COX-2 RNA INTERFERENCE BY OLIGONUCLEOTIDES ON A

NANOPLASMONIC CARRIER-BASED OPTICAL SWITCH

(ONCOS) IN HEPATOCELLULAR CARCINOMA

By

Uzma Azeem Awan

Regd. No. 2004-Gmdg-3517

Session 2012- 2015

Department of Biotechnology

Faculty of Sciences

University of Azad Jammu and Kashmir Muzaffarabad, Pakistan.

COX-2 RNA INTERFERENCE BY OLIGONUCLEOTIDES ON A

NANOPLASMONIC CARRIER-BASED OPTICAL SWITCH

(ONCOS) IN HEPATOCELLULAR CARCINOMA

By

Uzma Azeem Awan

(Regd. No. 2004-Gmdg-3517)

A Thesis

submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

In

Biotechnology

(Cancer Biology)

Session 2012- 2015

Department of Biotechnology

Faculty of Sciences

University of Azad Jammu and Kashmir Muzaffarabad, Pakistan.

vi

DEDICATION

This humble effort is sincerely dedicated to the people

who have inspired me to work hard no matter the circumstances

my loving & respectable Parents, Sisters, and

Husband

Thank you for your unconditional love and

support.

vii

LIST OF CONTENTS

CONTENTS Page No

List of Tables xii

List of Figures xiii

List of Abbreviations xix

Acknowledgements xxiii

Abstract xxvi

1 INTRODUCTION 1

1.1 Cyclooxygenase 1

1.1.1 Characteristics Structural Feature of Cyclooxygenase 1

1.1.2 Mechanism of Cyclooxygenase Reaction 2

1.1.3 Difference between COX-1 and COX-2 3

1.2 Role of COX-2 in Cancer 5

1.3 Non Steroidal Anti Inflammatory Drugs (NSAIDs) 6

1.4 Hepatocellular Carcinoma and COX-2 6

1.5 Nanomaterials (Optical Switches) 8

1.6 Photo-Triggered Gene Interference 9

1.7 Aims and Objectives 13

1.7.1 Specific Aims And Importance 13

1.7.2 Objectives Of The Study 13

2 REVIEW OF LITERATURE 14

2.1 Cyclooxygenases and Cancer 14

2.1.1 Cyclooxygenases and Hepatocellular Carcinoma 17

viii

2.1.2 Cyclooxygenases and Breast Cancer 18

2.2 Role of Non Steroidal Anti Inflammatory Drugs (NSAIDs) 19

2.3 Nanoparticles and their Biomedical Applications 20

2.4 Nanoparticles for Gene Knockdown 22

2.5 Photothermal Gene Release 22

3 MATERIALS AND METHODS 26

3.1 Materials 26

3.2 Plasmonic Nanoparticle Design 26

3.2.1 Gold Nanospheres Synthesis 26

3.2.2 Gold Nanorods Synthesis 27

3.3 Characterization of Nanomaterials 28

3.4 Nanoparticle Biofunctionalization 28

3.4.1 Conjugation with Poly Ethylene Glycol (PEG) 29

3.5 Effect of various Growth Parameters on the Synthesis of GNRs 29

3.6 Stability Studies of Gold Nanoparticles 29

3.6.1 Stability in various Environmental Conditions 29

3.6.2 Stability in Biological Media Solution and Proteins 30

3.7 Cell Biology 31

3.7.1 Cell Culturing and Sub Culturing 31

3.7.2 Cryogenic Storage of Cells 33

3.8 Cell Viability Assays 33

3.8.1 Mitochondrial Activity (XTT) 33

3.8.2 Haemotoxicity 34

3.8.2.1 Hemolytic Activity of GNRs 34

ix

3.8.2.2 Biocompatibility Assay on PBMCS 35

3.9 Conjugation of COX-2 Oligonucleotides 36

3.9.1 Oligos Reconstitution 36

3.9.2 Conjugation with old Nanospheres 37

3.9.2.1 Hybridization of Sense and Antisense Strands 37

3.9.2.2 Thiol Reduction of Phosphorothioate Protected Strand 37

3.9.2.3 Column Purification of Activated Strand 37

3.9.2.4 Conjugation with Particles 38

3.10 Characterization of Bioconjugate 38

3.10.1 UV-Vis Spectrophotometric Measurements 38

3.10.2 Fluorescence Measurements 38

3.10.3 Nanoparticle Dissolution with KCN Method 40

3.11 Conjugation with Gold Nanorods 40

3.11.1Characterization of Bioconjugate 41

3.12 Intracellular Localization and Uptake Studies 41

3.12.1 Live Cell Dark Field Imaging 41

3.12.2 Spectrophotometric Measurement of Relative Uptake 42

3.12.3 Cellular Uptake by Confocal Microscopy 42

3.13 Characterization of Immobilized Antisense Release from GNRs 43

3.13.1 Laser Power, Wavelength, Temperature and Exposure Time 43

3.14 COX-2 RNA Interference using Antisense Oligonucleotides 44

3.14.1 Transfection Experiments 44

3.15 Protein Expression Analysis 45

3.15.1 Protein Extraction 45

x

3.15.2 Protein Quantification 46

3.15.3 Protein Separation 47

3.15.3.1 Sample Preparation for Loading 47

3.15.3.2 SDS-PAGE 44

3.15.4 Coomassie Blue Staining 49

3.15.5 Reversible Protein Detection Method 49

3.15.6 Western Blotting 50

3.15.6.1 Densitometric Analysis 52

3.16 RNA Interference by ONCOS 53

3.17 Measurement of Prostaglandin-E2 Production Rate by ELISA 53

3.18 Cell Proliferation Assay 55

3.18.1 Apoptosis/necrosis by Flow Cytometry 55

4 RESULTS AND DISCUSSION 57

4.1 Plasmonic Nanoparticle Design 57

4.2 Effect of Various Growth Parameters on the Synthesis of GNRs 58

4.2.1 Effect of Seed Aging and Concentration 58

4.2.2 Effect of Silver Ion Concentration 60

4.2.3 pH Controlled Synthesis of GNRs 62

4.3 Stability of GNRs 65

4.3.1 Effect of Environmental Conditions 65

4.3.2 GNRs Stability at different pH 67

4.3.3 Effect of Temperature on GNRs Stability 69

4.3.4 Effect of Ionic Concentration GNRs Stability 70

4.3.5 Stability of GNRs in Cellular Media Solutions 71

xi

4.4 Cytotoxicity Assay 72

4.5 Hemolytic Assay 75

4.5.1 Cell Viability of PBMCS 76

4.6 Nanoparticles for Gene Knockdown Experiment 77

4.6.1 Gold Nanospheres 77

4.6.2 Gold Nanorods 79

4.7 Conjugation of COX-2 oligonucleotides 80

4.7.1 Characterization of Bioconjugates 80

4.8 Intracellular Localization and Uptake Studies 83

4.8.1 Live Cell Dark Field Imaging 83

4.8.2 Spectrophotometric Measurement of Relative Uptake 87

4.8.3 Cellular Uptake by Confocal Microscopy 90

4.9 Laser Power, Wavelength, Temperature and Exposure Time 92

4.10 COX-2 Interference using Antisense Oligonucleotides 93

4.11 Protein Expression Analysis 94

4.11.1 Protein Extraction and Quantification 94

4.11.2 Protein Separation 100

4.11.3 Western Blotting 101

4.12 COX-2 RNA Interference using ONCOS 103

4.13 Estimation of Prostaglandin-E2 Production Rate by ELISA 109

4.14 Apoptosis/necrosis assay on flow cytometry 110

SUMMARY 113

LITERATURE CITED 115

APPENDICES 139

xii

LIST OF TABLES

Table No. Title Page

3.1 Different concentrations of fluorescence labeled single stranded

antisense.

39

3.2 BSA standard solution preparation. 47

3.3 Preparation of different dilution of cell lysate solution. 47

3.4 Buffers Recipes for Western blot. 50

3.5 Primary and Secondary Antibody Dilutions. 52

3.6 Preparation of different concentrations of PGE2 Standard for ELISA. 55

3.7 PGE2 plate setup for ELISA. 55

4.1 Effect of Silver ions on GNRs aspect ratio 61

4.2 Protein concentrations of cell lysate after transfection with

nanocomposite (DNA conjugated gold nanospheres).

96

4.3 Protein concentrations of cell lysate after transfection with

nanocomposite (single and double stranded DNA conjugated gold

nanorods.

97

4.4 Protein concentrations of cell lysate after transfection with DNA

(single stranded, double stranded and control DNA).

98

4.5 Protein concentrations of cell lysate after transfection with

nanocomposite (DNA conjugated gold nanorods) followed by NIR

laser illumination.

99

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LIST OF FIGURES

Figure No. Title Page

1.1 Structure of two isoforms of COX presenting Isoleucine to valine

substitution responsible for hydrophobic side packet in COX-2

relative to COX-1.

02

1.2 The cyclooxygenase (COX-1 and COX 2) molecular pathway.

Prostaglandins (PG) are formed by specific isomerases from the

COX product PGH2.

03

1.3 Production of prostanoids through the COX induced metabolism

of arachidonic acid and their conversion to further metabolites by

specific synthases.

05

1.4 The COX-2/PGE2 pathway: key roles in the hallmarks of cancer. 06

1.5 Mechanism RNA interference by ONCOS. 12

1.6 Schematic diagram of optical control switch gene interference 12

3.1 Schematic diagram of GNRs synthesis 27

3.2 SDS PAGE Gel apparatus Assembly. 49

3.3 Western blot Cassette assembly diagram. 51

4.1 Characterization of synthesized GNRs. UV-Vis absorption

spectra of GNRs from shorter (682 nm) to longer (906 nm) LSPR.

TEM images show mono-disperse population with high yield.

58

4.2 UV-Vis absorption spectra and graph of gold nanorods prepared

with different concentrations of seed solution.

60

4.3 UV-Vis absorption spectra of gold nanorods prepared with

different concentrations silver ions.

61

4.4 UV-Vis absorption spectra of gold nanorods prepared at different

pH shows successful rod formation at slightly acidic and neutral

pH while no rod synthesis was observed at basic pH.

63

4.5 UV-Vis absorption spectra of CTAB coated gold nanorods and

PEG coated GNRs. Graph showing Zeta potential of CTAB

coated and PEG coated GNRs.

65

4.6 Stability studies of GNRs (a) PIP versus number of washes of 66

xiv

GNRs. (b) PIP versus time of GNRs kept in 0.001 M CTAB. (c)

UV-Vis absorption spectra of gold nanorods kept in 0.001 M

CTAB solution shows stability of GNRs upto 3 months.

4.7 Stability of GNRs with respect to pH. (A) UV–Vis spectra of

synthesized GNRs after 2 h at room temperature; (B) after 1

month at room temperature. Loss of Gaussian shape at pH 8-14 is

visible;(C) PIP values plot of GNRs stability after 2 h and 1

month shows particle degradation at pH 10-14 (PIP > 0.1) after 2

h. GNRs remain stable in acidic condition for 1 month (PIP <

0.1).

68

4.8 Plot of LSPR absorption peak wavelength in response to

temperature. Decrease in LSPR is observed when temperature is

raised above 60°C.Statistical analysis shows no significance

difference in LSPR when heated from 28 to 60°C for 2 min

(p<0.05). B). PIP versus temperature plot show less aggregation

of GNRs upto 60°C.

69

4.9 Stability of GNRs at different ionic concentrations. A) Plot of PIP

versus concentrations of NaCl shows stability at 5 to 10 mM and

100 to 150 mM. Aggregation of GNRs is observed between 10 to

100 mM and salt concentrations (PIP > 0.1); B).UV–Vis

absorption spectra of GNRs in response to different salt

concentrations at room temperature.

70

4.10 PIP values for CTAB coated and PEG coated GNRs incubated

with different cell culture media protein solutions. PEG coated

GNRs show higher stability in biological environment.

72

4.11 Dose-dependent viability of cells exposed to increasing

concentrations of CTAB-GNRs and PEG-GNRs (a) MCF-7 cells

(b) RD cells (c) MCF12-F cells. (d) Viability of HeLa cells after

laser irradiation for 2, 4, 6, 8 and 10. 10 min exposure.

74

4.12 Percentage hemolysis of CTAB coated and PEG coated GNRs on

whole blood. PEG coated GNRs show comparatively low

hemolysis in dose dependent fashion.

76

xv

4.13 Percentage Viability of CTAB coated and PEG coated GNRs on

PBMCs. PEG coated GNRs show comparatively low cytotoxicity

in dose dependent fashion.

77

4.14 Gold nanoparticles characterization (a) UV-Vis spectrum of GNS

with specific SPR of 530 nm (b) TEM of GNS shows

homogenous population.

78

4.15 Gold nanoparticles characterization (a) UV-Vis spectrum of

GNRs with specific LSPR of 785 nm (b) TEM of GNRs shows

homogenous population.

79

4.16 Positively charged CTAB coated GNRs (Blue color) with

negatively charged FAM labeled 21 mer DNA oligos (Green

color).

80

4.17 UV-Vis Spectra before and after FAM labeled DNA conjugated

gold nanospheres (GNS).

82

4.18 UV-Vis Spectra before and after FAM labeled DNA conjugated

gold nanorods (GNRs).

82

4.19 Live cell dark field imaging of HepG2 cells acquired by IX70

microscope using halogen lamp (a) untreated cells (b) cells with

nanoparticles (c) cells incubated with FAM labeled ssDNA

conjugated with GNRs (d) cells incubated with FAM labeled

dsDNA conjugated with GNRs.

85

4.20 Live cell dark field imaging of MDA-MB-231 cells acquired by

IX70 microscope using halogen lamp (a) untreated cells (b) cells

with nanoparticles (c) cells incubated with FAM labeled ssDNA

conjugated with GNS (d) cells incubated with FAM labeled

dsDNA conjugated with GNS.

86

4.21 MDA-MB-231 cells were incubated with DNA conjugated GNS

and imaged using inverted microscope with Nikon D200 digital

camera to observe cellular uptake (a) cells as a control sample (b)

cells with transfection agent and GNS (c) cells with dsDNA

conjugated GNS (d) cells with dsDNA conjugated GNS.

87

4.22 Cellular uptake measurements (a) UV-Vis spectrum of MDA- 88

xvi

MB-231cells supernatant after 24 h of incubation with

nanoparticles treatment solutions (b) percentage cellular uptake of

DNA conjugated and non-conjugated gold nanoparticles.

4.23 MDA-MB-231 cells were incubated with DNA conjugated GNRs

and imaged using inverted microscope with Nikon D200 digital

camera to observe cellular uptake (a) cells as a control sample (b)

cells with transfection agent and GNRs (c) cells with dsDNA

conjugated GNRs (d) cells with dsDNA conjugated GNRs.

89

4.24 Cellular uptake measurements (a) UV-Vis spectrum of cell

supernatants after 24 h of incubation with nanoparticles treatment

solutions (b) percentage cellular uptake of DNA conjugated and

non conjugated gold nanoparticles shows relatively increased

uptake with DNA conjugated GNRs.

90

4.25 Confocal images with 488 nm (FITC) excitation sources

presenting nanocomposite cellular uptake in HepG2 cells

91

4.26 Experimental characterization of ONCOS activation (a) FAM

labeled DNA conjugated GNRs carriers based on wavelength

specificity, a laser at the peak optical absorption (808 nm) was

compared to a laser outside the peak optical absorption (658 nm)

for photothermal oligos release. (b) Normalized fluorescence

versus time plots comparing fluorescence intensity with

increasing power densities of laser.

93

4.27 Corrected OD values at 595 nm were plotted against known BSA

standard concentrations. (a) Linear standard curve of known

concentrations of BSA. (b) Standards and samples in 96 well

plate presenting change in color depending upon protein

concentration.

96

4.28 Corrected OD values at 595 nm were plotted against known BSA

standard concentrations. (a) Linear standard curve of known

concentrations of BSA. (b) Standards and samples in 96 well

plate presenting change in color depending upon protein

concentration.

97

xvii

4.29 Corrected OD values at 595 nm were plotted against known BSA

standard concentrations. (a) Linear standard curve of known

concentrations of BSA. (b) Standards and samples in 96 well

plate presenting change in color depending upon protein

concentration.

98

4.30 Corrected OD values at 595 nm were plotted against known BSA

standard concentrations (a) Linear standard curve of known

concentrations of BSA. (b) Standards and samples in 96 well

plate presenting change in color depending upon protein

concentration.

99

4.31 Example of coomassie stained gel. (a) lane 1 contain molecular

weight standard, Kaleidoscope, lane 2 and 3 has the extracted

protein from untreated HepG2 cells, lane 4 and 4 has the

extracted protein from untreated MDA-MB-231 cells. (b)

molecular weight standard, Kaleidoscope (c) lane 1 contain

molecular weight standard, Kaleidoscope, lane 2 has untreated

cells lane 3 cells containing gold nanoparticles with laser

exposure, lane 4 cells with ssDNA conjugated gold nanoparticles

with laser exposure, lane 5 cells with dsDNA conjugated gold

nanoparticles illuminated with laser, lane 6 contain COX-2

positive lysate.

100

4.32 Western blot analysis and relative COX-2 protein density after 48

and 72 h of transfection.

103

4.33 Western blot analyses of MDA-MB-231 cells exposed to

treatment solutions of GNRs and DNA for photothermal COX-2

gene interference. (a) Relative protein density. (b) Relative

protein density after 72 h of transfection with nanocomposite and

DNA alone without laser exposure.

105

4.34 Western blot analyses of HepG2 cells exposed to treatment

solutions of GNRs and DNA for photothermal COX-2 gene

interference. (a) Relative protein density. (b) Relative protein

density after 72 h of transfection with nanocomposite and DNA

107

xviii

alone without laser exposure.

4.35 HepG2 cells were treated with the DNA conjugated

nanocomposite for 72 h and PGE2 levels in the culture medium

were measured using ELISA. (a) Cells were not exposed to laser

(b) cells were exposed to NIR (808 nm) laser after 12 h of

nanocomposite treatment.

110

4.36 Apoptosis/necrosis analysis after COX-2 knockdown on flow

cytometry.

112

xix

LIST OF ABRIVATIONS

µL Microliter

µM Micromole

5-FU 5-Fluorouracil

AgNO3 Silver Nitrate

Ag Silver

AIF Apoptosis Inducing Factor

ATP Adenosine Triphosphate

Au Gold

BSA Bovine Serum Albumin

bp Base Pair

cAMP Cyclic Adenosine Monophosphate

CCD Charge-Coupled Device

cDMEM Complete Dulbecco’s Modification of Eagle’s Medium

CTAB Cetyl Trimethylammonium Bromide

CRC Colorectal Carcinoma

CW Continuous Wave

CMC Critical Micelle Concentration

COX Cyclooxygenase

DAG Diacylglycerol

DNA Deoxyribonucleic Acid

DI Deionized

DTT Dithiothreitol

DMEM Dulbecco’s Modification of Eagle’s Medium

DMSO Dimethyl Sulfoxide

DLS Dynamic Light Scattering

xx

EDTA Ethylene Diamine Tetra Acetic Acid

EGFR Epidermal Growth Factor Receptor

EGF Enhanced Green Fluorescence

ELISA Enzyme-Linked Immunosorbent Assays

EPR Enhanced Permeation And Retention

FBS Fetal Bovine Serum

FITC Fluorescein Isothiocyanate

FGF Fibroblast Growth Factor

fs Femtosecond

GNPs Gold Nanoparticles

GNRs Gold Nanorods

GNS Gold Nanospheres

HBV Hepatitis B Virus

HCV Hepatitis C Virus

HCC Hepatocellular Carcinoma

HDAC Histone Deacetylases

HSA Human Serum Albumin

IgG Immunoglobulin G

IL-11 Interleukin-11

IP3 Inositol Trisphosphate

Fe Iron

LSPR Longitudinal Surface Plasmon Resonance

MAPK Mitogen-Activated Protein Kinase

MBD Membrane Binding Domain

MMP Matrix Metalloproteinases

mg Milligram

xxi

mL Milliliter

mM milli Mole

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium

Bromide)

NaCl Sodium Chloride

NaBH4 sodium Borohydride

NPs Nanoparticles

NIH National Institute of Health

NIR Near Infrared

NE Neuroendocrine

NO Nitric Oxide

nm Nanometer

nM Nano Mole

NSAIDS Non-Steroidal Anti-Inflammatory Drugs

OD Optical Density

ONCOS Oligonucleotides on Nanoplasmonic Carrier Based

Optical Switch

PEG Poly(Ethylene Glycol)

PI Propidium Iodide

PMSF Phenylmethylsulfonyl Fluoride

PTT Photothermal Therapy

PIP Phosphatidylinositol 4,5-Bisphosphate

PI3K Phosphoinositide 3-Kinase

PKC Protein Kinase C

PEI Polyethylenimine

KCN Potassium Cyanide

PTGS Prostaglandin- Endoperoxide Synthase

xxii

PG Prostaglandins

ROS Reactive Oxygen Species

RNAi RNA Interference

rpm Revolutions Per Minute

SD Standard Deviation

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel

Electrophoresis

SERS Surface Enhanced Raman Scattering

siRNA Small Interfering RNA

SPR Surface Plasmon Resonance

ssDNA Single-Stranded DNA

TBS Tris-Buffered Saline

TBST Tris-Buffered Saline and Tween

TEM Transmission Electron Microscope/Microscopy

TEMED Tetramethylethylenediamine

TGFβ1 Transforming Growth Factor-beta 1

TNF-α Tumor Necrosis Factor Alpha

TSPR Transverse Surface Plasmon Resonance

TXA Thromboxanes

UV-Vis Ultraviolet-Visible

VEGF Vascular Endothelial Growth Factor

XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2h-

Tetrazolium-5-Carboxanilide)

xxiii

ACKNOWLEDGEMENTS

I am ever grateful to Almighty ALLAH, to whom I owe my very existence,

who loves to explore His creation, who gave me the power to believe in my passion

and pursue my dreams by accomplishing this work. All respect and countless

Darood-o-Salam to the Holy Prophet Hazrat Mohammad (P.B.U.H), who enabled

us to recognize our Creator.

The work in this dissertation is the culmination of years of not only hard work

but also much personal and professional growth. I am indebted to my supervisor Dr.

Shaukat Ali Assistant professor, Department of Zoology for his patience,

motivation, consistent availability and scientific input during every step of my work.

Thank you for the advice, support, and willingness that allowed me to pursue

research on topics for which I am truly passionate. I would like to thank my Co-

supervisor Dr. Abida Raza, Principal Scientist, National Institute of Lasers and

Optronics (NILOP), Pakistan Atomic Energy Commission, who widened my horizon

for science and gave me the opportunity to exploit the exciting area of

nanotechnology. I am thankful to her for starting me on the path to this project and

for her immeasurable support, idea oriented discussion and providing me all the

necessary infrastructure and technical facilities for research at NORI.

I feel great pleasure in expressing my deep gratitude to my foreign

advisor Prof. Dr. Mostafa A. El-Sayed, Julius Brown Chair and Regents Professor,

Director Laser Dynamics Laboratory, School of Chemistry and Biochemistry,

Georgia Institute of Technology, USA. I highly appreciate that he gave me the

valuable opportunity to join Georgia Tech for my PH. D research. I learned a lot

from him. He always encourages me. I am really thankful to him for his unwavering

xxiv

support enthusiasm and funding for my scientific endeavors at Georgia Tech. Thank

you Dr. Sayed, your dedication to science and mentoring has been truly

inspirational. I would especially like to express my sincere gratitude to my foreign

co-advisor Dr. Steven Hira, Georgia Tech, USA who has provided me full time

tremendous support, continuous guidance and amazing scientific discussion

throughout the year. I appreciate his friendly mentorship and invaluable insights and

advice for my research. Thanks Dr. Steve for always motivating me and believing in

me to prove myself to be the scientist as I am today.

I feel great pleasure in expressing my deep gratitude to Dr. Basharat Ahmad,

Chairman, Department of Biotechnology. I would like to extend special thanks to M.

Phil advisor Dr. Saiqa Andleeb for always being so supportive. I would like to

thanks Dr. Ghazanfar Ali, Assistant Professor and all Biotechnology Department.

Special thanks to all the faculty members of Zoology Department, Dr. Nuzhat Shafi

Chairperson, Prof. Dr. Muhammad Nasim Khan, Dr. Muhammad Siddique

Awan (Associate Professor), and Dr. Abdul Rauf. I feel delighted in expressing my

sense of thankfulness to all the staff of Department of Biotechnology and Zoology.

