘8"
cat. NH4OH NP 1 > HZNMSI<oi oxrde
Amine-NP "
b) HO OH
HO OH H
H30 0 So N
O , e
M \/\,N N 0 .— \~.
Alkvne-NP ° ”3 4. TVOWKNMs— 0.1/111111 ‘
CuSO4.5H20, sodium ascorbate o H \ 0:893:
orvrso : H20 (4:1)
Man-MGNP 2
C)
H0552; 0
Amine-NP H 0 Ar \OMN HLYH \O oxrde
BOP, HOBT, DIPEA, DMF
Man-MGNP 3 R = 80 Gal-MGNP 4 R = How
Man - Ga?”

Figure 2-1. Syntheses of MGNPs.

Functionalization of the nanoparticles with o-mannose (Man) through
either a triazole linker (MGNP 2) formed by the [2+3] Huisgen reaction12 (Figure

2-1 b) or an amide linkage (MGNP 3) (Figure 2-1c) was then attained. We also

81

 

synthesized galactose (Gal) functionalized MGNP 4 via amide coupling. The
control of the shape, size, and dispersibility of MGNPs was accomplished by the
choice of a suitable surfactant, a proper surface-coating and an efficient
hydrophilic surface functionalization. With our covalent approach, all
carbohydrates are unifome oriented on the NP surface, which is crucial for high
performances in cell-capturing studies.13 All MGNPs were characterized by X-ray

diffraction (XRD), TEM, TGA, FT-IR and HR-MAS NMR.

2.3. Characterization of MGNPs
2.3. a. Transmission Electron Microscopy (TEM)

TEM images of MGNP indicate that the diameters of the nanoparticle core
are around ~ 6 - 15 nm (Figure 2-2). Samples were prepared by depositing 5 pl
of the particle dispersion onto 200 mesh carbon-coated copper grids. The
suspension was then allowed to settle for 1 to 2 minutes before excess liquid was

removed with a paper wick.

 

Figure 2-2. TEM image of MGNPs.

82

2.3. b. X-ray Diffraction (XRD)

XRD was carried out in a dry powder form to identify the nanocrystalline

structure of the NPs. The observed diffraction peaks indicates that the NPs are

indeed Fe304 magnetitems (Figure 2-3).

 

(311)

 

 

 

 

 

(220)

(111)

 

 

 

 

 

I l

d—Scale (A)

 

Figure 2-3. XRD of MGNPs.

2.3. c. Fourier Transform Infrared Spectroscopy (FT-IR)

FT -IR analysis was performed to characterize the surface nature of
MGNPs, as depicted in Figure 2-4. The bare Fe304 NPs were coated with TEOS
was proved by comparing the IR spectra of coated and uncoated NPs. The
spectrum of the bare NP shows two main bands at 577 and 453 cm‘1 related to
Fe—O vibrations. Moreover, an intense and broad band appeared in the region
3200-3600 cm‘1 region, corresponding to the O—H stretching vibration. Compared

with the bare Fe304 NP, NP 1 mainly possess additional absorption bands in the

83

region 980-1220 cm‘1 corresponding to stretching of Si-O-Si and Si-OH bonds.17
The IR spectrum of Man-MGNP 2 has additional alkyl C-H stretching at ~ 2900

-1

 

 

 

 

cm .
Silica Coated
60 ’x NP 1
so A
MGNP 3 1 Bare Fe304 NP
40- '
% T 1 /
30
C-H stretching vibration
20
J Si-O stretching vibration \_ F343 bond
10 l
l a
4000 3200 2400 1800 1400 1000 600

Wavenumber (cirr‘)

Flgure 2- 4. FT-IR spectra of bare Fe304 NP (brown), NP 1 (blue) and MGNP 3 (black).

2.3. d. Thermogravimetric analysis (TGA)

For therrnogravimetric analysis measurements, all nanoparticles were first
heated to 100 °C (heating rate = 10 °C/min) and then cooled to 50 °C under dry
nitrogen gas to remove all adsorbed water before any measurements were
recorded. NP 1 exhibited a weight loss of 5.5 %, while Man-MGNP 3 and Gal-
MGNP 4 have weight loss of 13 % and 11.5 % respectively (Figure 2-5),
implying the presence of organic molecules on the surface of MGNPs. The
number of mannose molecules on each MGNP 3 particle was estimated as

follows: The lattice volume of magnetite is 592 A3 and Z=8 (8 Fe304
84

 

molecules/lattice). The average diameter of MGNP is ~10 nm. Assuming the
MGNP is a sphere, the number of lattice in one MGNP particle is 884 and the
number of Fe304 (MW=232) molecules in each MGNP particle is 7072. From
TGA, the weight of Fe304 accounts for 88% of the MGNP and 6% of the MGNP is
due to the carbohydrates attached. Therefore, on average, there are 220

mannose molecules (Man + APTES, MW ~ 500) on each MGNP.

100 ‘

96*

 

924

   
  

Weight (%)

Gal-MGNP 4

 

88 — Man-MGNP 3

 

 

 

T

too 360 560 I 706 Y 906
Temperature (°C)

Figure 2-5. TGA curves for NP 1 (blue), Man-MGNP 3 (pink) and Gal-MGNP 4 (green).

2.3. e. High Resolution-Magic Angle Spinning (HR-MAS) 1H NMR

NMR is a critical tool for providing detailed ligand structural features.
Unfortunately, there is a serious limitation directly applying 1H NMR to MGNPs
due to the field inhomogeneity caused by the inherent superparamagnetism of
the particles. Drastic line broadening was observed in spectra recorded with a
conventional NMR probe, which led to undistinguishable 1H NMR spectra (Figure
2-6a). Recently, HR-MAS NMR was found to be a superior tool to overcome this

85

problem.” ‘9 indeed, HR-MAS ‘H NMR spectra of MGNPs gave solution-like
spectra with a completely resolved splitting pattern, including the correct signal
multiplicities and accurate integrations (Figure 2-6b). For instance, the anomeric
proton of the galactoside ligand on Gal-MGNP 4 was well resolved as a doublet
(J = 8.0 Hz) at 4.3 ppm, indicating the configuration of the carbohydrate was
unaffected during immobilization. The fact that only one set of peaks from
carbohydrates was observed suggested that the carbohydrate coating was
homogeneous on the particle surface.

1 -
HR-MAS‘H-NMR H NMR

 

 

 

 

”9 7 5 3 ' 1 -1 ppm
'l—"fi'*—F“fi—Tl'—fi"f" 'l""l"*—fil""T"r'TTfi
4.0 3.5 3.0 2.5 2.0 1.5 1 .0 0.5 ppm

Figure 2-6. a) 1H-NMR spectrum of Gal-MGNP 4 acquired in-solution with a conventional 5 mm
probe; and b) HR-MAS 1H NMR of the same sample.

2.4. Lectin Binding Assay: MGNP/Con A Interactions

To validate the efficiency of MGNPs and to insure their use as nanoprobes
to detect E. coli, we first tested carbohydrate-Iectin interactions. Hence, the
interaction between various MGNPs with a mannose-binding Iectin, Con A,20 was

first investigated. Carbohydrate-Iectin binding is central in designing our

86

biosensor.21 After incubating NPs with fluorescein-labeled Con A overnight, a
magnetic field was applied to the mixture through a handheld magnet inducing
aggregation of magnetic NPs on the side of the vial. The residual fluorescence of

supernatants was then recorded (Figure 2-7).

    

precipitation

magnet induced Aft. - ‘
>-i ‘:

 

Supernatant
Emission spectra
MGNP recorded

MGNP / Con A complex
Figure 2-7. Schematic representation of MGNP-Con A interactions.

With MGNP 3, the emission intensity of the supernatant decreased 87%
indicating that most Con A was removed by MGNP 3 (Figure 2-8a). Triazole
linked MGNP 2 was less efficient accounting for a 60% emission decrease
probably because of the low efficiency of the Huisgen reaction with immobilized
alkynes.12 NP 1 without carbohydrates (control) did not remove any Con A,
proving that the separation of Con A is due to its interaction with carbohydrates,
rather than the nonspecific absorption to NP surface.

To test the separation efficiency dependence on concentration, different
amounts of MGNP 3 (0.2 mg, 1 mg, 2 mg, 5mg) were mixed with fluorescein-

labeled Con A (100 ug/mL, 2 mL) at 4 °C for 12 hours. The particles were then

87

subjected to magnetic separation and the fluorescence spectra of the

supernatant were recorded.

   
 

 

 

 

 

  

 

 

 

a)
8 — NP 1 + ConA
A 6 —
D a
S.
c 4 ‘
.9
(I)
.92
LIEJ 2 ‘ MGNP3+ConA
0
\ buffer
500 550 600 650
Wavelength (nm)
b)
NP 1 (5 mg) + Con A
91
7- MGNP 3 (0.2 mg) + Con A
S
s _ MGNP 3 (0.5 mg) + Con A
c 5
o
'g \ MGNP 3 (2 mg) + Con A
._ 3.
E
LIJ
1 l -
W ‘ - -» __
_1 + Con A
500 550 600 650

Wavelength (nm)

Figure 2-8. Fluorescent emission spectra of the supernatants of fluorescein-labeled Con A
solutions (100 ug/mL, 2mL) after incubation a) with MGNPs or NP 1 (5 mg); b) with NP 1 and

various amounts of MGNP 3 (0.2 mg, 1 mg, 2 mg, 5mg) and subsequent magnet mediated
separation (km = 492 nm).

88

It was observed that with increasing the concentration of MGNP 3, the amount of
Con A removed by MGNP 3 increased, resulting in decreased fluorescence
emission intensity (Figure 2-8b). All these results reveal the multivalent appeal of
MGNP with ligand clustering leading to strong binding and gave us confidence to

use our MGNP system for E. coli detection.

2.5. E. coli Detection using MGNPs

One of the mechanisms of E. coli detection is mediated by interactions
between the bacteria and the specific carbohydrate receptors. Herein, E. coli
strain ORN178 was used to validate the specific binding of MGNP 3 to type 1
fimbriae (called Fim-H) of E. coli ORN178. Type 1 fimbriae are heteropolymeric
mannose binding proteinaceous appendages that obtrude from the surface of
many gram-negative bacteria and play a major role in the binding phenomena.
After incubating MGNP 3 (2 mg/mL) with solutions of an E. coli strain ORN178
(103-107 cellslmL in PBS buffer) for a few minutes, a magnetic field was applied
separating MGNP/E. coli aggregates (Figure 2-9). The supernatants were
carefully removed and the remaining aggregates were washed thoroughly,
stained with a fluoresecent dye (PicoGreen), transferred to a glass slide, and
imaged. Fluorescent microscopic imaging showed that E. coli can be reliably
detected with a limit of 104 cells/mL (Figure 2-10). With NP 1, no bacteria were

observed on the slides.

89

, Fluorescence

,' / Microscopy
/ I, //

Detection k Capture

7

/ _ Quantification EffiCier‘CY

Supernatant (SP)

Aggregate

    
 

1) MGNP O

8?”? r

2) Magnetic

1% Separation

 

 

 

 

 

 

Figure 2-9. Schematic demonstration of E. coli detection by MGNPs.

Following the same protocol, we enumerated bacteria in the aggregate
and supernatant using the fluorescent microscope. The capture efficiency was
calculated by dividing the number of E. coli in MGNP aggregate over the total
number of cells in both the supernatant and the aggregate. High capture
efficiencies up to 88% can be achieved with 45 minute incubation. Next, the
effect of incubation time was examined. Interestingly, even in just 5 minutes, E.
coli can be detected with a capture efficiency of 65%. Indeed, the capture
efficiency using MGNP 3 is much higher than the 10-30 % range typically

2' 3' 22 which can

observed with antibody or Iectin functionalized magnetic particles,
be difficult to fabricate because of challenges in immobilizing biomacromolecules.
Furthermore, the orientation of the antibody/Iectin on a NP surface is difficult to
control, which may affect their binding capacities.23

MGNP/E. coli complexes were then imaged by TEM with MGNP
aggregates observed on the surface, at the lateral ends and along the pili24 of E.
coli cells (Figure 2-11)., Although glyco-nanoparticles have been studied as

bioprobes for pathogens,” 26 this is the first time that MGNPs have been used

for bacterium detection and decontamination.

9O

 
  

 

Figure 2-10. Representative fluorescence microsc0pic images of captured E. coli. The
concentration of bacteria (cells/mL) incubated with MGNP 3 is indicated on each image.

I‘Vf‘n
a: .

Figure 2-11. TEM images of MGNP 3/E. coli complexes at different magnifications. Arrows
indicate where MGNP 3 binds to the pili, which were faintly stained in the pictures. A drop of 1%

phosphotungustic acid was used to negatively stain MGNP/E. coli aggregates. 7

2.6. Bacterial Differentiation
Although we presented a full account on the quantification and

enumeration of E. coli cells, we further investigated the possibility of bacterium

91

differentiation. It is known that several bacteria may bind with the same
carbohydrate albeit with various affinities.7 This provides a unique opportunity to
use a MGNP array system, where the selective binding of a microbe to various
carbohydrates will lead to different responses. The resulting characteristic

response patternsza' 29

will then allow differentiation of bacteria. As a proof-of-
principle, we investigated the usage of two MGNPs (Man-MGNP 3 and Gal-
MGNP 4) to rapidly detect and differentiate three E. coli strains: ORN178,
ORN208 a mutant strain with greatly reduced mannose binding affinity,30 and an
environmental strain (ES) isolated from Lake Erie with unknown carbohydrate
binding specificity. While 65% of ORN178 was captured by MGNP 3, only 15%
was caught by MGNP 4. The mutant strain ORN208 was trapped by both
MGNPs, although at lower levels. With the ES strain, capture efficiencies of 70%
and 75% were achieved by MGNPs 3 and 4, respectively, suggesting its strong
binding with both mannose and galactose. The response patterns of the three E.
coli strains to Man-MGNP 3 and Gal-MGNP 4 allowed us to easily determine the
microbial identity: ORN178 (Man strong, Gal weak), ORN208 (Man weak, Gal
weak), and ES (Man strong, Gal strong) (Figure 2-12). The ability to distinguish
pathogen strains can have clinical applications since the virulence of many
pathogens can be correlated with carbohydrate binding specificity.31 Moreover,

the nondestructive nature of MGNP binding can allow the concentration and

recovery of pathogens and further analysis by other techniques.32

92

 

J I

Capture efficiency by
MGNP 4 (%)
.h
0

20 J on" 208 0'“: 17°

 

 

o ' 30 I 40 ' so ' so
Capture Efficiency by MGNP 3 (%)

Figure 2-12. E. coli strain differentiation by MGNPs 3 and 4.

In general, we were able to explore a MGNP-based system to not only
detect E. coli within 5 min, but also remove up to 88% of the target bacteria from
the medium. Furthermore, the identities of three different E. coli strains were
easily determined on the basis of the response patterns to two MGNPs
highlighting their potential in biosensing. Thus, we demonstrate the potential of
sugar-coated magnetic nanoparticles for fast bacterial detection and removal,
which provides new endeavors in pathogen decontamination and diagnostic

applications.

2.7. Lipoic Carbohydrate Conjugates: Controlling Nonspecific Adsorption
at Biointerfaces for Biosensor and Biomedical Application

Nonspecific adsorption from serum components is a general problem in
biosensing and biomedical applications. Nonspecific adsorption of proteins or
human serum components on biosensor surfaces of diagnostic devices reduce
the accuracy of the bioanalysis and hence pose significant challenges for their
use and applications. Therefore, the mechanistic understanding of the protein

nonspecific adsorption on surfaces and the development of biointerfaces that

93

have the ability to resist nonspecific adsorption of protein have attracted
significant research interests.33 A number of blocking reagents have been
studied both experimentally and theoretically. For example, biointerfaces
modified with blocking reagents such as bovine serum albumin (BSA),“' 35
PEG,” 37 and oligo-ethylene glycol based groups,” 39 have been shown to
decrease nonspecific protein adsorption. Among them, the most commonly used
blocking agents are BSA and PEG containing agents. Herein, we hypothesize
that carbohydrates could be suitable candidates for reducing the nonspecific
adsorption from serum components as well as the nonspecific cell attachment,
and hence will play chief roles in enhancing the detection of harmful microbes
and toxins.

The SAM scaffold40 has recently appeared as one of the most promising
model systems for mechanistic study of multivalent interactions. Carbohydrate
SAMs offer extensive control over patterns, density, and orientation, which are of
tremendous importance to elicit a clear structure-activity relationship between
protein and carbohydrate recognition events.41 More importantly, SAMs have
been applied to label-free analysis methods such as QCM.42 QCM transducer
offers the benefits of real-time monitoring of protein adsorption to surface coating
and can be used to characterize the properties of carbohydrate biointerfaces.

Herein, in collaboration with Dr. Zeng’s laboratory from Oakland
University, SAMs of sugar-thiolates on gold platforms were fabricated. It is well
known that monolayers form spontaneously by adsorption of thiols from their

solutions onto gold surfaces.43 Six types of lipoic acid carbohydrate conjugates,

94

namely lipoic mannose (LMan), lipoic galactose (LGal), lipoic glucose (LGlc),
lipoic sialic acid (LSia), lipoic N-acetyl glucosamine (LGINAc), and lipoic fucose
(LFuc) were synthesized and their properties toward protein resistance were
measured using human serum samples, the most common clinical samples for
bioanalysis. Resistance toward Hela cell attachment was also investigated as an
example to understand the carbohydrate selectivity at cell surfaces.

As shown in Figure 2-13, the structures of the six lipoic carbohydrate
derivatives consist of three components. The first component is the cyclic
disulfide, which can adsorb strongly to the Au sensor surface via Au-S bond; the
second is the OEG chain which can enhance aqueous solubility and form a
monolayer; and the third is the carbohydrate linked through their respective
reducing end. Preparation of these compounds commenced from the
commercially available lipoic acid that was coupled to the Boc-protected diamine
using a standard amide coupling condition, followed by removal of the Boc group
promoted by trifluoroacetic acid (T FA) yielding lipoic amine (Figure 2-13a). The
functionalization of carbohydrates with carboxylic acids was performed by
reduction of the corresponding azido-propyl glycosides in the presence of
succinic anhydride leading to the O-acetyl protected sugar acids in 75-87% yields
(Figure 2-13b). These were then coupled to lipoic amine using HOBt/HBTU with
subsequent protective group removal affording the six lipoic carbohydrate

derivatives in good yields.

95

3 O

\m OI INZ/\/ \/\O/\/ .3 NBC. mfiwZ. OINQN
m + O ZIwoo 1'
NV 41>. GINO—N

 

 

r608 >oa woo 90693 923.8 36} 31:6 23 mfioum
s
o I 25 4:.” 7\0 o
> .,I/\ m. . r I
GO ,\,//O\/\/ZHw 1 >00 ‘I/O\/\/Z§OI

Emma I a

snap .032. 30838 0.33. acne. 85
um - 3.x. Seam

Ior/xo o I o
i / \/\/ Z/\/ \/\O/\/ m,
0 2 O z m
I O I
Coca moa omGogaBE am2<ma<mm
:0 cm. 0: 0:
gm 0 o
0 IO J.
08 CI
IO/l/\O rim: F603 2.2533 ram. Evan 9.5283
,oiw I
\w OI
IwO 0 %WO
CI 0 O/uu
IOOI ZI>O
rag—=36 F620

Eucn F608 manommv

288:: 9:032:53

INzx\/0\/\o/\/z\;/\/\/flm\,m
1

Coca misc

Coca mine

: :8:wa Semen
NV ZmOZm. 2.60:

am .. m._ .x. seam so. 3m 26 mfimum

 

 

rmmm Fiona mmm=o no.8

Figure 2-13. Synthesis of Lipoic Carbohydrate Conjugates.

96

The carbohydrate-derived lipoic acid derivatives were then studied as
protein and cell resistant biomaterials in collaborations with Dr. Zeng’s laboratory.
The six types of carbohydrates were examined for their abilities to reduce
nonspecific adsorption of human serum and Hela cells using QCM.“ The data
suggested that the structures of carbohydrates play an important role in resisting
nonspecific binding. Specifically, the resistance was found to increase in the
order LFuc < LMan < LGINAc < LGlc < LSia < LGal, where LGal derivative
resisted most nonspecific adsorption (Figure 2-14A). Furthermore, the
combination of lipoic galactose and BSA was the most effective in reducing the
adsorption of even undiluted human serum and the attachment of Hela cells
while allowing specific binding (Figure 2448). Several control experiments have
demonstrated that the resistant-ability of mixed LGal and BSA was comparable
to the best known system for decreasing nonspecific adsorption.“ The exact
reason for this phenomenon is still not known.

A)

 

-1oo -

AF (Hz)

-200 -

 

 

 

'300 l ' i ' i ' 1 ' l

3)

 

 

 

 

 

2 x 104 Hela cells 2 x 104 Hela cells

0 _.

-20 _
ii

33, -4o -
LL
<1

-60 _

-30 _

l ' l ' i ' i ' l
0.0 0.5 1.0 1.5 2.0
Time (h)

Figure 2-14. A) Blocking effect of various lipoic acid carbohydrate derivatives. (a) LGal, (b) LSia,
(c) LGIc, (d) LGlNAc, (e) LMan, (f) LFuc; B) Real-time frequency responses to the addition of
2x104 Hela cells onto (a) the mixture of lipoic galactose and BSA modified Au quartz and (b)

PEG-thiol modified Au quartz.

2.8. Experimental Section
2.8.a. Materials and Instrumentation

Unless otherwise indicated, all starting materials, reagents and solvents
were obtained from commercial suppliers and used as supplied without further
purification. Con A-FlTC was purchased from Molecular Probes. E. coli ORN178
and ORN208 strains were kindly donated by Prof. Orndorff (North Carolina State
University). All oxygen and moisture sensitive reactions were carried under
nitrogen. Air sensitive solvents were transferred via syringe. Column

chromatography was performed employing 230-400 mesh silica gels. Thin-layer

98

chromatography (TLC) was performed using glass plates pre—coated to a depth
of 0.25 mm with 230-400 mesh silica gel impregnated with a fluorescent indicator
(254 nm). All compounds were visualized by the use of UV light or a yellow stain
containing Ce(NH4)2(N03)6 (0.5 g) and (NH4)5M07024.4H20 (24.09) in 6 % H2804
(500 mL). Degassed and double deionized water (DDW) was used in all the
biological assays. .
NMR spectra were recorded on a Varian VXRS-400 or Inova-600
instruments and were referenced using Me4Si (0 ppm), residual CHCl3 (8 1H—
NMR 7.26 ppm), CDCI3 (5 13C-NMR 77.0 ppm), residual CHDZOD (6 1H-NMR
5.32 ppm), CD300 (8 13C-NMR 49.0 ppm). Assignments of proton and carbon
signals were carried out with the aid of gCOSY experiments. ESI mass spectra
were recorded on ESQUIRE LC-MS operated in positive ion mode. High-
resolution mass spectra were recorded on a Micromass electrospray TofTM ll
(Micromass, Wythenshawe, UK) mass spectrometer equipped with an orthogonal
electrospray source (Z-spray) operated in positive ion mode, which is located at
the Mass Spectrometry and Proteomics Facility, the Ohio State University. XRD
was recorded using Rigaku Ultima Ill multipurpose X-ray diffraction system.
FTIR spectra (400-4000 cm") were recorded as KBr pellet forms using FTIR
Perkin Elmer Spectrum GX with ATR capabilities. Fluorescence emission spectra
were recorded on an AMINCO BOWMAN ll Luminescence spectrometer
provided with a continuous (CW) high power Xe lamp as the excitation source
and an excitation wavelength of 492 nm and spectra-recording at 500-650 nm.

TGA was carried out on SDT 2960 Simultaneous DTA-TGA equipment and the

99

 

samples were heated at a constant heating rate of 10 °C/min from 50 to 1000 °C.
TEM images were obtained using a Philips CM10 Mawah-NJ software Image Pro
Plus 6.0, operating at 100 kV. Bacterium enumeration was carried out using an
epifluorescent microscope (Olympus BX-51) with a 50-W Osram UV mercury

lamp.

2.8.b. E. coli Detection using MGNPs

E. coli cells were grown overnight in LB media at 37 °C to an
approximately 109 cells/mL. The culture was then centrifuged and cells washed
with Phosphate Buffered Saline (PBS) buffer. MGNPs were added to a 4 ml vial
containing E. coli solution (103-107cells/mL, 1 mL) in PBS buffer, mixed well, and
incubated at room temperature with gentle shaking. After incubation, the
samples were left on a permanent external magnet for 1 minute. The supernatant
was carefully removed by pipetting and the resulting nanoparticles were washed
three times and then re-suspended with PBS to the original volume. It is worth
mentioning that the successive buffer washing cycles are crucial to remove
unbound cells and hence nonspecific binding. All samples were then fixed with
37 % para-formaldehyde (54 uUmL), sonicated for 2 min. and then stained with a
non-specific nucleic acid stain (PicoGreen, 2 pUmL sample) for 5 minutes. Since
aggregation of bacterial cells interferes with accurate optical quantification, the
cells were dispersed by sonication. E. coli detection and enumeration was carried
out using epifluorescent microscopy. Samples were vacuum-filtered through a
glass fiber prefilter and black polycarbonate filter (25-mm diameter; 0.22 pm).

The filter paper was then air dried, transferred to a microscope slide, covered

100

with a drop of immersion oil and a cover glass, and imaged. Individual cells were
quantified by counting randomly 20 fields per each run and a minimum of 200
cells. The total number of cells on each slide was calculated by multiplying the
average number of cell per field by the number of fields (46691) per slide. All
measurements were reproducible and the deviations were found to be within

10%.

2.8.c. Evaluation of Capture Efficiency Variation

Four solutions of E. coli cells (~ 1.5 x 106 celllmL, 1 mL) were incubated
with MGNP 3 (2 mg) each for 45 minutes and the capture efficiency (C. E.) for
each incubation was calculated to be 84, 80, 88 and 82 % for four separate
experiments. The average capture efficiency for this quadruplicate experiment is

83.5 i 3.4%.

Capture Efficiency (CE) = # of cells in MGNP aggregate
# of cells in MGNP aggregate + # of cells in supernatant

2.8.d. HR-MAS 1H NMR Analyses.

