GOLD NANOPARTICLE–BIOMOLECULE CONJUGATES:

SYNTHESIS, PROPERTIES, CELLULAR INTERACTIONS

AND CYTOTOXICITY STUDIES

A Thesis presented to the Faculty of the Graduate School University of Missouri

In Partial Fulfillment Of the Requirements for the Degree

Master of Science

By

SWAPNA MEKAPOTHULA

Dr. Chada S. Reddy and Dr. Kattesh V. Katti, Thesis Supervisors

MAY 2008

The undersigned, appointed by the dean of the Graduate School, have examined the thesis entitled

GOLD NANOPARTICLE–BIOMOLECULE CONJUGATES: SYNTHESIS, PROPERTIES, CELLULAR INTERACTIONS AND CYTOTOXICITY STUDIES

presented by Swapna Mekapothula,

a candidate for the degree of Master of Science,

and hereby certify that, in their opinion, it is worthy of acceptance.

Dr. Chada S. Reddy

Dr. Kattesh V. Katti

Dr. Wade V. Welshons

Dr. Stan W. Casteel

Dr. Raghuraman Kannan

Dedicated to my dear husband

ii

ACKNOWLEDGEMENTS

First and foremost I wish to thank Dr. Kattesh V. Katti for his support and

guidance throughout the project and for giving me this great opportunity to work in the

field of cancer treatment which is my goal in life. I am indebted to Dr. Chada S. Reddy

who has been a guide at every turn and was always there encouraging me with his very

valuable suggestions. This research would not have been fruitful without the

encouragement and in depth knowledge provided by Dr. Raghuraman Kannan.

Dr. Wade V. Welshons and Dr. Stan W. Casteel were gracious in providing me

with the laboratory facilities necessary to pursue the thoughts and ideas into practical

existence. John A. Flanders, Kavita Katti, Nripen Chanda, Nune Satish, Ravi Shukla,

Rajesh Kulkarni, Jeanne Muse, Randy Tindall and Cheryl Jensen were very helpful at

different stages of the project giving me a very good insight into my research.

It would have been a daunting task if it were not for the people in my personal

life. Constant encouragement and love of my parents and my dear sister always helped

me to believe in me and my dreams. Lastly, and most importantly, I wish to thank my

husband whose love and support made all this possible.

I cannot end without thanking Mr & Mrs Reddy and their family who made me

almost forget my home and no amount of gratitude is enough for them. I also wish to

thank my little angel Ankush, who has always put a smile on my face with his cute

mannerisms.

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...............................................................................................ii LIST OF ILLUSTRATIONS .............................................................................................vi LIST OF TABLES ..........................................................................................................viii LIST OF ABBREVIATIONS.............................................................................................ix ABSTRACT …....................................................................................................................x

CHAPTER 1 INTRODUCTION ………………………………………………………………………..1 1.1 Gold nanoparticles and their properties……………………………….....................1

1.2 Synthesis of Gold Nanoparticles …………………………………………………...4

1.3 Gold nanoparticle based targeted delivery systems………………………………...5

CHAPTER 2 BOMBESIN CONJUGATED GOLD NANOPARTICLES FOR TARGETTED DELIVERY…......................................................................................................................7 2.1 Introduction…………………………………………………………………………..7 2.2 Materials and Methods ………………………………………………………………8 2.3 Synthesis of Bombesin conjugated Gold nanoparticles ……………………………..9

2.3.1. Synthesis of Starch stabilized Gold Nanoparticles………………………....9 2.3.2. Filtration …………………………………………………………………..10 2.3.3. Conjugation of Thioctic acid-BBN conjugate to S-AuNps……………….10

2.4. Cell internalization procedure…………………………………………………….10 2.5. Results and Discussion ……………………………………………….. ………...12

iv

CHAPTER 3 ANNEXIN V – GOLD NANOPARTICLES AS APOPTOSIS DETECTION PROBES…………………………………………………………………………………25 3.1 Introduction……………………………………………………………………….25 3.2 Materials and Methods …………………………………………………………...26 3.3 Synthesis of Annexin V-Gold Nanoparticles ………………………….. ………..26