I am particularly indebted to Higher Education Commission (HEC),

Pakistan, for awarding me IRSIP scholarship and enabled me to get wonderful

research experience in world renowned Lab. I am eternally grateful to Georgia

Institute of Technology, USA for accepting me as a research scholar and providing

all facilities and funding to make my Ph.D. experience productive and stimulating. I

am thankful to Pakistan Science Foundation (PSF) for funding. Next, I would like

to convey my appreciation to my all research collaborator from Pakistan and USA. I

owe many thanks to friends and colleagues from UAJK, NORI, NILOP and the ones

xxv

in Georgia Tech for their encouragement and support especially at times when things

were going tough.

Lastly, but certainly not least, I would like to thank my family, my parents

and my sisters, you have never once stopped believing in me, even when I didn’t

believe in myself. You have sacrificed so much to help me succeed and your

unconditional love and support are the reasons I have accomplished my goals. I hope

I have made you proud and continue to do so.

I would like to acknowledge everyone that helped me during my journey of

pursuing the PhD degree. Thank you to everyone that gave me help, support, and

guidance along the way. Without your kindness, I could hardly finish all the work in

this dissertation.

Uzma Azeem Awan

xxvi

ABSTRACT

In this dissertation, nanoplasmonic optical antennae (gold nano particles) are

utilized as nanoplasmonic gene switches for on-demand and systematic gene

regulation in living systems. The plasmon resonance of nanoplasmonic gene

switches (gold nanorods) is specifically tuned to the near infrared spectral region

where cells and tissues are essentially transparent. Cyclooxygenase-2 (COX-2) is an

immediate early response gene in various cancers and is one of the most critical

enzymes in tumor metastasis. COX-2 non-selective inhibition as well as inhibition of

the constitutive isozyme COX-1 by selective and non selective Non-steroidal anti-

inflammatory drugs (NSAIDs), causes several adverse effects like renal, cardiac and

gastrointestinal toxicity. Therefore COX-2 inhibition should be localized and very

specific without inhibiting COX-1. Conventional gene-interfering techniques lack

spatial and temporal control.

This study employed RNAi to specifically knockdown endogenous COX-2

expression in the HepG2 (hepatocellular carcinoma) and MDA-MB-231 (breast

cancer) cell lines using nano plasmonic optical switches and NIR laser.

Nanoparticles, gold nanorods (GNRs) are successfully prepared with tunable

longitudinal plasmon resonance (LSPR) wavelengths between 682-906 nm. The

hydrodynamic diameter of the GNRs was estimated to be 20.90±2.15 to 50.5±2.13

nm. The zeta potential of the prepared GNRs was measured as +33.3± 0.34 -

40.1±0.71 mV. We tailored the aspect ratios of GNRs between 2.5 to 4.6 by

optimizing growth conditions that include concentrations of reactants continuous

stirring at acidic pH. Fine tuning of LSPR is achieved across a broad range by

varying silver ion and seed concentration. Colloidal stability of GNRs was studied

through UV-vis spectroscopy based particle instability parameter (PIP < 0.1). GNRs

xxvii

are stable at 28-60°C; however, prolonged high temperature (>60°C) and alkaline

pH can trigger colloidal instability. GNRs remain stable at higher salt concentration,

physiological and slightly acidic pH. GNRs can be stored in 0.001 M CTAB for 03

months without compromising their stability. PEG-GNRs are quite stable in cellular

media solution (PIP < 0.1). In vitro toxicological assays of CTAB-GNRs and PEG-

GNRs solutions with (human breast adenocarcinoma cell (MCF-7), human

Rhabdomyosarcoma cell line (RD) and non-tumorigenic epithelial mammary gland

cell line (MCF12-F) had confirmed that the toxicity is caused by free CTAB in

solution. PEGylation of GNRs substantially had reduced the cellular toxicity as

higher survival rates were observed in both normal and cancerous cell after

incubation with PEG-GNRs solution.

Gold nanorods, with aspect ratio 3.4 (λmax = 785 nm) and gold nanospheres

(GNS), with diameters of ~30 nm (λmax = 530 nm) were selected for gene

knockdown. Fluorescence labeled thiol reduced 21 mer DNA oligos directed to the

5′ end of COX-2 mRNA were conjugated with GNS and GNRs. UV-Vis

spectroscopy calculations reveal 225 DNA Strands per particle in case of GNS and

385 DNA Strands per particle in case of GNRs. The flow cytometric based

biocompatibility analysis for conjugated optical switches revealed neither significant

(p<0.05) level of apoptotic cells nor necrotic (approximately 95% healthy

population). The percentage cellular uptake of GNS with transfection agent is about

38% whereas, with ssDNA conjugated GNS the uptake increased to 56%, and it

increased more to 67% when dsDNA conjugated GNS were used. The percentage

cellular uptake of GNRs with transfection agent is about 26% whereas, with ssDNA

conjugated GNRs the uptake increased to 50.3%, and it increased more to 60.1%

xxviii

when dsDNA conjugated GNRs were used. Dark field microscopy and confocal

microscopy results for cellular uptake are consistent with UV-Vis spectroscopy.

After conventional RNAi technique using nanocarriers the COX-2 protein

expression level was reduced by 38% by gold nanospheres conjugated with COX-2

antisense oligos compared to their untreated counterpart after 48 h in MDA-MB-

231cells. Comparatively, the silencing efficacy of conjugated nanocarriers after 72 h

was significantly (p<0.05) increased to 78%. Average COX-2 protein level was

reduced with silencing efficacy of 98% after ONCOS activation in MDA-MB-231

cells. The silencing efficacy after ONCOS activation in HepG2 cells is 96% compare

to untreated cells. The decrease in protein level in the ONCOS-activated cells versus

the control sample indicates that ONCOS has effectively interfered with COX-2

protein expression. We may conclude optical switches can be conjugated with oligos

which can be released by remote optical extinction at desired intracellular location

ad at specific time to block the expression of target gene. We demonstrate a novel

gene-interfering technique presenting spatial and temporal control.

1

Chapter 1

INTRODUCTION

1.1 CYCLOOXYGENASES

Cyclooxygenases (COX), prostaglandin- endoperoxide synthase (PTGS) is the

enzyme that convert arachidonic acid (a polyunsaturated fatty acid, main precursor of

phospholipids domain of cell membranes) to prostaglandin H2, and related

compounds collectively known as eicosanoids by the peroxidase activity (Funk,

2001). Which then converted into a variety of Prostanoids including prostaglandins

(PGE2, PGF2a, PGD2, etc.), prostacyclin (PGI2) and thromboxanes (TXA2) by

specific prostaglandin and thromboxane synthases (Herschman et al., 2003;

Chandrasekharan and Simmons, 2004). These regulatory compounds modulate a

number of complex biological processes which trigger development and progression

of various types of tumors (Wang and DuBois, 2010). Two isoforms of enzyme,

COX-1, prostaglandin- endoperoxide synthase 1 (PTGS1) and COX-2 contain single

peptide of varying lengths (Kujubu et al., 1991).

1.1.1 Characteristics Structural Feature of Cyclooxygenase

Gene encoding COX-1 is present on the short arm of chromosome 9q32

-q33.2

and

COX-2 is present on chromosome 1q25.2-25.3

. Gene size for COX-1 is about 22 kb with

11 exons and 10 introns while COX-2 gene is about 8 kb having 10 exons and 9

introns. The length of primary mRNA transcript for COX-1 is 2.8 kb (length of

coding region includes 1,797 nucleotides) and for COX-2 is 4.5 kb (length of coding

region includes 1,812 nucleotides) (Smith et al., 2000; Tanabe and Tohnai, 2002;

Simmons et al., 2004). The COX-1 isoform contains 577 amino acids whereas COX-2

contains 581 amino acids. Two COX genes share 60-65% sequence identity at the

amino acid level having nearly super-imposable three dimensional structure (Hla and

2

Neilson, 1992). The isoforms of COX are membrane-bound, endoplasmic reticulum-

resident. (Rouzer and Marnett, 2003).

1.1.2 Mechanism of Cyclooxygenase Reaction

The role in COX conversion to PGG2 is to detain the choice for hydrogen (H)

abstraction and as a result dictate reaction stereochemistry. The COX dependent

catalysis initially requires its activation by peroxidase activity.

The EGF-like domain of the COX enzymes act as a dimerization domain, it

holds the monomers together via salt bridges, hydrophobic interactions and hydrogen

bonding. The membrane-binding domain incorporates into the lipid bilayer on the

luminal side of the endoplasmic reticulum (ER) and the nuclear envelope (Simmons et

al., 2004). The globular catalytic domain comprises the COX and the peroxidase

active sites(Marnett, 2000; Smith et al., 2000; Simmons et al., 2004).

Figure 1.1: Structure of two isoforms of COX presenting Isoleucine to valine

substitution responsible for hydrophobic side packet in COX-2 relative to COX-1.

3

1.1.3 Difference between COX-1 and COX-2

Structural and biochemical studies showed the main sequence dissimilarity

between two isoforms of COX happen in the membrane binding domains. A

distinctive feature of COX-2 is the insertion of six residues at the C-terminus of

amino acid 18 that is not found in COX-1. This insert marked COX-2 for proteolysis

consequently, the rapid cellular degradation of COX-2 protein occurs in conditions

where COX-1 protein preferentially remains stable (Spencer et al., 1999; Mbonye et

al., 2006). The other difference is that the COX-2 owes comparatively increased

volume of active site due to Substitution of isoleucine to valine(Kozak et al., 2002;

Warner and Mitchell, 2004). Expression profiles of both isoforms markedly different

regardless of their close resemblance in sub-cellular localization, structure and

catalytic function (Kang et al., 2007). The difference could be attributed to the

Figure 1.2: Molecular pathways of COX-1 and COX-2. Formation of

Prostaglandins by specific isomerases from the COX product PGH2 by

activation of transmembrane receptors (Fries and Grosser, 2005).

4

variability in the regulation of these isomers at the transcriptional as well as post-

transcriptional levels. Because of constitutive expression COX-1enzyme is

responsible for numerous physiological functions like cytoprotection of stomach,

production of proaggregatory prostanoid and vasodilatation in kidneys (Tanabe and

Tohnai, 2002).

1.2 ROLE OF COX-2 IN CANCER

Numerous in-vitro findings have showed the association of prostaglandins in

the pathogenesis of cancer. These oncogenes-induced enhanced prostanoids synthesis

via COX-2 pathway has been reported in different human malignancies including

those of the head and neck, skin, gastric, lung, liver, pancreas, prostate, breast,

bladder, renal and colon (Dubois et al., 1998; Thun et al., 2002) and the elevated

COX-2 expression is highly associated with overall decreased survival rate among

these patients (De Groot et al., 2007).

5

Several evidences indicate that COX-2 is one of the most critical enzymes in

tumor metastasis (Singh et al., 2005; Peddareddigari et al., 2010). High level of COX-

2 modulate apoptosis, increase angiogenesis (Chu et al., 2003; Zhao et al., 2007)and

suppress immune response (Casado et al., 2007; Greenhough et al., 2009). Results of

earlier studies demonstrated high COX-2 level in colorectal carcinoma tissues such as

in stromal cells, inflammatory cells and epithelial cells. On the other side adjacent

normal tissues express low or basal level of COX-2 .Transcriptional activation of

COX-2 occur early during tumorigenesis (Wu et al., 2010).

Figure 1.3: Prostanoids Production via COX induced metabolism of AA and

consequently their further conversion to respective metabolites by specific

synthases. PGE2 binds to the G-protein coupled receptors EP1, EP2, EP3 and

EP4. These receptors activate the downstream signaling pathways (ERK, STAT

and JNK) and also increase intracellular Ca2+

by altering cAMP levels (Lowry et

al., 2014).

6

1.3 NON-STEROIDAL ANTI-INFLAMMATORY DRUGS (NSAIDS)

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely being used as

COX inhibitors for pain, inflammation, fever and as anti- cancerous agents. COX-2

inhibition leads to restricted angiogenesis and helps in down-regulating the production

of angiogenic factors, like vascular epithelial growth factor (VEGF) and basic

fibroblast growth factor (FGF-2) and decrease the production of PGs (Masferrer et al.,

2000). COX non-selective NSAIDs inhibit COX-1 and COX-2. Many issues

including increased risk of renal failure, heart attack, thrombosis are involved with the

use of COX-2 inhibitors (Meek et al., 2010).

1.4 HEPATOCELLULAR CARCINOMA AND COX-2

Hepatocellular carcinoma (HCC) accounts for 90% of all liver cancers

(Verslype et al., 2012).The HCC is the fifth most prevalent cancers in the world, and

Figure 1.4: The COX-2/PGE2 pathway: key roles in the hallmarks of cancer

(Greenhough et al., 2009).

Hallmarks of Cancer

Evading ApoptosisTissue Invasion and

MetastasisSelf-Sufficiency in

Growth SignalsSustained

Angiogenesis

7

is the third leading cause of cancer-related mortality worldwide (Livraghi et al., 2011;

Yeh et al., 2011). It accounts for approximately 6% of all human cancers and up to 1

million deaths annually (Wu, 2006; Breinig et al., 2007). The incidence of HCC is

highest in under developed countries, this is largely due to the distribution and the

history of HBV and HCV (Bosch et al., 2004). The cell(s) of origin are believed to be

the hepatic stem cells and the tumors progress with local expansion, intrahepatic

spread, and distant metastases (Alison, 2005). Persistent hepatitis B virus infection

and specifically its X gene expression (HBx), hepatitis C virus core protein, alcohol

and exposure to aflatoxins B1 are clearly associated with the development of HCC

(Domínguez-Malagón and Gaytan-Graham, 2001). Inhibition of COX-2

(pharmacological or genetic deletion) suppresses hepatic tumorigenesis (Steinbach et

al., 2000).

High level COX-2 expression has been reported in hepatic carcinoma cells

compare to healthy hepatocytes suggesting a possible role for COX-2 in the initiation

or promotion of hepatic carcinogenesis (Enomoto et al., 2000; Mathurin et al., 2000).

Increased expression of iNOS catalysts, nitric oxide (NO) is an important regulator of

COX-2 in HCV positive HCC (Pérez-Sala and Lamas, 2001; Sasahara et al., 2002). A

HuR protein, an important mRNA stability factor bind to the AU rich element of

COX-2, extends the half-life of COX-2-mRNA eventually leading to the high COX-2

expression level in hepatoma cell lines (Sheflin et al., 2001). Cytochrome P450 and

activation of NF-kβ also regulate COX-2 expression in liver cells (De Lédinghen et

al., 2002).

COX-2 promotes angiogenesis through COX-2 induced VEGF in HCC

(Carmehet and DorY, 1998). COX-2 expression can modulate apoptotic pathways by

regulating Bcl-2 expression and P53 inactivation in cancer tissues (Lin et al., 2001).

8

COX-2 also influences cell cycle regulation via inhibiting cycline dependent kinase

inhibitor (Hung et al., 2000). Highly expressing COX-2 can induce an increased

production of reactive oxygen species (ROS) causing oxidative DNA damage and

favors the development of HCC. COX-2 expression may control the extra cellular

environment by inhibiting the level of E- Cadherin which is mostly under-expressed

in HCC (Okuda et al., 2002).

1.5 NANOMATERIALS (OPTICAL SWITCHES)

Nanotechnology has a valuable effect on various fields especially in

biomedical. Nanoparticles (NPs) with dimensions between 1-100 nm are being used

in different biomedical applications (Murthy, 2007; Dreaden et al., 2012). Due to

their smaller size, they can easily penetrate cell membrane and consequently, can

effectively attract with DNA, Proteins etc. In addition, they show numerous unique

properties compare to their bulk counterparts. Various shapes of gold nanoparticles

(GNPs) (Alkilany and Murphy, 2010; Huang et al., 2010; Tiwari et al., 2011) have

been researched extensively in the recent year, with emerging applications in

diagnostics and therapeutics (Chen, 2013; Zheng, 2015). Metallic nanoparticles, like

gold (Au), being inert and relatively less cytotoxic exhibits distinguishing properties

owing to their unique geometry dependent electronic, photothermal and optical

properties (Pissuwan et al., 2011).

In principle, gold nanoparticles (GNPs) are chemically reactive hence, can be

functionalized with different targeting moieties and stabilizing agents. Enhanced light

scattering and absorption (optical performance) is the one of the most important

properties. The free electrons oscillation on the surface of nanoparticle occurs by

radiative decay producing very strong scattering of light and by a non-radiative decay,

converting light energy into heat (Jain et al., 2008). Both mechanisms have been

9

utilized in diagnostic and therapeutic. Among commonly used gold based

nanoparticles spherical gold nanoparticles (GNS) gain much attention due to ease of

synthesis with high yield. Gold nanospheres can be synthesized by Turkevich’s citrate

reduction method (Faraday, 1857; Turkevich et al., 1951).

Gold nanorods (GNRs) can be a good choice as these can split SPR into two

distinct bands, the transverse surface plasmon resonance (TSPR) and the longitudinal

surface plasmon resonance (LSPR) (Garcia et al., 2010). GNRs exhibit strong optical

absorption and scattering at visible (520 nm) and near infrared (NIR) wavelengths

(600-1300 nm). Their biomedical applications can be broaden through change in

length to width aspect ratio based on their optical extinction spectrum that ultimately

tune the wavelength in NIR range (Park et al., 2014). Tissues remain transparent in

optical range of 650-1100 nm, hence can be a powerful tool in disease targeting (Chen

et al., 2006), therapeutic-delivery systems (Huang et al., 2006), photothermal therapy

(PTT) for gene/drug delivery (Chen et al., 2006), medical and biological imaging and

surface enhanced Raman scattering (SERS). (Conjusteau et al., 2011). Gold nanorods

(GNRs) are commonly synthesized by seed mediated method (Murphy et al., 2001).

1.6 PHOTO-TRIGERED GENE INTERFERENCE

Small interfering oligonucleotides, such as DNA, RNA and short interfering

RNA (siRNA), facilitate sequence-specific control over intracellular genes, but, lack

the temporal control. Current development in chemical biology, nanoscience, and

plasmonics enable new light-sensitive tools of sub-nanometer and nanometer size

scales to directly interface with intra-cellular processes. For example, nanoplasmonic

optical antennae can be used as carriers of oligonucleotide cargo (Lee et al., 2009a).

Initially, conjugated oligos functionality is in-activated. By means of NIR

illumination as a remote trigger to release oligos hence, activating their functionality,

10

endogenous intracellular genes can be silenced. Signal distortions can be minimized

since light-sensitive tools are activated from within the intracellular space, and

therefore, steps in the extracellular-to-intracellular cascade are bypassed at the time of

activation. Using new tools these approaches can control several dynamic activities

in a live cell which is not possible by conventional techniques (Lee et al., 2009a).

Gold nanorods with large absorption cross-section, facile tunability of their LSPR in

the near-infrared (NIR) spectral region, are attractive candidates for intracellular

control. The NIR wavelength regime is compatible for biological and biomedical

applications since tissues and cells are essentially transparent between 700-1300 nm

(biological window) (Svoboda and Block, 1994).

Due to their strong and sharp resonance peak in their optical properties,

nanoplasmonic optical switches (GNRs) are capable of photothermal conversion

(Cortie et al., 2005; Khlebtsov et al., 2006), when the incident light is matched to

their plasmon resonance wavelength (LSPR). In the presence of this incident light, the

conduction electrons of the nanomaterial oscillate and subsequently make collisions

with the metal lattice, thereby dissipating heat, producing photothermal effect (Link

and El-Sayed, 2000). Transfer of heat from the nanoparticle is highly localized and

decay only within a few nanometers (Skirtach et al., 2005; Lee et al., 2009b)

consequently, having minimum adverse effects on the cells.

It is presented in this dissertation that 21-mer single strand DNA, called

antisense, can be hybridized to a thiol modified complementary sense strand, bound to

a GNR’s surface by the Au-SH covalent bond, and photo-thermally de-hybridized

using continuous-wave (CW) NIR laser light, matched to LSPR of the GNRs. The

strategy of photothermal de-hybridization using continuous-wave NIR laser

illumination offers numerous remarkable advantages. First of all, no chemical

11

alterations are made to the antisense DNA strand itself as a thiol modified

complementary sense strand is directly conjugated to the GNR’s. Chemical

modifications of the oligos may interfere with DNA/RNA functionality and gene

silencing efficacy, therefore, unmodified antisense strand is highly desirable.

Moreover, Au-SH bonds are highly stable on laser irradiation, GNR’s surface remains

covered with the thiol modified complementary sense strands. This surface coating of

nanoparticle with sense strands upon and after laser illumination is critical for

biocompatibility (Jones et al., 2009).

In summary, nanocomposite comprising thiolated sense and fluorescence label

antisense oligos are conjugated to GNRs surface and then introduced into live cells.

After CW NIR laser irradiation, antisense strands de-hybridize (Barhoumi et al.,

2009) and release into the cytoplasm, whereas the sense strands remain attached to the

GNRs surface. The released fluorescein labeled antisense strand subsequently binds to

the corresponding mature mRNA of COX-2. Once the COX-2 mRNA/antisense DNA

heteroduplex is formed, it is recognized and degraded by RNase H enzymes, thereby

silencing the COX-2 gene expression (Figure 1.5 and 1.6).

12

Figure 1.6: Schematic diagram of optical control switch gene interference

Figure 1.5: Mechanism of RNA interference by ONCOS(Lee et al., 2009a).

13

1.7 AIMS AND OBJECTIVES

1.7.1 Specific Aims and Importance

Major objective of the current study is the development of optical control

switch technique for the delivery of antisense probe to have successful localized

suppression of COX-2 oncogene with high spatial and temporal control. This

therapeutic approach will render cancerous cells sensitive towards apoptosis, decreased

angiogenesis, tumor invasion, metastases and increased host immunity and will reduce

the duration of treatment when used as adjuvant with chemo and radiotherapy or alone.

It may also reduce the side effects of COX-2 selective inhibitors.

1.7.2 Objectives of the Study

1. To synthesize and characterize nanomaterials.

2. To prepare bioconjugates specific to COX-2 mRNA.

3. To determine cellular uptake studies of nanocomposite.

4. To evaluate cytotoxicity of bioconjugates on various cancerous and normal

cell lines.

5. To transfect liver and breast cancer cells with COX-2 specific probes

conjugated with gold nanoparticles.

6. To release COX-2 specific antisense probe from nanoparticles using NIR

laser.

7. To determine COX-2 expression after NIR illumination based RNAi with

nanocomposite (ONCOS).

8. To estimate molecular Profile before and after gene knock down.

14

Chapter 2

REVIEW OF LITERATURE

2.1 CYCLOOXYGENASES AND CANCER

Prostanoids are synthesized in more or less all mammalian tissues and are

responsible for numerous physiological and pathological responses (Grosser et al.,

2006; Liu et al., 2015). Generally, COX-1 expression is constitutive while COX-2 is

inducible in response to various stimuli (Kang et al., 2007). Though, COX-2 is

expressed constitutively in some parts of the body like kidney and brain (Harizi,

2015). The COX-2 expression is highly related to numerous pathological conditions

like inflammation and tumorigenesis (Rouzer and Marnett, 2009), since the last few

decades several studies have been conducted to explicate the mechanisms of

transcriptional regulation of COX-2 (Kang et al., 2007). It is also obvious that COX-

2 is regulated at post-transcriptional level (Mbonye et al., 2008). Several in vitro

studies have shown prostanoids synthesis is stimulated by growth factors, cytokines

and oncogenes. The AA metabolism, by COX dependent pathway is associated with

several human malignancies. Stimulation of COX-2 gene is highly responsible for this

fact (Zelenay et al., 2015).

Numerous hypothesis have been proposed to clarify association between

prostanoids and tumor hallmarks such as resistance to apoptosis, promotion of

angiogenesis, suppression of immune response and induction of tumor cell

proliferation (Wang and DuBois, 2006; Greenhough et al., 2009). Eberhart et al.

studied the expression of both isoforms of COX in normal and tumor tissues biopsies

and reported that COX-2 level is comparatively high in more than 80% of colorectal

cancer tissues compare to normal tissues where its expression is found to be at basal

level. (Eberhart et al., 1994). Same facts have also been reported in many other

15

malignancies like skin, lungs, head and neck, pancreas, gastric and bladder, cancers

(Thun et al., 2002), suggestive of COX-2 critical role in tumor initiation, promotion

and progression. Earlier reports have demonstrated that inducibly high COX-2 level is

one of the important contributors to tumorigenesis (Liu et al., 2001). Several in vitro

studies showed inhibition of apoptosis and increased tumor angiogenesis with COX-2

overexpression. High expression of COX-2 favors tumor metastasis by the

development of new vasculature and modulation of molecular pathways (Liu et al.,

2001).