HR-MAS experiments were carried out on a Bruker BioSpin FT-NMR
Avance 500 equipped with an 11.7 T superconducting ultrashield magnet
available at C.I.G.A. (Centro lnterdipartimentale Grandi Apparecchiature) of the
University of Milan. The HR-MAS probe with internal lock is capable of
performing either direct or indirect (inverse) detection experiments. MAS
experiments were performed at spinning rates of up to 15 kHz (15 kHz maximum
MAS rotation available) using a 50 pL zirconia rotor. All the samples were diluted

at different concentrations with deuterated solvents to find out the concentration

101

limit to the NMR signal broadening. HR-MAS 1H-NMR spectra were obtained
using 200-400 scans for each experiment. The sample temperature was

dependent on the rotation speed.

2.8.e. QCM Measurement

The QCM cell filled with 1 mL of PBS buffer was placed in a Faraday cage
at room temperature. Contents of the QCM cell were continuously stirred before,
during, and after the addition of analyte, which was added to the cell in 20 pL
volumes. The frequency and series damping resistance of the QCM were
monitored using a network/spectrum/ impedance analyzer (Agilent 4395A). The
relationship between the change in resonant frequency (AF) resulting from a
change in mass (Am), was given by the Sauerbrey’s equation, AF = -2nAmFg2
(pquq)"’2A'1, where n is the overtone number, pq is the shear modulus of the
quartz (2.947 x 1011 g.cm'1.s'2), and pq is the density of the quartz (2.648 g.cm'3).
According to the Sauerbrey’s equation, our fitted frequency change of 1 Hz
corresponds to a mass increase of 1 ng for the 10 MHz quartz crystal used in this

work.

102

2.8. f. Syntheses of MGNPs

1. Synthesis of sugar building blocks (Mannose and Galactose)

Synthesis of mannose amido-acid (M5).

AcO 03° 3 A00 OOAc b AcO 08°
Afigfig OAc 700/ A280 92% A2803 1
O
o’\/\cr o’\/\ N3
M1 M2 M3

Figure 2-15. Reagents and conditions: a) 3-chloro-1-propanol, BF3.Et20, DCM; b) NaN3, DMF,
80 °C; c) succinic anhydride, H2/Pd-C, THF, 3h; d) NaOMe, MeOH.

3-Azidopropyl 2,3,4,6-tetra-O-acetyI-a-o-mannopyranoside (M3). M3 was
prepared as previously described starting from pentaacetyl mannose M14547

Comparison of NMR spectra with those reported confirms its structure. 45“"

O-Acetyl protected mannose amido-acid (M4). A solution of M3 (0.650 g, 1.51
mmol), succinic anhydride (0.230 g, 2.30 mmol) and 10 % Pd/C (0.166 g) in dry
THF (20 mL) was stirred at room temperature under H2. After 3 hours, TLC
(EtOAc-hexanes, 1:1) showed complete conversion of starting material (R, = 0.5)
to a major product (Rf = 0.0). The reaction mixture was then filtered off through
Celite and concentrated. The residue was purified by column chromatography
starting from EtOAc-hexanes 1:1 increasing to MeOH-DCM, 1:10 to afford the
product M4 as a sticky white solid (0.67 g, 88 % yield). 1H NMR (CDCI3, 600
MHz): 5H 1.78-1.85 (m, 2H, OCHZCchHzNH), 1.97 (s, 3H, OAc), 2.03 (s, 3H,

103

 

 

OAc), 2.08 (s, 3H, OAC), 2.13 (s, 3H, OAC), 2.50 (t, J = 6.3 Hz, 2H,
COCH2CHZCOOH), 2.64 (t, J = 6.0 Hz, 2H, COCHZCHZCOOH), 3.35 (t, J = 6.0
Hz, 2H, OCH2CHZCH2NH), 3.47-3.50 (m, 1H, OCHHCHZ), 3.74-3.78 (m, 1H,
OCHHCHZ), 3.95-3.97 (m, 1H, H-5), 4.11 (dd, J = 2.4, 12.0 Hz, 1H, H-6a), 4.24
(dd, J = 4.8, 12.0 Hz, 1H, H-6b), 4.79 (d, J = 1.2 Hz, 1H, H-1), 5.21 (dd, J = 1.2;
2.4 Hz, 1H, H-2), 5.25-5.27 (m, 2H, H-3, H-4), 6.42 (bs, 1H, NH). 13C NMR
(CDCI3, 100 MHz): 6c 20.9, 21.0, 21.1 (40, CH3), 29.2 (1C, CHchzNH), 30.1
(1C, COCHZ), 31.0 (10, CHZCOOH), 37.6 (1C, CHZCHzNH), 62.8 (10, CHZOAC),
66.3 (10, CH), 66.8 (10, CH), 68.7 (10, CHZO), 69.4 (10, CH), 69.7 (1C, CH),
97.8 (10, Canomeric). 170.0 (10, CH30(O)O), 170.4 (10, CHgC(O)O), 170.5 (10,
CHgC(O)O), 171.2 (10, CH30(O)O), 172.9 (10), 175.9 (1C). ESl-MS m/z calcd.

for Cz1H31NN3013 [M + Na]+: 528.5; found: 528.4.

Mannose amido-acid (M5). A freshly prepared solution of NaOMe-MeOH (0.35
mL, 1 M) was added to a solution of M4 (0.350 g, 0.692 mmol) in dry methanol (8
mL) at room temperature under nitrogen. The mixture was stirred for 5 hours,
neutralized with Amberlite lR-120 (H‘) ion-exchange resin, filtered, and
concentrated. Flash column chromatography (MeOH-DCM, 1:1) of the residue
afforded M5 as a gel like white solid in 97% yield (0.23 g). 1H NMR (CD300, 600
MHz): 8H 1.76-1.80 (m, 2H, OCHZCHchzNH), 2.45 (t, J = 6.9 Hz, 2H,
COCH20H2COOH), 2.56 (t, J = 6.9 Hz, 2H, COCHZCH2000H), 3.23-3.29 (m,
2H, OCHchchzNH), 3.43-3.47 (m, 1H, OCHHCHz), 3.50-3.54 (m, 1H, H-5),
3.58 (t, J = 9.6 Hz, 1H, H-4), 3.68-3.71 (m, 2H, H-3, H-6a), 3.76-3.80 (m, 2H, H-

2, OCHHCHz), 3.84 (dd, J = 2.1, 11.7 Hz, 1H, H-6b), 4.74 (d, J = 1.2 Hz, 1H, H-

104

1). 13C NMR (C0300, 100 MHz): 60 29.1 (1C, CHchZCHzNH), 31.0 (1C,
COCHZCHZCOOH), 31.4 (1C, COCH20H2COOH), 36.4 (1C, CHZCHZNH), 61.7
(1C, CHon), 64.8 (10, CH2 CHzO), 67.5 (1C, CH), 71.0 (1C, CHzO), 71.4 (10,
CH), 73.5 (1C, CH), 100.5 (10, Canomen-c). 174.0 (10). HR-MS m/z calcd. for

C13H23NN309 [M + Na]+: 360.1271, found: 360.1273.

ACO OAc HO OH
O N OM O
89% a e. Hgfig

MeOH /\/\

o/\/‘N o N
M3 3 M6 3

Figure 2-16. Synthesis of Azido-mannose for Click Chemistry.

3-a-Azidopropyl o-mannopyranoside (M6). A solution of M3 (1.35 g, 5.13
mmol) in MeOH (10 mL) was treated with a solution of sodium (23 mg) in MeOH
(1 mL). After 1 hour, the solution was acidified with Amberlite lR-120H+ to pH 6.
Evaporation of the solvent under reduced pressure afforded M6 as a white
crystalline solid (0.783 g, 95%). 1H NMR (CD300, 600 MHz): 5H 1.83-1.88 (m,
2H, CHZCH2N3); 3.41 (td, J = 6.8, 3.6 Hz, 2H, CH2N3); 3.48-3.53 (m, 2H,
OCHHCH; + H-5); 3.60 (t, J = 9.6 Hz, 1H, H-4); 3.67 (dd, J = 3.2, 9.2 Hz, 1H, H-
3); 3.71 (t, J = 6.0 Hz, 1H, H’-6); 3.79-3.85 (m, 3H, H-2, H-6”, H-7”); 4.74 (d, J =
1.6 Hz, 1H, H-1). 13C NMR (C0300, 100 MHz): 8.; 28.8 (10, CH20H2N3); 56.2
(10, CH20H2N3); 61.7 (10, CH20H); 64.2 (10, CHZCHzO); 67.4 (10, CH); 71.0
(1C, OH); 71.4 (10, OH); 73.6 (10, CH); 100.5 (1C, Canomeric).

105

l'.‘

 

Synthesis of galactose amido-acid (G5).

ACO OAC AcO OAC ACO OAC
0 a 0 b $8
ACO OAC _—"’ AcO OW C I ——> AcO OW N 3
OAC 45% OAC 94% OAc
G1 (52 G3
OAC OH
—_‘* $590 N —* %
850/0 ACO 0Ac \/\/ OH 1000/o HO OH OWNWLOH
G4 0 GS 0

Figure 2-17. Reagents and conditions: a) 3-chloro-1-propanol, BF3.Et20, DCM; b) NaN3, DMF,
80 °C; c) succinic anhydride, H2/Pd-C, THF, 3h; d) NaOMe, MeOH.

3-Azidopropyl 2,3,4,6-tetra-O-acetyl-fi-o-galactopyranoside (G3). G3 was
prepared analogously as M3 starting from commercially available penta-acetyl
galactose G1.“ 48 Comparison of NMR spectra with those reported in the

literature confirms its structure. 47' ‘8

O-Acetyl protected galactose amido-acid (G4). A solution of G3 (0.510 g, 1.18
mmol), succinic anhydride (0.180 g, 1.80 mmol), and 10 % Pd-C (0.130 g) in dry
THF (15 mL) was stirred at room temperature under H2. After 3 hours, TLC
(EtOAC-hexanes, 1:1) showed complete conversion of starting material (Rf = 0.5)
to a major product (Rf = 0.0). The reaction mixture was then filtered off through
Celite and concentrated. The residue was purified by column chromatography
starting from 1:1 EtOAC-hexanes to 1:10 MeOH-DOM to afford the product G4 as
a sticky white solid (0.508 g, 85 % yield). 1H NMR (CDCI3, 600 MHz): 8H 1.74-
1.86 (m, 2H, OCHZCHZCHZNH), 1.99 (s, 3H, OAC), 2.05 (s, 3H, OAC), 2.08 (s,

3H, OAC), 2.17 (s, 3H, OAC), 2.47-2.70 (m, 4H, COCHzCHzCOOH), 3.31-3.38 (m,

106

 

1H, CHZCHHNH), 3.40-3.46 (m, 1H, CHZCHHNH), 3.56-3.61 (m, 1H, OCHHCHz),
3.92 (td, J = 1.2, 10.2 Hz, 1H, H-5), 3.98-4.03 (m, 1H, OCHHCHz), 4.10415 (m,
1H, H-6a), 4.19-4.24 (m, 1H, H-6b), 4.44 (d, J = 7.6 Hz, 1H, H-1), 5.05 (dd, J =
3.6, 10.4 Hz, 1H, H-3), 5.17 (dd, J = 8.0, 10.8 Hz, 1H, H-2), 5.40 (dd, J = 0.8, 3.2
Hz, 1H, H-4), 6.45 (t, J = 5.6 Hz, 1H, NH). 13C NMR (CDCI3, 100 MHz): 8,; 20.8,
21.9, 21.1 (4C, CH3), 29.1 (10, CHchzNH), 30.6 (10, COCHz), 30.7 (10, CH2-
COOH), 38.3 (1C, CH2CH2NH), 61.5 (10, CHzOAC), 67.2 (1C, CH), 69.3 (1C,
CH), 69.5 (10, CHzO), 70.7 (1C, CH), 71.0 (10, CH), 101.6 (10, Camisole). 170.3
(1C, CH30(O)O), 170.46 (10, CH3C(O)O), 170.54 (10, CHgC(O)O), 170.8 (10,
CH3C(O)O), 173.3 (10), 175.3 (1C). MS-ESI m/z calcd. for 021H31NNa013 [M +

Na]+: 528.5; found: 528.4.

Galactose amido-acid (GS). A solution of G4 (0.310 g, 0.613 mmol) in MeOH (5
mL) was treated with a solution of sodium (8.60 mg) in MeOH (0.35 mL) under
nitrogen. After 3 h, the solution was acidified with Amberlite lR-120 H+ to pH=6.
Evaporation of the solvent under reduced pressure afforded GS as a white sticky
solid in quantitative yield (0.206 g). 1H NMR (00300, 600 MHz): 8H 1.74-1.79 (m,
2H, OCHZCHZCHZNH), 2.44 (t, J = 6.6 Hz, 2H, COCH20H2COOH), 2.56 (t, J =
7.2 Hz, 2H, COCHZCH2COOH), 3.23-3.27 (m, , 1H, OCHZCHZCHHNH), 3.31-
3.35 (m, , 1H, OCHZCHZCHHNH), 3.45 (dd, J = 3.0, 9.6 Hz, 1H, ), 3.49-3.52 (m,
2H, H-2, H-5), 3.60 (dt, J = 6.0, 10.2 Hz, 1H, OCHHCHZ), 3.68-3.72 (m, 1H, H-
6a), 3.73-3.77 (m, 1H, H-6b), 3.81 (dd, J = 1.2, 3.3 Hz, 1H, H-4), 3.92 (dt, J = 6.0,
10.2 Hz, 1H, OCHHCHz), 4.20 (d, J = 7.8 Hz, 1H, H-1). 13C NMR (CD300, 100

MHZ): 5c 29.1 (1C, CH2CH2CH2NH), 32.6 (10, COCHz CHZCOOH), 33.1 (10,

107

COCHz CHzCOOH), 36.5 (1C, CHzCHzNH), 61.4 (1C, CHzOH), 67.2 (1C, CH2
CHzO), 69.3 (1C, CH), 71.3 (1C, CHzO), 73.8 (1C, CH), 75.5 (1C, CH), 103.8
(1C, Canomeric), 174.7 (10). HR-MS m/z calcd. for C13H23N09 [M + Na]+:

360.1271, found: 360.1273.

2. Synthesis of Alkyne-Siloxane (AS) Linker for Click Chemistry

/\/‘Si(OC H )
OH h{—(:\NH 2 5 3
72% o 60%

Propargyl Alcohol Alkyne-AcidH (AA) Alkyne-Siloxane (AS)

Figure 2-18. Reagents and conditions: a) succinic anhydride, DMAP, DCM, 6h; b) APTES, EDC,
DCM, 12h.

Alkyne-Acid (AA). To a solution of propargyl alcohol (2.06 g, 36.8 mmol) and
succinic anhydride (4.65 g, 46.5 mmol) in dry DCM (30 mL), 4-
dimethylaminopyridine (DMAP) (4.80 g, 39.3 mmol) was added and the reaction
mixture was stirred under nitrogen for 6 hours. After TLC showed complete
conversion, the solution was diluted with DCM, acidified with 0.1 M HCl till pH=2
and then extracted with DCM. The organic layer was then dried and concentrated
to afford 4.00 g (72 %) of AA as a white solid. 1H NMR (CDCIa, 600 MHz): 5H
2.47 (t, J = 2.4 Hz, 1H), 2.63-2.70 (m, 4H), 4.68 (d, J = 2.4 Hz, 2H, CHgO). 13C
NMR (CDCI3, 100 MHz) Sc 28.8, 29.0, 31.2, 52.6, 75.3, 171.6, 178.3. MS (ESI)

m/z calcd. for CyHeNaO4 [M + Nal“: 179.1; found: 179.1.

Prop-2-ynyl succinate amidopropyl triethoxysilane (AS). To a solution of acid

AA (0.550 g, 3.52 mmol) and 1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide

108

 

hydrochloride (EDC) (1.00 g, 5.22 mmol) in dry DCM (10 mL), APTES (0.942 g,
4.25 mmol) was added and the reaction mixture was stirred under nitrogen. After
12 hours, the solvent was removed under reduced pressure. The residue was
purified by column Chromatography from 1:1 EtOAc-hexanes to 1:10 MeOH-DCM
to give 0.75 g (60 %) of the desired linker AS as a clear white crystalline solid. 1H
NMR (CDCI3, 600 MHz): 6H 0.58 (t, J = 12.0 Hz, 2H, CHZCsti), 1.17 (t, J = 10.8
Hz, 9H, (OCH2CH3)3), 1.53-1.60 (m, 2H), 2.43 (t, J = 10.8 Hz, 2H), 2.45 (t, J = 4.2
Hz, 1H), 2.66 (t, J = 10.8 Hz, 2H), 3.18 (q, J = 10.8 Hz, 2H, NHCHZCHZ), 3.76 (q,
J = 10.8 Hz, 6H, (OCH2CH3)3), 4.63 (d, J = 3.6 Hz, 2H, CHgocO), 6.01(bs, 1H,
NH). 13C NMR (CDCI3, 100 MHz) 5.; 7.9, 18.5, 23.0, 29.5, 31.0, 42.1, 52.3, 58.6,
75.2, 77.7, 171.1, 172.4. MS (ESI) m/z calcd. for C15H29NN30551 [M + Naj“:

382.2; found: 382.3.

3. Synthesis of magnetic F9304 NPs and subsequent immobilization
Preparation of silica-coated magnetite nanoparticles (NP 1)

Fe304 NPs were prepared by slight modification of the co-precipitation
method.” ‘5' ‘9 Briefly, 1M Fe3+ and 1M Fe2+ were prepared by dissolving the
iron salts in 2M HCI solutions, respectively. Then 1 eq. of Fe” solution was
mixed with 0.5 eq. of Fe2+ solution in a degassed flask. NH4OH (30%) was
added until pH ~11 was reached where a black material was precipitated.
Adjustment of pH ~11-12 is critical to get 6 - 15 nm particle size (confirmed by
TEM). After vigorous stirring for 30 minutes at room temperature, the resulting
black NPs were isolated by applying a permanent external magnet (Lifesep® 50

SX magnetic separator), washed 6 times with double deionized water and 3

109

 

times with ethanol. in an effort to better suit biorecognition purposes, we used
PVP as the surfactant and TEOS as the stable coated silica shell. In a typical
experimental procedure,8 a solution of PVP (Mw ~ 55 KDa, 0.65 mL, 25.6 g/L)
was added to the bare Fe304 NPs suspended in DDW (150 mL). The mixture was
stirred overnight at room temperature. The resulting nanoparticles were then
separated, thoroughly washed by water-acetone mixture and re-suspended in 2-
propanol: water (4:1 WV, 150 mL). NH4OH solution (0.2 % v/v, 0.3 mL) was
added followed by dropwise addition of TEOS (1.5 mL) over 1 hour with
continuous stirring. The reaction mixture was stirred overnight, isolated and

washed repeatedly with diethyl ether and ethanol leading to silica coated NP 1.50

Preparation of Man-MGNP 2 using Huisgen [2+3] cycloaddition reaction
Alkyne—Siloxane (AS) (55 mg) was added to silica coated NP 1 (55 mg)
suspended in dry toluene (100 mL) (Figure 2-1 a). The mixture was sonicated for
12 hours and then stirred overnight. The resulting alkyne-NP were magnetically
isolated, washed with water, ethanol, diethyl ether, DMSO and redispersed in
DMSO:H20 (4:1). 3-Azidopropyl d-o-mannopyranoside M6 (55 mg),
CuSO4.5H20 (10 mg) and sodium ascorbate (20 mg) were then added to the
alkyne—functionalized NP in DMSOszO (4:1, 50 mL) under sonication (Figure 2-
1b). The reaction mixture was stirred at room temperature for 24 hours under
nitrogen. The resulting Man-MGNP 2 were then isolated and washed with water,

ethanol and ether successively.

110

Preparation of MGNPs 3 and 4 using amide (BOP) coupling

To prepare the amine-functionalized NP, NP 1 was suspeneded in dry
toluene (150 mL) and sonicated for 30 minutes. APTES (2 % w/v) was then
added and stirred at 60 °C overnight (Figure 2-1a). The resulting amine-NP was
isolated, washed and redispersed in ethanol. To immobilize the surface with
sugar using amide bond coupling, amine-NP (450 mg) was washed with DMF
twice, redispersed in DMF (150 mL) and sonicated for 30 minutes. The coupling
solution was prepared by mixing mannose amido-acid M5 (0.515 g),
benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate
(BOP) (1.1 g, 1.5 eq.), 1-hydroxybenzotriazole (HOBT) (0.25 g, 1.3 eq.), and
diisopropylethylamine (DIPEA) (1.0 mL, 4 eq.) in DMF (15 mL). The coupling
solution was then added dropwise to the amine-NP in DMF under sonication and
the reaction mixture was stirred overnight at room temperature (Figure 2-1c).
The resulting Man-MGNP 3 was then isolated and washed with ether and
ethanol. The same protocol was followed to synthesize the analogous Gal-MGNP

4 using galactose amido-acid G5

2.8.f. Synthesis of Lipoic Carbohydrate Derivatives

1. Synthesis of Lipoic Amine

 

HO 3’8
Li oic Acid 0 H '5
BocHN/VOwO/VNHZ O P > R/\/ \/\O/\/ S
a 80% O
, R=NHBoc,
Boc-protected diamine b 3% R=NH2, Lipoic Amine
0

Figure 2-19. Reagents and conditions: a) EDC, Et3N, DCM, overnight; b) TFA, DCM, 1 h.

111

 

Lipoic Amine. A solution of lipoic acid (0.50 g, 2.42 mmol), Boc-protected
diamine51 (1.40 g, 5.64 mmol), N-ethyl N,N’-dimethylaminopropyl carbodiimide
hydrochloride (EDC, 0.930 g, 4.84 mmol) and triethylamine (Et3N, 1.00 mL, 7.12
mmol) in anhydrous dichloromethane (DCM, 10 mL) was stirred under nitrogen at
room temperature for 12 hrs. The reaction mixture was rotary evaporated and
the resulting residue was purified by column chromatography eluting with 7%
MeOH in CH2CI2 to afford the protected lipoic amide as sticky yellowish oil (0.85
g, 80%). 1H NMR (500 MHz, CDCl3): 8... 1.42 (s, 9H, CH3), 1.40-1.50 (m, 2H,
COCHchchchZCHS), 1.60-1.72 (m, 4H, COCHZCHchZCHchS), 1.85-1.92
(m, 1H, CHgHCst), 2.18 (t, J = 7.5 Hz, 2H, COCHchZCHZCHchS), 2.40-2.47
(m, 1H, CHHbCst), 3.06-3.18 (m, 2H, CHQS), 3.28-3.32 (m, 2H, CONHCHz),
3.44 (dd, J = 5.5, 10.5 Hz, 2H, BocNHCHz), 3.52-3.57 (m, 1H,
COCHZCHchZCHZCHS), 3.54 (t, J = 5.0 Hz, 4H, OCHZCH20), 3.58-3.62 (m,
4H, CH200H2CH20CH2), 5.0 (bs, 1H, NH), 6.1 (bs, 1H, NH). 13C NMR (125
MHz, CDCI3): 60 25.6, 28.6, 29.1, 34.9, 36.6, 38.67, 38.69, 39.4, 40.45, 40.48,
40.55, 56.7, 70.16, 70.42, 70.47, 162.7, 173.1. HRMS mlz calcd. for
C19H37N205$2IM+H1+2 437.2018; found: 437.2009. Deprotection of lipoic amide
(0.85 g, 1.95 mmol) was completed with TFA (2 mL) in CH20l2 (3 mL) after 1 h.
The reaction mixture was evaporated and then purified by flash column
chromatography (25% MeOH in CHZClz) to furnish lipoic amine as a sticky
yellowish gelatin in 84% yield (0.55 g). 1H NMR (500 MHz, CD300): 6H 1.42-1.48
(m, 2H, COCHzCHchchchS), 1.60-1.78 (m, 4H, COCHZCHchZCHchS),
1.88-1.95 (m, 1H, CHaHCHZS), 2.24 (t, J = 7.0 Hz, 2H, COCHzCHzCHzCHzCHS),

112

2.45-2.52 (m, 1H, CHHbCst), 3.10-3.22 (m, 4H, CH23, CH2NH2), 3.38 (t, J =
5.5 Hz, 2H, CONHCHzCHzO), 3.58 (t, J = 5.5 Hz, 2H, CONHCHzCHzO), 3.58-
3.62 (m, 1H, COCHzCHzCHzCHzCHS), 3.66-3.71 (m, 4H, OCH2CH20), 3.74 (t, J
= 5.0 Hz, 2H, OCHgCHzNHz). 13C NMR (125 MHZ, CDgOD): Be 25.5 (10,
COCHzCHzCHzCHzCHS), 28.7 (10, COCHzCHzCHzCHzCHS), 34.6 (10,
COCHzCHzCHzCHzCHS), 35.7 (1C, COCHzCHzCHzCHzCHS), 38.2 (1C, SCH2),
39.0 (1C, CONHCHz), 39.5 (1C, NH20H2), 40.2 (1C, SCHCHzCst), 56.5 (10,
SCHCHz), 66.7 (1C, OCHzCHzNHz), 69.5 (1C, OCHzCHzNHCO), 70.12, 70.19
(2C, OCHchzO), 175.1 (1C, NHCO). HR-MS m/z calcd. for

C14H29N20382[M+H]+: 337.1620; found: 337.1628.

2. Synthesis of Lipoic Galactose (LGal)

0Ac %Ac H 0 OAc %Ac H O H s
moaoWNNOH a . Acog‘A/OWNNN’VOWONN S,
OAC O 61 0/o OAC O H O
Galactose amido-acld G4 LGalA

Figure 2-20. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA, DMF, 3hrs; b)
NaOMe, MeOH, 1h.