3.3.1 Synthesis of Thioctic acid analogue of Annexin V………………………....26 3.3.2 Starch coated AuNps………………………………………………………..27 3.3.3 Conjugation reaction of Annexin V-TA with S-AuNps………………….....27

3.4 Interaction of Annexin-V-AuNps with Apoptotic Cells…………………………..28 3.5 Scanning Electron Microscopy……………………………………………………29 3.6 Results and Discussion…………………………………………………………... 30 CHAPTER 4 GREEN GOLD NANOPARTICLES……………………………………………………37 4.1 Introduction………………………………………………………………………..37 4.2 Materials and Methods …………………………………………………………...38 4.2.1 Synthesis of Soybean Gold Nanoparticles …………………………………38 4.2.2 Synthesis of Tea Gold Nanoparticles ………………………………………38 4.2.3 Synthesis of Cinnamon gold nanoparticles ………………………………...39 4.3 Cellular Interaction studies …………………………………………………….39 4.3 Cytotoxicity Studies ………………………………………………………………39 4.4 Results and Discussion…………………………………………………………....40

v

CHAPTER 5

SUMMARY AND FUTURE WORK

5.1 Summary …………………………………………………………………………..53

5.2 Future work………………………………………………………………………...54 BIBILOGRAPHY………………………………………………………………………..55

vi

LIST OF ILLUSTRATIONS

Figure Page

1.1 Gold Nanoparticles and their electronic properties……………………………..1 1.2 Theory of Surface Plasmonics…………………………………………………..2

1.3 General Synthesis protocol of Gold Nanoparticles……………………………..5

2.1 Comparison of Amino acid sequences in Gastrin releasing peptide and

Bombesin…………………………………………………………………………7 2.2 Synthesis protocol of BBN-AuNps…………………………………………….14

2.3 a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Starch

Gold Nanoparticles ……………………………………………………………..15

2.4 a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Bombesin Gold Nanoparticles…………………………………………………..16

2.5 Different stages of the cellular uptake process of BBN-AuNps. TEM images of

PC-3 cells depicting the arrival of a BBN-AuNps at the cell membrane, binding of the nanoparticles to surface receptors, membrane wrapping of the Nps, and finally internalization into the cell nucleus……………………………………...17

2.6 TEM images of MCF-7 cells showing the internalization of BBN-AuNps into

the nucleus………………………………………………………………………18

2.7 TEM images of PC-3 cells showing no uptake of BBN-AuNps when the GRP-receptors are blocked by excess free BBN (0.035 mg)………………………….19

2.8 TEM images of MCF-7 cells showing no uptake of BBN-AuNps when the GRP-

receptors are blocked by excess free BBN (0.035 mg)……………………….....20

2.9 TEM Images of three different PC-3 cells showing uptake of Unconjugated-AuNps in to the lysosomes……………………………………………………...21

2.10 TEM Images of three different MCF-7 cells showing uptake of

Unconjugated-AuNps in to the lysosomes…………………………....................22

3.1 Synthesis of Thioctic acid analogue of Annexin-V…………………………….27 3.2 Conjugation reaction of Annexin-V-TA with S-AuNps………………………..28

vii

3.3: a) UV-Visible absorption spectrum, b) TEM image of Annexin V-Gold Nanoparticles………………………………………………………………………..30 3.4: a, b, c, d, e, f: Effect of increasing concentrations of Anx V-AuNps on FITC-Anx fluorescence signal in apoptotic Jurkat T-cells………………………………...32 3.5: Dose response curve for Anx V- AuNps in displacement of FITC-Anx from apoptotic Jurkat-T cells……………………………………………………………...34

3.7: Apoptotic Jurkat-T cell showing rough surface………………………………...35 3.8: a, b, c, d Back scatter SEM images showing surface binding of Anx V-AuNps to apoptotic Jurkat-T cells incubated with 278.4 µM Anx V-AuNps………………….36

4.1 a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Tea gold nanoparticles…………………………………………………………………...41

4.2 a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Soy gold nanoparticles…………………………………………………………………...42