A recent study illustrated the overexpression (≥50% positive cells) of COX-2

is associated with poor prognosis in malignant peripheral nerve sheath tumor

(MPNST) patients. Additionally a selective NSAID induced apoptosis in MPNST cell

line (FMS-1) by activating caspase-8, -9, and -3 (Hakozaki et al., 2014).

Successful inhibition of colon and breast tumors in rodents suggested the

participation of the high COX-2 expression level in chemical-induced tumorigenesis

(Reddy et al., 1987). These findings have been supported by the results of

epidemiological studies performed on humans where NSAIDs reduced incidence of

various cancers almost 40–50% including breast, colon, gastric, intestinal and bladder

cancers. Non selective COX inhibitor, aspirin, has been reported fairly effective

against sporadic colorectal adenomas (Thun et al., 2002; Dubé et al., 2007).

Clinical trials have reported COX-2 selective inhibitors (coxibs: Celecoxib

and rofecoxib) efficiently prevent tumor regression (Higuchi et al., 2003; Bertagnolli,

2007). Besides playing pivotal role in tumorigenesis, studies have also reported COX-

2 involvement in regulation of chemotherapy resistance in cancer (Harris et al., 1996).

High COX-2 expression is an indicative of an aggressive breast cancer phenotype that

is resistant to doxorubicin (Park et al., 2006). Significantly high expression of COX-2

16

in drug-resistant breast cancer cell lines such as (MDR1/Pgp170) indicates a strong

correlation between resistance to chemotherapy and COX-2 expression (Ratnasinghe

et al., 2000). In oral squamous cells COX-2 selective inhibition suppresses tumor

invasion by down regulating MMP-2-activating mechanism (Kinugasa et al., 2005).

Rahman and colleagues reported higher COX-2 expression in chemo-resistant

aggressive colorectal cancers (CRC) cells and tumor xenograft models. They improve

chemo-sensitivity in CRC cells by combination therapies of coxibs (celecoxib) and

anti-cancerous drugs (5-fluorouracil) (Rahman et al., 2012).

An earlier study reported that COX-2 protein overexpressed in 74% of

primary gastric adenocarcinoma patients and its intensity was significantly correlated

with lymph node involvement. Consequently these cases showed increased levels of

PGE2 in tumor cells in comparison with the normal gastric mucosa (Uefuji et al.,

2000). Highly expressed COX-2 and ultimately high production of PGE2 mainly

occurs in nearly all cancers of the gastrointestinal origin. High COX-2 expression is

associated with the irregular regulation by transcription factors, APC and iNOS which

can alter cellular responses related to carcinogenesis. (Wu et al., 2010).

In cholangiocarcinoma COX-2 expression is fairly restricted to tissue

macrophages and to recruited mononuclear phagocytes. The potential targets of COX-

2 selective and non selective inhibitors are not the tumor cells themselves, but the

tissue macrophages of the tumor micro-environment (Wójcik et al., 2012). In general,

PGE2 shows a strong immuno-suppression effect by inhibiting the natural killer cell

(T-cell) activity. Elevated level of PGE2 thus, induces cancer cell survival and

development of gastric cancer. In addition, this may also increase lymphatic invasion

by activating metalloproteinase activity (Uefuji et al., 2000). Overexpressed COX-2

17

gene is associated with the pathogenesis and high recurrence rate in primary ureteral

stump tumor (Chang et al., 2012).

2.1.1 Cyclooxygenases and Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the result of continuous injury and chronic

inflammation. Inducible COX-2 is the important mediator of inflammation. It has

been reported that COX-2 is persistently over-expressed in HCC (Cusimano et al.,

2009; Giannitrapani et al., 2009). In HCC, COX-2 selective inhibitors may have

potential therapeutic effects (Lampiasi et al., 2012). Hepato-carcinogenesis is highly

associated with chronically high COX-2 expression (Han et al., 2004; Cui et al.,

2005). PGE2, a widely used biomarker, has been revealed to stimulate cell

proliferation, angiogenesis and cell invasion. Studies confirmed that COX-2 selective

inhibitor, celecoxib, considerably inhibit growth of human HCC. COX-2

overexpression promotes G1-S transition of the cell cycle in human HCC xenograft

through a series of complicated signaling pathways. COX-2 deletion results in a

significant alteration in a cascade of cellular signaling pathways in HCC xenograft via

a complicated cell cycle modulation, PTEN/PI3K/Akt and histone deacetylases

(HDAC) signaling (Cui et al., 2007).

Selective COX-2 inhibitor, celecoxib and anticancerous drug, sorafenib was

used in combination for the treatment of HCC. The idea was based on the activation

of ERK1/2 and the inhibition of the MEK/ERK signaling pathway at the same time.

The results of this studied showed combined treatment of sorafenib and celecoxib

exhibited strong synergistic cytotoxic effects in HepG2 and Huh7 cell lines, and

displaying altered gene expression as compare to individual drug treatment (Cervello

et al., 2013).

18

Several studies revealed that COX-2 may be a rational therapeutic target in

HCC. Expression of COX-2 is comparatively higher in well-differentiated HCC to

less-differentiated HCC or in normal liver, signifying the involvement of COX-2 in

the early stages of hepato-carcinogenesis (Cervello et al., 2005). Additionally, it has

been reported that there is a significant correlation between COX-2 expression and the

presence of macrophages and mast cells and active inflammation in the adjacent non-

cancerous liver tissues (Morinaga et al., 2002). A positive correlation between COX-2

expression and tumor angiogenesis has been reported in patients with HCV- or HBV

associated HCC (Cheng et al., 2004). High COX-2 expression and elevated level of

PGE2 is associated with increased expression and activation of metalloproteinase-2

(MMP-2) that acquired increased metastatic potential in HCC cells (Mayoral et al.,

2005).

The COX-2 expression in human HCC was reported to be associated with the

levels of numerous important molecules involved in tumorigenesis like induction of

angiogenesis growth factors through vascular endothelial growth factor (VEGF)

(Cheng et al., 2004) and activation of Akt pathway by phosphorylation of Akt (Leng

et al., 2003). The Akt, a signal mediator, modulates cell survival and proliferation

(Vivanco and Sawyers, 2002). COX-2 up-regulation also inhibits apoptosis by

inducing the antiapoptotic genes Bcl-2 as well as activating antiapoptotic signaling

through Akt/PKB in liver cells (Wu et al., 2006). Chronic hepatitis C and B also leads

to HCC. In hepatocytes Hepatitis C virus (HCV) core protein stimulate COX-2

expression and causes oxidative stress leading to HCC (Nunez et al., 2004).

2.1.2 Cyclooxygenases and Breast Cancer

Breast cancer is one of the most common malignant tumors among female

worldwide. Earlier reports have demonstrated COX-2 might be a potential target for

19

breast cancer treatment due to its abnormally up-regulated expression in these cells

(Bos et al., 2009; Hu et al., 2009). The use of COX-2 selective inhibitor is related to a

considerable decrease in breast cancer risk (Liu et al., 2011).

The COX-2 expression occurs in premalignant epithelial cells of the breast

(Crawford et al., 2004). Genomic instability in breast cancer cells is induced by COX-

2 up-regulation (Singh et al., 2007b; Singh et al., 2008). Highly expressed COX-2 is

potentially associated with metastasis of breast cancer to bone which has been

reported in preclinical mouse model (Singh et al., 2005; Singh et al., 2006; Singh et

al., 2007a).

In breast cancer cells elevated expression of COX-2 is the reason of poor

prognosis (Kerlikowske et al., 2010). The PGE2 produced by COX-2 mediates

cellular responses by acting on a family of four G protein-coupled receptors

Prostaglandin E receptors. The EP4 expression has been found in a wide range of

epithelial malignancies including breast (Rundhaug et al., 2011; Wu et al., 2011a) and

its pharmacologic blockade inhibits proliferation and migration both in vitro and in

vivo (Ma et al., 2006; Thorat et al., 2013).

2.2 ROLE OF NON-STEROIDAL ANTI-INFLAMMATORY DRUGS

(NSAIDS)

Increased risk of vascular event associated with COX-2 selective inhibitors

prompted Merck and Dohme to remove their coxibs like Rofecoxib (Vioxx®) from

the market in 2004. Besides high cardiac toxicity, renal failure, increased ocular

pressure and infertility is also related with high dosage regimens of NSAIDs (Kearney

et al., 2006).

COX-2 plays a physiological role in many tissues and organs which

necessitate its localized and selective inhibition. In the kidney, constitutive expression

20

has been demonstrated for both isoforms. COX-2 inhibitor drugs, such as NSAIDs,

reduce sodium excretion, and may cause acute renal failure in patients in whom the

maintenance of adequate renal perfusion is prostaglandin-dependent. Therefore,

COX-2 selective inhibitors may also cause several adverse effects and cannot be used

in patients with predisposing diseases (Giovanni and Giovanni, 2001). Due to risk

associated with COX-2-selective and nonselective NSAIDs, there is an urgent

requirement to find out other strategies for down regulating COX-2 specifically in

cancer cells.

2.3 NANOPARTICLES AND THEIR BIOMEDICAL APPLICATIONS

Recently, metallic nanoparticles has been extensively use in biomedical

research. They are considered as ideal candidates for imaging applications because

they have tendency to produce quantum effects owing to their unique optical

properties. Commonly studied metal based nanoparticles comprise gold (Au), silver

(Ag), titanium oxide (TiO2) and iron (Fe) nanoparticles (El-Ansary and Al-Daihan,

2009). Among these metal particles, gold (Au) being inert and comparatively less

cytotoxic is widely used for various biomedical applications including drug and gene

delivery (Pissuwan et al., 2011). They can be functionalized using various ligands and

targeting moieties for their delivery to specific tissues.

Arrays of methods have been extensively used for the synthesis of

nanoparticles (NPs). The widely used methods mainly comprise gold salt reduction in

the presence of a stabilizing agent (Delehanty et al., 2010; Giljohann et al., 2010).

Size of synthesized product is mainly monitored by concentration of gold salt, growth

parameters and reaction temperature. Depending upon their applications, the different

size ranges are being synthesized. Modifications in the fundamental methods have

21

made it possible to synthesize and manipulate these NPs according to the specific

research objective requirements (Hung and Lee, 2007).

To achieve better physical and biochemical properties, NPs other than the

usual spherical shape have been synthesized. For example, rods, cubes and stars

shaped GNPs showing diverse optical and plasmonic properties (Tiwari et al., 2011).

Functionalized GNPs are widely being used to for drug and gene delivery. The PEG

functionalized GNPs were reported to have successful cellular uptake and nuclear

penetration in the HeLa cells without inducing significant cytotoxicity therefore they

can be used in biological applications (Gu et al., 2009). Drug carrying liposomes

encapsulated GNPs can be used for specific targeting (Chithrani et al., 2010). Trans-

gene expression was significantly enhanced by transfecting with GNPs conjugated

plasmid DNA encoding murine interleukin-2 (pVAXmIL-2) (Noh et al., 2007).

Oligos conjugated nanoparticles showed better uptake and transfection efficiency

(Tencomnao et al., 2011). Amino acid conjugated of GNPs can also reduce their non-

specific toxicity (Biswas et al., 2015).

GNPs conjugated with anticancerous drugs have shown efficient photothermal

effects against cancer cells (Wang et al., 2010). Nanoparticles can target tumor cells

by enhanced permeation and retention (EPR) phenomenon. Hence NPs have tendency

to selectively accumulate in the cancer cells at higher concentration compared to

adjacent normal cells. Better target cytotoxicity was achieved by gold coated iron

nano-shells without compromising healthy cells (Wu et al., 2011b).

In a previous finding liposomal release was efficiently achieved by irradiating

hollow gold nanoshells with NIR pulsed laser. Increased temperature on the gold

nanoshells surface favor microbubble formation and collapse which is responsible for

liposome disruption leads to spatially and temporally controlled drug release (Wu et

22

al., 2008). A study reported targeted gene regulation with gold nanospheres

conjugated siRNA against ribo-nucleotide reductase in human clinical trials (Davis et

al., 2010).

2.4 NANOPARTICLES FOR GENE KNOCKDOWN

Gold nanoparticles are now integral part of nano-science based research due to

their striking properties (Burda et al., 2005). Additionally, NPs can be stabilized with

different types of molecules due to their renowned chemistry involving alkyl thiol

adsorption on gold surface (Love et al., 2005). Particularly, thiolated oligos can easily

be conjugated with GNPs. The oligos conjugated GNPs have a wide range of

biological applications such as probes in various bio-diagnostic systems (Rosi et al.,

2006), like expression and regulation of genes and as nano-scale building blocks in

assembly strategies (Mirkin et al., 1996).

The research in nano-theranostic field reported the utilization of DNA

conjugated GNPs for intracellular gene regulation (Rosi et al., 2006) The NPs act as

biocompatible nano-carriers of antisense agent that can efficiently bind and scavenge

desired mRNA and block the expression within the cell (Lytton-Jean and Mirkin,

2005). Besides this, these oligos on the surface of the GNPs protects its degradation

by nucleases and allows the use of DNA functionalized GNPs in gene regulation

therapies (Hurst et al., 2006).

2.5 PHOTOTHERMAL GENE RELEASE

The appeal of gold nanorods in biological applications is amplified

exponentially due to their additional capability of converting absorbed photons into

heat by non-radiative pathways. Owing to this remarkable property, they serve as

widely used photothermal agents. NIR laser irradiation (20 mW for 1 min) of

polyurethane coated GNRs raised the temperature on the surface of nanomaterial by

23

100°C (Chou et al., 2005). The in vitro photothermal applications of GNRs on

different tumor cells have been reported in a number of studies (Huang et al., 2006;

Huff et al., 2007; Black et al., 2008). Dickerson et al., exploited EPR effect and

partial tumor resorption for the accumulation of PEGylated GNRs upon NIR

irradiation (Dickerson et al., 2008). Due to maximum absorbance intensities in NIR

region, GNRs capable of high opto-thermal conversion compare to other shapes and

types of nanostructures (Jain et al., 2006). At low photon densities, heat diffusion is

efficient which increase the temperature and cause local hyperthermia (Link et al.,

2000).

EGFR over-expressing malignant human squamous cell (HSC and HOC) were

treated with anti-EGFR conjugated GNRs and exposed to a continuous-wave (cw)

laser (800 nm). The results of study demonstrated high accumulation of GNRs in

malignant tumor cells which efficiently destroyed by photothermolysis (10 W/cm2)

compare to nonmalignant cells (Huang et al., 2006).

DNA conjugated gold nanorods (GNRs) have been utilized with subsequent

NIR laser irradiation for photo-triggered gene therapy. Yamada and co-workers made

a stable GNR-DNA complex irradiated NIR pulse laser. At higher power laser light

was absorbed by GNRs and converted into heat, the thermal effect transformed shape

of GNRs into spheres that favor DNA release from the surface (Takahashi et al.,

2005).

The application of photo-triggered DNA release from nanoparticle surface was

first established by Chen et al using fluorescence labeled probes for gene regulation

studies (Chen et al., 2006). To achieve combined chemotherapy and gene knockdown,

GNR-based nano-carrier has been used for proficient release of doxorubicin and small

interfering RNA against achaete-scute complex-like 1 in neuroendocrine (NE) cancer

24

cells. The study findings revealed strong anti-cancerous effect of combined

chemotherapy and RNA silencing using target nano-carriers in NE cancer cells (Xiao

et al., 2012).

Pulsed NIR laser-dependent release of gold nanoshells covalently bound to

thiol-modified siRNA recognizing EGFP have been studied for gene silencing in

living cells. The siRNA-gold nanoshells complex was internalized in EGFP

expressing mouse endothelial C166 cells. Photothermal melting was used to release

siRNA from the nanoshells. Nanoplasmonic gene silencing of EGFP expression was

found to be power and time-dependent (Braun et al., 2009). HeLa cells were

transfected with thiol-modified EGFP-N1 plasmid DNA conjugated GNRs followed

by NIR illumination for nanoplasmonic based induction of exogenous foreign genes.

Femto-second near-infrared (NIR) laser photo-thermally melted GNRs into spheres

and release EGFP-N1 plasmid DNA induced GFP expression was specifically

observed by immuno-fluorescence imaging (Chen et al., 2006).

Comparatively higher DNA de-hybridization induced by excitation of LSPR

on the NPs, relative to the thermal DNA de-hybridization has been achieved by

releasing single-stranded DNA (ssDNA) from the surface of gold nanoshells with

plasmon-resonant light (Barhoumi et al., 2009).

Light-controlled SPR-mediated and fully reversible release of nucleic acids

from nano-carrier was reported by photo-triggered de-hybridization of complementary

antisense strand from thiol modified sense strand conjugated with triangular gold

nanoprisms. The authors demonstrated that the Au-thiol bond between the thiol-

modified DNA oligonucleotides and the gold nanoprisms were chemically and

functionally stable after continuous-wave laser irradiation light illumination (Jones et

al., 2009). Selective photothermal release of covalently bound multiple species of

25

thiol-modified single-stranded DNA from the surface of GNRs based on aspect ratio

has been reported (Wijaya et al., 2008).

Localized gene interference by ONCOS has been reported in breast cancer

cells. Expression of ERBB2 gene was blocked at a specific time by photothermal

release of fluorescence labeled antisense oligos. The purposed method accomplished

gene knockdown with less than 100 nm resolution with minimal photothermal

damage.

26

Chapter 3

MATERIALS AND METHOD

3.1 MATERIALS

A list of all chemicals, supplies, instruments and software used in experiments

is provided in Annexure 1.

3.2 PLASMONIC NANOPARTICLE DESIGN

Gold nanospheres (GNS) and Gold nanorods (GNRs) are prepared by colloidal

synthesis method. All glassware and stir bars used throughout the synthesis process

were cleaned with activated (i.e. red and bubbling) aqua regia (3:1 HCl: HNO3), then

were rinsed with deionized (DI) water at least six times. The flask was considered

completely rinsed when chlorine gas could not be detected when fanning the air above

the opening of the flask toward your nose. Once the flask was rinsed, it was left to dry

upside down.

3.2.1 Gold Nanospheres Synthesis

Spherical shape gold nanoparticles with diameters of 30 nm were synthesized

by an optimized citrate reduction method (Turkevich et al., 1951). Tri sodium citrate

(18 mM) was prepared by dissolving 0.07935 g in 15 mL of DI water. Gold solution

(60 mM) was prepared by taking 1.8 mL from 100 mM stock and diluted it upto 3 mL

using DI water. After the solutions were prepared, 188 mL of DI water was added to

the 500 mL cleaned flask along with 2 mL of the 60.0 mM gold solution. The flask

was then covered with a watch glass and placed on a hot plate, with the settings:

temperature – high, stir rate – medium. The solution was heated with continuous

stirring until steady bubbles were seen rising from the stir bar (~ 100°C). When the

solution reached boiling point, immediately after steady bubbles were seen, 10 mL of

the 18 mM sodium citrate solution was added to the flask containing diluted gold

solution. The solution color changed as colorless→black→darkpurple→red wine. As

27

soon as the red wine color was seen, the flask was removed from the hot plate and

allowed to cool to room temperature. The GNS were then purified, to remove the

excess citrate, by centrifugation at 8000 rpm for 20 min and redispersed in DI water.

Purified GNS were characterized using UV-Vis spectroscopy, transmission electron

microscopy (TEM) and dynamic light scattering (DLS). Concentration of GNS was

calculated by Beer-Lambert Law using the extinction coefficients from literature.

3.2.2 Gold Nanorods Synthesis

Gold nanorods were prepared based on literature procedure reported earlier

(Nikoobakht and El-Sayed, 2003) with slight modifications. CTAB (0.1 M) was

prepared by adding 36.5 g in 1 L of DI water with slow stirring and medium heating

for 10 min. NaBH4 (10 mM) was prepared by dissolving 0.0023 g in 6 mL of DI

water; 10 mM of Ascorbic acid was prepared by adding 0.0704 g in 4 mL DI water;

10 mM of AgNO3 was prepared by dissolving 0.0085 g in 5 mL DI water and 10 mM

gold solution was prepared by taking 450 μL from 1 M stock diluted upto 45 mL.

Gold nanoseeds were synthesized by reducing 375 μL of 10 mM HAuCl4.3H2O using

900 μL of ice-cold freshly prepared 10 mM NaBH4 in the presence of 15 mL CTAB

(0.1 M) solution.

Figure 3.1: Schematic Diagram of GNRs synthesis

28

Growth solution was prepared by adding 10 mL of 10 mM HAuCl4.3H2O to

200 mL stirring solution of 0.1 M CTAB and 2 mL of 10 mM AgNO3 solution at

25°C. To maintain the stability of final product 0.5 mL of 1 M HCl was added,

followed by addition of 1.6 mL of 0.01 M ascorbic acid. Finely to the colorless

solution, seed solution (5.2 mL) was injected quickly. The reaction was left to proceed

at continue stirring for 4 h at 28ºC. Prepared GNRs were separated from spheres and

excess surfactants by centrifugation initially at 8000 g for 15 min then supernatant

was spun down by three-successive centrifugation at 14,000 g for 20 min and re-

dispersed in deionized water.

3.3 CHARACTERIZATION OF NANOMATERIALS

UV–visible absorption spectra were recorded(300-1100 nm) using Shimadzu

UV-1800 UV-Vis Spectrophotometer operated at a resolution of 1 nm with the

samples in 1 cm optical path quartz cuvette against deionized water as standard.

Spectra were acquired using UV Prob 2.34 and analyzed using Origin 8.0 software.

The hydrodynamic size distribution profile (nm) and the zeta potential (mV) of the

GNRs formulations were measured by Dynamic Light Scattering (DLS) system,

Nanotrac wave II Microtrac, GmbH.

Consistency in size and shape of GNRs was recorded by FEI Tecnai 12

Transmission Electron Microscope (TEM), Norway, with 80kV. TEM grids were

prepared by placing 10 µL of GNRs solution on carbon copper grids coated and

allowing the solvents to evaporate at room temperature. Images were acquired and

dimensions of GNRs were calculated from the average of 200 GNRs measurements

using ImageJ(Schneider, 2012).

3.4 NANOPARTICLE BIOFUNCTIONALIZATION

Gold nanoparticles and gold nanorods were functionalize as follows

29

3.4.1 Conjugation with Poly Ethylene Glycol (PEG)

For ligand exchange, GNRs pellet was redispersed in 1 mL of DI water. A 100

µL solution of 2 mM K2CO3 and 10 µL of 1mM mPEG5000-SH was added. Resulting

mixture was kept on a rocker at 180 rpm at 25°Cfor overnight. Excess mPEG5000-SH

was removed by two rounds of centrifugation at 12000xg for 20 min and finally

redispersed in DI water. GNRs were characterized using UV-Vis Spectrophotometer

and DLS techniques.

3.5 EFFECTOF VARIOUS GROWTH PARAMETERS ON THE SYNTHESIS

OF GOLD NANORODS

Various growth parameters were optimized to achieve GNRs of specific aspect

ratio. Different seed solution concentrations (10, 50, 100, 150, 200 and 250 μL) were

added to growth solution (5 mL aliquots), left at RT for 4 h and then purified. In order

to evaluate the influence of seed aging on LSPR of GNRs, freshly prepared seed was

added to growth solution after 30 min, 1 h, 2 h and 24 h and incubated for 4 h. Spectra

were recorded after purification. For optimization of silver ion concentration, silver

nitrate (10mM) was added to growth solution (5 mL aliquots) in different volumes,

while keeping in stirring for 4 h before purification. The effect of pH on synthesis of

GNRs was studied for 1 to14 pH of growth solution using different molarities of HCl

(1 M, 0.1 M, 0.01 M and 0.001 M) and NaOH (1 M and 0.01 M).

3.6 STABILITY STUDIES OF GOLD NANOPARTICLES

Stability of GNRs was recorded as a function of various parameters

3.6.1 Stability in Various Environmental Conditions

Stability as a function of repeated washes in water was characterized by

centrifugation-re-suspension cycles at 12000xg for 20 min up to six times. UV-Vis

spectra were acquired and particle instability parameter (PIP) was calculated for each

30

re-suspension. For choice of solvent re-dispersion, GNRs pellet was re-suspended in

deionized water, PBS, ethanol and different concentrations of surfactant (CTAB:

0.001-0.1 M) for a period of six months. Absorption spectra of GNRs were acquired

intermittently to record any shift in wavelength or change in absorption intensity.