General Procedures for O-acetyl sugar acids formation, amidation and
deprotection. To functionalize the carbohydrates, a solution of the azido propyl
glycoside, succinic anhydride (1.5 equiv.) and 10% Pd/C in dry THF was stirred
at room temperature under H2. After 3 h, TLC (EtOAc-hexanes, 1:1) showed
complete conversion of starting material to a major product. The reaction mixture

was then filtered through Celite and concentrated. The residue was purified by
113

column chromatography to afford the product O-acetyl protected sugar acids as a
gel like solid in 75 - 87%yield. A solution of O-acetyl sugar acid, 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium hexafluorophosphate (HBTU) (1
equiv), hydroxybenzotriazole (HOBT) (1 equiv), and DIPEA (3.5 equiv.) in
anhydrous DMF was stirred under nitrogen at room temperature for 5 min. Lipoic
amine (1.5 equiv.) was then added and the reaction mixture was stirred for 3 h.
The solvent was removed, and the resulting mixture was washed with water
followed by brine, dried over Na2$O4 and rotary evaporated. The residue was
purified by column chromatography eluting with 3 - 20% MeOH in DCM to afford
the lipoic sugar acetates. Then the solution of lipoic sugar acetate (0.2 M) in
MeOH (2 mL) was treated with a solution of 30% NaOMe in MeOH (0.2 mL)
under nitrogen. After 1 h, the solution was neutralized with Amberlite IR -120 H”
to pH=6 (For‘lipoic sialic acid synthesis, 0.2 mL of water was added to the
reaction mixture half an hour prior to neutralization). Evaporation of solvent under
reduced pressure afforded the requisite final lipoic carbohydrate derivatives

(LGal, LMan, LFuc, LGINAc, LGlc and LSia) in 45 - 61%yields for the two steps.

Lipoic Galactose Acetate (LGalA). LGalA (0.148 g, 0.180 mmol) was
synthesized in 61% yield following the general procedure for amidation starting
from galactose amido acid G4 synthesized before (0.150 g, 0.297 mmol). 1H
NMR (500 MHz, CDClg): 5H 1.40-1.48 (m, 2H, COCHchZCHZCHZCHS), 1.60-
1.73 (m, 4H, COCHZCchHZCchHS), 1.74-1.80 (m, 2H, COHZCHZCHzNH),
1.87-1.93 (m, 1H, CHaHCHZS), 1.98 (s, 3H, OCH3), 2.04 (s, 3H, OCH3), 2.07 (s,

3H, OCH3), 2.16 (s, 3H, OCHg), 2.20 (t, J = 6.5 Hz, 2H,

114

 

COCH2CH2CH2CH20HS), 2.42-2.48 (m, 1H, CHHbCst), 2.49-2.57 (m, 4H,
COCHchch), 3.08-3.13 (m, 1H, CchHaHS), 3.14319 (m, 1H, CHZCHHDS),
3.22-3.40 (m, 2H, OCHZCHZCHZNH), 3.41-3.46 (m, 4H, CONHCHz), 3.53-3.58
(m, 6H, HchCHZCHZOCHz, OCHaHCHZCHzNH, COCHZCHZCHZCHZCHS), 3.60
(s, 4H, H2COCH20HzOCH2), 3.91 (dt, J = 1.0, 6.0 Hz, 1H, H-5), 3.94-3.99 (m,
1H, OCHHbCH2CH2NH), 4.104.20 (m, 2H, H-6), 4.44 (d, J = 8.0 Hz, 1H, H-1),
5.03 (dd, J = 3.5, 10.5 Hz, 1H, H-3), 5.18 (dd, J = 7.5, 10.5 Hz, 1H, H-2), 5.40
(dd, J = 1.0, 3.5 Hz, 1H, H-4), 6.37, 6.41, 6.61 (t, 3H, NH). 13C NMR (125 MHz,
CDCI3): 250 20.8, 20.9, 21.1 (4C, CH3), 25.6 (1C, COCHZCHZCHZCHZCHS), 29.1
(10, COCHZCH20H2CH2CHS), 29.4 (10, OCHchchzNH), 31.8, 32.0 (2C,
HNCOCHZCHZCONH), 34.9 (10, COCH2CH20HZCH2CHS), 36.5 (1C,
COCH20H2CH2CH2CHS), 37.6 (1C, NHCHZCHZCHzo), 38.7 (10, SCHz), 39.45,
39.46 (2C, CONHCHz), 40.5 (1C, SCHCHZCst), 56.7 (10, SCHCHZ), 61.5 (1C,
C6), 67.2 (10, OCH20H20H2NH), 68.9 (10, CH), 69.2 (10, CH), 70.0 (1C, OH),
70.2 (2C, CH;OCH20HzOCH2), 70.46, 70.49 (2C, CH20CHZCHZOCH2), 70.9 (1C,
CH), 71.0 (10, CH), 101.5 (10, C1anomen'c). 170.1 (10, CH3C(O)O), 1703 (1C,
CHgC(O)O), 170.5 (10, CH30(O)O), 170.6 (10, CH3C(O), 172.5, 172.6, 173.2
(3C, NHC(O)CH2). HRMS m/z calcd. for 035H57N3NaO1582 [M + Na]*: 846.3129;

found: 846.3127.

Lipoic Galactose (LGal). LGal (0.0795 g, 0.121 mmol) was synthesized in
quantitative yield following the general procedure for deprotection starting from
LGalA (0.10 g, 0.121 mmol). 1H NMR (500 MHz, CDgOD): 8H 1.42-1.48 (m, 2H,
COCHZCHZCH20H2CHS), 1.60-1.75 (m, 4H, COCH20H2CH2CH20HS), 1.76-1.82

115

(m, 2H, COHchchzNH), 1.87-1.93 (m, 1H, CHaHCst), 2.22 (t, J = 7.5 Hz,
2H, COCHchZCHZCHZCHS), 2.44-2.48 (m, 1H, CHHbCHZS), 2.49 (dt, J = 4.5,
6.5 Hz, 4H, COCHZCHch), 3.08-3.14 (m, 1H, CHchaHS), 3.16-3.22 (m, 1H,
CHZCHHbS), 3.26-3.32 (m, 2H, OCHZCH2CH2NH), 3.34-3.40 (m, 4H, CONHCHz),
3.49 (dd, J = 3.0, 9.0 Hz, 1H, H-3), 3.52-3.54 (m, 2H, H-2, H-5), 3.53-3.57 (m,
5H, H2000H20H20CH2, COCHzCHZCHZCHZCHS), 3.58-3.62 (m, 1H,
OCHaHCHZCHzNH), 3.63 (s, 4H, HZCOCchHzocHz), 3.71-3.80 (m, 2H, H-6),
3.84 (dd, J = 1.0, 3.5 Hz, 1H, H4), 3.92-3.97 (m, 1H, OCHHbCHchzNH), 4.23
(d, J = 7.0 Hz, 1H, H-1). 13C NMR (125 MHz, C0300): ac 25.6 (10,
COCHZCHchZCHchS), 28.7 (10, COCHzCH2CH20HZCHS), 29.1 (10,
OCHZCH2CH2NH), 31.20, 31.23 (20, HNCOCHZCHZCONH), 34.6 (1C,
COCH2CHZCH2CHZCHS), 35.7 (1C, COCHZCHZCHZCHZCHS), 36.6 (10,
NHCHZCHZCHZO), 38.2 (10, SCHz), 39.16, 39.24 (2C, CONHCHZ), 40.2 (1C,
SCHCHZCHgs), 56.5 (10, SCHCHZ), 61.5 (1C, C6), 67.2 (1C, OCH2CHZCH2NH),
69.2 (1C, C4), 69.45, 69.50 (2c, CHZOCHZCHZOCHZ), 70.2 (2C,
CHZOCHZCH20CH2), 71.4 (1C, C2), 73.9 (10, C3), 75.6 (10, C5), 103.8 (1C,
Cum...) 173.42, 173.56, 174.95 (3C, NHC(O)CH2). HRMS mlz calcd. for

C27H50N301152 [M+H]+I 656.2887; found: 656.2880.

116

3. Synthesis of Lipoic Mannose (LMan)

ACO ACO

A O
280% O a b
H !
OWN
0H 54 % over 2 steps

O
Mannose amido acid M4

H0 051
69% H 0 H s
OWNNNNO$ONN S'
O

H o
LMan

Figure 2-21. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA, DMF, 3hrs; b)
NaOMe, MeOH, 1h.

Lipoic Mannose Acetate (LManA). LManA (0.0980 g, 0.119 mmol) was
synthesized in 60% yield following the general procedure for amidation starting
from mannose amido acid M4 synthesized before (0.100 g, 0.198 mmol). 1H
NMR (500 MHz, CDCI3): 6H 1.42-1.52 (m, 2H, COCHZCHZCHchZCHS), 1.58-
1.76 (m, 4H, COCHchgCHZCHchS), 1.78-1.87 (m, 2H, COHZCH2CH2NH),
1.88-1.96 (m, 1H, CHaHCst), 2.00 (s, 3H, OCH3), 2.06 (s, 3H, OCH3), 2.12 (s,
3H, OCH3), 2.16 (s, 3H, OCHa), 2.22 (t, J = 7.0 Hz, 2H,
COCHgCHZCHchZCHS), 2.44-2.50 (m, 1H, CHHbCst), 2.52-2.56 (m, 4H,
COCHchch), 3.10-3.21 (m, 1H, CHchzs), 3.33-3.38 (m, 2H,
OCHZCHZCHzNH), 3.42-3.48 (m, 4H, CONHCHz), 3.49-3.53 (m, 1H,
OCHaHCchHzNH), 3.54-3.60 (m, 5H, HZCOCHZCHZOCHz,
COCHZCHzCHZCHchS), 3.62 (s, 4H, H200CH20HZOCH2),3.72-3.78 (m, 1H,
OCHHbCHZCHzNH), 3.94-4.00 (m, 1H, H-5), 4.10 (dd, 1H, H-6a), 4.27 (dd, 1H,
H-6b), 4.80 (d, 1H, H-1), 5.20-5.24 (m, 1H, H-2), 5.24-5.29 (m, 2H, H-3, H-4),
6.30, 6.40, 6.60 (t, 3H, NH). 13C NMR (125 MHz, CDCl3): ac 20.8, 20.9, 21.1 (40,
CH3), 25.6 (10, COCH20H2CHZCHZCHS), 29.1 (1C, COCHZCHZCHZCHZCHS),

117

29.4 (1C, OCHzCHzCHzNH), 31.8, 32.0 (2C, HNCOCHzCHZCONH), 34.9 (1C,
COCHZCHZCH2CHzCHS), 36.5 (1C, COCHzCHzCHzCHzCHS), 37.6 (1C,
NHCHzCHzCHzO), 38.7 (1C, SCHz), 39.45, 39.46 (2C, CONHCHz), 40.5 (1C,
SCHCHzCst), 56.7 (1C, SCHCHz), 61.5 (1C, 06), 67.2 (1C, OCHzCHzCHzNH),
68.9 (10, CH), 69.2 (1C, CH), 70.0 (10, CH), 70.2 (2C, CHzOCHZCHzOCHz),
70.46, 70.49 (20, CHzOCHgCHzOCHz), 70.9 (10, CH), 71.0 (1C, CH), 101.5 (1C,
Gunmen-c), 170.1 (1C, CH3C(O)O), 170.3 (1C, CH3C(O)O), 170.5 (1C,
CH3C(O)O), 170.6 (10, CH3C(O), 172.5, 172.6, 173.2 (30, NHC(O)CH2). HRMS

mlz calcd. for C35H57N3NaO1582 [M + Naj‘: 846.3129; found: 846.3127.

Lipoic Mannose (LMan). LMan (0.029 g, 0.044 mmol) was synthesized in 90%
yield following the general procedure for deprotection starting from LManA
(0.040 g, 0.049 mmol). 1H NMR (500 MHz, CD30D): 8H 1.40-1.52 (m, 2H,
COCHZCHZCH2CHZCHS), 1.60-1.82 (m, 6H, COCHZCHchZCHchS,
COH2CH20H2NH), 1.87-1.93 (m, 1H, CHaHCHZS), 2.23 (t, J = 7.0 Hz, 2H,
COCHchZCHZCHZCHS), 2.44-2.52 (m, 1H, CHHbCHZS), 2.49 (td, J = 1.5, 5.5
Hz, 4H, COCHchch), 3.08-3.14 (m, 1H, CHZCHaHS), 3.16-3.22 (m, 1H,
CHZCHHDS), 3.24-3.30 (m, 2H, OCHZCHZCHzNH), 3.35-3.40 (m, 4H, CONHCHz),
3.44-3.49 (m, 1H, H-3), 3.52-3.62 (m, 7H, chOCHzCHZOCHg,
OCHaHCHZCHzNH, COCHchchZCHZCHS, H-2 or H-5), 3.63 (s, 4H,
HZCOCHZCH200H2 ) , 3.68-3.74 (m, 2H, H-6), 3.77-3.82 (m, 2H,
OCHHbCHchzNH, H-2 or H-5), 3.85 (dd, J = 2.5, 12.0 Hz, 1H, H-4), 4.75 (d, J =
1.5 Hz, 1H, H-1). 13C NMR (125 MHz, CDgOD): Sc 25.5, 28.7, 29.1, 31.09, 31.13,

34.6, 35.7, 36.5, 38.2, 39.13, 39.21, 40.2, 56.4, 61.8, 63.2, 64.9, 67.5, 69.44,

118

69.49, 70.1, 71.0, 71.5, 73.5, 100.47, 173.42, 173.59, 174.95. HRMS m/z calcd.
for 027H50N301182[M+H]+:656.2887; found:656.2899.
4. Synthesis of Lipoic Fucose (LFuc)
o
H3Cfizagrfik/IOH __f£___,

.46 %

over 2 ste s
ACO Fucose Amido Acid p

O 0 “MS
WNW \/\o’\/ s

M
N
H3CFQZCQI H o O
OH
OH

Lipoic Fucose (LFuc)

Figure 2-22. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA, DMF, 3hrs; b)
NaOMe, MeOH, 1h.

Lipoic Fucose Acetate (LFucA). LFucA (0.257 g, 0.336 mmol) was
synthesized in 50% yield following the general procedure for amidation starting
from fucose amido acid (0.300 g, 0.670 mmol). 1H NMR (500 MHz, CDCI3): 5H
1.14 (d, J = 6.0 Hz, 3H, CH3), 1.32-1.42 (m, 2H, COCHZCHZCHchZCHS), 1.52-
1.65 (m, 4H, COCHZCHZCHZCHZJCHS), 1.66-1.74 (m, 2H, OCH2CH2CH2NH),
1.79-1.85 (m, 1H, CHaHCst), 1.90 (s, 3H, OCH3), 1.98 (s, 3H, OCH3), 2.10 (s,
3H, OCH3), 2.12 (t, J = 7.5 Hz, 2H, COCH20H20H20H2CHS), 2.34-2.40 (m, 1H,
CHHbCst), 2.42-2.50 (m, 4H, COCH2CH200), 3.00-3.05 (m, 1H, CHchaHS),
3.07-3.11 (m, 1H, CHZCHHbS), 3.17-3.22 (m, 1H, OCHZCH2CH8HNH), 3.24-3.30
(m, 1H, OCHZCHchHbNH), 3.32-3.37 (m, 4H, CONHCHZ), 3.45-3.50 (m, 6H,
HZCOCHZCHZOCHz, OCHHbCchHzNH, COCHZCHZCH2CH2CHS), 3.52 (s, 4H,
HZCOCHchZOCHz), 3.75 (dq, J = 0.5, 6.5 Hz, 1H, H-5), 3.86-3.91 (m, 1H,
OCHaH CH20H2NH), 4.34 (d, J = 7.5 Hz, 1H, H-1), 4.94 (dd, J = 3.5, 10.5 Hz,
1H, H-3), 5.06 (dd, J = 7.5, 10.5 Hz, 1H, H-2), 5.15 (dd, J = 0.5, 3.5 Hz, 1H, H-4),

119

6.50 (dt, J = 5.5, 2H, NH), 6.71 (t, J = 5.5, 1H, NH). 13C NMR (125 MHz, CDCI3):
ac 16.2, 20.8, 20.9, 21.0, 25.6, 29.1, 29.3, 31.7, 32.0, 34.8, 36.4, 37.6, 38.6,
39.4, 39.4, 40.4, 53.7, 56.6, 68.7, 69.2, 69.4, 69.9, 70.1, 70.3, 70.4, 70.4, 71.2,
101.3, 170.1, 170.3, 170.8, 172.4, 172.6, 173.2. HRMS mlz calcd. for

033H55N3O1332[M]+I766.3255; f0Ul'le766.3259.

Lipoic Fucose (LFuc). LFuc (0.141 g, 0.220 mmol) was synthesized in 94%
yield following the general procedure for deprotection starting from LFucA (0.180
g, 0.235 mmol). 1H NMR (500 MHz, 00300): 6.4 1.28 (d, J = 6.5 Hz, 3H, CH3),
1.42-1.50 (m, 2H, COCHzCHzCHzCHzCHS), 1.60-1.74 (m, 4H,
COCHZCHchchchS), 1.76-1.82 (m, 2H, COH20H2CH2NH), 1.87-1.94 (m,
1H, CHaHCst), 2.22 (t, J = 7.5 Hz, 2H, COCHchzCHZCHZCHS), 2.44-2.48 (m,
1H, CHHbCHZS), 2.50 (t, J = 4.0 Hz, 4H, COCHZCHzco), 3.09-3.14 (m, 1H,
CHchaHS), I 3.17-3.21 (m, 1H, CHchHbS), 3.25-3.34 (m, 2H,
OCHchzCHzNH), 3.36-3.40 (m, 4H, CONHCHz), 3.48 (dd, J = 2.0, 4.5 Hz, 1H,
H-2, H-3), 3.55 (dd, J = 2.5, 5.5 Hz, 1H, H-4), 3.54-3.58 (m, 4H,
HchCHchzocHz), 3.58-3.60 (m, 1H, OCHaHCHchzNH), 3.60-3.62 (m, 1H,
COCHZCHZCHchchS), 3.63 (s, 4H, HchCH20H20CHz), 3.65 (td, J = 1.0, 7.5
Hz, 1H, H-5), 3.92-3.97 (m, 1H, OCHHbCHZCHzNH), 4.23 (d, J = 7.5 Hz, 1H, H-
1). 13C NMR (125 MHz, CDgOD): 6015.7, 25.6, 28.7, 29.1, 31.2, 31.2, 34.6, 35.7,
36.7, 38.2, 39.16, 39.24, 40.2, 56.5, 67.2, 69.46, 69.50, 70.17, 70.18, 70.8, 71.2,
71.9, 74.0, 103.6, 173.4, 173.5, 174.9. HR-MS m/z calcd. for 027H50N301082

[M+H]+: 640.2938; found: 640.2933.

120

5. Synthesis of Lipoic Glucose (LGlc)

AACO O H O b
3.
OAC O 46 %
Glucose Amido Acid over 2 steps

Rafi/OWNNNNOwO/VN S

OH 0 H 0

Lipoic Glucose (LGlc)

Figure 2-23. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA, DMF, 3hrs; b)
NaOMe, MeOH, 1h.

Lipoic Glucose Acetate (LGch). LGch (0.180 g, 0.218 mmol) was synthesized
in 50% yield following the general procedure for amidation starting from glucose
amido acid (0.220 g, 0.435 mmol). 1H NMR (500 MHz, CDCI3): 6H 1.38-1.48 (m,
2H, COCHZCH20H20H2CHS), 1.60-1.72 (m, 4H, COCH2CH20H2CH20HS), 1.72-
1.80 (m, 2H, OCH2CH2CH2NH), 1.85-1.92 (m, 1H, CHaHCst), 1.99, 2.01, 2.04,
2.08 (45, 4 x 3H, COCH3), 2.18 (t, J = 7.0 Hz, 2H, COCH2CHZCHZCH20HS),
2.40-2.48 (m, 1H, CHHbCHZS), 2.48-2.55 (m, 4H, COCHchZCO), 3.06-3.18 (m,
2H, CH2CH28), 3.21-3.35 (m, 2H, OCHzCHzCHzNH), 3.40-3.45 (m, 4H,
CONHCHZ), 3.51-3.58 (m, 6H, H2COCH20HZOCH2, OCHaHCHchgNH,
COCHZCHZCHchZCHS), 3.60 (s, 4H, H2COCH20HzOCH2), 3.67-3.71 (m, 1H,
H-5), 3.88-3.93 (m, 1H, OCHH), CHzCHzNH), 4.15 (dd, J = 2.5, 12.5 Hz, 1H, H-
6a), 4.25 (dd, J = 4.5, 12.5 Hz, 1H, H-6b), 4.47 (d, J = 8.5 Hz, 1H, H-1), 4.96 (dd,
J = 8.0, 9.5 Hz, 1H, H-2), 5.06 (t, J = 9.5 Hz, 1H, H-4), 5.20 ((t, J = 9.5 Hz, 1H, H-
3), 6.43 (t, J = 5.5 Hz, 1H, NH), 6.49 (t, J = 5.5 Hz, 1H, NH), 6.67 (t, J = 5.5 Hz,

1H, NH). 13C NMR (125 MHz, CDCI3): ac 20.83, 20.95, 21.0, 25.6, 29.1, 29.4,

121

31.8, 32.0, 34.9, 36.5, 37.4, 38.7, 39.43, 39.45, 40.5, 50.8, 56.7, 62.0, 68.55.
68.57, 70.0, 70.2, 70.43, 70.46, 71.5, 72.1, 72.8 101.0, 169.7, 169.9, 170.4,
171.0, 172.57, 172.58, 173.3. HRMS m/z calcd. for 035H58N3015$2 [M+H]*:
824.3309; found: 824.3305.

Lipoic Glucose (LGIc). LGlc (0.074 g, 0.11 mmol) was synthesized in 93% yield
following the general procedure for deprotection starting from LGch (0.10 g,
0.12 mmol). 1H NMR (500 MHz, CDaOD): 5H 1.42-1.52 (m, 2H,
COCHzCHZCHchZCHS), 1.60-1.76 (m, 4H, COCHZCH20H2CH20HS), 1.76-1.82
(m, 2H, COHchchzNH), 1.87-1.93 (m, 1H, CHaHCHZS), 2.22 (t, J = 7.5 Hz,
2H, COCH20H2CHZCHZCHS), 2.44-2.48 (m, 1H, CHHbCst), 2.50 (t, J = 4.0 Hz,
4H, COCHzCHch), 3.09-3.21 (m, 3H, CHZCst, OCHchZCHaHN'H), 3.27-3.32
(m, 2H, OCHZCHZCHHbNH, H-4), 3.33-3.39 (m, 6H, CONHCHZ,
COCHZCHZCHZCHZCHS, H-3), 3.54-3.62 (m, 6l-l, H2COCH2CH20CH2,
OCHaHCHZCHzNH, H-2), 3.63 (s, 4H, HZCOCH20H200H2), 3.64-3.69 (m, 2H, H-
5, H-6a), 3.88 (dd, J = 2.0, 10.0 Hz, 1H, H-6b), 3.92-3.96 (m, 1H,
OCHHbCHZCHzNH), 4.27 (d, J = 8.0 Hz, 1H, H-1). 13C NMR (125 MHz, CDgOD):
80 25.6, 28.7, 29.2, 31.11, 31.15, 34.6, 35.7, 36.5, 38.2, 39.1, 39.2, 40.2, 56.4,
61.6, 67.2, 69.43, 69.48, 70.2, 70.5, 74.0, 76.8, 76.9, 103.2, 173.45, 173.57,

175.0. HRMS mlz calcd. for C27H50N3011$2 [M+H]+: 656.2887; found: 656.2875.

122

6. Synthesis of Lipoic Sialic (LSia)

ACO

ACO H o 47 % over 2 stegs
Sialic Amido Acid

HO C02H H S
110% O HN/\/O\/\O/\/ N M
AcHN OMNM
HO H o 0
Lipoic Sialic (LSia)

 

Figure 2-24. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA, DMF, 3hrs; b)
NaOMe, MeOH, 1h, then 0.2 mL water.

Lipoic Sialic Acetate (LSiaA). LSiaA (0.085 g, 0.088 mmol) was synthesized in
52% yield following the general procedure for amidation starting from sialic amido
acid (0.11 g, 0.17 mmol). 1H NMR (500 MHz, CDClg): 5H 1.38-1.50 (m, 2H,
COCHZCH20H2CH2CHS), 1.60-1.76 (m, 6H, COCHZCH2CH20H2CHS,
OCHZCHchzNH), 1.87-1.95 (m, 2H, CHaHCst, ’H-3ax.), 1.86, 2.01, 2.02, 2.13
(5s, 5 x 3H, COCH3, NHCOCHg), 2.19 (t, J = 7.5 Hz, 2H,
COCH20H2CH2CH20HS), 2.40-2.48 (m, 1H, CHHbCHZS), 2.50 (bs, 4H,
COCH20H200), 2.63 (dd, J = 5.0, 10.5 Hz, 1H, H-3eq.), 3.07-3.18 (m, 2H,
CHchzs), 3.25-3.34 (m, 3H, OCH2CHZCH2NH, OCHaHCHchzNH), 3.40-3.45
(m, 4H, CONHCHz), 3.52-3.57 (m, 5H, chOCHZCHZOCHg,
COCHzCH20H20H2CHS), 3.60 (s, 4H, HZCOCHZCHzocHz), 3.74-3.78 (m, 1H,
OCHHbCHchzNH), 3.79 (s, 3H, COzCH3), 4.02-4.08 (m, 2H, H-5, H-9a), 4.10
(dd, J = 2.0, 11.0 Hz, 1H, H-6), 4.30 (dd, J = 3.0, 12.5 Hz, 1H, H-9b), 4.80-4.86
(m, 1H, H-4), 5.31 (dd, J = 2.0, 8.5 Hz, 1H, H-7), 5.35-5.38 (m, 1H, H-8), 5.48 (d,
J = 9.5 Hz, 1H, NHAC), 6.41 (t, J = 5.5 Hz, 1H, NH), 6.49 (t, J = 5.5 Hz, 1H, NH),

6.60 (t, J = 5.0 Hz, 1H, NH). 13C NMR (125 MHz, CDCI3): 80 21.05, 21.12, 21.4,

123

23.4, 25.6, 29.1, 29.4, 31.80, 31.96, 34.9, 36.5, 37.1, 38.1, 38.7, 39.4, 40.5, 49.5,
53.1, 53.7, 56.7, 62.8, 63.5, 67.5, 68.5, 69.2, 70.0, 70.2, 70.45, 70.47, 72.7, 99.0,
168.6, 170.4, 170.5, 170.6, 171.05, 171.16, 172.3, 172.5, 173.3. HRMS m/z

CfilCd. for C41H57N4O13$2IM+H1+I 967.2815; found I 967.2809.