Stability of GNRs was also studied as change in pH of re-dispersion solutions for a

period of one month.

Thermal stability of GNRs was studied from 28 to100°C and absorption

spectra were recorded. For GNRs ionic stability, GNRs pellets (prepared from 1 mL

aliquots) were re-dispersed in 1 mL NaCl solution at various concentrations ranging

from 2 to 500 mM at physiological pH. GNRs were left for 2 h at room temperature

before absorption measurements. GNRs dispersed in deionized water were used as

control. The pH (1 to 14) stability of GNRs was recorded after 2 h and then after 1

month at room temperature.

3.6.2 Stability in Biological Media Solutions and Proteins

Stability of CTAB-coated and PEGylated GNRs was investigated in serum

albumin (bovine serum albumin, BSA and human serum albumin, HSA), whole serum

(fetal bovine serum, FBS) and immunoglobulin protein, IgG. All these protein

solutions had a concentration of 1 mg/mL in 20 mM PBS. One mL GNRs aliquots

were spun down at 12000xg for 10 min and resultant pellets were redispersed in 2 mL

of each protein solution and incubated at 37°C for 24 h.

Particle instability parameter was used to monitor change in size, shape, yield

and aggregation using equation stated below (Ray et al., 2015). Change in peak height

(absorbance intensity), peak wavelength and spectral shape were recorded.

I =ΔI

𝐼°=

𝐼° − 𝐼𝑛

𝐼°

31

Where ‘I’ is change in absorbance intensity, ‘∆I’is difference between untreated (I°)

and treated (In) absorbance peak intensities. Change in peak wavelength was

calculated as follows:

λ = CΔλ = λ° − λn

λ°

‘λo’

is reference wavelength and ‘λn’is peak position after treatment, where ‘C’ is a

constant calculated as follows:

C =I ∗ thersh

λ ∗ thersh

We describe a suspension stable, if recorded absorption spectrum is less than 10% of

an initial peak. Hence, we define I* thresh as 0.1 and λ*thresh as 10, and an unstable

system is defined by a 10% change in peak absorbance intensity or a 10 nm shift in

wavelength. Final PIP was calculated:

𝑃𝐼𝑃 = I ∗2+ λ ∗2

3.7 CELL BIOLOGY

Human hepatocellular carcinoma (HepG-2), human breast adenocarcinoma

cell (MCF-7), human rhabdomyosarcoma cell line (RD), human breast

adenocarcinoma (MDA-MB-231) cells, human cervical carcinoma (HeLa Cells) and

non-tumorigenic epithelial mammary gland cell line (MCF12-F) were used in this

work. All worked was done in a sterile cell culture bio hood with all sterile lab

equipments and latex gloves rinsed with 70-95% ethanol to eliminate contamination.

Additionally, all materials used during cell culture experiments were disposed of in

biohazard waste boxes.

3.7.1 Cell Culturing and Sub Culturing

Complete cell culture media was prepared in a sterile cell culture bio hood

before starting all cell culture work. The RD cells were maintained in RPMI medium

32

supplemented with 10% fetal bovine serum (FBS), 100U/mL penicillin, 0.1 mg/mL

streptomycin and 25 mM HEPES. The MCF12-F cells were maintained in DMEM

with 10% FBS, 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 10μg/mL insulin, 100

U/mL penicillin, and 0.1 mg/mL streptomycin. All other cell lines utilized Dulbecco’s

Modification of Eagle’s Medium (DMEM) supplemented with 10% (v/v) FBS and

1% (v/v) antibiotic-antimycotic solution. All cell lines were maintained at 37°C with

5% CO2 in a 95% humidified atmosphere.

Prior to sub-culturing bio hood was wiped down properly using 70% ethanol

and the UV lamp was turned on for ~10-30 min. Trypsin and complete media (stored

in freezer) were placed in a 37°C sterile water bath. The flask containing cells to be

sub-cultured was then removed from the 37°C, 5% CO2 humidified incubator and

placed inside the cell culture bio hood. The cell culture media was removed from the

flask and placed in the bio waste container using a sterile 10 mL transfer pipet. Then 5

mL of sterile DPBS was added to the flask and was generously pipetted up and down

inside the flask to rinse the bottom of the flask and remove cell debris. Once the

bottom of the flask was rinsed 3 times with DPBS, 1.5 mL of trypsin was

administered to the flask and then returned to the 37°C, 5% CO2 humidified

incubator. Trypsin incubation times for all these cells were ~5-7. Successful

trypsinization was observed under inverted microscope. The flask then was placed

back into the cell culture bio hood and a sterile new serological pipet was used to add

1.5 mL of complete media to the flask. This 3 mL trypsin-complete media solution

was transferred to a 15 mL falcon tube and was centrifuged at 1500 rpm for 7 min.

After centrifugation supernatant was removed carefully using 10 mL serological pipet

and 5 mL of complete media was added into the tube containing the pellet. The media

was pippted up and down to thoroughly mix the cell solution, 0.5 mL of this cell

33

solution was added into a new labeled cell culture flask supplemented with 9.5 mL of

fresh complete media. The flask was then placed in the 37°C, 5% CO2 humidified

incubator. All cell lines were treated separately one by one. Flasks were labeled with

the experimenter’s name, date, and cell line name.

3.7.2 Cryogenic Storage of Cells

For long-term storage of cell lines, cells were placed on cryogenic storage. In

preparation for storage, a 90-100% confluent flask was trypsinized and centrifuged at

1500 rpm for 7 min. Cell pellet was redispersed in 950 μL of complete medium. The

cell solution was then transferred to the labeled 1 mL cryogenic tube and 50 μL of

sterile DMSO was added in it, after thoroughly mixing the tube was then placed in an

isopropanol freezing container and left overnight in a -20°C freezer. The next

morning the tube was transferred to a liquid nitrogen container or a -80°C freezer.

Same procedure was repeated for all cell lines sequentially.

3.8 CELL VIABILITY ASSAY

3.8.1 Mitochondrial Activity (XTT)

Cells were seeded in a 96-well culture plate (175 μL cell solution/well) to

achieve a final confluence of 70-75%. After cellular adherence, culture media was

removed and replaced with treatment solutions (100 μL nanoparticle solution/well)

with a control set of wells (cells without nanoparticles). Treated cells were allowed to

incubate for a desired time period (e.g. 24, 48 and 72 h). Activated XTT reagent

solution was prepared by adding 720 μL of the XTT reagent and 3.12 μL of the

activating reagent into 2.400 mL of fresh complete medium and kept in dark. After

which, media containing nanoparticles was replaced with an activated XTT reagent

solution (100 μL activated XTT/well) and incubated for 4-24 h. Activated XTT

solution without cells or media was also incubated in a set of wells as a background

34

reference samples. After desired incubation time the optical density was recorded on a

Biotek Synergy H4 multi-mode plate reader at the absorbance values of 450 nm

(maximum XTT absorbance) and 690 nm (baseline XTT absorbance). Absorbance

values were baseline corrected, mean and standard deviation (SD) was recorded.

Percentage cell viability was recorded using following formula

Percent Cell Viability =Absorbance of Sample

Absorbance of Control× 100

To determine the IC50, data was plotted as percentage cell viability vs. log of the

treatment concentration (M) and a logistic fit was applied.

3.8.2 Haemotoxicity

3.8.2.1 Hemolytic activity of GNRS

For haemolysis assay, after obtaining informed signed consent human whole

blood was collected in 1.8 mg/mL EDTA tubes from healthy volunteers. In 5 mL of

blood 15 mL of sterilized Phosphate buffer saline (PBS) was added and after slow

agitation tubes were centrifuged at 500×g for 10 min. Supernatant containing plasma

was aspirated and the buffy coat was washed thrice and diluted with normal saline to

a 50% packed cell volume (hematocrit). Various concentrations of CTAB-GNRs and

PEG-GNRs (5, 10, 25, 50, 100, 150, 200, 250, 500 and 1000 μL) were used for this

assay. 100 μL of these samples were incubated with 100 μL of RBCs suspension at

37°C in CO2 incubator for 4h. A 0.2% Triton ×100 solution was taken as positive

control and PBS as negative control in the current study (Dobrovolskaia et al., 2008;

Sadhasivam and Durairaj, 2014). After incubation, 50 μL of 2.5% glutaraldehyde was

added to the sample in order to stop the process of haemolysis. Samples were then

centrifuged at 1000×g for 10 min. Hemoglobin release induced by nanomaterials was

monitored at 562 nm using a microplate reader (Platos R496) by transferring

supernatant to 96 well plate (Nederberg et al., 2011; Laloy et al., 2014). Positive and

35

negative controls induced 100% and 0% absorbance respectively. Percentage

hemolysis was recorded using following formula (Zhao et al., 2011).

Percent Hemolysis =Sample absorbance − negative control absorbance

Positive control absorbance − negative control absorbance × 100

Every experiment was done in triplicate; data is presented as mean± standard

deviation (SD).

3.8.2.2 Biocompatibility assay of GNRS on human peripheral blood mononuclear

cells (PBMCS)

Human macrophages were isolated from human blood (by the consent of

volunteers) by a method of Almeida et al with slight modifications (de Almeida et al.,

2000). Process of isolation was carried out by ficoll-gastrografin gradient (density

1.070 g/mL). The density of this solution was achieved by dissolving 0.57 g of ficoll

in 9.5 mL deionized water. Solution was filtered using 0.22 µm syringe filters then

0.5 mL of gastrografin was added dropwise to achieve the density (1.064-1.070

g/mL). Blood was diluted three times with PBS solution and was layered over the

ficoll-gastrografin and centrifuged at 400×g for 30 min, plasma was removed down to

about 1cm above buffy coat. Approximately, 10 mL of buffy coat was aspirated

leaving the mononuclear cells undisturbed at interphase. Buffy coat was carefully

layered on top of the 5 mL ficoll solution without mixing both layers for the first

density gradient. This was then centrifuged at 400×g for 30 min at room temperature.

For each gradient white ring of peripheral blood mononuclear cells (PBMCs) was

collected which was located between the two phases and transfer to a new tube. Each

tube was filled with PBS and Centrifuged at 300xg for 10 min without brake at 20°C.

For complete removal of platelets, pellet was resuspended in 15 mL of PBS buffer

36

and centrifuged at 200×g for 10 min at 20°C. Supernatant was carefully removed and

cells were redispersed in appropriate amount of RPMI medium at 37°C with 5% CO2

supplemented with 15% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 25

mM HEPES buffer. Cells were counted using hemocytometer and cell viability was

determined by mixing 10 μL of trypan blue with PBMC solution 1:1 in a microtiter

plate. Viable cells were seeded in 96 well plates with a density of 1×106

cells per well

and incubated for 24 h with various concentrations of CTAB-GNRs and PEG-GNRs

using PBS (negative control) and 0.2% Triton×100 (positive control) at 37°C with

5%CO2, cytotoxicity was determined by MTT assay.

3.9 CONJUGATION OF COX-2 OLIGONUCLEOTIDES

Phosphorothioate oligonucleotides directed against 5' region of the COX-2

mRNA was purchased from Midland certified Reagent Ca. USA. Oligos comprised

COX-2 specific thiolated sense strands, fluorescein labeled antisense strands,

thiolated fluorescein labeled antisense strands and a set of scrambled thiolated sense

strands and fluorescein labeled antisense strands as a control. A detail of oligos is

provided in Annexure1 (Table 1A).

3.9.1 Oligos Reconstitution

All glassware and consumable were pre-autoclaved for all oligos experiments.

Sterilize Ultra- Pure DNase/RNase-Free distilled water was used throughout these

experiments. To photo-protect the fluorescent dye, all experiments utilizing antisense

strand were done in dark. Thiolated sense strands and fluorescence labeled antisense

strands were thawed at room temperature for several minutes. Oligos were spun down

slowly for few seconds then sterile Ultra- Pure DNase/RNase-Free distilled water was

added (in µL) 10 times the quantity of oligos in nmoles in order to get 100 µM stocks.

Tubes were spun down at low speed for 15 sec. Samples were then aliquoted into

37

minimum required volumes (25 µL per tube) tube with appropriate labeling. Sample

spun down for 15 sec then allowed to set for 2 min at room temperature and then

stored at -20°C.

3.9.2 Conjugation with Gold Nanospheres

3.9.2.1 Hybridization of sense and antisense strands

Sterilized DI water (800 mL) was added in a 1000 mL beaker containing stir

bar and left on hot plate with settings temperature high and stirring medium.

Temperature of this home-made water-bath was maintained at 90°C. To hybridize

sequences, 100 µL of 100 µM thiolated sense strand was mixed with 100 µL of 100

µM fluorescein-labeled antisense strand and 100 µL of Tris-NaCl (10 mM: 25 mM)

buffer. The sample was then kept in pre- maintained 90°C water-bath for 2 min and

then allowed to cool down slowly to 60°C in dark. To ensure maximum hybridization

sample was left at room temperature on rocker for 12 h and then stuck in fridge (4°C).

3.9.2.2 Thiol reduction of phosphorothioate protected strand

Thiol protected sense sequence was activated using DTT. Fresh DTT solution

was prepared in Tris-Nacl buffer to the final volume of 100 mM by dissolving 0.06 g

of DTT powder in 3.889 mL of buffer. Hybridized (sense/antisense) sequences were

kept at room temperature for several min followed by addition of 100 µL of 100 mM

DTT solution. Mixture was incubated at room temperature for 2 h in dark.

3.9.2.3 Column purification of activated strands

NAP-5 column was equilibrated first in order to purify the reduced oligos. Top

and bottom caps of the column were removed and allowed excess liquid to flow

through. Column was equilibrated by three complete refills of Tris-NaCl buffer. Per

column 200 µL of DTT- reduced hybridized sample and 300 µL of Tris-NaCl buffer

was allowed to enter the gel bed completely. UV handle lamp was used to monitor the

38

rate of flow of sample on the basis of fluorescent from antisense strand. Sample was

eluted using 500 µL of Tris-NaCl buffer when it reached the bottom of the column.

Reduced purified samples were then collected in pre-autoclaved labeled tubes and

stored at -20°C until next use.

3.9.2.4 Conjugation with particles

To conjugate GNS to the hybridized DNA, 185 µL of citrate-capped gold

nanospheres (3 nM) was added into the 500 µL of column-eluted double stranded

DNA and diluted upto 3 mL using Tris-NaCl buffer (10mM:25mM). In other tube 185

µL of PEGylated gold nanospheres (3 nM) was added into the second set of 500 µL of

column-eluted double stranded DNA and diluted upto 3 mL using Tris-NaCl buffer.

Sample was kept on rocker for 3 h at room temperature finely kept in the fridge for 48

h. To concentrate the double stranded DNA-conjugated GNS; samples were

centrifuged at 8000 g for 20 min and pellets were washed twice with Tris-NaCl

buffer.

3.10 CHARACTERIZATION OF BIOCONJUGATE

3.10.1 UV-Vis Spectrophotometric Measurements

After centrifugation pellets from DNA conjugated citrate-capped GNS and

PEGylated GNS were diluted to 200 µL. For UV analysis 8 µL of each sample was

taken and diluted upto 300 µL in Tris-NaCl buffer and the same buffer was used as

blank. Concentration of each sample was calculated by Beer Lambert Law using

extinction coefficient of particles.

3.10.2 Fluorescence Measurements

For fluorescence based studies of bioconjugated gold nanospheres three

different concentrations of dsDNA-conjugated citrate capped GNS was prepared by

adding 2.5, 1.25 and 0.63 µL of dsDNA-conjugated citrate capped GNS

39

independently into 325 µL of Tris-NaCl buffer. Equal volume of DTT (1.0 M in 0.18

M PBS) and FAM labeled DNA was used for displacing oligos from GNRs. After

overnight incubations GNRs were removed by centrifugation. For each dilution 65 µL

of sample was added per well into 384-well microtiter-plate. Six different

concentrations of fluorescence labeled single stranded antisense were used for

standard curve. Stock solution of control was prepared by adding 0.62 µL of

fluorescence labeled single stranded antisense into DTT and further dilutions were

prepared (Table 2.1). Fluorescence was recorded at excitation 495 nm.

For each dilution 65 µL of each sample was added per well. Fluorescence

measurements were recorded on Biotek Synergy H4 multi-mode plate reader with 485

nm excitation and 528 nm emission collection, 120 Gain. Measurements were taken in

this order, first at room temperature then on gradually heating (30, 37, 42, 47, 52, 57,

and 62°C) and finely on gradually cooling (62, 57, 52, 47, 42, 37 and 30°C). Data was

exported to Microsoft excel to determine the fluorescent intensity with increase and

decrease in temperature.

Table 3.1: Different concentrations of fluorescence labeled single stranded

antisense.

S.No ssDNA-FAM (µL) 1.0 M DTT buffer (µL)

1 65 0

2 32.5 32.5

3 16.25 48.75

4 8.13 56.87

5 4.06 60.94

6 2.03 62.97

40

3.10.3 Nanoparticle Dissolution with KCN Method

A solution of 200 mM of potassium cyanide (KCN) was prepared in water at

room temperature in fume-hood to dissolve the gold nanoparticles. Solution

containing 300 µL of dsDNA conjugated gold nanoparticles (6.2 nM) was dissolved

in KCN. After several min incubation at room temperature, gold nanospheres SPR

absorption band at 520 nm disappeared, resultant solution had absorption band at 495

nm (due to fluorescein labeled oligos) and at 260 nm (due to DNA). A linear

absorption versus concentration calibration curve was constructed by adding series of

known concentrations of fluorescence labeled ssDNA (antisense strand) to the

solution of citrate capped gold nanospheres and digested under the same conditions

described above. Surface coverage values were then calculated by standard curve

method. Experiment was performed in triplicate.

3.11 CONJUGATION WITH GOLD NANORODS

An aliquot of 100 µL thiol protected sense strand (100 µM) was thawed at room

temperature for few min then 100 µL of freshly prepared 100 mM DTT solution was

added in it. Mixture was incubated at room temperature for 2 h in dark. Sample was

purified using NAP-5 column as method described in section 3.9.2.3. GNRs were

PEGylated with 10% surface coverage by procedure outlined in section 3.4.1. The

purified sample was then added to 2 mL of PEGylated GNRs (5 nM) with 50 µL of

1X PBS. Sample was incubated at room temperature for 24 h then left at 4°C for 2 h.

Antisense strand was hybridized to GNRs-conjugated sense strand by the method

described in section 3.9.2.1. Conjugated GNRs were then concentrated by

centrifugation.

41

3.11.1 Characterization of Bioconjugate

DNA conjugated GNRs were characterized by UV-Vis Spectrophotometric

measurement as in section 3.10.1 LSPR peak was recorded at 780 nm. Fluorescence

based study was performed as described in section 3.10.2, KCN dissolution was

detected y method given in section 3.10.3.

3.12 INTRACELLULAR LOCALIZATION AND UPTAKE STUDIES

3.12.1 Live Cell Dark Field Imaging

Sterilized 18 mm diameter glass cover-slips were placed in a 12-well tissue

culture plate. Cells were seeded in complete medium (1.5 mL cell solution/well) on

the cover-slips to achieve 70-80% final confluence. Cover-slip was pushed down

gently with sterilized tweezers to ensure cell growth on the top of cover-slip. Cells

were adhered to the cover-slips after 24 h incubation. Old media was removed and

replaced with desired concentrations of gold nanoparticles and nanorods (500 μL NPs

solution/well) diluted upto 500 μL complete media solution. Each sample was added

in triplicate along with set of control wells (cells without treatment). After achieving

incubation for desired treatment time the nanoparticles solutions were removed and

washed thrice with PBS and replaced with fresh complete medium. Live-cell dark

field imaging system was set up to reach cell culture growth conditions (i.e. 37°C, 5%

CO2). The imaging system comprised an inverted microscope modified with a Fiber-

Lite MI-150 Illuminator, a live-cell imaging chamber, a temperature controlled water

system, and a CCD camera for image collection. The glass cover-slips were placed

inside the imaging chamber which then mounted carefully on the stage of the

microscope.

The incident white light, delivered by the fiber optic, was focused to the

sample such that white light illuminates the sample but does not enter the collection

42

cone of the microscope objective. The scattered light from the sample was collected

using a long working distance 40X objective. A digital camera was used to capture

the dark field pictures every two minutes. Images were collected for ~12-24 h.

3.12.2 Spectrophotometric Measurements of Relative Uptake

The relative uptake of DNA conjugated and non conjugated gold nanoparticles

were determined using UV-Vis spectroscopy. Cells were transfected with DNA

conjugated and non conjugated GNS following the procedure outlined in Section

3.12.1. Nanoparticle treatment solutions were also added to separate wells that did not

contain cells as these wells serve as reference samples. Final concentrations were

achieved using clear cDMEM as the diluent. Cells were incubated with particle

solutions for 24 h, after which, cell culture media and particle solutions (incubated

with and without cells) were collected and transferred to clean cuvettes for UV

analysis. Plates containing the cells were washed twice with PBS and incubated after

adding cDMEM. The optical density of the transferred media and nanoparticle

solutions was measured at the SPR peak of the nanoparticles (λmax = 530 nm), for

FAM dye (λmax =490 nm) and for DNA (λmax = 260 nm) using a Shimadzu UV-

spectrophotometer. Relative percent of nanoparticle uptake was recorded by

subtracting average optical density of control from average optical density of treated

sample multiplied by 100. Experiments were repeated at least three times and

averages from each experiment were used to obtain the mean ± standard error of the

mean (SEM). Statistical significance was determined using an unpaired t-test.

3.12.3 Cellular Uptake by Confocal Microscope

HepG2 cells were seeded in glass-bottom (optical) dishes at a density of 1 ×

104 cells/mL and incubated at 37°C for 48 h. Media was removed, cells were

washed with DPBS. Then the cells were transfected using serum free media as

43

described in section 3.12.1. Cells were incubated for 12 h, media was changed and

cells were washed and incubated in complete medium for additional 1 h. The same

test samples were also incubated without cells for analysis. Images were captured

with a Zeiss confocal microscope using the FITC filter (Ex 495 nm, Em 520 nm) and

Carl Zeiss LSM Aim software and were superimposed to determine the intracellular

localization of the nanoparticles conjugated with single and double stranded DNA.

Same procedure was followed for MDA-MB-231 cells.

3.13 CHARACTERIZATION OF IMMOBILIZED ANTISENSE STRAND

RELEASE FROM GNRS SURFACE

3.13.1 Laser Power, Wavelength Specificity, Temperature and Exposure Time

DNA conjugated GNRs were electrostatically attached to the glass slide to

visually monitored the release of antisense oligonucleotides from the surface of

nanoparticle. The conjugated GNRs solution was dispensed onto the glass slide and

then the solution was re-immersed in buffer solutions. Free nanoparticles were

removed by 3 times of washing steps; the conjugated GNPs remain attached to the

glass surface. An 808 nm laser was positioned above the immobilized DNA

conjugated GNRs on the glass slide with spot size of 2 mm. An inverted epi-

fluorescence microscope was used to visualize the optical control antisense strand

release. Wavelength specificity was characterized by using a 650 nm laser other then

808 nm fluorescence intensity of surrounding area was recorded as mentioned above.

Laser power was characterized using an optical power meter. Immobilized FAM

labeled DNA conjugated GNRs were illuminated at three different power intensities:

40, 60, 80, 120 and 160 mW/cm2 and temperature on the GNRs were also recorded

using a thermocouple. At 5 minute intervals, fluorescence intensity of surrounding

area was captured using a color CCD camera. Laser exposure time was characterized

44

in the same way by illuminating the conjugated switches for 2, 4, 6, 8, 10, 15, 20 and

30 min exposure time and fluorescence intensity of surrounding area was captured.

3.14 COX-2 RNA INTERFERENCE USING ANTISENSE

OLIGONUCLEOTIDES

3.14.1 Transfection Experiments

Escort TM

IV (neutral polycationic lipid) transfection agent was used to

transfect the cells. Before transfection several parameters were optimized to achieve

the highest possible transfection efficacy. Parameters included cell confluency, DNA

to lipid ratio and transfection time. Cells were counted and measured for density and

viability then cultured in 12-well plate (3.0 x 105 cells per well) in 1 mL of complete

medium to achieve 50-70% confluency at the time of transfection (in 24-48 hours).

Cells were incubated at 37°C with 5% CO2. After achieving desired confluency old

media was removed and replace with fresh complete growth media to a total volume

of 1 mL. To prepare the DNA: Escort TM

IV transfection complexes, Escort TM

IV,

dsDNA, ssDNA, GNRs conjugated dsDNA, GNRs conjugated ssDNA, and

serum/antibiotics free medium was warmed at room temperature for several min.

Serum/antibiotics free medium (98 µL) was added into 2 µL of GNRs conjugated

dsDNA (1.0 µg/µL) into a sterile microcentrifuge tube. Same was repeated for each

sample. Then 97.6 µL of serum/antibiotics free medium was added into 2.44 µL of

transfection agent into five sets. One set of this transfection agent was then added to

100 µL of dsDNA (supplemented with serum/antibiotics free medium), second set

was mixed with 100 µL of ssDNA (supplemented with serum/antibiotics free

medium), third set was mixed with 100 µL of GNRs conjugated dsDNA

(supplemented with serum/antibiotics free medium), fourth set was mixed with 100

µL of GNRs conjugated ssDNA (supplemented with serum/antibiotics free medium)

45

and fifth set was used as control without any sample. After thorough mixing with

gently pipetting, samples were left for 20 min at room temperature to allow the DNA/

liposome complexes to form, then 800 µL of serum/antibiotics free medium was

added in each tube followed by gentle mixing. Cells (50-70% confluency) were then

washed with serum/antibiotics free medium and supplemented with 500 µL of

reduced serum medium. The DNA/ liposome complexes (500 µL) were then

distributed to the cells by adding the complexes drop-wise to different areas of the

wells. Plate was then gently rocked back and forth and from side to side to evenly

distribute the DNA/ liposome complexes and samples were then incubated for 6-72

hours. Growth media was replaced 12-18 hours post transfection and cells were

monitored after regular interval for morphology changes. Same experiment was

repeated with GNS.

3.15 PROTEIN EXPRESSION ANALYSIS

3.15.1 Protein Extraction

After a desired incubation time, the cultured plate containing transfected cells

was placed on ice. Media from the plate was aspirated and cells were washed twice

using ice-cold PBS (300 μL per well). To prepare 1x Lysis buffer, 200 μL of 10x lysis

buffer was added into 1780 μL of DI water then supplemented with 20 μL of 100x

phenylmethylsulfonyl fluorides (PMSF), a protease inhibitor, to the final

concentration of 1 mM. Supplemented 1x lysis buffer was chilled on ice and then

added to the cells (250 μL per well). Cells were incubated on ice for 10 min and then

detached from the surface with ice cold sterilize rubber policeman. The cell lysis

solutions were collected and transferred to a chilled pre-labeled 1.5 mL

microcentrifuge tube. The lysis solutions were then sonicated (Branson Digital

Sonifier set at 50% amplitude with out-put control value 4) three times for four

46

seconds each with at least one minute rest on ice between each four-second pulses.

Lysates were incubated on ice for additional 15 min. Cell lysate solutions were

centrifuged at low speed 5000 g for 10 min at 4°C to break up cellular debris and

supernatants were then centrifuged at 13, 000 g for 15 min at 4°C. Tubes were gently

removed from the centrifuged and placed on ice, supernatants were collected in new

chilled pre-labeled micro-tubes. To avoid multiple freeze/thaw cycles, lysates were

aliquoted and stored at -20ºC.

3.15.2 Protein Quantification

The protein concentration of the cell lysates were then quantified using a

Bradford protein quantification assay (Bradford, 1976). The 1x Bradford dye reagent

was removed from the 4 °C refrigerator, allowed to warm at ambient temperature, and

inverted several time to ensure thorough mixing. BSA standards and different

dilutions of each lysate were then prepared in 1x PBS as shown in Table 3.2 and 3.3.

In a 96-well plate, 150 μL of sample lysate and the BSA standards were added (3

wells/sample). Once the sample and standards have been added, 150 μL of the 1x

Bradford dye reagent was added to each well. Solutions were mixed thoroughly and

allowed to sit at room temperature for 10-15 min. Absorbance measurements, at 595

nm, were recorded on a Bio-Tek Synergy H4 Multi-Mode Plate Reader and exported

into Microsoft Excel. The protein concentration of the cell lysate samples were then

calculated, the absorbance from the blank standard (i.e.0 μg/mL) was subtracted from

each BSA standard and cell lysate samples to give the corrected absorbance (OD-

corrected). Then, the OD-corrected values were plotted vs. their corresponding BSA

standard concentration. A linear trend line equation (y = mx + b) was then fit to the

data and used to calculate the diluted protein concentrations in the cell lysate samples.

47

Dilution calculations were then carried out to determine the original cell lysates

protein concentration.

3.15.3 Protein Separation

3.15.3.1 Sample preparation for loading

Sample loading buffer was prepared by adding 950 μL Laemmli sample buffer

and 50 μL β mercaptoethanol called supplemented Laemmli sample buffer solution.

The protein lysate samples were prepared by adding 1:1 of the lysate and

supplemented Laemmli sample buffer solution. The final total protein concentration

kept the same for each treatment sample. After which samples were boiled for 5 min

at 92°C in a pre maintained water bath and then immediately kept on ice and stored at

-20°C.

Table 3.2: BSA Standard solutions preparation.

Tube # Standard

Vol. (µL)

Source of

Standard

Diluent volume

(µL)

[BSA]Final

Conc. (µg/mL)

1 10 2 mg/mL stock 790 25

2 10 2 mg/mL stock 990 20

3 06 2 mg/mL stock 794 15

4 500 Tube 2 500 10

5 500 Tube 4 500 5

6 500 Tube 5 500 2.5

7 500 Tube 6 500 1.25

8 0 (blank) 0 500 0

Table 3.3: Cell lysate solution preparation.

Tube # Cell lysate Vol.

(µL)

Source (Cell lysates) Diluent volume

(µL)

1 10 unknown stock conc. 990

2 500 Tube 1 500

3 500 Tube 2 500

48

3.15.3.2 SDS-PAGE

The SDS-PAGE set-up (Figure 3.1) was assembled after the quantification of

lysates protein concentrations. A 10% APS solution (0.1 g APS in 1 mL of DI water)

was freshly prepared. After which, 54 μL of this solution and 4.5 μL of TEMED was

added to 9 mL of NEXT GEL (12%) solution in a 15 mL falcon tube. This solution

was inverted several times and immediately poured in between the glass plates to the

very top; a comb was then immediately inserted. The gel was then allowed to

polymerize (~20-25 min) and the comb was removed. The wells were rinsed with 1x

NEXT GEL Running Buffer and the gel tank was filled (to the line) with 1x NEXT

GEL Running Buffer.

The prepared protein lysate samples were loaded (20 μL) into the wells.

Additionally, 20 μL (10 μL of standard + 10 μL of DI water) molecular weight

standard Kaleidoscope and a 5 μL COX-2 (ovine) electrophoresis standard (50 ng), as

a positive control were loaded in two separate wells. For negative controls a cell

lysate from COX-2 negative cell line HSC-3 and loading buffer alone were loaded in

two independent wells. Once the samples, MW standard and controls were loaded, the

gel was run at 200 V for ~ 40 min (or until the dye reaches close to the bottom of the

gel). After the completion of running gel apparatus was disassembled carefully and

gel was allowed to cool for several min then removed from the glass plates to use in

further experiments.

49

3.15.4 Coomassie Blue Staining

For gel staining, staining and destaining solutions were prepared. For staining

solution 0.8 g of Coomassie Brilliant Blue R250 was dissolved into 400 mL of 40%

methanol (in DI water) and then filtered using 0.22 µm filter. Then 400 mL of 20%

acetic acid (in DI water) was added. Solution was mixed thoroughly and stored at

room temperature in dark. Destaining solution was prepared by adding 200 mL of

absolute methanol into 800 mL of DI water and 200 mL of acetic acid. Solution was

carefully mixed by inversion. Gel was placed in Coomassie Blue staining solution for

30 min with shaking then placed in destaining solution for 2-3 h with shaking.

Colorimetric images of gel were obtained on AmershamTM

Imager in both Epi and

Trans- illumination mode.

3.15.5 Reversible Protein Detection Method

ZiP™ Reversible Protein Detection Kit was used according to the instructions

of manufacturer. Kit contained equilibration solution, stain solution, developer

solution, and restore solution (10x each). Briefly, ZiP™ reagents were prepared to the

Figure 3.2: SDS PAGE Gel apparatus Assembly.

50

required concentrations. Each 10x reagent was mixed well then 50 mL of each reagent

(1x) was prepared by diluting the 10x concentrates 1:10 in deionized water

immediately before use. All wash and incubation steps were performed with constant,

gentle shaking on a rotating platform. Following electrophoresis, the gel was rinsed in

50 mL deionized water for 5 min then equilibrated in 50 mL of 1x equilibration

solution for 15 min. After equilibration gel was ready to stain, incubated in 50 mL of

1x stain solution for 30 min. Gel was rinsed in 50 mL of DI water for 1 min followed

by addition of 50 mL of 1x developer solution for 1 – 5 minutes or until bands

appeared and were clearly distinguishable. Gel was photographed using a dark

background. For further use e.g. in western blot, gel was completely destained by

adding 50 mL of 1x restore solution with gentle shaking until the gel became

translucent like an unstained gel.

3.15.6 Western Blotting

3.15.7

3.15.8

3.15.9

Table 3.4: Buffers Recipes for Western blot.

Buffer Component

1x Tris/Glycine transfer buffer 100 mL – 10x Tris/Glycine buffer

200 mL – absolute methanol

700 mL – DI water

10x Tris Buffered Saline (TBS) 24.2 g – Tris base

80 g – NaCl

1 L – DI water

adjust pH = 7.6 (w/ HCl)

1x TBS 100 mL – 10x TBS, pH = 7.6

900 mL – DI water

1x TBST 100 mL – 10x TBS, pH = 7.6

900 mL – DI water

1 mL – Tween-20 (100%)

Blocking buffer 900 mL – 1x TBST, pH = 7.6

45 g – Non-Fat dry milk

51

Gel was soaked in transfer buffer (Table 3.4) for 10-30 min, Whatman 3 mm

filter papers were cut to fit the dimensions of the gel and they were immediately

placed in transfer buffer, along with foam pads, for 10-30 min. The nitrocellulose

membrane was also soaked in the transfer buffer for ~5 min. After the gel, filter

paper, membrane and pads were thoroughly equilibrated in the transfer buffer; they

were removed and stacked as seen in Figure 3.2. There should be no air bubbles

between layers, as this will interfere with protein transfer.

The transfer cassette holder was then placed in the transfer tank with the latch

side facing up (the black cassette plate should face the black electrode plate). Transfer

buffer was added to the tank until the liquid reached the line and an ice pack was also

added to the tank. Blot was run at 25V for 2.5 h. After protein transfer, the cassette

was disassembled and the membrane was rinsed one time in 1x TBS. The membrane

was then incubated with a blocking solution (Table 3.4) under agitation for 1 h.

Efficacy of transfer was checked by either checking the color of standard

Kaleidoscope, by staining the gel with coomassie blue after transfer or by transferring

the proteins on two membranes sequentially. The membrane was then washed 3 times

Figure 3.3: Western blot Cassette assembly diagram

52

for 5 min with 1x TBST buffer (Table 3.4) and incubated with diluted primary

antibody solutions (Table 3.5) with gentle shaking at room temperature for overnight.

The house keeping β-actin was run each time as a protein loading control/reference.

After 12 h incubation, the membrane was washed 3 times for 5 min with 1x TBST

and incubated with the HRP-conjugated secondary antibody solution (Table 3.5) for 1

h at room temperature with gentle agitation. The membrane was then washed 3 times

for 5 min in 1x TBST and then incubated with the Pierce ECL Western Blotting

Substrate (3.940 mL detection reagent 1 + 3.940 mL detection reagent 2) for 1 min at

room temperature. The excess substrate was drained and the membrane was placed in

labeled sheet freezer bags.

The membrane was then imaged on AmershamTM

Imager with chemiluminescence.

3.16.6.1Densitometric analysis

Western blot protein bands were quantified using Image J software from

National Institute of Health (NIH) USA. Results were reported at average from three

independent trials.

Table 3.5: Primary and Secondary Antibody Dilutions.

Antibody Dilution Factor Antibody (µL) Dilution Buffer (mL)

Primary Antibodies

COX-2 1:400 50 9.950

β-actin 1:1000 10 9.990

P53 1:1000 10 9.990

p38 MAPK 1:1000 10 9.990

Caspase-3 1:1000 10 9.990

HtrA2/Omi 1:1000 10 9.990

AIF 1:1000 10 9.990

Secondary Antibodies

Anti-rabbit IgG HRP 1:2000 5 9.995

Anti-mouse IgG HRP 1:2000 5 9.995

53

3.16 RNA INTERFERENCE BY ONCOS

Cells were plated on glass cover-slips in the 6-well plates to achieve 50-60%

confluency. After achieving desired confluency, old media was removed and cells

were washed with 1x PBS. Cells were then transfected for 8 hours according to

procedure outlined in section 3.15.1 with GNRs conjugated dsDNA and GNRs

conjugated ssDNA. In addition to these, wells containing dsDNA, ssDNA, PEGylated

GNRs and untreated cell were used as control. After 8 hours, the cover-slips

containing cells were removed carefully from the 6-well plate and were washed three

times with serum free medium. The cover-slip containing cells were then placed in a

live cell imaging chamber slide and the chamber was filled with media. Samples were

illuminated using the focused NIR 808 nm laser positioned above the sample with

spot size of 5 μm diameter at 120 mW/cm2for 1 minute. Images were captured before

and after activation using epi-fluorescence inverted microscope. The cells are then

incubated at 37°C for 48 hours. After desired incubation protein expression was

checked by method outlined in section 3.16.

3.17 MEASUREMENT OF PROSTAGLANDIN-E2 (PGE2) PRODUCTION

RATE BY ELISA

The ELISA buffer and wash buffer were dilute to desired working

concentration prior to use. Cells were transfected following the method described in

section 3.15.1. After desired incubation cell were centrifuged at 500 g for 7 min. Cell

culture supernatant was collected and diluted upto 2 folds with ELISA buffer for

Prostaglandin E2 (PGE2) measurement. The PGE2 ELISA standard was reconstituted

in 1 mL of ELISA buffer. The concentration of this stock is 10 ng/mL; stock was then

aliquoted and stored at -20°C. Serial dilution of PGE2ELISA standard was prepared

(Table 3.6) and used within 24 h. The PGE2 tracer was reconstituted in 6 mL of

54

ELISA buffer and stored at 4°C. The PGE2 monoclonal antibody was reconstituted in

6 mL of ELISA buffer and stored at 4°C. A plate set up (Table 3.7) was done using

96-well plate (included in the kit). Each sample was plated in triplicate. Plate was then

covered with thin film and incubated at 4°C for 18 h. For the development of the plate

Ellman's reagent was reconstituted (immediately before use) in 20 mL of Ultrapure

water. At desired incubation time after emptying the wells they were rinsed five times

with wash buffer. A 200 µL of Ellman's reagent was added to each well and 5µL of

tracer was added into TA well. The plate was then covered with a plastic film, placed

on orbital shaker in dark for 60-90 min. Absorbance was checked periodically and the

plate was read at wavelength 420 nm when the absorbance intensity reached the range

0.3-1.0 for B0 well. Data was processed by subtracting the average absorbance reading

for NSB well from B0 well, this is the corrected B0 value. For standard and sample

average absorbance was first subtracted from average absorbance of NSB and then

divided by corrected B0 value final values were then multiplied by 100 to get

percentage values. A linear standard curve equation (y = mx + b) was then fit to the

data and used to calculate the PGE2 concentration per sample.

55

3.18 CELL PROLIFERATION ASSAYS

3.18.1 Cell Viability by Flow Cytometry

Cells were seeded in 6-well plates as explained in the cell preparation method

above. After achieving desired confluency, cells were transfected as explained in

section 3.15.1. After 24 hours, the cells were washed with serum free media and

placed back in complete media. The 808 nm laser was used to illuminate cells at 120

mW/cm2 for 1 min. The cells were then incubated at 37°C for 48 h and 72 h. After 48

h and 72 h cells were prepared for analysis with flow cytometry. The cells were then

trypsinized and collected by centrifugation (500 g for 3 min) followed by re-

suspension in 1x PBS which contained 2 μM Calcein AM. As a control, un-adhered

dead cells were collected from the media in the 6 well plates and sonicated for 10

Table 3.6: PGE2 Standard preparation.

Tube # Standard

Vol. (µL)

Source of

Standard

Diluent volume

(µL)

Final Conc.

(pg/mL)

1 100 10 ng/mL stock 900 1000

2 500 Tube 1 500 500

3 500 Tube 2 500 250

4 500 Tube 3 500 125

5 500 Tube 4 500 62.5

6 500 Tube 5 500 31.3

7 500 Tube 6 500 15.6

8 500 Tube 7 500 7.8

Table 3.7: PGE2 plate setup for ELISA.

Wells ELISA

buffer

(μL)

Standard/

Sample

(μL)

Tracer

(μL)

Antibody

(μL)

Blank 0 0 0 0

Total activity (TA) 0 0 5 0

Non-specific Binding (NSB) 100 0 50 0

Maximum-Binding (B0) 50 0 50 50

Std/Sample 0 50 50 50

56

seconds. These dead cells were then collected using centrifugation (500 g, 3 min), and

re-suspended in 1x PBS which contained 2 μM Calcein AM for 20 min. These

samples were then immediately transferred to FACS tubes for flow cytometry

analysis. Their fluorescence was analyzed by flow cytometry using the Coulter EPICS

XL flow cytometer. Approximately 10,000 events (cells) were evaluated for each

sample.

57

Chapter 4

RESULTS AND DISCUSSION

4.1 PLASMONIC NANOPARTICLE DESIGN

The formation of GNRs via the seed mediated growth method has received

significant interest due to its simplicity and homogeneous yield (Liu and Guyot-

Sionnest, 2005). A strong nucleating agent, sodium borohydride, was used in the first

step that reduced gold ions to form spherical NPs acting as seed for GNR synthesis

(Nikoobakht and El-Sayed, 2003). During seed preparation, upon rapid addition of

NaBH4 to HAuCl4.3H2O containing CTAB solution, change in color from yellow to

light brown was observed. The nucleation was initiated upon the addition of

suspension of the seed particles to the growth solution containing CTAB that bind to

the surface of NPs and stabilized gold complex in the presence of ascorbic acid which

act as a weak reducing agent and silver nitrate, shape directing agent. Figure 1 showed

the visible spectra (682-906 nm). The hydrodynamic diameter of the GNRs was

ranged between 20.90±2.15 to 50.5±2.13 nm with the zeta potential of 33.3± 0.34 -

40.1±0.71 mV. TEM images displayed GNRs with good monodispersity with 2.5 to

4.6 aspect ratios (Figure. 4.1).

58

Figure 4.1: Characterization of synthesized GNRs. UV-Vis absorption spectra of

GNRs from shorter (682 nm) to longer (906 nm) LSPR. TEM images show mono-

disperse population with high yield.

4.2 EFFECT OF VARIOUS GROWTH PARAMETERS ON THE SYNTHESIS

OF GOLD NANORODS

The most interesting aspect of GNRs is their plasmonic properties that can be

modified during synthesis process. Fine-tuning GNRs shapes and sizes define their

morphology and consequently their plasmonic properties.

4.2.1 Effect of Seed Aging and Concentration

Homogenous population of small aspect ratio GNRs are formed after 30

minutes of addition of gold seed soln. Gold seed addition at different time points (30

min, 1 h, 2 h and 24 h) show a notable increase in average GNRs size and

heterogeneous population, as the seed ages. This may be due to decrease in LSPR and

59

an increase in TSPR, corresponding to more spheres than rods(Jiang and Pileni,

2007; Zhang and Lin, 2014). Fresh seed solution having ultrasmall gold seed NPs is

crucial for the formation of GNRs in high yield (Koerner et al., 2012). The aspect

ratio of the GNRs was highly influenced by the concentration of seed solution.

Smaller aspect ratio rods were synthesized with lowest seed concentration (10 µL)

having LSPR at 680 nm (Figure. 4.2).

We have reported that low seed concentrations as well as addition of seed

beyond the critical values (100 µL) resulted in low aspect ratio rods. As the gold

concentration in growth solution increased, the peak absorption and wavelength

decreased because of higher concentration of reduced gold in the solution that led to a

larger diameter but shorter length GNRs. A red shift in LSPR was observed with

increase in seed concentration upto100 µL however, after 100 µL the LSPR of GNRs

decreased on further addition of seed concentration, corresponding to low aspect ratio

GNRs. CTAB capped seed act as a template for gold ions to reduce onto. The larger

nanoparticle seed template may not be able to interact with the cylindrical CTAB

micelles hence the diameter become larger and their growth could not be directed

anisotropically (Gole and Murphy, 2004; Watt et al., 2015). Our results are consistent

to that of previous reports according to which increasing the seed concentration

resulted in lower aspect ratio rods (Stacy, 2012; Ward et al., 2014).

60

4.2.2 Effect of Silver Ion Concentration

With an increase in silver nitrate (10 mM) from 30 to 36, 40, 46, and 50 µL

the corresponding aspect ratios increase. GNRs with low aspect ratio (2.4) are

observed at low silver ion concentration (30 µL). Initial increase in silver nitrate

concentration cause red shift (increase in aspect ratio) but beyond certain limit (50 µL

of 10 mM in 5 mL total volume), addition of more silver ions inhibits the growth of

rods hence blue shift in LSPR is observed (decrease in aspect ratio).A blue shift in the

LSPR is observed on further increase in silver nitrate concentration (56 and 60 µL)

indicating formation of low aspect ratio GNRs. The decrease in LSPR at high Ag+ ion

has been attributed to the effect of ionic strength. Current findings show optimum

400 500 600 700 800 900 1000 1100

0.0

0.5

1.0

1.5

2.0

2.5

Ab

so

rba

nc

e (

A.U

)

Wavelength (nm)

10 µL

50 µL

100 µL

150 µL

200 µL

250 µL

Figure 4.2: UV-Vis spectroscopic analysis of GNRs as a function of seed

concentration. An initial positive correlation between the amount of seed and

the resultant LSPR peak can be seen (formation of larger rods) until a

threshold reaches (100 µL) beyond which a blue shift in peak (decreased

aspect ratio, formation of smaller rods) was observed with further increase in

seed concentration.

61

silver concentration for GNRs is 46-50 µL, since this concentration favors growth at

{111}and {100} facet (Figure 4.3) (Table 4.1).

We have observed the aspect ratio of GNRs first increased and then decreased

with increasing amount of silver nitrate. These further indicate that the silver ions play

important role in regulating growth rate on different planes of GNRs and promoting

one-dimensional crystal growth. Some contradictory results have also been reported

Table 4.1: Effect of Silver ions on GNRs aspect ratio

S.No Silver nitrate

concentration (µL)

LSPR GNRs

aspect ratio

1 30 673 2.4

2 36 722 3.2

3 40 735 3.4

4 46 765 3.8

5 50 801 4.6

6 56 703 2.9

7 60 675 2.5

300 400 500 600 700 800 900 1000 1100

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Ab

so

rba

nc

e

Wavelength (nm)

30 µL

36 µL

40 µL

46 µL

50 µL

56 µL

60 µL

673nm

721nm

735nm765nm

801nm

703nm

675nm

Figure 4.3: UV-Vis absorption spectra of GNRs prepared with respect to silver

nitrate concentrations. Initial increase in silver nitrate concentration cause red shift

in the LSPR peak but beyond certain limit addition of silver ion inhibit the growth

accordingly blue shift in LSPR is observed.

62

stating the aspect ratio decrease with increase of silver nitrate (Li et al., 2013). Silver

ion concentration has a critical effect on the synthesis of GNRs. Addition of AgNO3

stabilize the growth process and enhance yield significantly (Kang et al., 2015).

During GNRs growth silver ions form complex (AgBr) with bromine ions of CTAB

(Sau et al., 2010). These complexes deposited on specific facets mainly {110} of rod

surfaces at the Au-CTAB interface, retard Au ion reduction onto these facets

ultimately playing dominant role in the synthesis of stabilized GNRs. This

phenomenon facilitates directed growth of GNRs from a specific facet which is less

densely covered by AgBr or CTAB, consequently hampering unregulated deposition

of Au atoms on the entire seed surface (Cai et al., 2010).