Lipoic Sialic Acid (LSia). LSia (0.047 g, 0.059 mmol) was synthesized in 95%
yield following the general procedure for deprotection starting from LSiaA (0.060
g, 0.062 mmol) except that 0.2 mL of water was added to the reaction mixture
half an hour prior to neutralization. 1H NMR (500 MHz, CDgOD): 8H 1.42-1.52 (m,
2H, COCHZCHZCHZCHchS), 1.60-1.80 (m, 7H, COCHZCHchchchS,
COHchchzNH, H-3ax.), 1.87-1.94 (m, 1H, CHaHCHZS), 2.00 (s, 3H,
NHCOCH3), 2.23 (t, J = 7.5 Hz, 2H, COCHgCHzCHZCHZCHS), 2.44-2.48 (m, 1H,
CHHbCHZS), 2.50 (bs, 4H, COCH2CH2CO), 2.71 (dd, J = 5.0, 12.5 Hz, 1H, H-
3eq.), 3.09-3.21 (m, 2H, CHZCHZS), 3.25-3.28 (m, 2H, OCHzCHZCHzNH), 3.36-
3.40(m, 4H, CONHCHz), 3.48-3.53 (m, 1H, OCHaHCHZCHzNH), 3.54-3.61 (m,
7H, HfiOCHZCHrZOCHg, COCHZCHchZCHZCHS, H-5, H-8), 3.62-3.66 (m, 1H,
H-7), 3.63 (s, 4H, HZCOCHgCHzOCHz), 3.70-3.80 (m, 2H, H-4, H-6), 3.82-3.88
(m, 3H, H-9, OCHHbCH2CH2NH). 13C NMR (125 MHz, CD30D): 6c 19.6, 21.5,
25.6, 28.7, 29.2, 31.2, 34.6, 35.7, 36.4, 38.2, 39.2, 39.3, 40.2, 40.5, 52.7, 56.4,
61.4, 63.5, 67.6, 69.0, 69.43, 69.48, 70.16, 71.7, 73.8, 98.7, 170.8, 173.5, 173.6,
174.0, 174.2, 175.0. HRMS mlz calcd. for ngH57N4014SZ [M+H]*: 785.3034;

found : 785.3030.

124

7. Synthesis of Lipoic N-acetyl glucosamine (LGINAc)

OAC O
A O H a,b
Afifi/OWN 0H ——_*
O

45% over 2 steps
Glucosamine Amido Acid

0
o H H ,s
NHAC H O
O O
Lipoic N-acetyl glucosamine (LGINAc)

Figure 2-25. Reagents and conditions: a) Lipoic amine, HBTU/HOBT, DIPEA, DMF, 3hrs;
b) NaOMe, MeOH, 1h.

Lipoic N-acetyl Glucosamine Acetate (LGNAcA). LGlNAcA (0.114 g, 0.139
mmol) was synthesized in 50% yield following the general procedure for
amidation starting from glucosamine amido acid (0.140 g, 0.278 mmol). 1H NMR
(500 MHz, CDCI3): 8H 1.40-1.50 (m, 2H, COCHZCHchZCHZCHS), 1.60-1.72 (m,
5H, COCHZCHchZCHZCHS, OCHZCHaHCHzNH), 1.72-1.80 (m, 1H,
OCHZCHHDCHgNH), 1.85-1.92 (m, 1H, CHaHCHZS), 1.92, 1.99, 2.00, 2.06 (4s, 3
x 3H, COCH3, 3H, NHCOCH3), 2.19 (t, J = 7.5 Hz, 2H, COCHgCHZCH2CH20HS),
2.40-2.48 (m, 1H, CHHbCHZS), 2.48-2.58 (m, 4H, COCHZCHZCO), 3.06-3.18 (m,
3H, CHZCHZS, OCHZCHZCHaHNH), 3.40-3.47 (m, 5H, CONHCHg,
OCHaHCHZCHzNH), 3.51-3.58 (m, 6H, chocHZCHZOCHg, OCHchZCHHbNH,
COCHchZCHZCHchS), 3.60 (s, 4H, HzCOCHZCHZOCHz), 3.68-3.72 (m, 1 H,
H-5), 3.94-4.00 (m, 2H, H-2, OCHH), CHchzNH), 4.11 (dd, J = 2.5, 12.5 Hz, 1H,
H-6a), 4.25 (dd, J = 4.5, 12.0 Hz, 1H, H-6b), 4.54 (d, J = 8.5 Hz, 1H, H-1), 5.06 (t,
J = 10.0 Hz, 1H, H-4), 5.16 ((t, J = 9.5 Hz, 1H, H-3), 6.35 (bs, 1H, NH), 6.65 (m,
2H, NH), 6.92 (d, J = 8.5 Hz, 1H, NHAC). 13C NMR (125 MHz, CDCI3): 50 20.86,

20.96, 21.0, 23.4, 25.6, 29.1, 29.4, 31.80, 31.83, 34.9, 36.53, 36.57, 38.7, 39.46,
125

39.54, 40.5, 54.5, 56.7, 62.3, 67.9, 68.9, 69.9, 70.2, 70.46, 70.50, 72.0, 73.3,
101.6, 169.6, 170.99, 171.08, 171.14, 172.66, 172.73, 173.3. HRMS mlz calcd.
for C35H53N4Na01482 [M+Na]*: 845.3875; found: 845.3868.

Lipoic N-acetyl Glucosamine (LGINAc). LGINAc (0.046 g, 0.066 mmol) was
synthesized in 90% yield following the general procedure for deprotection starting
from LGINAcA (0.060 g, 0.073 mmol). 1H NMR (500 MHz, CDgOD): 6H 1.42-1.52
(m, 2H, COCHZCHchchchS), 1.60-1.72 (m, 4H, COCH20H20H20H20HS),
1.72-1.78 (m, 2H, COHZCH2CH2NH), 1.87-1.94 (m, 1H, CHaHCH2S), 2.01 (s, 1H,
NHCOCHa), 2.23 (t, J = 7.0 Hz, 2H, COCH20H2CH20HZCHS), 2.44-2.48 (m, 1H,
CHHbCst), 2.50 (t, J = 3.0 Hz, 4H, COCHchch), 3.09-3.21 (m, 3H,
CHZCst, OCHZCHZCHaHNH), 3.27-3.34 (m, 2H, OCHZCHZCHHbNH, H-4), 3.35-
3.40 (m, 5H, CONHCHz, COCHZCHchZCHchS), 3.45 (dd, J = 8.5, 10.5 Hz,
1H, H-3), 3.53-3.62 (m, 6H, chOCHzCHZOCHz, OCHaHCHZCHzNH, H-2), 3.63
(s, 4H, HchCHchgocHz), 3.64-3.72 (m, 2H, H-5, H-6a), 3.88-3.94 (m, 2H, H-
6b, OCHHbCHchzNH), 4.40 (d, J = 8.5 Hz, 1H, H-1). 13C NMR (125 MHz,
CD30D): 8c 21.9, 25.6, 28.7, 29.3, 31.11, 31.14, 34.6, 35.7, 36.3, 38.2, 39.1,
39.2, 40.2, 56.3, 56.4, 61.7, 66.9, 69.44, 69.48, 70.15, 70.17, 71.0, 75.1, 76.8,
101.6, 172.8, 173.45, 173.57, 175.0. HRMS mlz calcd. for ngH52N4 Na 01182

[M+Na]+: 719.3804; found: 719.3806.

126

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131

CHAPTER 3

Magnetic Gcho-Nanoparticles: A Tool to Detect, Differentiate,
and Unlock the Glyco-Codes of Cancer via MRI

3.1. Cancer Facts

Cancer is the second leading cause of death in the United States and is
expected to surpass cardiovascular diseases in 2012.1 Statistically, nearly half of
all men and over one third of all women in the United States will contract cancer
during their lifetimes, and one in four will die from it.1 The estimated annual cost
of cancer to the United States is around ~ $110 billion. Thus, early detection and
prevention are the most effective means for the control and treatment of the
disease.

Cancer usually appears as a tumor, while not all tumors are cancerous.
Noncancerous tumors (benign) do not spread or metastasize to other parts of the
body and are rarely life-threatening. Cancerous tumors (malignant) develop when
cells in the body begin to grow and metastasize in an uncontrolled manner (out-

of-control growth of abnormal cells).

3.2. Cancer Detection and Diagnosis

Conventional cancer diagnosis depends on the cell pathology and
histological assessment where biopsy, endoscopy, and imaging are employed.2
Imaging techniques including X-ray, computed tomography (CT), ultrasound (US),
positron emission tomography (PET), optical coherence tomography (OCT),
laser-induced fluorescence endoscopy, and MRI are usually applied.3 MRI uses

radio-frequency waves and a strong magnetic field (~ 1.5-3 T) to provide

132

remarkably clear and detailed pictures of internal organs and tissues.‘ The MRI
technique has proven very valuable for the diagnosis of a broad range of
pathologic conditions including cancer, heart, vascular, and stroke problems.5
Molecular, proteomic and genomic technologies, on the other hand, offer
the promise of a comprehensive understanding of cancer at the molecular level.6
These technologies determine how the genes, proteins and DNA in cancer cells
behave and interact by expressing these molecular signatures as patterns
enabling personalized cancer medicine through analysis of gene-expression
blueprints.”9 Indeed, cancer cells have diverse biological capabilities that are
conferred by numerous genetic aberrations. Mining the cancer genome for
aberrations and alternations has become a major activity in cancer research,
because it is widely understood that these aberrations provide potential clues to
identify molecular biomarkers (biological indicators) that aid in early detection,
diagnosis, and targeted therapy.7 Detection of cancer-specific DNA mutations,
abnormal DNA methylation patterns, and DNA replication proteins have been
explored. Molecular alterations in tumors can be revealed using technologies that
assess changes in the content or sequence of DNA, its transcription into mRNA,
the production of proteins or the synthesis of various metabolic products.9 The
challenge in this approach, however, is to discover the correct cancer biomarkers
and to identify which molecular abnormalities contribute to cancer and which are
simply ‘noise’ at the genomic level. Although there has been success in the

genomic approach, the translation of emerging insights from the genome into the

133

clinical practice of cancer medicine has not yet been fully attained.8 New and
better diagnostic tools to attain treatments are certainly needed.

Nowadays, nanotechnology is revolutionizing the biotechnology industries,
clinical and medical fields at both the diagnostic and therapeutic fronts. The use
of functionally-targeted nanoparticles in medicine is one of the important

directions that bio-nanotechnology is taking. Indeed, their applications in

16-18 19-22

imaging,”12 targeting,”15 drug delivery, cancer cell diagnostics and
therapeuticsz‘ns have been active fields of research. Although NPs exhibit similar
size dimensions to many common biomolecules such as proteins and DNA,
complete understanding of the bio-conversation between those entities and its
effective clinical translation is still to be explored.27 Research concerning the
interactions between organic/inorganic nanocomposites and biological cells will

enable new developments in nano-biotechnology to reach their fullest potential.

3.3. Gcho-conjugates as Cancer Biomarkers

Cancer is a complex group of diseases. Each malignant cell type has
molecular signatures that distinguish it from the healthy counterpart. The
availability of simple and fast methods to identify the unique cellular
Characteristics can greatly benefit cancer treatment and improve the clinical
outcomes for patients?" 283° Currently, the majority of methods for cancer
detection target biomarkers such as mutated DNA/RNA and over-expressed
antigens?“33 This requires extensive prior knowledge on the presence of the

34

specific markers,“ which can be very time consuming to acquire.

Furthermore, as tumor cells have high tendencies to mutate,” 36 their antigenic

134

variants may escape the detection leading to false negative results. An appealing
alternative is to take advantage of cell surface receptor mediated recognition
events. Receptor binding is often critical to cell functions and usually cannot be
abolished without affecting cell viability. In addition, cell surface receptors would
be easier to target without requiring the probes to cross the cellular membrane as
compared to intracellular markers.

An attractive target for receptor mediated interaction is carbohydrates and,
in particular glycoconjugates, which play important roles in cancer development
and metastasis.”42 Carbohydrates are uniquely suited for encoding biological
information becaUse of their rich structural variations.‘“”‘45 Aberrant glycosylations
on tumor cell surfaces have been extensively probed by antibodies and plant
derived Iectins.“ 46' ‘7 This led to the identification of characteristic tumor

40. 42

associated carbohydrate molecules, which has greatly facilitated the

development of carbohydrate based anti-cancer vaccine studies.” ‘8' ‘9 In
comparison, the understanding of carbohydrate-binding properties of tumors is
not as advanced. Cancer cells can interact with the extracellular matrix in their
microenvironment through endogenous receptors binding with carbohydrates.”
52 These interactions vary depending on the physiological state of the cells, as
supported by the ground breaking histological studies of tumor tissues.“55
Therefore, the ability to Characterize and distinguish carbohydrate binding
profiles of a variety of cells can expedite both the mechanistic understanding of

their roles in disease development and the expansion of diagnostic and

therapeutic tools.”58 As the distinctions among cancer cell subtypes and

135

malignant vs. normal cells can often be subtle, a suitable tool is needed to
quantitatively analyze the fine characteristics in carbohydrate binding of various

cell types.

3.4. Nanoparticles for Cancer Detection

As mentioned earlier, nanotechnology has begun to play increasingly
important roles in cancer research.34 Using antibody immobilized nanoparticles,
various types of cancer cells were detected both in vitro28 and in viva.59 Recently,
instead of relying on the specific antibodies, structurally related cationic gold
nanoparticles bearing fluorescent polymers on the surface were prepared.21 The
differential electrostatic and hydrophobic interactions between the gold
nanoparticles and cells were reflected in changes of fluorescence intensity upon
cell binding, which allowed the differentiation of tumor from normal cells as well
as closely related tumor cells. Herein, we explore the possibility of using a MGNP
based system to detect and profile various cell types based on their carbohydrate
binding abilities. MGNPs provide an appealing platform for biological detection.
The spherical nanoparticles have large surface areas, which allow the
attachment of multiple carbohydrates leading to enhanced avidity with
carbohydrate receptors through multivalent binding.“ 6°' 6‘ Unlike the toxic heavy
metal containing nanoparticles such as quantum dots,” 63 the magnetite
nanoparticles have been approved for clinical uses with minimum cytotoxicity.“
65 Furthermore, the super-paramagnetic nature of this MGNPs can allow cell

detection via MRI without the need to pre-label the cells.“68

136

One challenge, however, in using MGNPs and carbohydrates for
molecular recognition is that multiple cell types may bind with the same
carbohydrate structure albeit having different affinities. To address this issue, we
envision that by pooling the responses from an array of MGNPs, the various cell
types may be differentiated through pattern recognition?" 69’" Furthermore, the
information obtained on the physiologically relevant carbohydrate-receptor
interaction can not only enhance our understanding of the roles carbohydrate
play in cancer but also guide the development of potential therapeutics such as
agents against cancer adhesion. Although glyco-nanoparticles have been
previously employed for elegant studies of carbohydrate-mediated biological
interactions,”83 MGNPs have not been utilized to detect and systematically

profile mammalian cells.

3.5. Synthesis and Characterization of MGNPs

The synthesis of MGNPs commenced from the tetraethoxysilane coated
magnetite nanoparticle (NP 1) prepared earlier on which amino groups were
introduced via silanization with APTES (Figure 3-1 a). Carboxylic acid derivatives
of four types of naturally occurring monosaccharides, namely, Man, Gal, fucose
(Fuc), and sialic acid (Sia), were then immobilized onto the amine functionalized
nanoparticles through amide coupling reactions leading to MGNP 2 - 5

respectively.

137

APTES
1) (Et0)3Si\/\/NH2

 

2) Man , Gal , Fuc or Sia 0 Amide coupllng

/\/\
HOV/{$4 O H

O

o
1) (EtO)3SiWnNu’\/O\/\o’\/ N3
0 8 _

2) GlcNAc 7 OH Click coupling

3;. 99:3
“30 "Q

 

Sugar-i NHAC
OH HO
HO cg OH 2. OH C02H
O H C 0 HOW 1‘
O 3
3&4 Hog» g. @9244 AM o o
Man-MGNP 2 Gal-MGNP 3 Fuc-MGNP 4 Sia-MGNP 5

Figure 3-1. Syntheses of MGNPs.

Sugar

,\--J..

\f ‘
Sugar \( If?" ‘./ Sugar

V

Sugar
MGNP 2-6

0“ OCH3
110%;er
HO ‘3
NHAC
GlcNAc-MGNP 6

In an alternative method, we synthesized GlcNAc-MGNP 6 via click

chemistry. To this end, we explored the possibility of using a native unprotected

sugar. The free reducing sugar GlcNAc was chemoselectively ligated with a

methoxy amine linker producing GlcNAc derivative 7 containing a terminal alkyne

at the reducing end (Figure 3-2).8“'36 NP 1 was then modified with the azido

siloxane derivative 8, which was subsequently coupled with alkynyl-GlcNAc 7

through the copper catalyzed Huisgen Click reaction"'89 to yield GlcNAc-MGNP 6

(Figure 3-1b). It is advantageous to use the native unprotected sugar as this

opens up an avenue for future incorporation of natural polysaccharides without

extensive synthetic manipulations.

138

a b C QCH3 d 0% QCH3
H300‘NH2 .HCI ——’——'———> HN\/é ——'o—> HfimNj
80 % over 3 steps 92 /° NHAC

GlcNAc 7

Figure 3-2. a) Benzoyl chloride, pyridine, CHzclz, 0 0C to r.t.,2 hrs; b) 3-bromopropyne, K2003,
acetone, reflux for 8 hrs; c) 6% MeOH in HCI, reflux for 2 hrs; d) N-acetyl glucosamine, 0.1 M
acetate buffer (pH=6.5) / DMF (3:1), 50 °C, 24 hrs.

As usual, all MGNPs assembled were characterized by a variety of
techniques, including TEM, NMR, TGA, FT-IR, and XRD. TEM images of MGNPs
indicated that the diameters of NPs were around ~ 6 nm and TGA showed that
about 8% of the dry weight of MGNPs was due to carbohydrates. XRD showed
the expected composition of magnetite Fe3O4 NPs and the patterns of the
nanocrystals were assigned to the reflections of the structure of Fe3O4. FT-IR
analysis was consistent with the expected peaks discussed before. Moreover,
HR-MAS 1H-NMR spectra of MGNPs gave solution-like spectra indicating that
the NPs are indeed functionalized with the requisite sugars and that the

carbohydrate-coating was homogenous on the particle surface.90

3.6. Validation of MGNP Binding Specificities
Although glyco-nanoparticles have been previously utilized to probe

carbohydrate-receptor interactions,”83

it is important to validate that the
carbohydrates immobilized on MGNPs retain their biological recognition
specificity. This was probed using four Iectins, i.e., Concanavalin A (Can A, a
Man selective Iectin),91 Wheat Germ Agglutinin (WGA, a GlcNAc and Sia

selective Iectin),92' 93 a Bandeiraea Simplicifolia isolectin (BS-l, a Gal selective

Iectin)94 and Lotus Tetragonolobus Purpureas Agglutinin (TPA, a Fuc selective

139

Iectin).95 Upon incubation of a fluorescently labeled Iectin with a MGNP, if the
MGNP can bind with the Iectin, subsequent application of an external magnetic
field to the sample would remove the fluorescent Iectin from the solution leading
to a reduction of fluorescent intensity of the supernatant. Con A is a well
characterized Man selective Iectin with weak Gal binding affinities.91 Incubation of
Man-MGNP 2 (1 mg/mL) with FITC-labeled Con A (100 uglmL) followed by
magnetic separation led to a 89% reduction in fluorescent intensity of the
solution, while the same amount of the weakly bound Gal-MGNP 3 was able to
remove only 8% of the Con A (Figure 3-3a). This is consistent with the known
carbohydrate binding preferences of Con A.91 The addition of a solution of free
mannose (18 mg/mL, 100 mM) to Man-MGNP 2/Con A mixture did not increase
the intensity of residual emission of the supernatant after magnetic separation
and high concentration of mannose (180 mg/mL, 1 M) was required to partially
disrupt the Con A/Man-MGNP 2 complex. These results reveal that multivalent
display of carbohydrate ligands on MGNP resulted in strong Iectin binding.

In contrast to Can A, the Gal selective FITC-BS-l9‘1 strongly bound with
Gal-MGNP 3, producing 91% reduction of the solution emission intensity (Figure
3-3b). At the same time, as BS-I has weak affinities with mannose,94 incubation
of Man-MGNP 2 with F lTC-BS-l only decreased emission intensity a little. The
same phenomena were observed with WGA and TPA.90 WGA bound tightly with
GlcNAc-MGNP 6 and Sia-MGNP 5 but not with Gal-MGNP 3 and TPA interacted
strongly with Fuc-MGNP 4 as predicted based on their known binding

specificities.” 93' 95' 96

140

a) ConA Binding

   
 
    
   
  
 

Emission (AU)

 

 

   
      
    
   

45° ‘ ConA or NP1 +ConA
400 1
j . Gal-MGNP3
350 \
GIcNAc—MGNP6
300 -
250 . Fuc-MGNP4
200 ~
Sia-MGNP5
150 1
100 .
Man-MGNP2
50 ~/__\
01% _T—’"""" *‘w—f ' ’_' I “7 _ A _' W I”
502 522 542 562 582
Wavelength (nm)
b) BS-l Binding
450
‘00 l BS-l or NP1 +BS-l or GIcNAc-MGNP8 +BS-I
350 I Man-MGNP2
5 300 J, Fuc—MGNP4
5 l
g 250
.2
a
2 200
“E, 150

 

o I f fl T ' V" —'7_—

T fi

502 512 522 532 542 552 562 572 582 592
Wavelength (nm)

Figure 3-3. Fluorescent emission intensities of supernatents of FITC-Iabeled a) Can A, b) BS-I

(100 ug/mL) upon incubation with various MGNPs 2-6 or control NP 1 (1 mg/mL) followed by
magnet mediated separation (km = 494 nm).

In addition, NP 1 devoid of carbohydrates on the surface could not remove

any proteins from the solution suggesting that the non-specific absorption on NP

141

surface is minimal. These observations coupled with the good specificities
obtained in the E. coli detection experiments (section 2.5) unequivocally
demonstrated that the MGNPs are not promiscuous in binding and the
carbohydrates immobilized on MGNPs maintain the same biological recognition

preference as the free forms in solution.

3.7. Monitoring MGNP Binding by MRI

After establishing the specificity of MGNPs using fluorescence
experiments, we moved onto examine the utility of MRI to monitor the interaction
of MGNPs with their biological targets. MRI measures the relaxation time of
water protons in a magnetic field, which is commonly used to non-invasively
visualize the internal structures and functions in viva. Using MRI to monitor
MGNP allows multiple samples to be measured simultaneously within a single
scan,97 thus enabling rapid response time and shortening muIti-analyte data
acquisition. Furthermore, when the MRI methodology for MGNP monitoring is
established, it can be translated to in viva applications without the need to
develop a new detection technology.

Magnetic NPs can serve as MRI contrast agents, where they decrease the
transverse relaxation time (T 2) producing a negative contrast from the
environment by virtue of signal reduction.“ 98' 99 In the presence of a crosslinking
receptor, due to the small sizes of MGNPs, multiple MGNPs can bind with the

receptor assembling into clusters (Figure 3-4).

142

Dispersed, Long T2 Aggregated, Short T2
a) . . . ConA
Q . 63E}
9 Q 0 1::
O , _ O

   

Man-MGNP 2
Bright image Man-MGNP 2/Con A aggregates

Dark image

Can A + Man-MGNP 2

 

 

   
 

 

 

Can A + NP 1
c) 350 1
u—F I I I
300 J NP1
250 ‘
’5?
g 200 -
£1 Man-MGNPZ
150 . R2=0.97
100 T T I 1 i 1
0 20 40 60 80 100 120
ConA(pg/mL)

Figure 3-4. a) Incubation of Man-MGNP 2 (shown as pink balls) with Con A, a tetrameric
mannose selective Iectin (shown as blue rectangles) resulted in the formation of aggregates,
leading to shorter T2 relaxation time and consequently a darkened MRI image. b) T2 weighted
MRI images of Man-MGNP 2 (20 pg/mL) and NP 1 (20 ug/mL) upon incubation with increasing
concentrations of Con A. c) T2 Changed linearly upon incubation of Man-MGNP 2 with increasing
concentrations of Con A, while incubation with NP 1 devoid of carbohydrates did not lead to any

143

With their increased sizes, the aggregates create larger local magnetic field
gradients and thus become more efficient at dephasing the spins of surrounding
water protons as the motional averaging condition is satisfied at the nanoparticle
size regime.1°°‘ ‘01 This leads to lowering of T2 relaxation times as described by
the magnetic relaxation switching theory.

Our detection assay was first tested using Con A. As one Can A contains

four mannose binding sites,102

it can crosslink multiple mannose containing Man-
MGNP 2, leading to NP aggregation, which should result in the reduction of T2
relaxation time (Figure 3-4a). Due to the superparamagnetic nature of the
magnetic nanoparticles, only 20 pg/mL of MGNP was needed for detection.
When Man-MGNP 2 was mixed with increasing concentrations of Con A, the
binding equilibrium was shifted more towards the aggregates. This led to a
sequential decrease of the brightness of the corresponding T2 weighted MR
images (Figure 3-4b). Quantification of the images showed an excellent linear
correlation between Con A concentration and T2 relaxation time, with as little as
0.1 pglmL (1 nM) of Con A detected (Figure 3-4c). In contrast, the control NP 1
devoid of mannose did not cause any change in T2, signifying that the contrast
change was due to the specific binding between Man-MGNP 2 and Con A. In
addition, incubation of the GlcNAc selective WGA with GlcNAc-MGNP 6 led to a
linear decrease of T2 relaxation time with increasing concentrations of WGA,

while the mixture of WGA with the non binding Gal-MGNP 3 did not produce any

T2 changes. These results corroborated the fluorescence studies and confirmed

144

MGNP binding specificities which gave us great confidence to apply this

technology to cancer cell study.