4.2.3 pH Controlled Growth of GNRS

The pH of the reaction solution is considered as one of the most dominant

parameters for GNRs synthesis. At acidic pH 1-3, UV-Vis spectrum of the growth

solution illustrates both TSPR and LSPR peaks. At this specific range of pH, the

Gaussian shape of LSPR is sharpened, indicating the formation of uniform and

monodispersed GNRs (Figure 4.4). With increase in pH of growth solution from 1to

2, shift in LSPR shows formation of high aspect ratio GNRs. But increase in pH from

2-4 shows blue shift in LSPR, indicative of low aspect ratio rods. When pH is

increased above 4, LSPR peaks become broad, represent the formation of

polydispersed GNRs. At alkaline pH 12 to 14, only TSPR peak is visible indicate

absence of GNRs. Current study specifies the formation of fairly uniform and low

aspect ratio GNRs at low pH whereas at alkaline pH rod’s shape no longer remains

(Figure 4.4).

63

At acidic pH of growth solution, the reduction potential of ascorbic acid is

very mild. Therefore, rate of reduction of Au ions (Au+3

to Au+1

then to Au0) on

addition of seed solution; (Murphy et al., 2005) is very slow, which yield less amount

of Au atoms that can deposit on the {111} and {110} facets of the GNRs. Secondly,

due to weak reducing power, ascorbic acid cannot reduce silver ions that favor the

formation of AgBr (Scarabelli et al., 2015). CTAB bilayer coated with AgBr absorbed

onto the different facets of GNRs with variable affinities towards CTAB. When the

pH of the growth solution is low, compared to the {111}facet, the CTAB bilayer

coated with AgBr primarily absorbed onto the {110} facet preferably because of high

surface energy and less stability on this facet (Xiang et al., 2008). This well defined

CTAB bilayer coated with AgBr on the {110} facet should prevent more Au atoms

from depositing on this plane, so the Au atoms then deposit onto the {100} and {111}

400 500 600 700 800 900 1000 1100

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Ab

so

rba

nc

e (

A.U

)

Wavelength (nm)

pH 1

pH 2

pH 3

pH 4

A

300 400 500 600 700 800 900 1000 1100

0.0

0.5

1.0

1.5

2.0

2.5

Ab

so

rba

nc

e (

A.U

)

Wavelength (nm)

pH 14

pH 13

pH 12

pH 11

B

Figure 4.4: UV-Vis absorption spectra of GNRs prepared at different pH. Successful

rod formation at slightly acidic and neutral pH while no rods are produced at basic pH.

64

preferentially to form long rod-shaped NPs (Sau and Rogach, 2010). When the pH of

the growth solution is increased, firstly, the reduction potential of ascorbic acid and

rate of reduction of Au ions were amplified likewise, secondly, the CTAB bilayer on

GNRs surface collapse resultant in an unstable {110} facets that would most likely

susceptible to reaction solution. Therefore the Au atoms constantly deposited onto

{111} facet causing reduction in aspect ratio and change in morphology of GNRs

(Song et al., 2005). Similarly, on further increasing pH, there is decrease in the

electrostatic attractive force between CTAB molecules and the Au surface that will

sparse absorbed CTAB bilayer. This sparser CTAB layer allows deposition of Au

atoms not only onto the {100} but also onto the {110} facet, resultant change in shape

and morphology of GNPs (Wei et al., 2008).

In alkaline conditions instead of bromide ions ascorbate ions or hydroxyl ions

preferentially absorb on the CTAB micelle surface leads to smaller micelles formation

(Wang et al., 2005). Meanwhile at higher pH silver ions would be reduced to Ag0

by

ascorbic acid so AgBr no longer facilitate GNRs formation because of deposition of

Au atoms onto all facets of Au nanoseeds, so only spherical shape particles formed

(Liu and Guyot-Sionnest, 2005).

PEGylated GNRs were settled down at the bottom of eppendorf tube and then

were completely redispersed in DI water without any aggregation indicating

successful loading of mPEG5000-SH. LSPR of GNRs was similar to that obtained

before PEGylation, indicating no aggregation or interaction during PEGylation.

Spectra shows no noticeable shift (<5 nm) for LSPR peak, only 1 nm red shift was

detected after pegylation. According to previous studies both (a small) red and blue

shifts were observed after pegylation of GNRs (Zhang and Lin, 2014). Zeta potential

65

of CTAB coated GNRs was positive (36±2 mV) whereas that of PEGylated GNRs

reduced to -1.2±1 mV (Figure 4.5).

4.3 STABILITY OF GNRs

Stability of GNRs is one of the major concerns. The stability of gold nanorods

was determined by following the LSPR peak in the NIR region of the absorbance

spectrum as a function of various parameters.

4.3.1 Effect of Environmental Conditions

Once GNRs are synthesized, first step is removal of surfactant and re-

dispersion in appropriate solvent. In order to find out on/off equilibrium effect of the

CTAB bilayer, the gold nanorods were washed multiple times and redispersed in DI

water. Change in absorption spectra of CTAB-GNRs was observed with increase in

number of washes. Absorption intensity decreased considerably and LSPR slightly

broadened and blue-shifted, indicating aggregation of GNRs due to loss of CTAB

from their surface. This shows that only two to three times washing is suitable for the

400 500 600 700 800 900 1000

0.0

0.1

0.2

0.3

0.4

0.5

Ex

tin

cti

on

(a

. u

.)

Wavelength (nm)

CTAB-GNRs

PEG-GNRs

CTAB-GNRs PEG-GNRs-5

0

5

10

15

20

25

30

35

40

Ze

ta P

ote

nti

al

(mV

)

Figure 4.5: UV-Vis absorption spectra of CTAB coated gold nanorods and PEG coated

GNRs presenting no significance difference between LSPR peak absorption intensity

Graph showing Zeta potential of CTAB coated and PEG coated GNRs. CTAB coated

GNRs show positive zeta potential (36±2 mV) whereas this reduced to -1.2±1 mV for

PEG coated GNRs.

66

stability of GNRs (Figure 4.6). Kah et al also reported the same but described the

results as aggregation index which is a qualitative analysis (Kah et al., 2012). Here we

have presented quantitative analysis in terms of particle instability parameter as

colloidal stability index. PIP requires two spectra; a reference spectrum of untreated

nanoparticle and the data spectrum of nanoparticle with the analyte of interest while

the FP and AI methods are based on averaged changes in a spectral profile.

1 2 3 4 5 6

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Pa

rtic

le I

ns

tab

ilit

y P

ara

me

ter

(PIP

)

Number of Washes

A

1 2 3 4

0.0

0.1

0.2

0.3

0.4

Pa

rtic

le I

ns

tab

ilit

y P

ara

me

ter

(PIP

)

Time (Months)

B

700 800 900 1000 1100

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Ab

so

rba

nc

e (

a.u

)

Wavelength (nm)

0 Month

1 Month

2 Months

3 Months

4 Months

817 nm 817 nm

813 nm

810 nm

810 nm

C

Figure 4.6: Particle instability parameter (PIP) value as a stability index. A). PIP

versus number of washes. Less aggregation upto 3 time washing is observed; B) PIP

versus time (months) plot; GNRs stability study upto 04 months. GNRs in 0.001 M

CTAB solution are stable for 3 months at room temperature (22-25°C) without

compromising considerable change in the LSPR peak intensity (PIP > 0.1). UV-Vis

67

spectroscopic analysis shows decrease in peak intensity without a notable wavelength

shift with LSPR peak (810-817 nm).

The next step was redispersion of GNRS in proper solvent to maintain their

integrity at different time intervals. GNRs remained stable in growth solution but only

for short period of time (less than a week). After synthesis, if GNRs are not removed

from growth solution, they will result in large size and short aspect ratio due to slow

continual growth (Rostro-Kohanloo et al., 2009). When GNRs were kept in DI water

for few days (approximately, for 1-2 weeks at 4°C) LSPR peak remained stable while

the absorbance intensity of GNRs was decreased in PBS and ethanol. GNRs remained

stable in 0.001M CTAB solution for at least three months (PIP> 0.1). There was no

shift in wave length (785 nm) and absorption intensity of GNRs was observed after

one month (Figure: 4.6). Optimum concentration of CTAB is 0.001 M for storage of

GNRs. This concentration is not too high to become susceptible to crystallization, if

room temperature dropped and not too low to cause aggregation of GNRs due to

equilibrium shift between CTAB in the solution and that on the surface of GNRs

(John et al., 2013; Merrill et al., 2013).

4.3.2 GNRS Stability at Different pH

We have studied GNRs for their stability in acidic and alkaline environment

initially for 2 h and then for 1 month. Absorption spectra after 2 h present stable

GNRs at almost all pH except 14. Under acidic conditions GNRs are stable after one

month. However, absorption intensity decreases considerably at neutral pH with slight

blue shift (decrease in size). At alkaline conditions GNRs aggregate after 1 month.

PIP values greater than 0.2 indicate aggregation of particles at alkaline pH. At

alkaline pH, considerable uncertainty in the stability of GNRs is observed. Therefore,

it may be assumed that acidic and neutral pH conditions are suitable to keep GNRs

68

stable. Extracellular environment of tumor tissue is often acidic (Kato et al., 2013).

Gold nanorods for use in cancer theranostics must not aggregate at acidic and

physiological pH in order to deliver their payload at the site of action. In previous

studies GNRs aggregate at physiological pH (Ding et al., 2007) but in current study

GNRs are stable at acidic and physiological pH, even after one month (Figure 4.7).

400 500 600 700 800 900 1000 1100

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Ab

so

rba

nc

e (

a.u

)

Wavelength (nm)

pH 1

pH 2

pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

pH 14

A

400 500 600 700 800 900 1000 11000.0

0.2

0.4

0.6

0.8

Ab

so

rba

nc

e a

.u

Wavelength (nm)

pH 1

pH 2

pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

pH 14

B

2 4 6 8 10 12 140.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Pa

rtic

le I

ns

tab

ilit

y P

ara

me

ter

(PIP

)

pH

2 Hours

1 Month

C

Figure 4.7: Stability of GNRs with respect to pH. (A) UV–Vis spectra of synthesized

GNRs after 2 h at room temperature; (B) after 1 month at room temperature. Loss of

Gaussian shape at pH 8-14 is visible;(C) PIP values plot of GNRs stability after 2 h

and 1 month shows particle degradation at pH 10-14 (PIP > 0.1) after 2 h. GNRs

remain stable in acidic condition for 1 month (PIP < 0.1).

69

4.3.3 Effect of Temperature on GNRs Stability

Gold nanorods are stable at 28-60°C with no significant change in absorbance

intensity and wavelength at 2 minutes heating (PIP < 0.1) making them a good

candidate for heat assisted external stimuli responsive experiments. Earlier study

reported GNRs instability above 40°C (John et al., 2013). However, significant

change in absorbance at 90-100°Cis recorded (Figure 4.8). LSPR blue shift is

observed, probably due to disruption of CTAB bilayer. Both length and width become

broader after thermal treatment. Surface melting phenomenon may be involved in

shortening of LSPR and increase in average diameter. Possible explanation may be

that tips of GNRs are more reactive than the body, hence at higher temperature the

tips of smaller GNRs would like to dissolve first, the resultant cluster deposit on the

body of the larger GNRs (Zou et al., 2010).

28 37 40 50 60 70 80 90 100

650

700

750

800

850

900

Temperature (°C)

Wa

ve

le

ng

th (

nm

)

*

**

**

20 30 40 50 60 70 80 90 100 110

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Pa

rtic

le I

ns

tab

ilit

y P

ara

me

ter

(PIP

)

Temperature (°C)

B

Figure 4.8: A). Plot of LSPR absorption peak wavelength in response to temperature.

Decrease in LSPR is observed when temperature is raised above 60°C.Statistical

analysis shows no significance difference in LSPR when heated from 28 to 60°C for 2

min (p<0.05). B). PIP versus temperature plot show less aggregation of GNRs upto

60°C.

A

70

4.3.4 Effect of Ionic Concentration on GNRs Stability

GNRs must be stable in buffer solution at physiological pH for best use in

biological systems. In current study two plasmon bands of GNRs change slightly from

5 to 10 mM NaCl concentration indicative of particles stability (PIP < 0.1), while

aggregation is observed from 10-100 mM. Minimal aggregation of GNRs is observed

when salt concentration is above 100 mM (PIP > 0.1) (Figure 4.9). Minimal

aggregation of the GNRs in solution was observed when salt concentration was above

100 mM. At salt concentration <100 mM, the Cl- anions present electrostatically bind

to the surface of positively charged GNRs, result in cross linking of material and

consequently aggregation.

0 20 40 60 80 100 120 140 160 180 200

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Pa

rtic

le I

ns

tab

ilit

y P

ara

me

ter

(PIP

)

NaCl Concentrations (mM)

A

400 500 600 700 800 900 1000

0.0

0.5

1.0

1.5

2.0

2.5

Ab

so

rba

nc

e a

.u

Wavelength (nm)

0

5mM

10mM

20mM

40mM

60mM

80mM

100mM

150mM

200mM

300mM

400mM

500mM

B

Figure 4.9: Stability of GNRs at different ionic concentrations. A) Plot of PIP versus

concentrations of NaCl shows stability at 5 to 10 mM and 100 to 150 mM.

Aggregation of GNRs is observed between 10 to 100 mM and salt concentrations (PIP

> 0.1); B).UV–Vis absorption spectra of GNRs in response to different salt

concentrations at room temperature.

At higher salt concentration (i.e.>100 mM), the Cl-anions are sufficient

enough to entirely bind the positively charge surface of GNRs, the phenomena leads

to neutralize surface charges. Consequently, this double layer on the GNPs surface

creates a repulsive force between the nanoparticles hence minimizes aggregation and

71

could potentially lead to stabilization of GNRs (Sethi et al., 2009; Kah et al., 2012).

GNRs remain resistant to extreme low and high salt concentrations. We may assume

that GNRs show non-monotonic behavior in ionic solution (Sethi et al., 2008; Ferhan

et al., 2010).

4.3.5 Stability of GNRS in Cellular Media Solutions

Studies have showed that GNPs are rapidly attracted by biological fluids

(Lynch et al., 2009; Monopoli et al., 2012). These biomolecules mostly include

proteins that adsorb to the nanoparticle surface through electrostatic interactions, Au-

N and Au-S bonding (Liu et al., 2012). In this study, four proteins are investigated

including serum albumin (BSA and HSA), whole serum (FBS) and one

immunoglobulin protein, IgG at physiological pH. The wavelength and absorption

intensity of GNRs showed slight changes in the presence of FBS, BSA and HSA.

However, GNRs are more stable in immunoglobulin protein. It is hypothesized that

these proteins have ability to form a stable protein corona on the GNRs surface which

prevent nanorods aggregation CTAB-GNRs are positively charged while serum

albumins are negatively charged at neutral pH. Hence, the electrostatic interaction

between oppositely charged proteins and nanoparticles may cause CTAB bilayer

disruption. Immunoglobulin are membrane proteins, these may bind to the GNRs by

initially inserting their Fc region of the antibody into CTAB bilayer and then further

interact with GNRs. GNRs surface layer is not disrupted during protein adsorption

(Liu et al., 2012). PEG coated GNRs are more stable in proteins solution than CTAB

coated GNRs (Figure 4.10). The non-specific protein adsorption on the surface of

GNRs cannot be fully prevented due to lose coverage of PEG chain. This may cause

interaction between hydrophobic surface of GNRs and plasma proteins (Khlebtsov et

al., 2006).

72

BSA

HSA

FBS

IgG

0.0

0.1

0.2

0.3CTAB-GNRs

PEG-GNRs

Culteur Media Proteins

Part

icle

In

sta

bil

ity P

ara

mete

r (P

IP)

******

Figure 4.10: Stability studies of GNRs in cellular media. Significant difference

between CTAB coated and PEG coated GNRs is observed at p< 0.05 in case of three

serum proteins.

4.4 CYTOTOXICITY ASSAY

In spite of the immense potential of GNRs for their medical applications, their

possible toxicity has become a key issue. Hence we have performed a study on the

cytotoxicity of bare and coated GNRs. The CTAB coated GNRs illustrated dose-

dependent cytotoxicity in MCF-7 (Figure. 4.11a), RD (Figure. 4.11b) and MCF12-F

cells, even though centrifugation was performed twice for the removal of CTAB in

the sample. MCF-7 and MCF12-F cells were highly responsive to both GNRs and

PEG-GNRs than RD cells. GNRs at 250 µg/mL concentration decreased the cell

viability of MCF-7, MCF12-F and RD cells to 27±1% (Figure 4.11a), 28± 3.01

(Figure 4.11c) and 31±1.04% (Figure 4.11b), respectively. Therefore, the CTAB-

GNRs showed notable cytotoxicity in these cell lines. PEGylated GNRs have

significantly improved biocompatibility, as it had not much effect on cell viability of

MCF-7, MCF12-F and RD cells, even at a 500 µg/mL concentration. The CTAB-

GNRs at 500 µg/ml concentration killed more than 80% of MCF-7 (Figure 4.11a),

79% of MCF12-F (Figure 4.11c) and 75% of RD cells (Figure 4.11b). A dose-

73

dependent trend toward increase of cell death can be seen for three cell lines.

According to previous studies CTAB-GNRs can kill living cells in a sufficient dose

due to the presence of free CTAB (Hauck et al., 2008). So the coating of surfactant in

the bilayer would reduce the toxicity of these GNRs because surfactant desorption

would be retarded. A dramatic decrease in the toxicity of GNRs after polymerization

of the surfactant bilayer was observed in a previous finding (Alkilany et al., 2010).

Niidome and colleagues demonstrated CTAB-GNRs were extremely cytotoxic

at low concentration of 0.05 mM leaving only 20% of viable cells. But whenever

mPEG-SH was added to replaced CTAB, 95% of cell viability was observed

(Niidome et al., 2006b). PEGylated GNRs were more biocompatible than PSS-coated

GNRs (Rayavarapu et al., 2010). Replacing cytotoxic CTAB with mPEG-SH may

renders the colloid stability of the particles in cell culture medium (Rayavarapu et al.,

2010).

Some previous studies demonstrated the role of nanoparticle surface charge in

cytotoxicity (Hoffmann et al., 1997). Work of Alkilany presented 50% cell toxicity

with CTAB coated particles (Alkilany et al., 2009). PEG-modified GNRs showed a

negative surface potential (Rostro-Kohanloo et al., 2009), as well as low level of

cytotoxicity in vitro (Niidome et al., 2006a), and can therefore be used for biomedical

applications (Liao et al., 2006).

74

Compared to other metals, one of the important advantages of gold

nanomaterials is that the gold core itself is not inherently toxic (Lewinski et al.,

2008). However, synthesis of different shapes like GNRs by most commonly used the

seed-mediated growth method required cationic surfactant like CTAB, which is

cytotoxic even at very low concentrations (Alkilany et al., 2009). CTAB molecule is

necessary for the stability of GNRs as it forms a bilayer assembly on the surfaces of

GNRs (Murphy et al., 2005). The apparent cytotoxicity of the GNRs solution is

mainly attributed to free CTAB molecules desorption because of the dynamic nature

of CTAB bilayer (Connor et al., 2005; Alkilany et al., 2009). Complete removal of

the desorbed CTAB from the surface of GNRs will result in aggregation because of

the lack of a repulsive interaction between the GNRs. Due to this fact it is hard to

Figure 4.11: Dose-dependent cell viability; with cells exposed to increasing

concentrations of CTAB-GNRs and PEG-GNRs (a: upper left) Viability MCF-7 cells

(b: upper right) viability of RD cells (c: lower left) viability of MCF12-F cells. MCF-

7 and MCF12-F cells were more sensitive to the both CTAB-GNRs and PEG-GNRs

than RD cells. (d: lower right) Viability of HeLa cells after laser irradiation for 2, 4,

6, 8 and 10. 10 min exposure decrease the viability to 40% compared to untreated

control.

0

10

20

30

40

50

60

70

80

90

100

5 10 25 50 100 150 200 250 500 1000

Ce

ll vi

abili

ty (

%)

Concentrations (μg/mL)

CTAB-GNRs

PEG-GNRs

0

10

20

30

40

50

60

70

80

90

100

5 10 25 50 100 150 200 250 500 1000

Ce

ll vi

abili

ty (

%)

Concentrations (μg/mL)

CTAB-GNRsPEG-GNRs

0

10

20

30

40

50

60

70

80

90

100

5 10 25 50 100 150 200 250 500 1000

Ce

ll vi

abili

ty (

%)

Concentrations (μg/mL)

CTAB-GNRs

PEG-GNRs

10

30

50

70

90

2 4 6 8 10

Ce

ll vi

abili

ty (

%)

Time (min)

75

remove all of the unbound CTAB without aggregation of GNRs. For GNRs

biomedical applications, it is obligatory to modify it with coating agent to stabilize the

NRs, and reduce their cytotoxicity.

Among variety of compounds like PSS, phospholipids and polyelectrolyte,

PEG is considered to be more widely used polymer for surface coating due to its

biocompatibility and stability (Niidome et al., 2006a; Takahashi et al., 2006; Khanal

and Zubarev, 2007; Yu and Irudayaraj, 2007; Yu et al., 2007; Parab et al., 2009). PEG

stabilize nanoparticles, decrease their non-specific binding, increase their circulation

time with enhanced biocompatibility (Roberts et al., 2012; Jang et al., 2011;

Vigderman et al., 2012). We have extended cell-toxicity studies by applying PEG-

GNRs to a two different cancerous cell lines and one non-tumorigenic cell line.

PEGylated GNRs showed higher compatibility than bare GNRs.

Cell viability after photothermal treatment was recorded on PEG-GNRs

treated HeLa cells. Cells were exposed with an 808 nm NIR laser (power density =

1.5 W cm-2

for 2, 4, 6, 8, 10 min). Cell viability decreased in a time dependent

fashion. As we increased the laser exposure time to 10 min the cell viability reduced

to 40% (Figure 4.11d). The control experiment containing cells treated with

nanomaterials and without laser irradiation showed more than 90% viability.

4.5 HEMOLYTIC ASSAY

If nanoparticles interact with RBCs in the blood stream they can cause

hemolysis. Therefore hemolytic properties and interaction with RBCs are main

parameters for the biocompatibility of nanoparticles. CTAB-GNRs showed a

considerable hemolytic potential; as the values at higher concentrations are equal to

those of the positive control (0.2% Triton X-100). Results of current study indicated

76

hemolysis of over 70% for CTAB-GNRs at 1000 μg/mL (Figure 4.12). For both

samples the hemolysis percentage of RBCs increases in a dose-dependent manner.

The test experiments revealed good biocompatibility for PEG-GNRs as

quantified by concentration of hemoglobin in supernatant of GNPs-RBCs mixture by

monitoring absorbance intensity at 570 nm. PEG conjugated GNRs showed low

hemolysis compare to CTAB coated GNRs as shown in Figure 4.12 (Alkilany et al.,

2012). Marked reduction in percentage hemolysis for these samples is associated with

the existence of PEG, as surface of GNRs not comes in contact with the RBCs

because of completely passivated by PEG coating. Minimum hemolytic toxicity was

observed below 10 μg/mL for both samples. Previous reports suggested that gold

particles are more haemocompatible compared to silver and other; they also not

agglutinate erythrocyte and did not have precipitation properties (Asharani et al.,

2010).

4.5.1 Cell Viability of PBMCS

Cell viability of nanoparticles against PBMCs was compared to the control

group (Figure 4.13), and the percent viability was higher for PEG-GNRs (IC50 289.1

μg/mL) than CTAB-GNRs (IC50 151.1 μg/mL). Approximately 100% cell viability in

Figure 4.12: Percentage hemolysis of CTAB coated and PEG coated GNRs on

whole blood. Data presents hemolysis in dose dependent fashion.

0

20

40

60

80

100

5 10 25 50 100 150 200 250 500 1000

% H

aem

oly

sis

Concentrations (μg/mL)

CTAB-GNRs

PEG-GNRs

77

negative control (PBS) and 20% cell viability in positive control group (treated with

0.2% Triton ×100) confirmed the accuracy of the assay. Biocompatibility studies of

GNRs demonstrated that CTAB-GNRs have harmful effects on human PBMCs and

significantly affected cytotoxicity status (4.13).

The knowledge of GNRs interaction with human blood components is of

extreme importance if they are planned to be used for biomedical applications that

might require their intravenous (I.V) administration (Lin and Haynes, 2010;

Khlebtsov and Dykman, 2011). Blood is one of the most common translocation routes

to different organs. Nanoparticles possess fast systemic translocation rates on

exposure (Mills et al., 2009). Hemolysis can lead to several pathological disorders

like jaundice anemia etc (Dobrovolskaia et al., 2008). Therefore in vitro

hemocompatibility evaluation of nanoparticles is necessary parameter for early

preclinical development. We have adopted universal testing criteria for evaluating the

1 .0 1 .5 2 .0 2 .5 3 .0

0

2 0

4 0

6 0

8 0

1 0 0

L o g C o n c µ g /m l

Ce

ll V

iab

ilit

y%

P E G -G N R s

C T A B -G N R s

2 8 9 .1

1 5 1 .1

Figure 4.13: Percentage Viability of CTAB coated and PEG coated GNRs on

PBMCs. PEG coated GNRs show comparatively low cytotoxicity in dose

dependent fashion.

78

hemolytic properties of the CTAB-GNRs and PEG-GNRs at various concentrations in

human red blood cells (RBCs). Biological applications of GNRs have to consider

their interactions with the cells of the reticuloendothelial system (RES). RES

comprised of phagocytes and endothelial cells. Monocytes are the common progenitor

of macrophages and dendritic cells (Bartneck et al., 2012).

4.6 NANOPARTICLES FOR GENE KNOCKDOWN EXPERIMENT

4.6.1 Gold Nanospheres

Gold nanospheres (GNS), with diameters of ~30 nm, were synthesized by an

optimized citrate reduction method (Kimling et al., 2006). Gold salt was reduced by

adding 18 mM sodium citrate on continuous stirring and heating (~ 100°C). As the

sodium citrate solution was added to the gold solution, color changed from colorless

to black dark purple red wine, indicating the successful formation of GNS.

UV-Vis spectroscopy of synthesized nanoparticles showed peak absorption intensity

at λmax = 530 nm (Figure 4.14). The prepared GNS were characterized using, UV-

Vis spectroscopy, TEM and DLS (4.14). Extinction coefficient used for these particles

was 3.0x109(Darbha et al., 2008).

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Figure 4.14: Gold nanoparticles characterization (a) UV-Vis spectrum of GNS with

specific SPR of 530 nm (b) TEM of GNS shows homogenous population.

79

4.6.2 Gold Nanorods

Gold nanorods (GNRs) with aspect ratios of 3.4 were synthesized using the

seed-mediated growth method (Nikoobakht and El-Sayed, 2003) as optical switches

for gene knock down. The synthesized material showed peak absorption intensity at

λmax = 785 nm and TEM analysis showed monodispersed GNRs with high yield

(Figure 4.15). The extinction coefficient used for these GNRs was 1.9x108(Darbha et

al., 2008).

The size of these synthesized GNRs was particularly designed for use in

optical experiments. Rod shaped nanoparticles were specifically selected for ONCOS

owing to their stability and biocompatibility (Tang et al., 1998; Loo et al., 2005). Rod

shape geometry favors proficient photothermal conversion that triggers oligos release.

Plasmon resonant wavelength of GNRs can be adjusted to a light source that can

activate optical switches by selecting their aspect ratio. A variety of different optical

switches can remotely be activated in live cells by carefully selecting their size,

geometry and tuning their optical transmission frequency according to light source.

Additionally, the GNRs can carry hundreds of oligos per particle due to large surface

400 500 600 700 800 900 10000.0

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Figure 4.15: Gold nanoparticles characterization (a) UV-Vis spectrum of GNRs with

specific LSPR of 785 nm (b) TEM of GNRs shows homogenous population.

80

area and can down-regulate gene expression by the optical activation of a single GNR

(Lee et al., 2009a).

4.7 CONJUGATION OF COX-2 OLIGONUCLEOTIDES

A 10% PEGylated optical switches, GNRs with aspect ratio 3.5 and λmax =

785 nm were used for oligos conjugation. Fluorescence labeled thiol reduced 21 mer

DNA oligos directed to the 5′ end of COX-2 mRNA was purified using NAP-5

column to separate non reduced and inactivated oligos. Since 90% of the GNRs

surface was coated with surfactant that gave overall positive surface charge from the

quaternary ammonium surfactant head-group (Nikoobakht and El-Sayed, 2003) so

negatively charged, fluorescence labeled DNA oligos attached to the CTAB-GNRs,

allowing visualization of complex on fluorescent microscope.

The first step in signifying biocompatible nanocomposite involved 10%

surface covering with 1mM methoxy PEG5000thiol. After synthesis, excess CTAB

from the GNRs was removed by centrifugation and then were PEGylated as described

earlier.

4.7.1 Characterization of Bioconjugation

Concentration of GNRs and number of oligos loaded on per particle was

calculated by Beer Lambert Law using UV-visible spectroscopy. (Haiss et al., 2007).

4.16: Positively charged CTAB coated GNRs (Blue color) with negatively

charged FAM labeled 21 mer DNA oligos (Green color).

81

Nanoparticle concentration calculated was 11.84 nM. A 100-fold dilution of

lyophilized fluorescent labeled DNA strand was prepared in DNase/RNase free water.

Absorbance at 260 nm (A260) of the diluted oligonucleotides was recorded. DNA

concentration in the stock solution was calculated using a rearrangement of Beer’s

Law

co = (A260 ×100)/(εo× b)

Where 100 is the dilution factor, b is the path length of the cuvette (typically 1

cm), and εo is the extinction coefficient of the oligonucleotide at 260 nm. The number

of oligonucleotides per particle for each sample was calculated by dividing the

concentration of fluorescence labeled DNA oligonucleotides by the concentration of

nanoparticles. The DNA conjugated gold nanoparticle concentration calculated by

above method was observed about 6.2 nM. Fluorescence based quantification results

are consistence with UV-Vis results. KCN dissolution method shows 225 DNA

Strands per particle in case of GNS and 385 DNA Strands per particle in case of

GNRs (4.17b).

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Figure 4.17: (a) UV-Vis Spectra before and after FAM labeled DNA conjugated gold

nanospheres (GNS). Peak absorption intensity for non conjugated GNS showed at 530

nm (Black), FAM labeled ssDNA shows characteristic peak at 260 nm and other at 490

nm for FAM dye (Red), FAM labeled ssDNA conjugated with GNS showed peak

absorbance at 269 nm (DNA) and a broad peak at 502 nm, presenting combination of

particle and FAM (Blue). (b) After KCN dissolution GNS peak disappeared and only

DNA (260 nm) can be seen.

a b

83

4.8 INTRACELLULAR LOCALIZATION AND UPTAKE STUDIES

4.8.1 Live Cell Dark Field Imaging

For cellular uptake studies, HepG2 cells were transfected using Escort TM

IV

with fluorescence labeled DNA conjugated GNRs for 24 h and imaged using a dark-

field microscopy using halogen lamp. The system consists of an Olympus IX70

microscope equipped with a homemade live-cell imaging chamber and a CCD camera

and a Fiber-Lite MI-150 Illuminator. A heating fan was set-up 20 cm from the stage

to maintain the temperature of the samples at 37°C. A dark field condenser was

secured underneath the microscope stage for imaging.

FAM labeled DNA oligos without nanoparticles were also incubated with

HepG2 cells for same time and cells alone were also used as control. Dark field

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Figure 4.18: UV-Vis Spectra before and after FAM labeled DNA conjugated gold

nanorods (GNRs). (a) Peak absorption intensity for non conjugated GNRs showed at

785 nm (Black), FAM labeled ssDNA shows characteristic peak at 260 nm and other

at 490 nm for FAM dye (Red), FAM labeled ssDNA conjugated with GNRs showed

peak absorbance at 269 nm (DNA) and peak at 780 nm, presenting LSPR of GNRs

and peak at 500 shows combination of TSPR of GNRs and FAM (Blue) (b) After

KCN dissolution GNRs peak disappeared and only DNA (260 nm) can be seen.

a b

84

images shows the cells incubated with nanoparticles revealed strong light scattering as

compared to control samples. Images shows greater cellular uptake for DNA

conjugated nanoparticles then particles alone. FAM labeled ssDNA and dsDNA

conjugated with GNRs show brighter images prove greater scattering by the presence

of higher number of nanoparticles consequently, a higher relative uptake (Figure

4.19). DNA conjugation helps in cell penetration and uptake. Gold nanoparticles show

enhanced Rayleigh (elastic) scattering owing to their LSPR property resulted at the

plasmon resonance frequency. The light scattering of nanoparticles mainly depend

upon their shape and size (Jain et al., 2007; Jain et al., 2008). Gold nanospheres and

large size gold nanorods possess strong scattering therefore can be utilized in

biological imaging and as sensing agent. They have several advantages such as they

have stronger scattering efficacy compare to fluorophores and also not sensitive to

photo-bleaching (Gibbs-Flournoy et al., 2011).

85

In the next experiment MDA-MB-231 cells were transfected with DNA

conjugated gold nanospheres and cellular uptake was recorded by dark field

B

D

Figure 4.19: Live cell dark field imaging of HepG2 cells acquired by IX70 microscope

using halogen lamp (a: upper left) untreated cells showing some scattering (b: upper right)

cells with nanoparticles, GNRs shows good scattering and cellular uptake (c: lower left)

cells incubated with FAM labeled ssDNA conjugated with GNRs, brighter images shows

greater scattering by particles consequently, better uptake (d: lower right) cells incubated

with FAM labeled dsDNA conjugated with GNRs, much brighter images proves greater

scattering by the presence of higher number of nanoparticles and cells show a higher

relative uptake of dsDNA conjugated GNRs when compared to cells alone and cells with

nanoparticles

86

microscopy using the same procedure. The results revealed that gold nanospheres

showed greater scattering as compared to gold nanorods.

Figure 4.20: Live cell dark field imaging of MDA-MB-231 cells acquired by IX70

microscope using halogen lamp (a: upper left) untreated cells showing some scattering (b:

upper right) cells with nanoparticles, GNS shows good scattering and cellular uptake (c:

lower left) cells incubated with FAM labeled ssDNA conjugated with GNS, brighter images

shows greater scattering by particles consequently, better uptake (d: lower right) cells

incubated with FAM labeled dsDNA conjugated with GNS, much brighter images proves

greater scattering by the presence of higher number of nanoparticles and accordingly, greater

cellular uptake due to more surface coverage of particles with DNA strands.

87

4.8.2 Spectrophotometric Measurements of Relative Uptake

The relative uptake of DNA conjugated and non conjugated gold nanoparticles

were determined using UV-Vis spectroscopy. The optical density of the transferred

media and nanoparticle solutions showed minimum absorbance intensities at the SPR

peak of the nanoparticles (λmax = 530 nm), for FAM dye (λmax = 490 nm) and for

DNA (λmax = 260 nm) (Figure 4.21). This fact indicated higher cellular uptake of

treatment solutions after 24 h of incubation. After the removal of treatment solutions,

cDMEM was added and the cells were observed under inverted microscope. Images

presents good cellular uptake of nanoparticles conjugated with DNA (Figure 4.21).

Figure 4.21: MDA-MB-231 cells were incubated with DNA conjugated GNS and imaged

using inverted microscope with Nikon D200 digital camera to observe cellular uptake (a:

upper left) cells as a control sample (b: upper right) cells with transfection agent and GNS

(c: lower left) cells with dsDNA conjugated GNS (d: lower right) cells with dsDNA

conjugated GNS presenting better cellular uptake

88

The average optical density (ODAVG) for each experimental sample was

determined and then ODAVG from media only and cells only were subtracted from the

ODAVG of the nanoparticle solutions that were incubated with (ODNP+) and without

(ODNP-) cells. Once these values were obtained, the OD of the nanoparticles taken up

by the cells (ODuptake) was found by subtracting the ODNP+ from the ODNP- for all

samples. The ODuptake value is then divided by the background corrected ODNP- value

and multiplied by 100 to attain the relative percent of nanoparticle uptake.

Standard deviations for each step of mathematical analysis were determined

using error propagation formulas. Experiments were independently repeated at least

three times and averages from each independent experiment were used to obtain the

mean ± standard error of the mean (SEM). The percentage cellular uptake of GNS

with transfection agent is about 38% whereas, with ssDNA conjugated GNS the

uptake increased to 56%, and it increased more to 67% (Figure 4.22) when dsDNA

conjugated GNS were used. Increased uptake is attributed to the presence of lipid

complex (transfection agent) and negatively charged DNA oligos.

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Figure 4.22:Cellular uptake measurements (a: left) UV-Vis spectrum of MDA-MB-231cells

supernatant after 24 h of incubation with nanoparticles treatment solutions (b: right)

percentage cellular uptake of DNA conjugated and non conjugated gold nanoparticles shows

10 fold increased uptake with DNA conjugated GNS.

89

In case of gold nanorods the optical density of the transferred media and

nanoparticle solutions showed minimum absorbance intensities at the LSPR peak of

the nanoparticles (λmax = 785 nm), for FAM dye (λmax = 490 nm) and for DNA

(λmax = 260 nm) (Figure 4.23). This fact indicated higher cellular uptake of treatment

solutions after 24 h of incubation.

A B

Figure 4.23: MDA-MB-231 cells were incubated with DNA conjugated GNRs and

imaged using inverted microscope with Nikon D200 digital camera to observe cellular

uptake (a: upper left) cells as a control sample (b: upper right) cells with transfection

agent and GNRs (c: lower left) cells with dsDNA conjugated GNRs (d: lower right)

cells with dsDNA conjugated GNRs presenting better cellular uptake.

90

The percentage cellular uptake of GNRs with transfection agent is about 26%

whereas, with ssDNA conjugated GNRs the uptake increased to 50.3%, and it

increased more to 60.1% (Figure 4.24) when dsDNA conjugated GNRs were used.

Increased uptake is attributed to the presence of lipid complex (transfection agent) and

negatively charged DNA oligos.

4.8.3 Cellular Uptake by Confocal Microscopy

Cellular uptake of nanocomposite was also confirmed by confocal microscopy.

Confocal images were taken using a Zeiss LSM 700 multi-photon excitation confocal

microscope with 405 (DAPI) and 488 nm (FITC) excitation sources. Optical images

acquired using nanoparticle treatment solutions without cells were used as reference

samples. Confocal results shows good cellular uptake of DNA conjugated

nanoparticles (Figure 4.25).

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Figure 4.24: Cellular uptake measurements (a: left) UV-Vis spectrum of cell supernatants

after 24 h of incubation with nanoparticles treatment solutions (b: right) percentage

cellular uptake of DNA conjugated and non conjugated gold nanoparticles shows

relatively increased uptake with DNA conjugated GNRs.

91

Figure 4.25: Confocal images with 488 nm (FITC) excitation sources presenting

nanocomposite cellular uptake in HepG2 cells (a) cells with FAM labeled DNA

conjugated GNRs (b) cells with transfection agent and GNRs (c) cells with FAM

labeled DNA and transfection agent (d) GNRs without cells (e) FAM labeled DNA

and transfection agent without cells (f) Transfection agent and FAM labeled DNA

conjugated GNRs without cells.

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4.9 LASER POWER, WAVELENGTH SPECIFICITY, TEMPERATURE AND

EXPOSURE TIME

The first indicative step of the efficacy of optical experiments implicated

authenticating the wavelength specificity of laser to remotely control photothermal

release of oligos. FAM labeled double-stranded oligos conjugated GNRs were

immobilized onto a glass surface. All the experiments were done on attached GNRs

on the glass surface. In the nanocomposite, the antisense oligos were fluorescently

labeled hence, release was visually monitored by recording fluorescence intensities.

Wavelength specificity was measured using two different lasers one at peak

optical absorption and other outside peak optical absorption 808 and 685 nm

respectively for photo-triggered oligos dehybridization. The entire nano-complex was

irradiated using each of above laser and ONCOS based oligos release was visualize

by inverted fluorescence microscope. Photo-triggered oligos dehybridization was

recorded by measuring the fluorescence intensity of an area away from the conjugated

GNRs. The initial fluorescence intensity was set to 0. The antisense strand

dehybridized from GNRs conjugated sense strand at their melting temperature

consequently, increased fluorescence intensity was observed. Current study findings

showed better release of oligos from the surface of GNRs when illuminated with 808

nm laser (Figure 4. 26).

In order to photo-toxicity, we optimized laser power for ONCOS. Immobilized

fluorescently labeled antisense conjugated GNRs were irradiated with different laser

power densities 40, 60, 80, 120 and 160 mW/cm2. Figure 4.26 shows fluorescence

intensity rose with increasing power densities of laser, consequently, increased

photothermal efficiency. The laser power 120 mW/cm2 shows better release without

increasing surrounding temperature above threshold. On the basis of these preliminary

93

experiments laser wavelength of 808 nm with power density of 120 mW/cm2 was

selected for photothermal and ONCOS experiment in order to achieve better release

along with minimal photo-damage to the surrounding healthy cells.

4.10 COX-2 RNA INTERFERENCE USING ANTISENSE

OLIGONUCLEOTIDES

Prolonged COX-2 mRNA stability is one of the main reasons for resistance

against anticancer drugs in COX positive cancers. Non specific inhibition of COX-2

causes several adverse effects. We investigated localized suppression of COX-2 gene

at the site of action in HepG2 and MDA-MB-231 cell lines. The cells were treated

with the various concentrations of nanoparticle treatment solutions using Escort TM

IV

transfection agent when cell confluency reached 50-60% for 48 and 72 h. Afterward

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60 mW/cm2

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160 mW/cm2

Figure 4.26: Experimental characterization of ONCOS activation (a: left) FAM labeled

DNA conjugated GNRs carriers based on wavelength specificity, a laser at the peak

optical absorption (808 nm) was compared to a laser outside the peak optical absorption

(658 nm) for photothermal oligos release. (b: right) Fluorescence intensity versus time

of exposure plots comparing fluorescence intensity with increasing power densities of

laser, consequently, increased photothermal efficiency.

94

whole protein was extracted from treated and untreated cells. After quantification

protein was separated on SDS-PAGE, COX-2 protein expression was checked by

western blotting after RNA interference.

4.11 PROTEIN EXPRESSION ANALYSIS

4.11.1 Protein Extraction and Quantification

Over the past few decades, solvent precipitation methods have been in use for

protein extraction, which efficiently removes the non-protein materials like cell

organelles, nucleic acids and cellular debris. These methods are usually used to

achieve higher protein yield, facilitating protein based studies. The first step in protein

extraction from the tissue is selection of an optimal extraction buffer to solubilize cell

membranes with better efficiencies (Schuck et al., 2003).

Sonication was done to rupture cell membrane in order to get the maximum

amount of protein from every sample. Initially, the output of these extraction

experiments was observed by measuring the concentration of extracted protein by

Bradford assay and by recording the intensity of prepared and separated protein bands

on SDS-PAGE gels, stained with coomassie blue (R-250). The protein concentration

of the cell lysate samples for each experiment was calculated by subtracting the

absorbance of the blank standard (i.e.0 μg/mL) from each BSA standard and cell

lysate samples to get the corrected absorbance (ODcorrected). Subsequently, the

ODcorrected values were plotted vs. their corresponding BSA standard concentration. A

linear trend line equation (y = mx + b) was then fit to the data and used to calculate

the diluted protein concentrations in the cell lysate samples. The protein concentration

recorded for various experiments is given in Tables below. Results of quantification

shows almost equal amount of protein was extracted each time.

95

Rapid and sensitive methods are frequently required for the quantification of

purified protein. Herein we used standard Bradford assay (Bradford, 1976) which

utilize Coomassie Brilliant Blue G-250 dye. The dye exists in three forms: cationic

(brownish red), neutral (green), and anionic (blue). Under acidic conditions, the dye is

primarily in the doubly protonated red cationic form (λmax = 470 nm). When the dye

binds to protein, it is converted to a stable un-protonated blue form (λmax = 595 nm)

(Sedmak and Grossberg, 1977). The resultant protein-dye complex has a high

extinction coefficient consequently leading to great sensitivity in measuring the

protein concentration. The binding of the dye to protein is a very quick process

(roughly 2 min), and the protein-dye complex remains dispersed in solution for a

fairly long time (approximately 1 hr), therefore making the procedure very fast though

not requiring critical timing for the assay (Reisner et al., 1975).

96

Figure 4.27: Corrected OD values at 595 nm were plotted against known BSA standard

concentrations. A linear fit was used to determine the diluted protein lysate concentration.

Protein stock concentration was then determined by multiplying with dilution factor. (a)

Linear standard curve of known concentrations of BSA showing gradual increase in

absorbance intensity with increase in protein concentration. (b) Standards and samples in

96 well plate presenting change in color depending upon protein concentration. Lighter to

deep blue color indicates protein concentration from lower to higher.

Table 4.2: protein concentration of cell lysate after transfection with nanocomposite (DNA

conjugated gold nanospheres).

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Figure 4.28: Corrected OD values at 595 nm were plotted against known BSA standard

concentrations. A linear fit was used to determine the diluted protein lysate concentration.

Protein stock concentration was then determined by multiplying with dilution factor. (a)

Linear standard curve of known concentrations of BSA showing gradual increase in

absorbance intensity with increase in protein concentration. (b) Standards and samples in

96 well plate presenting change in color depending upon protein concentration. Lighter to

deep blue color indicates protein concentration from lower to higher.

Table 4.3: protein concentration of cell lysate after transfection with nanocomposite (single

and double strand) DNA conjugated gold nanorods.

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Figure 4.29: Corrected OD values at 595 nm were plotted against known BSA standard

concentrations. A linear fit was used to determine the diluted protein lysate concentration.

Protein stock concentration was then determined by multiplying with dilution factor. (a)

Linear standard curve of known concentrations of BSA showing gradual increase in

absorbance intensity with increase in protein concentration. (b) Standards and samples in

96 well plate presenting change in color depending upon protein concentration. Deep to

light blue color indicates protein concentration from higher to lower.

Table 4.4: protein concentration of cell lysate after transfection with single and double

stranded and control DNA.

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Figure 4.30: Corrected OD values at 595 nm were plotted against known BSA standard

concentrations. A linear fit was used to determine the diluted protein lysate concentration.

Protein stock concentration was then determined by multiplying with dilution factor. (a)

Linear standard curve of known concentrations of BSA showing gradual increase in

absorbance intensity with increase in protein concentration. (b) Standards and samples in

96 well plate presenting change in color depending upon protein concentration. Lighter to

deep blue color indicates protein concentration from lower to higher.

Table 4.5: protein concentration of cell lysate after transfection with nanocomposite (DNA

conjugated gold nanorods) and illuminated with 808 nm Laser for 1 mint.

100

4.11.2 Protein Separation

After the protein lysate quantification, equal amount of protein (25 µg) was

loaded on 12% SDS-PAGE to separate the proteins on the basis of molecular weight.

Gel was then stained with Coomassie Blue for 30 min on shaking images were taken

after destaining the gel. Results shows quite lighter band for dsDNA conjugated with

gold nanorods and illuminated with NIR laser (Figure 4.31).

Figure 4.31: Example of coomassie stained gel. (a: left) lane 1 contain molecular weight

standard, Kaleidoscope, lane 2 and 3 has the extracted protein from untreated HepG2 cells,

lane 4 and 4 has the extracted protein from untreated MDA-MB-231 cells. (b: middle)

molecular weight standard, Kaleidoscope (c: right) lane 1 contain molecular weight

standard, Kaleidoscope, lane 2 has untreated cells lane 3 cells containing gold

nanoparticles with laser exposure, lane 4 cells with ssDNA conjugated gold nanoparticles

with laser exposure, lane 5 cells with dsDNA conjugated gold nanoparticles illuminated

with laser, lane 6 contain COX-2 positive lysate. Distinct band at approximately 72-75 kDa

(for COX-2 protein) is obvious in all samples. Comparatively band intensity for lane 6

sample is lighter than other, indicating negative expression of COX-2 protein after laser

exposure.

101

4.11.3 Western Blotting

In order to quantify COX-2 protein expression after RNA interference proteins

were transferred from SDS-PAGE gel to nitrocellulose membrane and stained with

COX-2 specific primary antibody. Previous studies showed high basal level of COX-2

in MDA-MB-231 (Singh et al., 2006). To identify optimal time point for mRNA

inhibition of COX-2, experiment was planned with three different time points (24, 48

and 72 h).