3.8. Mammalian Cells Selectively Bound with MGNPs as Detected by MRI

Building on the success of Iectin and E. coli detection, we evaluated the
utility of MGNPs in monitoring mammalian cell interactions and cancer cell
detection. The use of carbohydrates as the recognition elements can provide
functional information on cell surface active carbohydrate receptors. This is
complementary to the usage of antibodies, as the latter monitor the presence of
particular antigenic structures, which can be absent in some cancer cell mutants.
In addition, an antibody is commonly limited to binding a specific target, while
carbohydrate ligands can be used to monitor a range of cells, thus reducing the
number of reagents required for study.

A normal breast cell line 18435 and nine types of representative cancer
cells were used for our study including human ovarian adenocarcinoma SKOV-3,
colon HT29, kidney A498, lung A549, and breast cancer MCF-7/Adr-res and the
closely related murine melanoma cell lines B16F10 and Bl6F1, mammary
adenocarcinoma TA3-ST and TA3-HA. Each type of cells at two concentrations
(105 and 106 cells/mL) was incubated with MGNP 2-6 or the control NP 1, and
the T2 relaxation times of all the samples were recorded. Thirty samples were
measured at the same time with our MRI setup. While no significant T2 changes
were observed with any cells upon mixing with the NP 1, the ten cell lines

produced a large variation in T2 reductions upon MGNP incubation with the T2

145

changes normalized against the largest AT2 within each MGNP category (Figure
3-5).

The decrease of the absolute values of T2 upon MGNP incubation can be
explained due to particle agglomeration upon cell binding. At a higher cell
concentration (106 cells/mL, Figure 3-5b), more MGNPs were bound leading to
larger AT2 compared with lower cell concentration (105 cells/mL) for each cell line
(Figure 3-5a). Furthermore, when the cells were pre-incubated with a solution of
a free monosaccharide (100 mM) and then treated with the MGNP bearing the
same carbohydrate, T2 changes were less due to the competitive binding of the
free monosaccharide with cells. These observations were consistent with the

notion that T2 reduction was induced by the specific MGNP/cell interactions.

3.9. Biological Implications of Carbohydrate-receptors on Cancer Cells
Based on the MR responses, in most of the cell lines examined, bindings
with Fuc-MGNP 4 and Sia-MGNP 5 were observed (Figure 3-5), suggesting
these cell lines have active fucose and sialic acid receptors.56 Bl6F 1, B16F10,
MCF-7/Adr-res, and SKOV-3 were found to interact with fi-galactoside. This is of
special interest since it confirmed the previously reported galactoside binding of
the B16F1056 presumably through galectins, a family of galactose specific Iectins

3 cells.

as well as the high expression level of galectins on MCF-7/Adr-res10
Furthermore, HT29, MCF-7/Adr-res, 18435, A498, and SKOV-3 express
functioning GlcNAc receptors as suggested by their interactions with GlcNAc-
MGNP 6. Moreover, MCF-7/Adr-res, 18485, SKOV-3, B16F10, B16F1, A549

and A498 bind mannose.104

146

a) 1005 cells/mL
12° ‘ IMan-MGNPZ IGaI-MGNPB IFuc-MGNP4 ISIa-MGNPS IGIcNAc-MGNP6 ITEOS-NPI

100*
80‘

601

%ATZ

40 a

201

 

 

HT29 A549 TA3ST TA3HA 816F1 816F10 MCF-7 Breast A498 SKOV3
Adr-Res Normal
18485

V 11111

HT29 A549 TA3ST TA3HA 816F1 816F10 MCF-7 Breast A498 SKOV3
Adr-Res Normal
18485

120 -

b) 1046 cells/mL

100 ‘

80 '1

%ATZ
8

40.

20.

   

Figure 3-5. a) Percentage changes of T2 relaxation time (% AT2) obtained upon incubating
MGNPs 2-6 or the control NP 1 (20 ug/mL) with ten cell lines (105 cells/mL). The AT2 was
calculated by dividing the T2 differences between MGNP and MGNP/cancer cell by the
corresponding highest AT2 from each MGNP category. b) % AT2 obtained upon incubating
MGNPs 2-6 or the control NP 1 (20 pg/mL) with ten cell lines (10° cells/mL). The above data
represent the averages of 8 individual measurements with the error bars showing standard
deviations.

147

The sugar free nanoprobe NP 1 did not bind to any cells in our study, evidence to
the importance of intrinsic carbohydrate-protein interactions involved and the
exclusion of nonspecific interactions as the cause of binding. The wealth of new
information generated on the sugar binding preferences of these cell lines can be
very useful for cancer research. Although some of the receptors responsible for
binding are not determined yet, the magnetic nature of MGNPs can help facilitate
the enrichment and identification of carbohydrate receptors on these cells in the

future through magnet mediated separation.

3.10. Establishment of MR Responses as Molecular Signatures for Full
Differentiation of All Ten Cell Lines via LDA Analysis

With the diverse MR signature in hand, we examined whether it was
possible to differentiate all ten cell lines. This was a particularly stringent test due
to the large number of cell lines being analyzed using only five types of MGNPs.
In order to accomplish this, linear discriminant analysis (LDA), a statistical
method for classification of groups of objects, was employed.70 LDA converts the
AT2 values of each cell line to canonical linear discriminants (LDs), which are
linear combinations of the original data weighted by coefficients producing the
greatest analyte discrimination. LDA is a powerful technique, which has been
successfully applied to the detection of a variety of targets including
carbohydrates, proteins, and cells.“ 70' 71' 105107 All T2 changes (6 types of NPs,
10 cell types, 8 repetitions) at each cell concentration were submitted to LDA and
LDs were generated. Based on the LDA patterns, the ten cell lines were easily
clustered into ten respective groups (Figure 3-6). At the 105 cells/mL

concentration, the first three LDs contain 49.5, 25.3 and 17.5% of the variations

148

respectively, which account for 92.3% of the total variations. Validation of the
LDA was carried out using a jackknife matrix method,108 where all but one
measurement out of each group was treated as a new training set. The group
memberships of the omitted observations were then predicted based on the new
training set, which were accurately Classified in all the cases tested. This
highlights that despite the simple structures of the monosaccharides utilized, the
T2 changes of the MGNP array can be employed as Characteristic molecular

signatures for each cell line.

-2

.4

0
LD1(49.5/o) 444240 -3 6

-5-10
1510 5‘ 0 .2

.li‘.""‘=‘

   
   

 

 

 

Figure 3-6. LDA plots for the first three LDs of AT2 patterns obtained with the MGNP array upon
binding with the ten cell lines (105 cells/mL). Full differentiation of the ten cell lines was achieved.

149

3.11. Detection of Cancer Cells vs Normal Cells based on the MR Signature
A major hurdle for cancer treatment and early cancer detection is the
identification of pertinent cellular signatures to allow the differentiation of normal
cells from their cancerous counterparts. We envision that this can be achieved by
analysis of the respective cellular characteristics towards carbohydrate binding.
As a proof-of-principle, the AT2 of the breast cancer MCF-7lAdr-res cells vs the
normal breast endothelial cells 18435 upon MGNP binding were examined in
detail. As the trend of binding is the same qualitatively at both cell
concentrations, we focused mainly on T2 changes at 105 cells/mL. The
interactions with Man-MGNP 2, Fuc-MGNP 4 and GlcNAc-MGNP 6 were found
to be very similar between the two cell lines (Figure 3-7a). However, the MCF-
7/Adr-res cells caused a significantly larger AT2 upon binding with Gal-MGNP 3
as compared with the normal breast cells 18485, which enabled easy detection
of breast cancer cells. This corroborated with literature that MCF-7/Adr-res cells
contain the cancer-specific galactoside binding galectins which are absent in
non-cancer cell lines.103 Modification of the galactoside ligand structures”9 as
well as optimization of ligand density and nanoparticle surface chemistry can

further improve the selectivity in binding for future in viva applications.

150

P < 0i00001

I I
100 100 4
a) b)
80 l 80 - P< 0100001
I l

60 1, 60 _ P< 0.00001
E ' I l I
a! 40 "'

20 ‘

0 -

 

 

 

)z__l

 

 

 

Breast Cancer Normal Breast Cell TA3ST TA3HA
MCF-7/Adr-Res 18485
0 ..

10 C)

80 I Man-MGNP 2

’ <
I P 0100001 I I Gal-MGNP 3
60 - I Fuc-MGNP4
40 ._ I Sia-MGNP S
I GlcNAc-MGNP 6

20 I NP 1

"I 0 4 ~
816F1 816F10

Figure 3-7. % AT2 obtained upon incubating MGNPs 2-6 or the control NP 1 (20 ngmL) with a)
breast cancer MCF-7/Adr-res vs normal breast cell 184B5; b) TASST vs TA3HA cell lines; and c)
B16F1 vs B16F 10 (105 cells/mL). Significant differences in binding with MGNPs were observed
differentiating these cell lines.

3.12. Differentiation of Closely Related lsogenic Cancer Cells including
Metastatic Cancer Cells

Besides the differentiation of cancer vs normal cells, the MR data also
enabled the distinction between closely related isogenic sublines of cancer cells.
lsogenic cancer cells are derived from the same parent cell line, presenting
significant Challenges for identification. One example is the mouse mammary
carcinoma cells TA3HA and TA3ST. These two types of cells originated from the
same parent cell line with TA3HA expressing the mucin like cell surface

151

glycoprotein epiglycanin absent in TA3ST.110 Despite this subtle difference,
TA3HA showed significantly stronger interactions with the Fuc-MGNP 4 and Sia-
MGNP 5 (Figure 3-7b).

Another example of closely related cells are the widely used B16F10 and
B16F1 mouse melanoma cells, where no qualitative differences in protein
composition, galactose or sialic acid content on the cell surfaces or the
membrane fluidity were observed before.111 The quantitative nature of the MGNP
approach uncovered the subtle difference between these two cell lines with
B16F10 showing larger T2 change (P<0.00001) upon binding with Gal-MGNP 3
as compared to B16F1 (Figure 3-7c), which is likely due to the higher level of
galectins expressed on B16F10 cells.”5 This is consistent with the observation
that galactoside mimetics were more potent in preventing the adhesion of
B16F10 to extracellular matrix component than that of B16F1.56 B16F10 also
caused larger AT2 with Man-MGNP 2 and Fuc-MGNP 4 at 105 cells/mL. These
results indicate that despite the overall similarity, there are quantitative changes
in carbohydrate binding between the B16F1 cells and its metastatic variant

B16F10.

3.13. Cellular Uptake and Bio-compatibility of MGNPs

In order to gain insights into how MGNPs interact with cells, cellular
staining experiments were performed. Besides their properties as MRI contrast
agents, MGNPs can be visualized by Prussian blue staining, which yields an
intense blue color upon reaction with the magnetite core of MGNPs allowing easy

tracking of the particles.

152

Man-MGNPZand 816F10 Gal—MGNP3and Bl6FlO Fuc-MGNP4and816F10

 

GIcNAc-MGNPGand B16F10 NP1 and Bl6F10 Bl6F10

Figure 3-8. Prussian blue staining images of 20 pg/mL of a) Man-MGNP 2; b) Gal-MGNP 3; C)
Fuc-MGNP 4; d) GlcNAc-MGNP 6; and e) control NP 1 incubated with B16F10 cancer cells after
unbound particles were removed by washing and f) B16F10 cells. The images clearly indicated
the high intracellular uptake of Man-MGNP 2, Gal-MGNP 3 and Fuc-MGNP 4. No Prussian blue
stains were visible with the non-binding GlcNAc-MGNP 6 and the control NP 1 proving the
selectivity in binding.

As an example, B16F10 cells were incubated with MGNPs (20 pg/mL), washed
extensively with buffer to remove all unbound nanoparticles and stained with
Prussian Blue. As Man-MGNP 2, Gal-MGNP 3 and Fuc-MGNP 4 caused the
biggest T2 changes when incubated with B16F10, strong blue stains were
observed both on cell surface and inside the cells suggesting a significant cellular
surface binding and cellular uptake (Figure 3-8a-c). In contrast, the non-binding
GlcNAc-MGNP 6 and control particle NP 1 without any carbohydrates did not
show much staining (Figure 3-8d-f). The same phenomena were observed with
other cell lines as well. The correlations between Prussian Blue staining and AT2
changes proved that the MR changes were indeed due to NP binding with the
cells. The selective uptake and intracellular accumulation of specific MGNPs in

cancer cells and the ability of MGNPs to differentiate normal cells from tumor

153

cells bode well for further development of MGNPs as vehicles for targeted drug

112,113

delivery and magnetic induced hypertherrnia therapy of cancer.114

3.14. Cytotoxicity of MGNPs
Next, we examined the toxicity of the MGNPs towards the cells by 3-(4,5-

dimethylthiazol-Z-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays.
Incubation of cells with different MGNPs for one week did not show any negative
effects on cell viability as compared with untreated cells (Figure 3-9). This
demonstrates that the MGNPs are biocompatible and can be used as safe MRI
contrast agents.

120 a

 

100‘

Cell Viability (%)

20-

 

 

Nodrug MGNP MGNP MGNP MGNP MGNP Adriamycin
0.2ug/mL Zug/mL Sug/mL 10ug/mL 20ug/mL 10ug/mL

Figure 3-9. As an example, MTT assay of A498 cells incubated with Fuc-MGNP 4 showed no
cytotoxicity after one week incubation, while Adriamycin killed most of the cells.

3.15. Anti-adhesive Properties of MGNPs

Tumor metastasis is associated with poor prognosis of cancers.47 One of
the critical steps in metastasis is the adhesion of circulating tumor cells to
endothelium at the target location. It has been demonstrated that cell adhesion

inhibitors can be a potential treatment for metastatic diseases.""*117

154

The MRI signature from MGNPs provides detailed information on how
tumor cells interact with each carbohydrate, which is valuable for guiding the
development of anti-adhesive agents, as the strongly binding MGNPs can reduce
the adhesion of cancer cells to the matrix by blocking the cell surface receptors.
According to the MRI signature, Gal-MGNP 3 interacted strongly with B16F10
cells, which led us to measure its anti-adhesive properties as a proof-of-principle
study. Upon incubation of B16F10 cells with Gal-MGNP 3, the number of cells
adhered to the surface was reduced by more than 50% (Figure 3-10). In
contrast, the non-binding NP 1 showed little effect on cell adhesion. Modification
of the monosaccharide ligands immobilized on MGNPs may strengthen the

binding with the cells, further enhancing the anti-adhesive effects.

   
  
  

 

 

6 a
5 _ -O-GaI-MGNP 3
3
E -I-NP1
s‘ 4 -
3 +815r10
X
33 2 4
<
£
8 1 -
0 r i i T i
0 10 20 30 40 50
Time (min)

Figure 3-10. The adhesion of mouse melanoma B16F10 (~ 5 x 104 cells/mL) to the surface was
significantly reduced by incubation with Gal-MGNP 3 (blue line), while the control NP 1 (green
line) had no effect on cell adhesion as compared to cells without treatment with any NPs (red
line). Error bars indicate standard deviations (triplicate readings).

155

in conclusion, a new approach based on the multi-channel MR responses
of MGNPs to qualitatively and quantitatively map the carbohydrate binding
characteristics of a variety of cancer cells was developed. Validated through
binding with a series of Iectins and a well characterized E. coli system, the
carbohydrates immobilized on MGNPs were found to retain their biological
recognition and binding specificities. Although the monosaccharides utilized in
this study are fairly simple in structures and multiple cells may bind with the same
carbohydrate, the selective carbohydrate-receptor binding with the MGNP array
amplified the small structural differentials. The resulting combined array
responses allowed the detection of cancer cells as well as the differentiation of
closely related isogenic cancer cell subtypes without detailed prior knowledge on
endogenous carbohydrate receptors, while the wealth of information generated
and magnetic nature of the MGNPs can facilitate future identification of the
receptors. The LDA pattern recognition method was applied to decipher the
glyco-code of tumor cell binding, which may be a useful and general protocol to
analyze carbohydrate-receptor interactions.

The strongly binding MGNPs were found to be internalized by tumor cells,
and they significantly reduced the cancer cell adhesion. As the MGNP array
measures the physiologically related carbohydrate-receptor interactions, which
are involved in a variety of cellular functions including endocytosis, cell-matrix
and cell-cell communications, the knowledge gained from this new addition to the
glyco-nanotechnology toolbox can enhance our understanding of cancer cell

functions. This can provide leads for further ligand optimization to improve the

156

specificity in carbohydrate receptor recognition, which in turn can enable the

application of MGNPs for in vivo cancer detection through MRI in the future.

3.16. Experimental Section

3.16. a. Cells and Culture Conditions

Unless otherwise indicated, all starting materials, reagents and solvents
were obtained from commercial suppliers (Sigma-Aldrich or Fisher Scientific) and
used as supplied without further purification. All fluorescein labeled lectins were
purchased from Aldrich. All cell lines were purchased from the American Type
Culture Collection (ATCC) [Cell line designation (Catalog No.), type] unless
otherwise noted: 18485 (CRL-8799), normal breast cell; A498 (HTB-44), kidney
cancer; A549 (COL-185), lung cancer; HT29 (HTB-38), colon cancer; SKOV—3
(HTB-77), ovarian cancer; B16-F10 (CRL-6475), metastatic mouse melanoma;
B16—F1 (CRL—6323), less metastatic mouse melanoma. The MCF-7/Adr-res
(breast cancer Adriamycin resistant) cell line was obtained from the National
Cancer Institute. Two murine mammary carcinoma cell lines (T A3-HA, TA3-ST)
were kind gifts from Dr. John Hilkens, Netherlands Cancer Institute. All cell lines
were grown as monolayer cultures on tissue culture dishes in phenol red free
DMEM (lnvitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum
(FBS) (Sigma), penicillin G (Sigma, 61.4 pg/mL), streptomycin (Sigma, 100
pg/mL) and L-Glutamine (Sigma, 292 pg/mL) at 37°C in an atmosphere of 5%
C02 and 95% air. All cells were grown to log-phase, trypsinized with trypsin-

EDTA solution (0.25 % trypsin, 1 mM EDTA) to detach the cells, washed twice by

157

centrifugation to remove any residual trypsin and re-suspended in the

appropriate media.

3.16. b. MRI and Relaxivity Measurement

Magnetic resonance imaging studies were carried on a 3T Signa® HDx
MR scanner (GE Healthcare, Waukesha, WI). Thirty test tube samples were
scanned simultaneously in a standard quadrature birdcage head coil. For T2
measurements, a multi-echo fast spin—echo sequence (time of repetition = 500
ms, receiver bandwidth = :I: 31.25 kHz, field of view = 20 cm, slice thickness = 3
mm, gap = 3 mm, number of excitation = 1, and matrix size = 256 x 128) was
used to simultaneously collect a series of data points at seven different echo
times (TE) 15-60 ms with an increment of 7.50 ms at two slice locations. For
each sample, the region of interest (ROI) (circles of 4.5 mm radius) was drawn at
the center of each test tube at both slice locations. The T2 was calculated based
on the semi-log linear regression of the mean signal intensity values at the ROI
and the corresponding TEs. Specifically, 1/T 2 = - [(InSn — InSm)/(TEn-TEm)],

where Sn and 8m are voxel signal intensity values at TE values of TE“ and T5,“.

3.16. c. Detection of Cancer Cells using MGNPs

Cell suspensions (105 or 106 cells/mL) were prepared in phenol red free
DMEM media supplemented with 0.20 % bovine serum albumin. Aliquots of
these cultures were placed in sterile tubes and MGNPs 2-6 (final concentration
20 pg/mL) were added. NP 1 without any carbohydrates was used as the control.

The tubes were incubated with gentle mixing at 37 °C. The T2 values MGNP/cell

158

suspensions and MGNP in the absence of cells were then recorded via MRI. MRI
experiments were performed 8 times for each cell line at both cell concentrations.
The largest T2 value change upon binding with cells for each MGNP was set as
100%. The AT2 % for each cell line was calculated as the percentage of change
relative to the largest T2 changes for the respective MGNP. LDA was performed

using the statistical computing and graphics software R.

3.16. d. Prussian Blue Staining

Different cancer cell lines were seeded onto 24-well plate. After incubation
for 12 hrs at 37 °C, nanoparticles were added to the plate in a final concentration
of 20 ug/mL per well. After 12 hrs, the supernatant was removed and cells were
washed three times with PBS, treated with 10% formalin solution (0.50 mL) for 5
minutes to fix the cells, and then washed with PBS. Prussian blue staining was
then performed. To each well was added a 1:1 mixture of 4% potassium
ferrocyanide(ll) trihydrate and 2% HCI solution (0.50 mL), and cells were
incubated for 30 minutes at 37 °C in the dark, counterstained with nuclear fast
red for 3 min, and then washed three times with PBS. The Prussian blue staining

images were assessed by an inverted light microscope.

3.16. e. Cell Viability

Adherent cancer cells (10’5 cells/mL) were plated in each well (100 pL/well)
of a 96-well plate. MGNP at the desired concentrations (from 0.2, 2, 5, 10, 20
pg/mL per well) as well as Adriamycin (10 pg/mL) were then added respectively.

After incubation for 5 days at 37 °C in 5% 002, aqueous MTT solution (5 mg/mL,

159

20 pL) was added to each well. After incubation for 4 hrs at 37 °C, the

supernatant was removed from each well and DMSO (100 pL) was added to
dissolve the converted dye. Absorption at 550 nm was then measured on a plate
reader to determine the cell viability. Each result was the average of triplicate
readings for each concentration. The absorbance from the untreated cells was
set as 100% viability. Cell viabilities with various concentrations of MGNPs and
Adriamycin were calculated by dividing the respective absorbance by that from

the untreated cells.

3.16. f. Anti-adhesive Assay

Cancer cells were detached using 2 mM EDTA in PBS, washed with PBS,
counted to final concentration of ~ 5 x 104 cells/mL and incubated with Gal-
MGNP (20 ug/mL) for 3 h at 37 °C in DMEM containing 10 % F88. The cells
were then immediately seeded in a 24-well plate and incubated at 37 °C. After
10, 20, 30 and 45 minutes of incubation, the medium and the floating cells were
carefully removed by aspiration, and the attached layers were washed twice with
PBS. The firmly attached cells were then counted under an inverted light
microscope. Cell adhesion curves were generated after counting triplicate wells

(20 different homogeneous fields per each well).

160

3.16. 9. Preparation of MGNPs

1. Syntheses of Carboxylic Acid-functionalized Sugars

a- Synthesis of Fucose Amido-acid (Fuc)

0Ac Br

/\/\
“30 OAc __a__, HSCF-QiOAc _b__. “30 03c Br._c__. H30 ORZVNC‘
OAchc OACc):A° 81 % OAC-3A0 97 % 0Ac
over 2 steps 0Ac
L-Fucoee Acetate Fuc1 RM
0.36 (1.5 : 1) FUC3
d M OH e /\/\ H
_. o N ——> N
,5 Hac 0A 0A. (”or g. Hacagfofi H 0
GAO c Ol-P
Fuc4 Fuc

Figure 3-11. a) 33% HBr in AcOH, CHZCIZ, 2 hrs; b) 3-bromo-1-propanol, MS 4A, A92003,
CHZCI2, 18 hrs; c) NaN3, DMF, 80 0C, 18 hrs; d) succinic anhydride, H2/Pd-C, THF, 3 hrs; 6)

. NaOMe/MeOH, 1h.

Bromopropyl 2,3,4-tri-O-acetyI-fi-L-fucose (Fuc2). To a cooled solution of L-
fucose tetraacetate118 (1.50 g, 4.51 mmol) in dry CH20I2 (15.0 mL), 33% HBr-
AcOH (9 mL) was slowly added, and the mixture was stirred for 2 hrs at room
temperature under nitrogen. The reaction mixture was concentrated and the
resulting orange oil was azeotroped with toluene (5x). Then, a solution of the
residue in dry CHZClz (20 mL) containing activated molecular sieves (4 A; 1.5 g)
was stirred for 15 min at room temperature under nitrogen. 3-Bromo-1-propanol
(0.750 mL, 8.63 mmol) was added and stirring was continued for another 30 min.
Silver carbonate (1.65 g, 5.98 mmol) was then added, and the obtained
suspension was stirred overnight. The reaction mixture was then diluted with
CHch2 and filtered through Celite. The filtrate was washed with saturated aq.

NaHCOa and brine repeatedly, dried over NaZSO4, filtered, and concentrated.

161

Column chromatography (30% EtOAc in Hexane) of the residue afforded Fuc2
as a white foam (1.50 g, 81% over two steps). 1H NMR (500 MHz, CDCI3): 6H
1.18 (d, J = 6.5 Hz, 3H, CH3), 1.93-2.00 (m, 1H, OCH2CHHbCHzBr), 1.94, 2.03,
2.12 (3s, 3 x 3H, CH3CO), 2.10-2.18 (m, 1H, OCH20HaHCHzBr), 3.44 (dd, J =
5.5, 7.5 Hz, 2H, OCHZCHZCHgBr), 3.59-3.64 (m, 1H, OCHaHCHZCHzBr), 3.78
(dq, J = 1.0, 6.5 Hz, 1H, H-5), 3.94-3.98 (m, 1H, OCHHb CH2CHzBr), 4.41 (d, J =
7.5 Hz, 1H, H-1), 4.98 (dd, J = 3.5, 10.0 Hz, 1H, H-3), 5.12 (dd, J = 7.5, 10.5 Hz,
1H, H-2), 5.19 (dd, J = 1.0, 3.5 Hz, 1H, H-4). 13C NMR (125 MHz, CDCI3): be
16.2, 20.8, 20.9, 21.0, 30.5, 32.5, 67.3, 69.2, 69.3, 70.4, 71.5, 101.5, 169.8,
170.4, 170.9. HRMS m/z calcd. for C15HzaBrNa08 [M+Na]*: 433.0474; found:
433.0472.