We had examined the degree of COX-2 knockdown at the protein level in

MDA-MB-231 cells using GNS-based nanocarriers with and without COX-2

antisense oligos complexation. The relative density for COX-2 expression has been

recorded by densitometric analysis of the blot and resultant values were compared

against the β-actin housekeeping gene to normalize protein loading. There was no

significant change in protein expression of COX-2 after 24 h. As Western blot

analysis demonstrates (Figure. 4.32), the COX-2 protein expression level was reduced

by transfection of MDA-MB-231cells with nanocarriers complexed with COX-2

antisense oligos (i.e., ssDNA-GNS) having silencing efficacy of 38% compared to

their untreated counterpart. A small reduction in COX-2 protein level was recorded

for ssDNA (12%), importantly, no significant difference was observed for other tested

samples, transfection agent, GNS and control DNA sequence. However, COX-2

protein was effectively silenced only by ssDNA-GNS. The results were further

analyzed after 72 h of transfection. The COX-2 protein expression level was

significantly reduced by transfection (72 h) of MDA-MB-231 cells with nanocarriers

conjugated by COX-2 antisense oligos (i.e., ssDNA-GNS) (72%) compared to their

untreated counterpart. A significant reduction in COX-2 protein level was recorded

for ssDNA (34%), no significant difference was observed for other tested samples,

102

transfection agent, GNS and control DNA sequence (Figure 4.32). This result is

consistent with the previous results that gold nanoparticle-based carriers possess an

excellent capability of entering cancer cells and delivering DNA for gene knockdown

(Zhang et al., 2011). Gold nano rods conjugated siRNA was successfully delivered to

BON cells to block ASCL1 expression (Xiao et al., 2012).

According to our results ssDNA (antisense strand) conjugated with GNS

showed much better COX-2 silencing compared to unbound ssDNA. In

conventional antisense technique, oligos were used to hybridize with target messenger

RNA to knockdown the expression of the resultant proteins (Draz et al., 2014). In

vitro Study demonstrated comparatively higher resistance of the nanoparticle

conjugated antisense oligos to intracellular DNase then free oligos (Rosi et al., 2006).

Successful inhibition of GFP in prostate cancer cells was carried out using

GNPs conjugated with siRNA (Lee et al., 2008). Potential oncogene such as cell-

cycle kinase were significantly down-regulated by GNPs conjugated siRNA in breast

cancer cells (Song et al., 2010). Gold nanorods complex with siRNA was used to

reduce galectin-1gene expression. Successful inhibition of target gene was achieved

using nanoparticles conjugated with corresponding sequences (Reynolds et al., 2012).

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Figure 4.32: Western blot analysis of COX-2 protein expression in MDA-MB-231

after RNAi silencing by DNA conjugated GNS. (a) COX-2 protein expression was

measured after 48 h of transfection to elucidate a possible gene knockdown by

nanocomposite (b) COX-2 protein expression was significantly reduced after 72 h of

transfection with ssDNA conjugated GNS nanocomposite. (c) Relative protein density

after 48 h of transfection with nanocomposite illustrate cells treated with ssDNA-GNS

shows significant low expression of COX-2 compared to untreated cells. (d) Relative

protein density after 72 h of transfection with nanocomposite illustrate cells treated

with ssDNA-GNS shows significant low expression of COX-2 compared to untreated

cells. The protein level is low after 72 h compare to 48 h. The β-actin was used as the

protein control in all experiments. Data is expressed as mean ± SE of three

independent experiments. Statistical significance is indicated by * (p<0.05).

4.12 COX-2 RNA INTERFERENCE USING ONCOS

The nanocomposite comprising COX-2 oligos conjugated GNRs were

incubated with cultured MDA-MB-231 cells. The cells were exposed to NIR laser

after 12 h of transfection and then incubated for 72 h. the set of control experiments

COX-2

β actin

48 h 72 h

a b

c d

104

were designed in order to make sure optical release was the main factor that cause

gene knockdown. The control experiments include (1) cells treated with single (20%)

and double stranded DNA (7%) conjugated GNRs without laser (2) cells s irradiated

with NIR laser without GNRs (0%) and (3) cells with non-specific sequence

conjugated GNRs followed by NIR exposure (0%) (Figure 4.33). Cells activated by

ONCOS express significantly low level of COX-2 compare to all control samples.

This fact proves that ONCOS is responsible for targeted COX-2 gene knock down.

Quantitatively analysis of COX-2 inhibition was recorded by measuring relative

protein density using Western blot technique. Figure 4.33 shows that the significant

low level (86%) of COX-2 protein in cells treated with ONCOS compare to their

untreated counterpart.

105

Figure 4.33: Western blot analyses of MDA-MB-231 cells exposed to treatment

solutions of GNRs and DNA for photothermal COX-2 gene interference. (a & b)

COX-2 protein expression was significantly reduced after 72 h of transfection with

dsDNA conjugated GNRs and laser irradiation. (c) Relative protein density illustrates

cells treated with dsDNA-GNRs followed by NIR laser irradiation show complete

loss of COX-2 protein expression after 72 h of transfection. Significantly low

expression of COX-2 has been observed with ssDNA-GNRs followed by laser

illumination compared to untreated cells. (d) Relative protein density after 72 h of

transfection with nanocomposite and DNA alone without laser exposure illustrate

cells treated with ssDNA-GNRs show significant low expression of COX-2 compared

to untreated cells. The β-actin was used as the protein control in all experiments. Data

is expressed as mean ± SE of three independent experiments. Statistical significance

is indicated by * (p<0.05).

Same experimental setup was design for COX-2 gen interference by ONCOS in

HepG2 cells. Cells were illuminated with focused NIR laser after 12 h of

a b

c d

COX-2

β-actin

106

internalization of conjugated GNRs within cultured HepG2 cells then incubated for 72

h. The set of control experiments were conducted as mentioned above. Protein

expression level was then quantified by analyzing western blot membrane using

ImageJ. Cells activated by ONCOS express significantly low level of COX-2 compare

to all control samples. This fact proves that ONCOS is responsible for targeted COX-

2 gene knock down in HepG2 cells. Quantitative analysis of COX-2 inhibition was

recorded by measuring relative protein density using Western blot technique. Figure

4.34 shows that the significant low level (93%) of COX-2 protein in cells treated with

ONCOS compare to cells treated with conventional gene interference technique.

107

Figure 4.34: Western blot analyses of HepG2 cells exposed to treatment solutions of

GNRs and DNA for photothermal COX-2 gene interference. (a & b) COX-2 protein

expression was significantly reduced after 72 h of transfection with dsDNA

conjugated GNRs and laser irradiation. (c) Relative protein density illustrates cells

treated with dsDNA-GNRs followed by NIR laser irradiation show complete loss of

COX-2 protein expression after 72 h of transfection. Significantly low expression of

COX-2 has been observed with ssDNA-GNRs followed by laser illumination

compared to untreated cells. (d) Relative protein density after 72 h of transfection

with nanocomposite and DNA alone without laser exposure illustrate cells treated

with ssDNA-GNRs show significant low expression of COX-2 compared to untreated

cells. The β-actin was used as the protein control in all experiments. Data is expressed

as mean ± SE of three independent experiments. Statistical significance is indicated

by * (p<0.05).

a b

c d

COX-2

β-actin

108

On illumination GNPs photo-thermally release their cargo to the site of action.

Oligos directed to gene of interest has been photo-thermally dehybridized from gold

nanoshells (Barhoumi et al., 2009) and gold nanoprisms (Jones et al., 2009) using

continuous-wave illumination.

By systematically perturbing with extracellular stimuli, specific internal

connections can be mapped and the resulting cell function can be observed in

response to the single environmental change. GNPs enable “nanoplasmonic control”

of genetic activities with sequence-specificity and spatiotemporal resolution. GNPs

carrying genetic cargo are internalized in living cells by endocytosis. While attached

to their carriers, the cargo is temporarily “inactive”. The cargo is also protected from

degradation by nucleases (Rosi et al., 2006). Laser irradiation of specific wavelength

“activate” cargo by photo-thermally disrupting encapsulating endosomes (Febvay et

al., 2010) and photo-thermally releasing free cargo into the cytosol. In this way,

endogenous intracellular genes expression can be blocked.

Nanoplasmonic induction of exogenous EGFP expression has been

demonstrated in human HeLa cervical carcinoma cells (Chen et al., 2006).

Nanoplasmonic based inhibition of endogenous expression of ERBB2 gene by

antisense DNA has been demonstrated in human BT474 breast carcinoma cells (Lee

et al., 2009a). The siRNA can be used for the degradation of specifically targeted

mRNA; however, degradation is through a different mechanism compared to

antisense DNA. Nanoplasmonic silencing of EGFP expression using siRNA has been

shown in mouse endothelial C166 cells stably expressing EGFP (Braun et al., 2009).

Nanoplasmonic silencing of endogenous genes using siRNA has also been

demonstrated in vivo. Thiol-modified siRNA targeting NF-κΒ p65 are covalently

bound to hollow gold nanoshells, internalized in HeLa cells, and transplanted in mice

109

as xenografts. Pulsed illumination is used to photo-thermally melt gold hollow

nanoshells, thereby destabilizing the thiol-gold bond and releasing the siRNA(Lu et

al., 2010) .

4.13 MEASUREMENT OF PROSTAGLANDIN-E2 (PGE2) PRODUCTION

RATE BY ELISA

The prostaglandin E2 (PGE2) is a primary product of the COX-2 enzyme,

which represents COX-2 activity. As a further demonstration of the efficiency of the

COX-2 knockdown mediated by ONCOS, we also found a significant decrease (0.9

pg/mL) of PGE2 production in HepG2 cells after treatment with dsDNA conjugated

GNRs followed by 808 nm laser exposure compared to untreated counterparts

(7pg/mL) (Figure 4.35b). Similarly, cells treated with single strand conjugated GNRs

followed by laser illumination showed comparatively reduced level of PGE2 (3.6

pg/mL). Cells treated with conventional RNA interference showed significantly

higher level PGE2 production compared to cells treated with ONCOS. Since we

found that the transfection of exogenous synthetic oligos directed to the 5 prime end

of COX-2 mRNA is capable of blocking the expression of endogenous COX-2

expression in HepG2cells, the following aim was to demonstrate whether an

endogenous and constitutive production of PGE2 is reduced with COX-2 knockdown

110

Figure 4.35: HepG2 cells were treated with the DNA conjugated nanocomposite for

72 h and PGE2 levels in the culture medium were measured using ELISA. (a) Cells

were not exposed to laser (b) cells were exposed to NIR (808 nm) laser after 12 h of

nanocomposite treatment shows significant reduction in PGE2 level in dsDNA

conjugated GNRs. Results were expressed as mean± S.E.M. from at least three

independent experiments*(P<0.01).

Previous studies demonstrated that the COX-2 selective inhibitor decreased

the level of PGE2 in animal model. The PGE in hepatocytes increased the levels of

proteins involved in apoptotic/anti-apoptotic pathway thus, modulating the normal

cell death consequently, leads toward various malignancies. Liu et al described the

combination treatment of COX-2 inhibitor and a Chinese herb to decrease

prostaglandin level in breast cancerous cells (Liu et al., 2011).

4.14 APOPTOSIS/NECROSIS ASSAY ON FLOW CYTOMETRY

After successful gene knockdown by ONCOS, samples were also analyzed for

apoptosis/necrosis on flow cytometer. The purpose of the experiment was to check

weather COX-2 knockdown in HepG2 cells render these cells sensitive towards

apoptosis or cell death. The conjugated optical switches were when internalized into

a b

111

the cells and illuminated with NIR (808 nm) laser. Samples, consisting of at least

10,000 cells, were immediately run on a BD LSR II flow cytometer with a 488 nm-

excitation laser and fluorescence detection in the R-Phycoerythrin (PE) and

Fluorescein (FITC) channels. The flow cytometric analysis revealed n significant

level of apoptotic cells in case of double strand DNA conjugated GNRs followed by

NIR exposure. At least two tubes were recorded for each treatment sample. Cells

were separated into four quadrants (Q1-4), with Q1, Q2, Q3 and Q4 representing

necrotic, necrotic, apoptotic and viable cell populations, respectively. In control

(untreated cells, DNA without nanoparticles, nanoparticles alone and scrambled

sequence) samples, the whole population of cells present in Q4 quadrant, representing

viable population. A significant cells population treated with ONCOS is present in Q2

(0.8%) and Q3 (1.02%) compare to untreated cells Q2 (0.06%) and Q3 (0.1%) (Figure

4.36).

112

The COX-2 knockdown thus, sensitized cells towards cell death through either

apoptosis or necrosis. The results of the current study revealed that high level of

COX-2 protein may interfere with hallmarks of apoptotic pathway by changing the

level of pro-apoptotic and anti-apoptotic proteins in downstream regulation.

Suppression of COX-2 gene at translational level sensitized these cells towards cell

death. The fact is also proven by earlier reports (Mayoral et al., 2008).

Control (Cells)

ssDNA

GNR-dsDNAGNR-ssDNA

dsDNA GNRs

GNR-ssDNA + Laser GNR-dsDNA + Laser Laser

Figure 4.36: Flow cytometric analysis of apoptosis/necrosis after COX-2 RNA

interference.

113

SUMMARY

In this dissertation, on-demand gene silencing of endogenous intracellular

COX-2 gene using antisense DNA was demonstrated. Biologically functional cationic

phospholipid-gold nanoplasmonic carriers that are compatible with and capable of

carrying siRNA cargo were fabricated. Current optimized conditions have great

potential for controlled production of monodispersed and tunable GNRs. Fine tuning

of LSPR can be achieved by varying silver ion and seed concentration. LSPR spectral

response against various parameters can be used for quantitative analysis of GNRs

instability via PIP values. We have optimized the conditions for long shelf life,

thermal exposure and GNRs stability at physiological conditions, making them a good

candidate for a wide array of biomedical applications.

A transcriptional pulse of target gene expression was generated using COX-2

interfering oligos conjugated nanoplasmonic gene switches of desirable aspect ratios

to selectively and temporally manipulate the activities of repressors and activators

upstream from the target gene.

The current findings substantiate the major role of nanoparticle based RNA

interference technology owing to its high efficiency and biocompatibility. Selectively

targeting an oncogene is one of the novel techniques in cancer management.

Cyclooxygenase-2 is potential target in cancer therapy. Results reported here indicate

an easy-to-use, powerful and high selective nanoparticle based optical control method

to knockdown COX-2 gene in a stable and long-lasting manner, in hepatocellular

carcinoma and breast cancer cells. Furthermore, they open up the possibility of an in

vivo application of this anti-COX-2 ONCOS, as therapeutic agent for human cancers

over-expressing COX-2. We observed a significant reduction in COX-2 expression

and PGE2 level after ONCOS treatment in comparison with conventional RNAi

114

technology. Using ONCOS nanoplasmonic particles can directly probe several

dynamic activities within the living cell that were impossible to detect using

conventional RNA interference techniques.

115

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139

APPENDICES

Table 1a: Chemicals used in experimental Methods

Item Supplier/Cat No. Experimental use

Hydrochloric acid (HCl) VWR

BDH3028-2.5LG

NP synthesis/cleaning

Nitric acid (HNO3) VWR

BDH3046-2.5LPC

NP synthesis/cleaning

Gold (III) chloride trihydrate

(HAuCl4)

Sigma-Aldrich

520918-5G

GNP synthesis

Trisoidum citrate dihydrate Sigma-Aldrich

S1804-500G

GNP synthesis

Hexadecyltrimethylammonium

bromide (CTAB)

Sigma-Aldrich

H6269-250G

GNP synthesis

Silver nitrate (AgNO3) Sigma-Aldrich

209139-25G

GNP synthesis

L-Ascorbic acid

Sigma-Aldrich

255564-100G

GNP synthesis

Sodium borohydride (NaBH4)

Sigma-Aldrich

452173-100G

GNP synthesis

Doxorubicin hydrochloride

(DOX)

Sigma-Aldrich

D1515-10MG

NP functionalization

Methoxy-PEG-Thiol, MW =

5000 (m-PEG-SH)

Laysan Bio, Inc.

MPEG-SH-5000-5g

NP functionalization

poly(sodium-4-styrenesulfonate)

PSS

Sigma-Aldrich

243051-5G

NP functionalization

DPBS w/out calcium &

magnesium, sterile

VWR

45000-434

Cell culture

Trypsin EDTA 0.25% VWR 45000-664 Cell culture

Red DMEM VWR 45000-304 Cell culture

Clear DMEM VWR 45000-336 Cell culture

FBS VWR 45000-734 Cell culture

Antibiotic/Antimycotic VWR 45000-616 Cell culture

DMSO ATCC 4-X Cell culture

Ethanol VWR 89125-164 Cell culture

XTT reagent kit VWR 89138-908 Cell Viability

Glycerol

VWR 0854-1L Dark field & confocal

microscopy

RNase Sigma-Aldrich

R6513-10MG

Flow cytometry

(cell cycle)

Propidium iodide (PI) VWR 89139-064 Flow cytometry

Cell lysis buffer 10x Cell Signaling Tech. SDS-PAGE

140

9803S Western blot

Ammonium per-sulfate

VWR 97064-884 SDS-PAGE Western

blot

TEMED VWR 97064-686 SDS-PAGE/Western

blot

NEXT GEL Poly-achrylamide

gel12.5% solution

VWR

97063-030

SDS-PAGE

Western blot

Tris-Glycine 10x

VWR

97063-776

SDS-PAGE

Western blot

Tween 20

VWR

97063-872

SDS-PAGE

Western blot

TRIS (base), ultra pure

VWR

JT4109-4

SDS-PAGE

Western blot

Laemmli sample buffer Bio-Rad

161-0737

SDS-PAGE

Western blot

Precision Plus Protein

Kaleidoscope Standards

Bio-Rad

161-0375

SDS-PAGE

Western blot

Quick Start™ Bradford Protein

Assay

Bio-Rad

500-201

SDS-PAGE

Western blot

Coomassie brilliant blue, R-250 Bio-Rad

161-0400

SDS-PAGE

Western blot

β-mercaptoethanol VWR

EM-6010

SDS-PAGE

Western blot

Methanol VWR

BDH1135-4LP

SDS-PAGE

Western blot

Pierce ECL Western Blotting

Substrate

VWR

PI32209

SDS-PAGE

Western blot

DL-Dithiothreitol DTT Sigma Aldrich

43815

Oligos Reduction

EDTA Sigma Aldrich

E5134

NP Conjugation

NaCl Sigma Aldrich

S7653

General

Escort™

IV Transfection

Reagent

Sigma Aldrich

L3287

Cell Transfection

ZIP™ Reversible Protein

Detection Kit

VWR 97064-932 Protein Detection

Triton x100 Sigma Aldrich T878 SDS-PAGE/Western

blot

Cox-2 electrophoresis standard Cayman Chemical

360120

SDS-PAGE

Western blot

Bovine serum albumin (BSA) Rockland Immuno-

chemicals BSA-50

Western blot

141

Milk, Nonfat Powdered,

Proteomics Grade

VWR 97063-958 Western blot

Tris HCl VWR B85827 SDS-PAGE Western

blot

Prostaglandin E2 ELISA Kit -

Monoclonal

Cayman Chemicals

514010

PGE2 Concentration

Measurement

Table 1b: COX-2 Oligos used in Experimental Methods

Oligos Sequence MW

(g/mol)

TM

Sense 5'-(Thiol S-S)CCTTCTCTAACCTCTCCTATT-3' 6544.3 50.42

Antisense 5'- AATAGGAGAGGT(Fluorescein-dt)AGAGAAGG-3' 7144.1 49.73

Antisense 5'- AATAGGAGAGGTAGAGAAGG-3' 6632.6 50.42

Antisense 5'-(Thiol S-S) AATAGGAGAGGT(Fluorescein-dt)AGAGAAGG-3' 7244.1 49.73

Sense 5'-(Thiol S-S)GCTCCGGACTAGATGATATC-3' 6443.3 51.78

Antisense 5'-GATATCATCTAGT(Fluorescein-dt)CCGGAGC-3' 7122.1 50.1

Antisense 5'-GATATCATCTAGT CCGGAGC-3' 6443.3 51.78

Sense 5'-(Thiol S-S)GCTCCGGAACTAGATGATATC-3' 6443.3 51.78

Antisense 5'-GATATCATCTAGTCCGGAGC(Fluorescein) -3' 6715.6 51.78

Antisense 5'-GATATCATCTAGTCCGGAGC-3' 6117.1 51.78

142

Table 1c: Antibodies used in Protein Expression Analysis

Antibody Supplier/cat#

COX-2 monoclonal antibody (clone 12C10) Cayman Chemicals. 20198

β-actin mouse mAb Cell Signaling Tech. 3700

Anti-mouse IgG, HRP linked antibody Cell Signaling Tech. 7076

143

Table 1d: Materials/supplies used in experimental Methods

Item Vendor/Cat no Experimental Use

NAP-5 Column GE- 17-0853-01 Oligos Purification

200 μL Gel-loading pipet

tips

VWR 37001-152 SDS-PAGE/ Western blot

Grade No. 3MM

Chromatography Paper

VWR

21427-456

SDS-PAGE

Western blot

200 Mesh Cu

Formvar/Carbon

(TEM grids)

Ted Pella, Inc.

01801

NP synthesis

CellStar cell culture flasks,

sterile

VWR

82050-856

Cell culture

10 mL serological pipets,

sterile

VWR

82050-482

Cell culture

1.5 mL microcentrifuge

tubes

VWRCA1700-GT Cell culture

15 mL falcon tubes, sterile VWR 89039-666 Cell culture

50 mL falcon tubes, sterile VWR 89039-658 Cell culture

1000 μL pipet tips, sterile VWR 29442-706 Cell culture

200μL pipet tips, sterile VWR 1001-318 Cell culture

10 μL pipet tips, sterile VWR 89136-570 Cell culture

12-well tissue culture

plates, sterile

VWR

29442-040

Cell culture

96-well clear tissue culture

plates, sterile

VWR

29442-060

Cell culture

96-well black tissue culture

plates, sterile

VWR

82050-742

Cell culture

Fine tip pipets VWR 414004-020 Cell culture/General use

General pipets VWR 414004-004 Cell culture/General use

18 mm diameter cover-

slips

Fischer Scientific, Inc. 12-

545-100

Dark field, confocal &

live cell Raman

microscopy

22x50 mm rectangular

cover-slips

Fischer Scientific, Inc.

12-544-18

Dark field, confocal &

live cell Raman

microscopy

35 mm glass bottom dishes

MatTek Corp.

P35G-1.5-14-C

Live cell & confocal

microscopy

75x25mm confocal glass

slides

VWR

16004-368

Confocal microscopy

5 mL round bottom snap

cap tubes

VWR

60819-310

Flow cytometry

40 μm filters VWR 21008-949 Flow cytometry

CellStar tissue culture

plates, 15 cm

VWR

82050-916

SDS-PAGE

Western blot

144

Table 1e: Instruments used in experimental Methods

LDL= Laser Dynamics Labs

GT= Georgia Institute of Technology, USA

Instrument Use Location Contact Person

Ocean Optics UV-Vis

spectrometer

Optical density LDL, GT Mostafa A. El-

Sayed

VWR Clinical 200

centrifuge

NP purification Boggs 1-28,

GT

Mostafa A. El-

Sayed

Eppendorf Centrifuge

5424

NP purification LDL, GT Mostafa A. El-

Sayed

JEOL 100CX-2 TEM NP size RBI, GT Yolande Berta

Malvern Zetasizer

Nano

DLS MoSE, GT Rick Sullivan

Baker SterilGARD

III Advance

Cell culture Boggs 1-28,

GT

Mostafa A. El-

Sayed

Olympus IX70

microscope w/

dark field condenser

(U-DCW)

Dark field

imaging,

Live cell imaging

LDL, GT Mostafa A. El-

Sayed

Biotek Synergy H4

multi-mode

plate reader

Cell viability

assays,

NP uptake

IBB, GT Andrew Shaw

BD LSR II flow

cytometer Flow

cytometry

assays

IBB, GT N. Boguslavsky

Zeiss LSM 700

confocal microscope

Confocal

microscopy

IBB, GT Andrew Shaw

Ti:Sapphire laser

(CW) (NIR: 808 nm)

Photothermal

studies

LDL, GT Mostafa A. El-

Sayed

Mini-PROTEAN

Tetra Cell, Mini

Trans-Blot Module,

PowerPac HC Power

Supply

SDS-PAGE,

Western blot

Boggs 1-28 Mostafa A. El-

Sayed

Renishaw In Viva

Raman Microscope

Live cell Raman

imaging

LDL, GT Mostafa A. El-

Sayed

AmershamTM

Imager Western blot

imaging

IBB, GT N. Boguslavsky

Branson Digital

Sonifier

Cell lysis Boggs Mery Peek

145

Table 1f: Software used in Result Analysis

Software Experimental Use

Origin 8.5 Data analysis

Graph pad Prism Data analysis

Image J NP analysis, Western blot analysis

FlowJo 7.6.1 Flow cytometry analysis

ZEN 2012 (Blue Edition) Confocal data analysis

WIRE 3.3 Raman data analysis

EndNotex5 References