Azidopropyl 2,3,4-tri-O-acetyl-B-L-fucose (Fuc3). Bromopropyl 2,3,4-tri-O-
acetyl-B-L-fucose Fuc2 (0.920, 2.24 mmol) and NaN3 (1.16 g, 17.8 mmol) were
dissolved in DMF (15.0 mL), and refluxed for 18 hrs at 80 °C under nitrogen. The
mixture was then concentrated in vacuo, diluted with CH20I2 and washed with
water, dried over NaZSO4, filtered, and concentrated affording the product Fuc3
as a white foamy solid (0.810 g, 97%). 1H NMR (500 MHz, CDCI3): 6H 1.22 (d, J
= 6.0 Hz, 3H, CH3), 1.77-1.93 (m, 2H, OCHzCH2CHzBr), 1.98, 2.06, 2.17 (3s, 3 x
3H, CHacO), 3.35-3.39 (m, 2H, OCHchchzBr), 3.56-3.60 (m, 1H,
OCHHbCHZCHzBr), 3.81 (dq, J = 1.0, 6.5 Hz, 1H, H-5), 3.95-3.99 (m, 1H, OCHaH
CHZCHzBr), 4.43 (d, J = 8.0 Hz, 1H, H-1), 5.01 (dd, J = 3.5, 10.5 Hz, 1H, H-3),
5.18 (dd, J = 8.0, 10.5 Hz, 1H, H-2), 5.23 (dd, J = 1.0, 3.5 Hz, 1H, H-4). 13C
NMR (125 MHz, CDCI3): 60 16.3, 20.8, 20.9, 21.0, 29.2, 48.2, 66.5, 69.2, 69.4,

162

70.5, 71.5, 101.4, 169.8, 170.4, 170.9. HRMS mlz calcd. for C15H2403N3[M+H]+:

374.1563; found: 374.1565.

O-Acetyl fucose amido-acid (Fuc4). A mixture of fucoside Fuc3 (0.770 g, 2.06
mmol), succinic anhydride (0.300 g, 3.00 mmol) and 10 % PdlC (0.250 g) in dry
THF (20.0 mL) was stirred at room temperature under H2. After 3 hrs, the
reaction mixture was filtered off through Celite and concentrated. Purification by
column chromatography eluting 5% MeOH in CH20I2 afforded Fuc4 (0.690 g,
75% yield). 1H NMR (500 MHz, CDCI3): 6H 1.21 (d, J = 6.5 Hz, 3H, CH3), 1.70-
1.84 (m, 2H, OCH20H2CH2NH), 2.16, 2.05, 1.96 (33, 3 x 3H, CHacO), 2.51-2.67
(m, 4H, OCCHgCHchOH), 3.26-3.32 (m, 1H, OCHZCHchHbNH), 3.36-3.42 (m,
1H, OCHZCHzCHaHNH), 3.52-3.56 (m, 1H, OCHHDCHZCHzBr), 3.81 (dq, J = 1.0,
6.5 Hz, 1H, H-5), 3.96-4.00 (m, 1H, OCHaHCHZCHzNH), 4.40 (d, J = 7.5 Hz, 1H,
H-1), 5.02 (dd, J = 3.5, 10.5 Hz, 1H, H-3), 5.13 (q, J = 7.5, 10.5 Hz, 1H, H-2),
5.22 (dd, J = 1.0, 3.5 Hz, 1H, H-4), 6.56 (t, J = 5.5 Hz, 1H, NH). 13C NMR (125
MHz, CDCI3): 60 16.2, 20.8, 20.9, 21.1, 29.1, 30.3, 30.7, 38.2, 69.3, 69.4, 69.5,
70.4, 71.2, 101.4, 170.4, 170.5, 170.9, 173.0, 176.2. HRMS mlz calcd. for

C19H30011N [M'I'Hri 448.1819; found: 448.1817.

Fucose amido-acid (Fuc). Deprotection of O-acetyl fucose amido-acid Fuc4
(0.200 g, 0.447 mmol) using NaOMe/MeOH (0.50 mL, 5.40 M) in dry methanol
(2.00 mL) afforded the final product Fuc as a foamy white solid in 94 % yield
(0.135 9) after flash column chromatography (30 % MeOH in DCM). 1H NMR
(CD30D, 500 MHz): 6H 1.27 (d, J = 6.5 Hz, 3H, CH3), 1.76-1.82 (m, 2H,

OCHchchzNH), 2.48 (t, J = 7.0 Hz, 2H, COCHzCHzCOOH), 2.58 (t, J = 7.0
163

Hz, 2H, COCHZCHchOH), 3.24-3.38 (m, 2H, OCHZCHZCHZNH), 3.49-3.51 (m,
2H, H-2, H-3), 3.56-3.63 (m, 2H, H-4, OCHHbCHz), 3.64-3.67 (m, 1H, H-5), 3.88-
3.93 (m, 1H, OCHaHCHz), 4.21 (d, J = 7.5 Hz, 1H, H-1). 13c NMR (coaoo, 500
MHz): 60 15.7, 29.1, 29.9, 30.8, 36.6, 67.1, 70.8, 71.2, 71.8, 74.0, 103.5, 173.6,

176.1 . HRMS mlz calcd. for C13H23NNaOa [M + Na]": 344.1321; found: 344.1313.

b- Synthesis of Sialic Amido-acid (Sia)

AcO 0Ac STol AcO OAc 002Me

A c0 __, AcoACO 9A0 (302% _b> A c0 /\/\
ACH'XC o 002”“? 70 % ““ch 0 C' 98 % ACHIXCO 0 N3
sun 3'82 $193
a :5 (3:1)
c AcOACO 9A0 CCLZMB WWW HA0 HO OH COzifl:/\O
43 % a :1? mixture ACHNACO 95 d% HO O HWOH
41 % a only 5'“ Sia

Figure 3-12. a) 3-chloro-1-propanol, NIS, TfOH, 3A MS, CHZCI2:CH30N, -40 °C to r.t. 3 his; b)
NaNa, DMF, 80 °C, 12 hrs; c) succinic anhydride, H2/Pd-C, THF, 3 hrs; d) NaOMe/MeOH, 2hrs.

O-Acetyl sialic chloride (Sia2). s1a11119 (3.01 g, 6.32 mmol) and 3-chloro-1-
propanol (0.840 g, 10.1 mmol) were dissolved in a mixture of anhydrous
CH2C|2:CH3CN (30:30 mL) under N2 in the presence of 3 A molecular sieves.
After stirring at room temperature for 30 minutes, N-iodosuccinimide (NIS) (1.42
g, 6.32 mmol) was added and the mixture was stirred for additional 30 minutes at
room temperature. The reaction was cooled down to -40 °C and
trifluoromethanesulfonic acid (TfOH) (0.0700 g, 0.880 mmol) was slowly added.
A change of color to dark red was observed. After 3 hrs, the reaction mixture was
diluted with CH2C|2 washed with 20% Na28203 (x2), and then water (x3). The

organic phase was then dried and concentrated. Column chromatography

164

(hexanes—EtOAc, 1:1) yielded Sia2 (2.514 g, 70% yield). The chemical shift for
H-3eq of the a anomer was characterized by the downfield shifted 6H = 2.51 (dd,
J = 4.5, 12.5 Hz, 1H, H-3eq.), as opposed to H-3eq. of the B anomer with 6H =
2.40 (dd, J = 4.5, 12.5 Hz, 1H, H-3eq.).‘2° 1H NMR (500 MHz, Cock.) for a
anomer: 5.. 1.86-1.94 (m, 1H, H-3ax.), 1.90-1.96 (m, 2H, OCHgCHgCHZCI), 1.80,
1.96, 1.97, 2.06, 2.07 (5s, 5 x 3H, COCH3), 2.51 (dd, J = 4.5, 12.5 Hz, 1H, H-
3eq.), 3.33-3.37 (m, 1H, OCHaHCHZCHZCI), 3.54 (t, J = 6.5 Hz, 2H,
OCHchzCHzcl), 3.73 (s, 3H, COzCHa), 3.76-3.80 (m, 1H, OCHHbCH20H2Cl),
4.00-4.08 (m, 3H, H-5, H-6, H-9a), 4.26 (dd, J = 3.0, 12.5 Hz, 1H, H-9b), 4.74-
4.80 (m, 1H, H-4), 5.26 (dd, J = 2.5, 8.5 Hz, 1H, H-7), 5.30-5.40 (m, 1H, H-8).13C
NMR (125 MHz, CDCI3): dc 20.88, 20.95, 20.98, 21.21, 23.23, 32.76, 38.13,
41.55, 49.39, 52.92, 61.49, 62.63, 67.61, 69.09, 69.36, 72.79, 98.93, 168.58,
170.27, 170.29, 170.49, 170.82, 171.06. HRMS mlz calcd. for CzaHuClNNaOn

[M+ Na]+: 591.1923; found: 591.1915.

O-Acetyl sialic azide (Sia3). O-Acetyl sialic chloride Sia2 (0.650 g, 1.14 mmol)
and NaN3 (0.595 g, 9.15 mmol) were dissolved in DMF (15.0 mL), and refluxed
for 12 hrs at 80 °C under nitrogen. The mixture was then concentrated in vacuo,
diluted with CHZCI2, washed with water, dried over Na2804, filtered, and
concentrated to yield the compound as a 0:8 (3:1) mixture (0.650 g, 98%). The
chemical shift for H-3eq. of the a anomer was characterized by the downfield
shifted 6H= 2.55 (dd, J = 4.5, 12.5 Hz, 1H, H-3eq.), as opposed to H-3eq. of the
,8 anomer with 6”: 2.42 (dd, J = 4.5, 12.5 Hz, 1H, H-3eq.). 1H NMR (500 MHz,
CDCI3) for o anomer: 6H 1.77-1.83 (m, 2H, OCH20H20H2N3), 1.86-1.94 (m, 1H,

165

H-3ax.), 1.85, 2.00, 2.02, 2.11, 2.12 (5s, 5 x 3H, cocm), 2.55 (dd, J = 4.5, 12.5
Hz, 1H, H-3eq.), 3.28-3.33 (m, 1H, OCHaHCHchzNa), 3.36 (t, J = 6.5 Hz, 2H,
OCHzCHZCHgNg), 3.78 (s, 3H, COzCH3), 3.79-3.84 (m, 1H, OCHHbCHZCHzNa),
4.02414 (m, 3H, H-5, H-6, H-9a), 4.30 (dd, J = 3.0, 12.5 Hz, 1H, H-9b), 4.76-
4.84 (m, 1H, H-3), 5.30 (dd, J = 2.5, 8.5 Hz, 1H, H-7), 5.36-5.40 (m, 1H, H-8).
13c NMR (125 MHz, CDCI3): 6c 20.95, 20.98, 21.02, 21.04, 21.08, 21.2, 21.3,
23.3, 28.9, 29.3, 31.6, 36.7, 37.6, 38.2, 48.3, 48.4, 49.4, 49.5, 52.9, 53.0, 60.8,
61.8, 62.7, 67.6, 68.80, 68.84, 69.1, 69.3, 72.1, 72.4, 72.7, 98.8, 98.9, 162.8,
167.6, 168.6, 170.2, 170.3, 170.5, 170.78, 170.85, 170.90, 171.1. HRMS mlz

calcd. for C23H34N4Na013 [M+ Na]? 597.2020; found: 597.2003.

O-Acetyl sialic amido-acid (Sia4). A mixture of Sia3 (0.240 g, 0.420 mmol),
succinic anhydride (0.0750 g, 0.750 mmol) and 10 % Pd/C (0.0500 g) in dry THF
(10.0 mL) was stirred at room temperature under H2. After 3 hrs, the reaction
mixture was filtered off through Celite and concentrated. Purification by column
chromatography using 5-20 % MeOH in CHZClz afforded Sia4 as 028 (2.5:1)
mixture (0.115 g, 43 % yield). Elution at 25 % MeOH in CHZCIZ and increasing
gradually to pure MeOH yields exclusively a product as a foamy white solid
(0.110 g, 41 % yield). Overall yield 84 %. 1H NMR (500 MHz, CD300): 6H 1.72-
1.78 (m, 2H, OCH20H2CH2NH), 1.78-1.88 (m, 1H, H-3ax.), 1.85, 1.99, 2.02, 2.11,
2.15 (55, 5 x 3H, COCH3), 2.49 (t, 4H, J = 4.0 Hz, OCCHZCH2COOH), 2.63 (dd, J
= 4.5, 12.5 Hz, 1H, H-3eq.), 3.22-3.28 (m, 2H, OCH2CHZCH2NH), 3.30-3.36 (m,
1H, OCHaHCH2CH2NH), 3.76-3.82 (m, 1H, OCHHbCHZCHzNH), 3.83 (s, 3H,

C020H3), 3.96 (t, J = 10.5 Hz, 1H, H-5), 4.08 (dd, J = 5.5, 12.5 Hz, 1H, H-Qa),

166

4.18 (dd, J = 2.0, 10.5 Hz, 1H, H-6), 4.30 (dd, J = 2.5, 12.5 Hz, 1H, H-9b), 4.76-
4.84 (m, 1H, H-4), 5.34 (dd, J = 2.0, 9.0 Hz, 1H, H-7), 5.38-5.42 (m, 1H, H-8). 13c
NMR (125 MHz, c0300): be 19.56, 19.60, 19.8, 20.1, 21.6, 29.3, 31.8, 32.1,
36.6, 38.0, 52.2, 62.4, 62.6, 67.5, 68.4, 69.6, 72.1, 98.9, 168.5, 170.4, 170.60,
170.65, 171.3, 172.3, 174.5. HRMS m/z calcd. for Cz7H40N2NaO16 [M + Na]":

671.2276; found: 671.2273.

Sialic amido-acid (Sia). Sia4 (32.0 mg, 0.0493 mmol) was de-O-acetylated in
dry MeOH (2.00 mL) containing 0.200 mL of freshly prepared 1M NaOMe in
MeOH. The reaction was complete after 2 hrs at room temperature. The solution
was carefully neutralized with Amberlite 120 (H*) resin till pH=6.5. The reaction
mixture was then filtered and evaporated. Flash column chromatography (40 %
MeOH in DCM) afforded the final product Sia (22.0 mg) in 95 % yield. 1H NMR
(500 MHz, CD3OD)I 6H 1.70-1.78 (m, 3H, H-3ax., OCHZCHchzNH), 2.02 (s, 3H,
NHCOCH3), 2.45 (bs, 4H, OCCHchchOH), 2.67 (dd, J = 4.5, 13.0 Hz, 1H, H-
3eq.), 3.25 (t, J=6.5 Hz, 2H, OCHZCH2CH2NH), 3.36 (s, 3H, COzCH3), 3.42-3.46
(m, 1H, OCHHbCHZCHzNH), 3.51 (d, J = 8.5 Hz, 1H, H-9a), 3.62 (d, J = 10.0 Hz,
1H, H-9b), 3.64-3.72 (m, 2H, OCHHaCHZCHgNH, H-5), 3.78-3.88 (m, 4H, H-4, H-
6, H-7, H-8). 13C NMR (500 MHz, CDgOD): be 21.7, 29.3, 32.7, 36.2, 40.3, 48.7,
52.3, 52.5, 61.7, 63.5, 67.3, 69.2, 71.3, 73.6, 99.0, 169.9, 173.9, 174.9, 180.2.

HRMS mlz calcd. for C19H32N2NaO12 [M + Na]+: 503.1853; found: 503.1848.

167

2. Synthesis of Alkynyl-GlcNAc (7) for Click Chemistry

0
a H O b VHF;
/NH2 .HCI ___, N_/( __, N/

/

CH3O 95 % CH3o’ P“ 84% CH30

H/ Ol'cl) 9CH3
c d /
___> N —.> kN/
100 % CH3O/ 92 % I190 NHAc
GlcNAc 7

\

Figure 3-13. a) Benzoyl chloride, pyridine, CH2Cl2, 0 °C to r.t.,2 hrs; b) 3-bromopropyne, K2CO3,
acetone, reflux for 8 hrs; c) 6% MeOH in HCI, reflux for 2 hrs; d) N-acetyl glucosamine, 0.1 M
acetate buffer (pH=6.5) / DMF (3:1), 50 °C, 24 hrs.

N-2-propynyI-O-methoxyamine. N-2-propynyl-O-methoxyamine was
synthesized by slight modification of literature proceduresm' 122 Briefly,
protection of N-methoxyamine hydrochloride with benzoyl chloride, N-alkylation
with 3-bromopropyne, followed by benzoyl deprotection yielded the product as
hydrochloride salt. 1H NMR (500 MHz, D20): 6H 2.87 (t, J = 2.5 Hz 1H), 3.80 (s,
3H), 4.06 (d, J = 2.5 Hz, 2H).13C NMR (500 MHz, 020): be 39.2, 62.2, 72.0,
78.4. HRMS mlz calcd. for C4H3NO [M + H]*: 86.0606; found: 86.0603.

N-2-propynyl-N—(8-D-glucosamine)-O-methylhydroxylamine (7). A solution of
GlcNAc (0.200 g, 0.904 mmol) and N-2-propynyI-O-methoxyamine (0.300 g, 2.53
mmol) in a mixture of 0.1 M aqueous sodium acetate buffer pH 6.5 I DMF (3:1)
was stirred at 50 °C for 24 h. The solvent was evaporated and flash column
chromatography (15 % MeOH in DCM) afforded alkynyl-GlcNAc 7 as exclusively
8 anomer (92 %, 0.240 g). Repeating the same procedure without adding DMF
decreased the yield to 30 %. 1H NMR (500 MHz, CD30D): 6H 1.98 (s, 3H,
NHCOCH3), 2.57 (t, J = 3.0 Hz, 1H, CHZCECH), 3.16-3.20 (m, 1H), 3.33 (s, 1H),

3.39 (t, J = 9.0 Hz, 1H), 3.57 (s, 3H, OCH3), 3.66 (dd, J = 6.0, 12.0 Hz, 1H), 3.70
168

(dd, J = 3.0, 16.0 Hz, 1H), 3.81-3.85 (m, 2H), 3.90 (t, J = 9.5 Hz, 1H), 4.30 (d, J =
10.0 Hz, 1H, H-1). 13c NMR (500 MHz, 00300): 60 21.9, 41.4, 52.6, 61.45,
61.67, 70.5, 72.3, 76.6, 78.5, 79.5, 90.5, 172.7. HRMS mlz calcd. for

C12H20N2NaOs [M + Na]+: 311.1219; found: 311.1207.

3. Synthesis of Azido Siloxane Derivative (8)

O
75 % o H
Azido succinic acid

H O
——3—>(02H50)3Si\/\/N\"/\/‘LN/\/O\/\O/\/N3
70 % o H
Azido siloxane derivative 8

Figure 3-14. a) Succinic anhydride, DMAP, CHZClg, 12 hrs; b) APTES, EDC, CHZClz, 8 hrs.

Azido succinic acid. A solution of azido amine derivative123 (0.200 g, 1.15
mmol), succinic anhydride (0.140 g, 1.40 mmol), 4-dimethylaminopyridine
(DMAP) (0.210 g, 1.72 mmol) in dry CH2CI2 (10.0 mL) was stirred under nitrogen
at room temperature for 12 hrs. The solvent was evaporated to afford a yellowish
solid (0.250 g) in 75 % yield. 1H NMR (CDCI3, 600 MHz): 6H 2.49 (t, J = 6.0 Hz,
2H), 2.61 (t, J = 6.0 Hz, 2H), 3.35 (t, J = 4.0 Hz, 2H), 3.41 (q, 2H), 3.52 (t, J = 3.5
Hz, 2H), 3.58-3.62 (m, 4H), 3.64 (t, J = 4.0 Hz, 2H), 6.82 (bs, 1H, NH). HRMS

mlz calcd. for C10H13N4Na05 [M + Na]+: 297.1175; found: 297.1177.

Azido siloxane derivative (8). To a solution of azido succinic acid (0.200, 0.689
mmol) and EDC (0.300 g, 1.56 mmol) in dry CHzclz (10.0 mL), APTES (0.280 g,

1.27 mmol) was added and the reaction mixture was stirred at room temperature

169

under nitrogen. After 8 hrs, the solvent was removed under reduced pressure.
The residue was purified by column chromatography (2 to 5 % MeOH in DCM)
to afford 0.230 g of azido siloxane derivative 8 as a white solid in 70 % yield. 1H
NMR (CDCI3, 500 MHz): 6... 0.59 (t, J = 8.0 Hz, 2H, CHZSi), 1.19 (t, J = 7.0 Hz,
9H, (OCHZCH3)3), 1.53-1.61 (m, 2H), 2.47 (t, J = 3.5 Hz, 4H, COCHZCH2CO),
3.18 (q, J = 6.4 Hz, 2H), 3.40 (dt, J = 5.0, 17.2 Hz, 4H), 3.51 (t, J = 2.8 Hz, 2H),
3.58-3.63 (m, 4H), 3.66 (t, J = 5.2 Hz, 2H), 3.78 (q, J = 7.2 Hz, 6H, (OCH20H3)3),
6.38 (bs, 2H, NH). HRMS mlz calcd. for C19H39N5Na07$i[M + Na]“: 500.2571;
found: 500.2575.

4. Synthesis of MGNPs (2-6)
Amine-NP. MGNPs were prepared by slight modification of the co-precipitation
method described earlier. Briefly, 0.22 micron filtered 1M aqueous solution of
ferric chloride hexahydrate FeCl3.6H20 (20.0 mmol, 5.41 g, 5.00 mL) and
polyvinylpyrrolidone PVP (Mw ~ 55 KDa) (0.0256 g/mL, 0.650 mL) were mixed
with vigorous stirring under nitrogen in a degassed flask for 15 min. To the
above mixture was added a freshly prepared 0.22 micron filtered solution (10.0
mmol, 2.00 g, 2.50 mL) of ferrous chloride tetrahydrate (FeCl2.4H20).
Ammonium hydroxide NH4OH (30%) (~20.0 mL) was then added until pH of ~11-
12 was reached. Black precipitates started to form indicating nanoparticle
formation. After vigorous stirring for 1 hr at 80 °C, the resulting black NPs were
isolated by applying a permanent external magnet (Lifesep® 50 SX magnetic
separator), washed 6 times with DDW and 3 times with ethanol. To further

protect the magnetite core, we coated the nanoparticles with a silica shell using

170

tetraethoxysilane (T EOS). In a typical experimental procedure, the nanoparticles
were re-suspended in 2-propanol: water (4:1 WV, 150 mL). NH4OH solution (0.2
% v/v, 0.300 mL) was added followed by dropwise addition of TEOS (4.50 mmol,
1.00 mL) over 1 hr with continuous stirring. The reaction mixture was stirred
overnight, isolated and washed repeatedly with diethyl ether and ethanol to yield
silica coated magnetic nanoparticle 1 (T EOS-NP 1). To prepare the amine
functionalized NP, TEOS-NP 1 was suspended in ethanol (150 mL) and DDW
(1.50 mL) and sonicated for 30 minutes. 3-aminopropyl triethoxysilane (APTES)
(2 % wlv, 12.8 mmol, 3.00 mL) was then added and stirred at 60 °C overnight.
The resulting Amine-NP was isolated, washed and re-dispersed in ethanol.

MGNPs (2-5). To immobilize the surface with carbohydrates (e.g. fucose) using
the amide bond linkage, amine functionalized NP (80.0 mg) was washed twice
with DMF, re-dispersed in DMF (50.0 mL) and sonicated for 30 minutes. The
coupling solution was prepared by mixing fucose amido-acid Fuc (110 mg, 0.342
mmol), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate (BOP) (230 mg, 0.520 mmol, 1.50 eq.), 1-
' hydroxybenzotriazole (HOBT) (50.0 mg, 0.370 mmol, 1.10 eq.), and
diisopropylethylamine (DIPEA) (0.230 mL, 1.39 mmol, 4 eq.) in DMF (10.0 mL).
The coupling solution was then added dropwise to amine NP 2 in DMF under
sonication. The reaction mixture was stirred overnight at room temperature. The
resulting nanoparticles were isolated by magnet, washed 3x with ethanol and 3x
with water to afford Fuc-MGNP 4 (~ 80.0 mg dry weight). The same protocol was

followed to synthesize the analogous Man, Gal and Sia-MGNPs. For Sia-

171

MGNPs, the last step involved deprotection of methyl ester using 0.2 M aqueous
NaOH (100 pl) to afford Sia-MGNP 5 (~ 15.0 mg dry weight).

Azide NP. The synthesis of Azide NP was carried out in a similar manner to the
previously prepared Amine NP employing the azido siloxane linker instead.
Briefly, azido siloxane derivative 8 (70.0 mg, 0.150 mmol) was added to NP 1
(100 mg) suspended in THF: Ethanol (100: 25 mL) and stirred at 60 °C for 48 hrs.
The resulting Azide NP (130 mg) were isolated by magnet and washed several
times with ethanol, water and diethyl ether.

MGNP 6. Alkynyl-GlcNAc 7 (150 mg, 0.520 mmol), CuSO4.5H2O (6.5 mg, 0.026
mmol) and sodium ascorbate (50.0 mg, 0.260 mmol) were added to Azide-NP 9
(100 mg) in t-BuOH : H20 (1 :1, 40 mL). The reaction mixture was stirred at room
temperature for 24 hrs, isolated and washed with water and ethanol successively

to afford GlcNAc-MGNP 6 (160 mg).

3.16. h. Characterization of MGNPs

1. TEM

 

Figure 3-15. TEM images of MGNP shows that NP core is ~ 5 - 10 nm.

172

2. FT-IR

FT-lR spectra for MGNP 2-5 were identical to those reported previously.
FT-lR spectra for azido-NP and GlcNAc-MGNP 6 are shown below in Figure 3-
16. The fact that azido-NP was indeed coated with azide was proven by the
appearance of a strong absorbance at 2095 cm'1 due to N=N=N stretching. The

success of click reaction was confirmed by the disappearance of the azide peak

 

 

in GlcNAc-MGNP 6 spectra.
60 i
501 \
N=N=N
% T antisymmetric stretching
40 1
4 a \
30 ' after click chemistry
1
20 ‘ b
10 1
4000 1 3200 ' 2400 Y 1800 . 1400 7 1000 ' 660

Wavenumber (cm")

Figure 3-16. FT -IR spectra of a-) Azido-NP b-) GlcNAc-MGNP 6.

3. TGA

As shown in Figure 3-17, all MGNPs (2-5) exhibited a weight loss between
11 and 14% compared to NP 1 with a weight loss of only 5.5%, implying the
presence of organic molecules on the surface of MGNPs. TGA analysis showed
that ~ 8% of the dry weight of the particles was due to the carbohydrate moieties,

which led to an estimate of 500 copies of carbohydrates on each particle. On the

1.73

other hand, Azido-NP showed a weight loss of 13% compared to the GlcNAc-

MGNP 6 with a weight loss of 19%, indicating that the MGNP surface was indeed

functionalized with GlcNAc.

 

   
   
 
 

 

 

 

   

 

    

 

99 1
97 1
. 95 ~
5 TEOS-NP1
3:" 93 «
5
3 91 1
G l-MGNP3
89 - a
Fuc-MGNP4
87 - Si-MGNPS
Man-MGNPZ
85 1 T T 1 1
0 200 400 600 800 1000
TemperatumPC)
100 -
98 4
96 _
TEOS-NP1
94 ..
25 92
3:"
9 90 1
0
g 88 .1
86 1
84 ..
82 i GlcNAc-MGNP6
so --———————e --—---—-- - - - — --

 

200 400 600 800 1000
Temperature ( °C)

Figure 3-17. a) TGA curves for TEOS-NP 1 (light blue), Man-MGNP 2 (dark blue), Gal-MGNP 3
(red), Fuc-MGNP 4 (violet), and Sia-MGNP 5 (green). b) TGA curves for TEOS-NP 1 (blue),
Azide-NP 9 (red), and GlcNAc-MGNP 10 (green). All samples were heated at a constant heating
rate of 10 °C/min from 50 to 1000 °C under nitrogen.

174

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185

CHAPTER 4

Hyaluronan Magnetic Gcho-Nanoparticles (HA-MGNPs) for
Detection and Molecular Imaging of Atherosclerosis

4.1. Introduction

Atherosclerosis is a silent systemic disease that is characterized by the
build-up of patchy deposits of fatty materials (atheromas or atherosclerotic
plaques) within the walls of medium and large-sized arteries, leading to reduced
or blocked blood flow. With time, the plaques weaken and rupture leading to
thrombosis and occlusion and thus can cause heart attacks or strokes. Although
enormous clinical efforts and drug trials have had a major impact on decreasing
coronary events,1 it will be necessary to better understand atherogenesis to
provide a rational basis for the design of novel diagnostics and therapeutics to
further reduce the risk. Until recently, there has been no effective way to detect
the presence of atherosclerosis in patients unless it has reached a relatively
advanced stage. Thus, it is imperative to better understand the secrets behind
this disease so as to assess prevention and treatment methods.

Atherosclerosis is a very complex disease. The pathogenesis of
atherosclerosis is not well understood. Previous studies indicate that hyaluronic
acid or hyaluronan (HA) are present in regions of atherosclerotic lesions and its
principal receptor, CD44, participate in many stages of atherosclerosis.“ In fact,
the pathogenesis includes recruitment of inflammatory cells to the vessel wall
and activation of vascular cells. CD44 and HA represent a significant receptor-
carbohydrate ligand pair mediating an activation-dependent pathway of

lymphocyte / endothelial cell adhesion.2 It has been demonstrated that HA is

186

upregulated in atherosclerotic lesions and that CD44, expressed on both
inflammatory and vascular cells, promotes the recruitment of macrophages to
atherosclerotic lesions via multiple mechanisms.5 These findings place HA at the
beginning of the inflammatory response,6 a critical step in the formation of the
atherosclerotic lesion. Not only is HA important in the initial stages of leukocyte
extravasation but also its accumulation in the early lesions may promote
inflammatory cell retention by serving as an anchor for these cells. All this imply
that inhibition of CD44 binding with HA may provide an effective means for
reducing the development of atherosclerotic lesions.

The unique interactions between CD44 and HA have been exploited for
targeted delivery of agents to tumor cells."'10 HAICD44 binding has been shown
to facilitate CD44 mediated uptake killing tumor cells,7 target and deliver genes
into the ocular epithelial cells with high transfection levels,11 decrease breast
cancer metastasis, increase antitumor activity in syngeneic and xenograft tumor
models10 and image cancer.12 However to date, CD44 has not been explored for
atherosclerotic plaque binding. In this work, we will examine the possibility to use
CD44 as a molecular target for site specific delivery of imaging and therapeutic
agents to study atherosclerosis. We hypothesized that superparamagnetic
polyvalent HA-functionalized magnetic glyco-nanoparticles can be utilized for the

detection and imaging of atherosclerotic plaques.

187

4.2. Biological Significance of Hyaluronan and CD44

HA is a naturally occurring, non-sulfated glycosaminoglycan GAG (a class
of negatively charged polysaccharides) found predominantly in extracellular
matrices (ECM), cartilages and the vitreous body of eyes.” it plays vital roles in
biological events such as cell adhesion, cell migration, inflammation and also
mediates cell proliferation making it not only a structural component of tissues,
but also an active, signaling molecule.13 HA is comprised of linear, unbranched,
repeating units consisting of glucuronic acid-8-1 ,3-N-acetyl glucosamine (GlcU-B-
1.3-GlcNAc) connected through 8-1,4 glycosidic linkages (Figure 4-1). HA, along
with the other GAGs present in the ECM, provides compressive strength to
tissues and promote wound repair.6' 1‘ Its negative charge, hydrophilicity and
long polymer length result in large amounts of water being bound with the
matrix."5 In addition to providing a hydrated space around cells, it regulates the
traffic of growth factors and other signals due to its pore size and charge density.
HA can be transformed in vivo into oligosaccharide fragments (sHA) ranging from
di— to deca-saccharides with tandem repeats of the 8-1,4-GlcU-8-1,3-GlcNAc
where each respective size is characterized by its own function and properties."*
‘8 In general, HA attracts considerable attention due to its apparent ability to

influence cell behavior.

Hyaluronic acid (HA)
OH OH
«“0 CWT-évvofl/o . O~~~
HO HO \ NHAC
OH NHAC OH

Figure 4—1. Structure of hyaluronic acid (HA).

188

The cell-HA interaction is a true receptor-ligand interaction where the
principal receptor in question is identified as CD44,” which is a transmembrane
glycoprotein present richly on plasma membrane domains, facing open
intercellular spaces, rich in HA. CD44 is expressed on both inflammatory and
vascular cells and can mediate adhesion of T lymphocytes to endothelium and
smooth muscle cells (SMCs), release of inflammatory mediators from
macrophages and T lymphocytes, and proliferation of vascular SMCs.20 A few
years ago, research concerning CD44 has expanded rapidly and CD44 has been
implicated in cell migration during angiogenesis, tumor invasion and
metastasis.21 CD44 has a large HA-binding domain (HABD) that is necessary for
its functional activity.22 The majority of the proteoglycan-rich pericellular matrix
can be displaced by reagents that compete with CD44-HA binding such as anti-
CD44 antibodies and sHA hexasaccharides (HA5). Competitive binding analyses
with sHA showed that the smallest HA unit that could competitively bind HA
polymers is a hexasaccharide (HA6), but that octasaccharide (HA3) to
dodecasaccharide (HA12) are more efficient competitors.22 This confirms that the
minimum size of oligosaccharide required to displace high molecular HA from cell
surface CD44 is between 6 and 10 saccharide units (HAe-HAm) depending on
the cell background.19 Recently, the crystal structure of HA binding to CD44 has

been revealed (Figure 4-2).23

189

 

GlcNAc2

Figure 4-2. Crystal structure of CD44 complexed with HA.

It was shown that hyaluronan binding at the cell surface is a complex
interplay of multivalent binding events affected by the size of the multivalent
hyaluronan ligand, the quantity and density of cell surface CD44.19 Since binding
of a CD44 containing cell to HA substrate involves multiple weak receptor-ligand
interactions, we will assess ligand binding of CD44 by preparing multivalent HA

functionalized on magnetic nanoparticles.

190

4.3. Atherosclerotic Plaque Detection using HA-MGNPs
4.3. a. Plaque Imaging via MRI

Numerous imaging modalities have been applied to detect the progression
of atherosclerotic plaque.24 Among them, MRI is emerging as a non-invasive,
nondestructive technique with high spatial resolution and 3—dimensional
capability.25 MRI has minimal side-effects as it does not involve the use of
radioactive compounds or invasive procedures and can be repeated sequentially
over time to monitor disease progression. MRI can provide high-resolution
images of multiple vascular regions which may aid early intervention in the
treatment of vascular diseases.26

In principle, MRI relies on the detection of the water proton’s relaxation
rate in a magnetic field.27 The variations of water environment in body tissues
create MR images. Briefly, in MRI, the subject is placed in a strong high-extemal
magnetic field which aligns the protons. The MR image is based on the
radiofrequency signal, typically from water protons, following administration of an
RF pulse. The emitted signal varies according to the water concentration and to
the spin-lattice longitudinal (T 1) and spin-spin transverse (T 2) relaxation times.
Fortunately, the T1 and T2 relaxation times vary among different types, providing
a highly useful means of generating image contrast. Although MRI has good
spatial resolution, but it has relatively low sensitivity compared to other
techniques. However, resolution can be improved by the use of contrast agents

that target specific cells or molecules improving the sensitivity of MRI.28

191

An important feature of MRI imaging is that it can improve the precision of
diagnosis and enable earlier detection of the disease, perhaps allowing
intervention even “before symptoms occur” or “in'eparable damage ” has been
inflicted (i.e. what we refer as “early detection”). In general, effective imaging
necessitates the proper density of the target molecule, the high affinity of the
ligand to its receptor, and the payload of contrast that can be delivered to it.
Using a combination of MRI contrast generated in so called T1-weighted (T 1w)
and T2-weighted (T2w) images, it has been possible to determine both plaque

anatomy and composition in experimental animals” 3°

and carotid artery of
humans.31 However, identifying suitable biomarkers and imaging the plaque at
early stages to locate the disease is still not translated into clinical applications.
As a more precise understanding of the plaque formation and evolution, MRI
studies are required to be assessed. This objective necessitates the
development of novel contrast agents targeting molecules within cells or within
the extracellular matrix of the evolving plaque.

In fact, atherosclerosis presents a number of potential targets, including,
endothelial cell adhesion molecules, extracellular matrix, thrombosis,
neovasculature 0,83 integrins, oxidized LDL, matrix metalloproteinases, and
macrophages.” ”‘35 Nevertheless, there are challenges to implement these
targeted technologies into the clinical practice. Molecular imaging using targeted
contrast agents to cell adhesion molecules upregulated on the endothelial

surfaces of the arteries is a promising approach and has the potential of

detecting the atherosclerotic lesions non-invasively. The expression of CD44,

192

 

predominantly on endothelial cells, mediates leukocyte recruitment to sites of
inflammation and is an early event in atherogenesis.” 33 Their endothelial
location makes such molecules potentially accessible to targeting by

intravascular imaging agents.

4.3. b. Nanoparticles as Imaging Agents

The promise offered by nanotechnology and the recent development
achieved in molecular imaging open a great deal of opportunities on both the
diagnostic and therapeutic fronts.36 Indeed, imaging molecules and cells involve
signal enhancement through the use of purpose-built contrast agents. The
contrast agent must identify the target with high specificity and should possess
physical properties that permit its sufficient localization to the target. This
consequently provides intense signal enhancement within the imaged volume so
as to be distinguishable from the unenhanced tissue.28 Other considerations of
such agent include its fast clearance from the body, cytotoxicity, biodegradability
and immunogenicity.37

Direct imaging of the atherosclerotic plaques by MRI has been studied.“
38 The quality of MR images can be greatly enhanced by delivering a
paramagnetic contrast agent to the region of interest, with the most popular
contrast agents being paramagnetic Gadolinium (Gd) compounds such as Gd-
DTPA.39 To further improve detection, Gd has been attached onto ligands to
target several different components of plaques.26 However, the recent
association of Gd with nephrogenic systemic fibrosis, an untreatable disease,

has spawned great interests in alternative agents for MR molecular imaging.40

193

 

Moreover, specific targeting was typically achieved with peptides or monoclonal
antibodies.41 However, such approaches are limited by potential toxicity,
immunogenic responses and larger sizes compared to HA-MGNPs. Indeed, the
most successful approaches have involved the synthesis of nanocomposites that
combine target specificity with the capacity to carry a substantial payload of

superparamagnetic agent.42

Functionalized iron oxide nanoparticles have proven
to be effective T2fT 2* imaging contrast agents,“‘°"“5 where they shorten relaxation
times producing a sharp negative contrast by virtue of signal reduction. They can
be utilized as targeted-packaged units and thus concentrated at specific sites.
Recently, SPIONs have received much attention because of their applications in
a variety of fields such as biosensing, imaging, drug delivery and therapy.46 Their
applications in MRI have ranged from non-targeted to targeted detection.47 For
instance, cross-linked dextran-coated superparamagnetic iron oxide
nanoparticles have been functionalized with different biomolecules and used for
in vitro detection of different targets including oligonucleotides,48 proteins and
intracellular labeling,49 viruses,50 and enantiomeric impurities.51 For in vivo
purposes, studies have shown that modified nanoparticles can both target and
image the atherosclerotic biomarkers.”56 Indeed, researchers have shown that
MRI can noninvasively detect atherosclerotic plaques in living mice,“ 58 rabbits30
and humans.59 SPIONs have been shown to be more taken up by macrophages
than other cells.6°' 6‘ In recent studies, injection of ultrasmall SPIONs into

hyperlipidemic rabbits resulted in significant T2* decrease in the aortic wall after

5 days, which after histopathology showed accumulation in macrophages

194

embedded in the atherosclerotic plaque?4 Similar observations have been
reported in human carotid arteries where ultrasmall SPIONs were accumulated in
macrophages.62 This is indeed a solid proof that USPION-enhanced MRI can be
utilized for in vivo detection of macrophages in human plaques. MRI has also
been used to monitor monocyte recruitment into developing atherosclerotic
plaquesf?‘3 Imaging of apolipoprotein E-deficient (ApoE"') mice 6 days after
administration of SPION demonstrated the localization of iron particles to the
regions of accumulating macrophages in the diseased sections of aorta.
Combined with their high contrast enhancement abilities, low toxicities and
flexible surface chemistry, SPIONs can be excellent MRI probes and effective
delivery vehicles.64 Several SPIONs such as Feridex and Ferumoxtran61 have
been approved by FDA for use in humans as passive MRI contrast agents.
Herein, we hypothesized that certain features associated with plaques’
vulnerability including inflammation, endothelial adhesion molecule expression,
macrophage recruitment, and the role CD44/HA plays in those chief events may
allow us to use novel HA-MGNP as potential contrast probes to detect
inflammation early and image the atherosclerotic plaque. Thus, highly colloidal,
biocompatible and well dispersed HA—MGNPs will be prepared. Taking
advantage of the iron oxide core for imaging and CD44/HA binding events for
targeting, their potential use in targeted-detection and enhanced-imaging of

atherosclerotic plaque will be [investigated

195

 

4.4. Synthesis of HA-MGNPs

HA-MGNPs are made of Fe304 core coated by hyaluronic acid polymer
(~ 16 KDa) rendering the NPs dispersed, colloidal and thus appropriate for in vivo
imaging.

We showed in the previous chapters the successful utilization of MGNPs
immobilized with carbohydrates for pathogen and cancer detection in vitro. As
discussed earlier, the characteristics and properties of the nanoparticles are
extremely important, as those factors determine their use for in vivo or in vitro
applications. For in vitro, the size restrictions are not so severe as for in vivo. For
in vivo application, magnetic nanoparticles should be stable in water at neutral
pH and physiological salinity. The colloidal stability will depend mainly on the
dimensions (sufficiently small so that precipitation will not occur), charge and
surface of the nanoparticles (steric and electrostatic stabilization).

Despite the large number of reports on the synthesis of superparamagnetic
nanoparticles with different methods, including co-precipitation and thermal

decomposition,65' 65

we investigated the use of a sol-gel co-precipitation process
at elevated temperatures to form colloidal, stable and polymeric coated
superparamagnetic nanoparticles. Particularly, for the preparation of HA-MGNPs,
we used a modified synthetic procedure with controlled passivation of the
polymeric sugar on the surface of the nanoparticle.” 68 Briefly, the synthesis of
colloidal superparamagnetic iron oxides by the cold gelation process involves two
steps: (i) the neutralization of iron salts (Fe2+ and Fee”) with base (NH4OH) at 0°

C to form a weakly paramagnetic gel, followed by, (ii) heating to convert the

paramagnetic gel to a superparamagnetic colloid. The newly devised

196

nanoparticles are composed of magnetic iron oxide core, encapsulated by a 10
kDa dextran coating. To develop more stable and amino-functionalized
nanoparticles, the dextran coating has been cross-linked with epichlorohydrin to
yield magnetic dextran-coated nanoparticles (MDNP), and then treated with
ammonia to provide functional amino groups on the surface affording amine-
MGNP (Figure 4-3). Amino groups can then react with the acid functionality of
HA polymer (~ 16KDa) in aqueous media using triazine-activated amidation69

allowing attachment of hyaluronan polymer onto the NP surface.

1) NH4OH, dextran

 

   

 
  

 
 

 

Fe3" + Fe” 2) Heating 75-85 °C NaOH
7 o
LK/C'
epichlorohydrin
O
HA/K HA
H N NH NH N
2 \/ 2 I ”_ O
NH OH . x
Y ‘~
‘ \ HA
H2N NH2 H2N NH2
Amlne-MDNP HA-MGNP

Figure 4-3. Synthesis of HA-MGNP.

HA-MGNPs were thoroughly characterized by a variety of techniques
including FT-IR, TEM, TGA and NMR. TGA analysis demonstrated that ~ 90% of
the particle weight was from dextran and HA coating (Figure 4-4). TEM images
showed that the HA-MGNPs were highly mono-dispersed with the average core
diameter of ~ 6 nm (Figure 4-5a). HR-MAS NMR spectrum of our HA-MGNP

197

 

gave solution like resolution, with the molar ratio of 1:3 between dextran to HA
(Figure 4-5b). With the HA coating, it is most likely dextran is completely
shielded by the HA, thus not interfering with the desired biological recognition of
NP through HA. HA-MGNPs are highly colloidal, monodispersed, can be simply

prepared in large-scales, thus suitable for MRI targeted applications.

100 1

HA-MGNP

9O 7
80 ‘ .
70 7

col

 

50 7

Weight (%)

40J
30-

20 *

~10°/o

 

10 1

 

0 100 200 300 400 500 600 700 800 900 1000

Temperature ( °C)

Figure 4-4. TGA for HA-MGNP showed that ~ 90% of the particle weight was from dextran and
HA coating. Sample was heated at a constant heating rate of 20 °C/min from 50 to 1000 °C under
nitrogen.

198

 

O

V. :
t") ‘- l
,2

[x

‘1

('0
2

1‘ I
...... UIWJ“ Kmflmz

0.07017 1.46 13.13
:1: L =
ml'j—TTIWIIWITI I llITrllIl ITIIIIIII IIIIII 'TTrTI1IIUUTI'IIIIUI'III]'UIT'TIITIIIIIIUIIIIIIIII1

I l
30 55 5b 45 4D 35 30 25 20 L5 L0

 

gift '7qu

 

 

Figure 4-5. a) TEM of HA-MGNP (scale bar is 20 nm) and b) HR-MAS ‘H-NMR of HA-MGNP.

4.5. HA-MGNPs as Nanoprobes for Diagnosis and Imaging of Injured Rabbit

In order to examine imaging of atherosclerotic plaques, we first incubated
HA-MGNP with both normal rabbit artery tissue and rabbit atherosclerotic artery
tissues in vitro. The unbound HA-MGNP was removed from the tissue by
thorough washing. The presence of the NPs in the tissues was first detected by
Prussian blue staining with nuclear fast red counterstaining. The healthy rabbit
artery tissue showed little Prussian blue staining demonstrating that it did not
bind much with HA-MGNP (Figure 4-6a). In contrast, HA-MGNP bound to the
atherosclerotic tissue strongly while the vessel wall remained unstained (Figure

4-6b,c). Incubation of HA-MGNP with atherosclerotic tissue in the presence of

199

 

high concentration of free HA completely abolished the HA-MGNP binding.
Furthermore, the control MDNP without HA coating did not stain the
atherosclerotic tissue either, suggesting the HA-MGNP/plaque tissue interactions
were due to specific binding with HA. The presence of HA-MGNP on plaque
tissue was easily detected by MRI, as the selective darkening of the plaque was
observed due to the adhesion of NPs to the tissue (Figure 4-6d). This indicates

that HA-MGNP can be used to selectively target and image the atherosclerotic

     

plaques.
..."... “1... 1 selective uptake
'3- of the particles in
l the plaque
vessel wall
a. Healthy tissue after b Atherosclerotic tissue C. CI'OSS SGCfiOfl Of the
counter staining surfam after wunterstaining atherosclerotic tissue d MRI image of the

atherosclerotic tissue

Figure 4-6. Binding of HA-MGNP with rabbit artery tissues after removal of unbound particles. a)
healthy non-atherosclerotic tissue surface; b) atherosclerotic tissue surface; c) cross section of
the atherosclerotic tissue; and d) MRI image of the atherosclerotic tissue.

With the promising ex vivo imaging results in hand, we moved to
demonstrate the feasibility of in vivo imaging using an injured atherosclerotic
rabbit model. Full plaques will develop in rabbit aortas injured through a balloon
de-endothelialization process followed by feeding with a high (1%) cholesterol
diet for six months. The de-endothelialization mimics the ox-LDL induced arterial
injury, recruiting macrophages to the injury site as in the early stage of plaque
development.” 7‘ In order to examine whether we can detect early plaque
formation, a rabbit’s aorta was injured through the balloon-catheter procedure

and fed it the high cholesterol diet for three weeks. This is an injured rabbit model

200

where full plaque did not have time to completely develop. Our hypothesis is that
if we can detect early inflammatory lesions of the disease (i.e. the key for the
progression of the disease), we might be able to stratify clinical risks. The earliest
molecular changes and protein signaling occur on the endothelial surfaces of the
aorta and vasculature and are chief contributors to initiation of atherosclerosis.
Thus, targeting CD44 upregulated on the endothelium under inflammatory
conditions is ideal for next generation of diagnostics and therapeutics. HA-MGNP
(1.5 mg Fe total/kg of body weight) was hence injected into the ear vein of the
injured rabbit. Excitingly we observed selective darkening of the rabbit artery wall
by high-resolution 3D T2*-weighted MRI with a voxel size of 0.2 mm3 fifteen
minutes after HA-MGNP injection (Figure 4-7a,b). It is worth mentioning that
there was only a little plaque developed at this stage without much stenosis, and
that pinpoints the advantages of our approach. The rabbit was then sacrificed
with its aorta imaged ex vivo by high-resolution 3D T2*-weighted MRI (Figure
4-7c) with a voxel size of 0.06 mm3. It should be emphasized that the dosage in
our study is much lower than that ~ 56 mg Fe/kg34 and 11 mg Fe/kg72 adapted in
two studies utilizing SPION as a passive contrast agent to image the
atherosclerotic plaques in rabbit aorta. This highlights the advantage of active

targeting.

201

 

corresponding

b) Effect of NP on arteial wall C) slicelocation ‘

  

‘ 1/1 is»
,1 I?
wag:

HA-MGNP E)

Injection

   

Figure 4-7. MRI images of rabbit artery. 3) in vivo image before HA-MGNP injection; b) in vivo
image after HA-MGNP injection; and 0) ex vivo image following HA-MGNP injection and artery
removal with the arrow showing corresponding in vivo slice location.

The same study was repeated as shown in Figure 4-5. Interestingly,
injection of the control Feridex (FDA approved dextran coated nanoparticle) at
the same dose showed no affinity for endothelial cells with no darkening in the
arterial wall (Figure 4-8a,b). Selective darkening of the rabbit artery wall after
HA-MGNP injection was observed as before (Figure 4-8d). This proves the
reliability of our results and the selectivity of HA-MGNP as targeted-disease
specific molecular imaging agents, which can have great impacts on

atherosclerosis treatment.

202

No Effect of NP
on arteial wall

Effect of NP
on arteial wall

 

Figure 4-8. MRI images of rabbit artery. a) in vivo image before Feridex injection; b) in vivo image
after Feridex (1.5 mg Felkg) injection; c) in vivo image before HA-MGNP injection; d) in vivo
image after HA-MGNP (1.5 mg Felkg) injection.

Moreover, in a separate study, HA-MGNP was injected into two rabbits to
test toxicity. It is known that both the magnetite NP and HA are highly
biocompatible. Indeed, in our study, no toxicities or apparent adverse effects on
these rabbits were observed two months following NP injection. Achieving good
successful results for the injured rabbit, detection of atherosclerotic plaque at

more advanced stages seems promising

4.6. Current Studies and Future Insights
Recent studies point to evidence that indicates dextran-NPs are not as
proficient as currently believed.” 7“ One of the main limitation of typically

synthesized and dextran-functionalized iron oxide for biomedical applications is

203

the presence of a thick polymer that increases the overall hydrodynamic radii
(~ 150 nm) causing not only possible dampening of the MR signal but also
shorter blood-circulation times. They do not present sufficient cellular uptake to
enable cell tracking because of a relatively inefficient fluid phase endocytosis
pathway.73 Targeted biodistribution of these nanoparticles remains a challenge
because of a quick process known as opsonization that renders the particles
recognizable by the body’s major defense system, the reticulo-endothelial system
(RES) leading to decreased circulation blood half-life.73 Moreover, surprisingly,
some cases point to situations where dextran-magnetite nanoparticles resulted in
cell damage and reduced cell proliferation.73 As a result, for in vivo applications,
there is a need to explore novel nanoparticles with proper surface modification
that would ensure the particles to be non-toxic, biocompatible and stable to the
RES.

As discussed earlier, magnetic nanocomposites are not bioCompatible
without the aid of a protective hydrophilic layer on the surface to serve as a buffer
for bio-cellular interactions. However, sometimes this layer will be thick affecting
the overall hydrodynamic size of the particles in media and hence their in vivo
efficacy. Ideally, the size of the nanoparticles should be small enough to escape
capture by macrophages in RES, mainly liver and spleen, but large enough to
prevent their rapid leakage into blood capillaries, making it a challenging issue for
the synthetic chemists. Indeed, the size of the nanoparticles should be ~ 50 nm
to reach tumor tissues by passing through these 2 particular vascular structures.

Moreover, functionalizing the surface with hydrophilic compounds is important as

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it protects them from opsonization by repelling plasma proteins. Thus, it is of
immense value to design MNPs that satisfy the above 2 requirements as those
NPs will have the ability to circulate longer in the bloodstream62 and greater
chance of reaching targeted tumor tissues.” 76 As a result, there is a push for
finding and developing new surface-modifying and targeting contrast nano-
agents.

Consequently, we planned the synthesis of HA-coated magnetic
nanoparticles in an alternative method without using the dextran layer. Instead of
using the co-precipitation or sol-gel method explored earlier, we anticipated the
use of the thermal-decomposition technique that usually offers better control over
crystallinity, monodispersity and uniformity of nanoparticles and thus higher
relaxivity. The hydrophobic inorganic nanocrystals (coated with oleic acid) formed
will be then transferred to aqueous solutions through a “robust ligand exchange”
method that uses the hydrophilic HA polymer to directly replace the original
hydrophobic ligands (oleic acids) at an elevated temperature (~ 110 °C) in a 2-
solvent system. This method offers several advantages: a) more exchange
efficacy at elevated temperature; b) ideal dispersant characteristics (HA is a long
hydrocarbon polymeric chain with carboxylate and hydroxyl groups); c) strong
coordination to the nanocrystal surface through multiple anchoring groups; (I)
excellent solubility in water (abundant uncoordinated groups facing water); 9)
good stability; f) direct immobilization of the target molecule HA; 9) two-phase

reaction system and h) fast, simple and scalable.

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Experimentally, a basic solution of polymeric HA dissolved in basic
aqueous solution (pH > 9) will be rapidly injected into a pre-heated toluene
solution containing oleic-Fe3O4 nanocrystals (prepared by the thennal-
decomposition method discussed in chapter one) and refluxed. The composition,
size, and morphology of the as synthesized particles can all be tailored, which, in
combination with their nanoscale magnetic phenomena, makes them potentially
suitable for in vivo applications. Preparation and characterization of such
particles and mechanistic investigation onto their size, shape and quality-control
is in progress.

Both HA-MGNPs and the newly optimized directly-HA coated
nanoparticles will be evaluated for their abilities to image atherosclerotic plaques
in vivo. As HA—MGNP targets CD44, a marker for inflammation, we hypothesize
that we will be able to use HA-MGNP to follow the plaque development. Our
preliminary imaging results suggest that detection of early stages of
atherosclerosis development can be achieved with HA-MGNP, which would be
difficult to accomplish using the traditional methods such as angiography as there
is no significant stenosis. In the preliminary in vivo studies, MR images were
acquired 15 minutes after injection and the rabbit was sacrificed. NPs were found
to be close to the surface of the injury. In the fully developed plaque rabbit model,
we will increase the circulation time between injection and MR imaging to test
whether the NP will circulate longer in the blood enabling penetration deep inside
the plaques. We will also study the biodistribution and rate of clearance of NPs

from the body.

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Overall, the innovation of our work lies in the development of a nano-
based platform technology that can not only detect early and unstable plaques
but also have the potential to be functionalized with therapeutic agents. Image-
based therapeutics will provide conclusive evidence that the drug is reaching the
desired site and the molecular effect can be easily monitored. The advantages of
our approach are: 1) CD44 is a novel plaque component for targeting. By using
HA-MGNPs, active targeting can greatly reduce the amount of agents required,
lowering the risk of side effects; 2) the NP system can be used to deliver a
variety of therapeutic agents with the capability to non-invasively monitor the
effects of the therapeutics (in process); 3) due to the small sizes of NP, it can
potentially penetrate plaques deeply allowing drug and imaging agent delivery to

the core; and 4) the NP system is highly biocompatible.

4.7. Conclusion

In conclusion, as a part of our carbohydrate-based nano-program, we
devised "smart" nanoparticles that can be used as sensing, imaging, and
targeting agents for pathogen detection, cancer differentiation and plaque
progression. We believe that carbohydrate-coated nanoparticles are an important
addition to the stock of diagnostic and disease-fighting nanoparticles. We would
have been missing vast opportunities not to recruit the “sugar-code” to cellular
information transfer and clinical applications. Indeed, we proved that designed
carbohydrate-functionalized magnetic bio-nanocomposites can be valuable not
only as MR contrast enhancement and imaging agents, but also as excellent

targeting and sensing vehicles. Although the development of discrete, highly-

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colloidal, ultrasmall, biocompatible, stable, high contrast and targeted magnetic
glyco-nanoparticles probes is still in its early stages, they have a massive
potential to bring significant advances to the biomedical field at both the
diagnostic and therapeutic fronts. Target selectivity and receptor specificity is the
single most significant factor if MRI is to establish itself in the next decade. We
believe that tailored MGNPs could pave the way to in vivo diagnosis of the early
stages of diseases. Ultimately, the goal is to enable clinicians to better identify
people at the highest risk of the disease and to fine-tune therapies to reduce the
saddle of diseases in affected patients. To achieve this aim, we should start from
ourselves, from the chemistry labs and let me conclude by what Prof. Gregory M.
Lanza said: “We should not be sitting in the lab generating nanoparticles and
then looking for what they could be used for.... We should seek a medical

problem and ask what kind of particle might overcome it and then try to create it”.

4.8. Experimental Section

4.8. a. Synthesis of Colloidal and Monodispersed HA-MGNPs
1) Preparation of Crosslinked Dextran-coated Fe304 Nanoparticles (MDNPs)
FeCI3.6H20 (0.32 g, 1.2 mmol) and 4.5 g dextran (~ 9-11 KDa) were
mixed in 0.22 pm filtered aqueous solution (10 mL), vigorously stirred and cooled
to 0 °C. FeCl2.4H20 (0.13 g, 0.65 mmol) was then dissolved in 0.45 mL of 0.22
pm filtered aqueous solution and added slowly to the above cooled mixture.
While being rapidly stirred, cooled 30% NH4OH solution (0.45 mL) was added
dropwise to the above acidic solution. At this stage, the greenish suspension was

heated to ~85° C for 90 min. The mixture was then cooled, resulting in the

208

 

 

formation of black superparamagnetic colloidal suspension. Ammonium chloride
and excess dextran were removed by extensive dialysis (14 KDa cutoff), followed
by ultrafiltration (30 KDa Mw cutoff membrane). After several washes, the
colloidal product was concentrated by ultrafiltration and 0.22 pm filtered to a total
volume of 45 mL (3.0 mmol Fe total). To the superparamagnetic colloid (1.2
mmol Fe, 2 mL), was added 5 M NaOH (10 mL), distilled water (4 mL), and
epichlorohydrin (51 mmol, 4 mL). The mixture was stirred at room temperature
for 24 hrs. Excess epichlorohydrin was then removed by dialysis (14 KDa cutoff)
against 10 changes of distilled water to yield MDNP. Amination was achieved by
the addition of 30% NH4OH solution (8 mL) to MDNP (30 mL, ~80 mg total
weight), followed by heating at 37° C for 36 hrs. The resulting mixture was then
dialyzed (14 KDa cutoff) against 10 changes of distilled water to afford amine-
MGNP.
2) Preparation of HA-coated Magnetite Nanoparticles (HA-MGNP)
Triazine-activated amidation of HA was performed using previously
reported procedure.69 Briefly, 100 mg of hyaluronic acid sodium salt (~ 16 KDa)
was dissolved in distilled water (5 mg/mL), mixed with Amberlit H+ and stirred at
room temperature for 4 hrs until pH ~3. The resulting mixture was then filtered
and rotary-evaporated under high vacuum to obtain hyaluronic acid in protonated
form. 4-methylmorpholine (NMM) (50 pL, 0.45 mmol) was added to protonated
hyaluronic acid (83 mg, 0.22 mmol carboxylic acid) dissolved in waterzacetonitrile
(ratio 3:2, 6.5 mL) mixture. The above solution was then cooled to 4 °C, and 2-

chloro¥4,6-dimethoxy-1,3,5-triazine (30 mg, 0.17 mmol) was added and stirred at

209

 

 

room temperature for 1 h. Amine-MGNP (10 mL, ~30 mg total dried weight) was
then added and stirring continued for 24 hrs at room temperature. The reaction
was then neutralized with resin and filtered. Extensive dialysis (14 kDa Mw cutoff)
of the solution against deionized water, followed by ultrafiltration (100 KDa Mw

cutoff membrane) and 0.22 pm filtration afforded HA-MGNP (~55 mg Fe total).

4.8. b. HR-MAS 1H NMR

HR-MAS NMR experiments were carried out on a Varian Inova-500 NMR
spectrometer equipped with a 4 mm gHX Nanoprobe (Variannmr Inc., Palo Alto,
CA) available at the University of Tennessee Health Science Center (Memphis,
TN). The HR-MAS probe with internal lock is capable of performing either direct
or indirect (invese) detection experiments. MAS experiments were performed at
spinning rates of up to 2.5 kHz using a 40 pL glass rotor. All HA-MGNP samples
were dissolved in D20 solvent and were further diluted at different concentrations
with 020 to find out the concentration limit to the NMR signal broadening. HR-
MAS 1H NMR spectra were obtained using 100-600 scans for each experiment.

The sample temperature was regulated with an accuracy of :01 °C.

4.8. c. Injured Atherosclerotic Rabbit

New Zealand White rabbits weighing between 2 and 3.5 kg were exposed to the
following preparatory regime: the control group consisted of normal rabbits (n=3)
that were fed a regular diet for 8 months. Atherosclerotic rabbits (n=3) underwent
balloon-induced arterial wall injury, then were fed a diet of 1% cholesterol
(Harlan-Sprague Dawley, Inc, Indianapolis, Ind) for 2 months followed by a

regular diet for 2 months for a total of 4 months. Under general anesthesia

210

(ketamine 50 mg/kg and xylazine 20 mg/kg IM), balloon-induced de-
endothelialization of the aorta was performed with a 4F Fogarty arterial
embolectomy catheter (Baxter Healthcare Corp, Irvine, Calif) introduced via a
right femoral artery cutdown. The catheter was advanced in a retrograde fashion
to the ascending aorta, inflated with 1 mL of air, and pulled back to the iliofemoral
artery. This was repeated 3 times; the femoral artery was then ligated, and the

skin incision was sutured. Details of model have been reported previously.” 78

4.8. d. Magnetic Resonance Imaging

Imaging was performed before injection, 15 min, 3 hrs, and 6 hrs after
intravenous injection in the marginal ear vein of either HA-MGNP or the control
Feridex (1.5 mg Fe total, 5 mL total volume). Under general anesthesia
(ketamine 50 mg/kg and xylazine 20 mg/kg IM), the rabbits were held in a supine
position on plastic boards with medical tape to minimize motion artifact and were
placed inside the MRI coil for scanning. MRI rabbit experiments were carried on
a GE 3T Signa® HDx MR scanner (GE Healthcare, Waukesha, WI) with an 8-
channel knee coil. After a 3-plane Iocalizer, we first used a 2D time-of-flight
(TOF) pulse sequence to localize the rabbit aorta with the following parameters:
Axial scan, anterior-posterior frequency direction, flow compensation, flip angle =
60 degree, echo time (TE) = 4.4 ms, time of repetition (TR) = 23 ms , receiver
bandwidth (rBW) = +/- 15.6 kHz, field of view (FOV) = 14 cm, slice thickness = 2
mm, # of slices = 79, acquisition matrix = 256 x 128, number of excitation (NEX)
= 1, and scan time = 4 min 4 sec. Then we acquired high-resolution 30 fast

spoiled gradient recalled echo (FSGR) images to cover the rabbit aorta with the

211

 

center around the kidney and liver regions with the following parameters: Axial
scan, anterior-posterior frequency direction, flip angle = 15 degree, TE = 9.3 ms,
TR = 18.9 ms, rBW = +/- 7.8 kHz, field of view = 12 cm, slice thickness = 1 mm, #
of slices = 90, acquisition matrix = 256 x 256, NEX = 2, and scan time = 14 min
31 sec. T2*-weighted ability of the FSPGR sequence was used to detect the
effect of the nanoparticles before injection, 15 minutes after injection, three hours

and six hours later at the same locations.

4.8. e. Histology and Tissue Staining

All procedures were performed according to the Michigan State University Animal
Care and Use Committee of the Deaconess Hospital. Rabbits were killed by an
overdose of intravenous pentobarbital and potassium chloride. The aorta and
iliofemoral arteries were dissected and excised, and the intimal surface was
exposed by an anterior longitudinal incision of the vessel. Cross-sectional tissue
samples (1 cm in length) were taken from the thoracic aorta, 3 and 6 cm distal to
the aortic valve; from the abdominal aorta, 7 and 4 cm proximal to the iliac
bifurcation; and from the iliofemoral arteries. The samples were fixed, serially
dehydrated in alcohol, and embedded in paraffin. Inverted light microscopy was
performed on tissue sections mounted on glass slides and subsequently scanned
for the presence of iron after standard histochemical staining (Prussian blue iron
staining). Briefly, healthy non-atherosclerotic rabbit artery tissue and
atherosclerotic rabbit artery tissue were incubated with HA-MGNPs or the control
MDNP (1 mg/mL) for 12 hrs. After 12 hrs, iron-oxide labeled tissues were

washed thoroughly, fixed with formalin, incubated for 30 min at 37 °C in the dark

212

 

with Perl’s reagent (2 % potassium ferrocyanide(ll) trihydrate and 3.7 "/0 HCI
solution), and counterstained with nuclear fast red (for making contrast between

blue iron particles and red nucleus).

 

213

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221

 

 

Appendices

Supplementary Materials
A.1. NMR spectra for Chapter 2

1H-NMR (CDCI3, 600 MHz)
AcO 0Ac
O
”28% 0 OH
Ome

H
M4 0

 

 

 

 

 

 

 

 

 

 

13C-NMR (CDCI3, 100 MHz)

 

 

 

 

 

 

Tr‘r‘r-rw—r‘r‘rr—mfir‘r—f' rj‘Y—TrTW—WTYYTT’W’Ymm-r'ymfifrfl-fiwfiwfirvrrtvrthw -Tw—fr - r :1

200 180 160 140 120 100 80 60 40 20 0

222

1H-NMR (CD300, 600 MHz)

HO
wagog if\/\n,OH

 

CYA\’/\N
M5H o

 

 

 

 

 

 

13c-NMR (CD3OD, 100 MHz)

 

 

 

H0 05'
+96% 0
cr”\«”‘NJL~/“jrC"‘
M5H ' c>

 

 

EH! I‘l

 

T v—y 1 v w . WT—v T . , HWTWV’VWW Fr---

26o 160 160 140 120 160 30 so 40 20 o

223

1H-NMR (CD3OD, 600 MHz)

 

HC) (¥§

“Bo

O/\/\ N3

 

 

 

 

 

 

 

1o 9 a 7 6 5 4 3 2 1 o
13C-NMR (c0300, 100 MHz)

9&3,

o/\/\N
M6 3

 

 

 

 

 

MW- , *, ‘_;;JLJ:.l ‘LL;M ”ff: LL“. *1 LL‘;_“‘ *'

I I I I I I I I I ‘I I I I I I I I I I V I l

200 180 160 140 120 100 80 60 40 20 O

 

 

 

224

1H-NMR (CDCI3, 600 MHz)

 

AcO 0Ac O

0 H

OAC O

 

 

 

 

 

 

 

* fi.fi.f¢.7~wv~. ...—.ww—r—cw'van.fi—. fiy—r-F. r~—-r-

10 9 8 7 6 1

- ~—-¢—.—r .vvv-.-—y—- .—¢—'—v . Ofi—v --.—.—v .

I ,

- _ . _L___LL1L__1JIIL». Ilkagm
'W'IVIEHWHIII 3 2 1
13C-NMR (CDCI3, 100 MHz)
AcO 0Ac

O
o H

OAC O

 

 

 

 

 

 

 

 

wf—fl-Y-v—fl—v—rT—vfy—T—y v—v— wr'v—Vfi—v-v—vavyvr-w w—v—v—vwvvvrTvvyfi-y—v—v‘rV-irvv—y-vr‘v—r—ywvy—ry-r—v fvrf'VvY‘ y’V rwf-y ffi‘ vaTfi~yv

2'00 160 160 1210 1730 160 66 6'0 4'0 '2'0

225

 

‘H-NMR (CD3OD, 600 MHz)

 

HO OH
$0 H 0
HO OWNWLOH

OH
G5 0

 

 

 

13C-NMR (00300, 100 MHz)

HO 0”
fig 2 °
HO OH W No”
es 0

 

 

 

I I

, _ _ 1.‘

‘>vT—Y v—r-7—y 1"fi Y '_T y T—Y—y v y y Y -v«r—vAr—v—y—fi—r yo. 7 v y——»-q—7——Y—T~—T—T———va—7Tv71~r—v-T rwfrrw~v~wlrwrv
l . I

200 180 160 140 120 100 80 60 2:0 20 o

 

226

1H-NMR (CDCI3, 600 MHz)

 

2%

OH
Alkyne-Acid (AA)

 

 

 

 

 

Alkyne-Acid (AA)

 

 

 

 

 

2

 

 

 

 

 

 

1H-NMR (CDCI3, 600 MHz)

0 '
O

o
\\

 

 

 

 

 

 

 

 

 

Alkyne-Siloxane (AS)
I
I
I
_ i L _I __ L,“ x - 2 L" .‘\,l\ _ L_J A k‘ a
10 9 8 7 6 5 4 3 2 1 0

13o-NMR (CDCI3, 100 MHz)

0 '
O

o
\\

Alkyne-Siloxane (AS)

 

 

 

 

 

 

 

 

LII. _ .U . II

 

 

I I r

.... .f.,.r. ,
200 180 160 140 120 100 80 60 40 20 0

I I I Y T

228

 

 

H
H2N/\/O\/\O/\/N S
O
Lipoic Amine

 

 

 

 

 

229

 

 

 

 

 

_ .
# L.
_
._ .m
_ .
4 ~
. m
_ ...
u . ,m u L
I LII I r L L. F». r r I}

 

Moo

Hmo

Hmo

woo

HMO

Poo

mo

230

mo so 35

 

 

H 0 H s
OWNNNNOwO/VN s’
o H o

Lipoic Galactose (LGal)

 

 

 

 

 

 

add

231

 

 

 

_ . v

. .

Irllhlu
(I

 

 

 

 

who

MNo

Moo

Hmo

Hoe

who

HMO

mo

NO

232

 

H 0 H ,8
H3C OCR-{W NNNNO¢ONN S
o H o H o
Lipoic F ucose (LFu)

 

 

 

 

 

 

 

 

 

add

233

 

 

 

 

 

 

 

'—
on
0
Pg..
H
m.
o
H
L
o
p
I"
N
o
1
,I
....
O
o
a)
O
,.,,
r
O!
O
5....
a.
0
it?
4F::”“'"‘ ‘
N
o
L-..
I
',§ ..

234

 

 

 

0 H

O\/\/N NNNOwO/V N

H
Llpolc Mannose (LMan)

,S
M
O

 

 

nldd

 

235

 

0811

091

091:

031

001'.

08

09

OZ

add

236

 

 

 

 

 

OH

Wflvownfl

NHAc

Lipoic N-acetyl Glucosamine (LGINAc)

H

H S
N /\/O\/\O/\/ N M
O

 

 

uldd

La.
I

 

I

i

M

II

I

I
I
I
I
I

I‘- m'

I,

I
I

I

if

I

 

‘P‘I‘rfiqfi’l ”W1

115
t

i

V.“ 'W'F‘
n ~

237

 

DWI

 

 

 

 

 

 

”a.“
Mac

Hmo

Hgo

“be

#00

mo

238

 

 

WSEVVOWNNNNOwO/VN S,
O O

 

 

 

OH H
Lipoic Glucose (LGIc)
4
m
u: -- I
I
I

 

 

 

fi
w ..
N .
H

v

 

 

239

 

081:

OST

09'!

0812

0012

08

 

 

 

09
I
I

 

 

0'

 

OZ

 

odd

 

240

 

 

 

 

 

 

 

 

Ho H 0 H ,8
WOWNNNNOwO/VN S
HO H
O O
Llpolc Sialic (LSia)
q
I
cn
U I
I
b i l"; q..-
u q I.
,. I:
. F.;- “““““““““““ “—
I.
I" ..
N { 121‘ '5 .. .-
t”.—
H

 

241

V II t I O I

«#4 I J «11

 

242

A.2. NMR spectra for Chapter 3

 

H3C
OA‘BAc

 

C)
(mAc

Fuc2

MB

f

 

 

0—

 

 

V

 

 

j'w

 

243

NOO

.....

who

who

HMO

HOO

 

so

 

 

v3

244

 

 

H3C
0A8Ac

(DAc

Fuc3

 

 

add

r
I
I
i

 

I

I

I

V‘IT‘IW‘

I

l I
I

 

—_

 

 

 

TWINS“

 

—‘ w

 

245

_ _ ‘- ms». t -.__' ..

 

 

.w
w
n
a
a
m
w-
. ...
_ . L
m M . ..
I. III? by I. .F.IIII Li) I II. F.,»Irilr VLF I» - -i LI IL Lil; Ir-
4 4 .11 II I 11.4 II ‘3 4 1‘

 

IIIIIIILII LII- rfhill'} It I'!‘ 7| ' I? II! III! IE III...
IAIlIlt. 4 1 II t 441111411: (11.11! 1141.1 11114111411 1.1111(11 III 41 4 I. 1 [14 III: 1 ‘I 1 I4 I 4: I"
l ..-. I .III 11.-11].. ‘rI’t‘TIIIlI 1‘ III 11' IIIIIII. 1:0.IOIIIIII1I91 ‘lI'l’I‘IfiAItVII‘r‘Pi‘I arr .QIlltlI III‘IEJI E51,} I’ll" IIIIIIII. 1.1.! It: 114i! .1111!!!)llllll I‘lIi Illiltl»4!.rl..l.l‘.al‘ 1‘11: ll] u... I J I 11 IIILII t: J! I 11] (ll. 9. Illall‘rII! .1 It: II'III

”8 :3 H8 :0 5o poo 8 8 3 No 28.

246

 

 

H

OMN
Fuc4

(JAc

H3C
6A8

 

 

247

 

 

 

 

 

 

 

...-L.

 

 

 

248

 

 

0
OH
0
or?”

Fucose amido-acid (Fuc)

 

 

 

 

 

 

 

 

s)
m
i
I
I
l
I
u- i.‘
I
I
I: _'
a I
I)!“
I
Zia?
.. I
l
I
'1- «6.....-—
., I
I
a ,' Inns—-
)9
I
I“ I»
I
I
I
I
r
O
i
I

249

 

 

 

aaaaaa

.....

NO

 

Us

250

 

 

 

 

 

 

COzMe O
H
Sia4

\
.

ACO 0Ac
AcHN
AcO

AcO

-rn—u-n.. . ................. . .......... .....L. ...,........L L... ..L ,.

 

 

 

 

 

 

251

,,,,,,,,,,,,

- - amp-m.

 

252

 

 

Ho OH cone 0

 

 

 

-‘ OH
HO O
Sialic amido-acid (Sia)

I
.1
G
0| I
I
§
IL?“
{‘ y.
“' I; _-
In,»
I.
.. I...
1‘73:
I.
,. I
I
I
I
6.

 

253

07 09 08 00! DZ! 07'! 09! 08'! 002 02"! 072

OZ

 

 

254

 

 

OH

(IDCH3
+19% N\///
NHAc
GlcNAc 7

 

 

I00

255

 

 

08!

09'!

0"!

02'!

00'!

08

09

0'

OZ

 

 

256

 

 

 

H/

CH3O/
N-Z-propynyl-O-mothoxyamino

 

 

 

4
m
I
9‘ .
r
I
b I 5
F
g.
I
w I
1‘ “v
I
N
p

257

Hwo

“4| .-

 

ma

.0

258

 

TEOS-NPs

 

 

 

 

’-
I
/:
'I
I - I _
A w J: “AA . H A _ 4'12" ’kfi...’ 42....
5' " l I "I I *1 l l‘ I" l"“‘l'""‘

 

 

 

Fuc-MGNP

 

 

I
I" I
,
.i , I l

I , T ’ II I I I , K
E 5 ‘ ; k | h. ' '.' £ :5!
. .' 1 g . ‘ I, I!“ "I 2 I {I i\ '. I". m '51,, I "‘5‘
I“, VMW .‘f/ .‘W V bde—nh/Uw/ hand: v...‘ V. W, ‘ "‘9..'/"\./‘ W

 

y-y"---vflf-Tv'v-‘v--Q v-r-v-f~- v qnv'v rv-v-rY-v~f-7-—y?-?v I'Ay-y f-vAyv-rwwyf‘v 91'. '7 7“? :- Q'v—v u~r-v-v ‘f '—v’ o TV" v v r- v¢ ' ' .-y 0" c

4.5 4 3.5 3 2.5 2 1.5 1 0.5

259

  

HO "
defi 0 H 8.10 "

Man-MGNP

 

 

 

, I
..g 22 2 UIILwIIIImL

H' MW fl V—fi] ”TfirfiT l'—'_'l 'Tff 'T "
8.5 8.0 7.5 7.0 65 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

 

 

 

 

 

 

u-J

 

 

 

 

 

3.5 3.0 2.5 2.0 1.5 1.0 ppm

260

OH OH O

H _/
HO O OWHNMNWS'
OH 0

 

Gal-MGNP

 

 

 

 

   

 

_ 2‘1 WV~WQ:A:M: M + l LJJ
T""I"' l' ' T'H'I'fi'Iher' l ' I" 'fij' Inwm‘

 

 

 

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3. 2.5 2.0 1.5 1.0 0.5 pp

 

 

 

 

 

261

U

 

”IIIIIIIIIIIIIIIIIILIIIIIIIIIIII“