4.3 a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Cinnamon Gold Nanoparticles………………………………………………………43

4.4 a, b, c: TEM Images of different MCF-7 cells showing uptake of Tea-AuNps in to the lysosomes……………………………………………………………………..45

4.5 a, b: TEM Images of different MCF-7 cells showing uptake of Soya -AuNps in to the lysosomes……………………………………………………………………..46

4.6 a, b: TEM images of MCF-7 cell showing uptake of Cin-AuNps in to the lysosomes……………………………………………………………………………47

4.7 a, b: Dose dependent cytotoxicity of Tea-AuNps in cultured PC-3 and MCF-7 cells after 24 hrs of exposure using MTT assay…………………………………….49

4.8 a, b: Dose dependent cytotoxicity of Soy-AuNps in cultured PC-3 and MCF-7 cells after 24 hrs of exposure using MTT assay…………………………………….50

4.9 a, b: Dose dependent cytotoxicity of Cin-AuNps in cultured PC-3 and MCF-7 cells after 24 hrs of exposure using MTT assay………….........................................51

4.10: Dose dependent cytotoxicity of NaAuCl4 in cultured PC-3 and MCF-cells after 24 hrs of exposure using MTT assay………………………………………….52

viii

LIST OF TABLES

3.6: Table showing the effect of increasing concentrations of Anx V-AuNps on FITC-Anx fluorescence signal in apoptotic Jurkat T-cells…………………………………34

ix

LIST OF ABBREVATIONS

AuNps Gold nanoparticles

Anx V Annexin V

Anx V-AuNps Annexin V-Gold nanoparticles

BBN Bombesin

Cin-AuNps Cinnamon gold nanoparticles

DI De-Ionized Water

DMF Dimethylformamide

GA Gum Arabic

GRP Gastrin releasing peptide

HSA Human serum albumin

RS Raman Scattering

S-AuNps Starch stabilized Gold nanoparticles

SERS Surface Enhanced Raman Scattering

Soy-AuNps Soy Gold nanoparticles

TA Thioctic Acid

T A-BBN Thioctic acid-Bombesin conjugate

TEM Transmission Electron Microscopy

THPAL Tris hydroxyl phosphine alanine

T-AuNps Tea Gold nanoparticles

x

GOLD NANOPARTICLE–BIOMOLECULE CONJUGATES:

SYNTHESIS, PROPERTIES, CELLULAR INTERACTIONS

AND CYTOTOXICITY STUDIES

Swapna Mekapothula

Dr. Chada S. Reddy and Dr. Kattesh V. Katti, Thesis Supervisors

ABSTRACT

In recent years, Gold nanoparticles (AuNps) have been widely used in diverse biomedical

applications because of their efficient optical and electronic properties. Gold

nanoparticles also posses a strong surface chemistry which renders them suitable for

attachment with biomolecules. Researchers are considering the use of gold nanoparticles,

as potential contrast enhancement agents in X-ray Computed Tomography and Photo

Acoustic Tomography imaging techniques, useful in the early detection of specific

tumors. Fundamental to any further clinical developments in gold nanoparticulate based

imaging or drug design is the ability to synthesize gold nanoparticles conjugated with

proteins/biomolecules that impart high affinity to target various disease pathologies. This

research focuses on conjugation of two major bio-molecules i.e., Bombesin and Annexin

V, to gold nanoparticles and studying their target specificity. The target specificity of

gold nanoparticles coated with Bombesin, a GRP (Gastrin releasing peptide) receptor

specific protein was tested using two cancer cell lines (MCF-7, breast cancer cells; and

PC-3, prostate cancer cell line) that over-express GRP receptors. Both cell types exhibit

xi

significant uptake of Bombesin gold nanoparticles, internalizing them through a highly

specific receptor mediated endocytosis pathway.

Binding of AuNps coated with a phospholipid-binding protein Annexin V with

high affinity towards apoptotic cells was tested in Jurkat-T- lymphocytes. Annexin V-

gold nanoparticles showed excellent affinity towards the apoptotic Jurkat-T lymphocytes

binding to the cells in a manner similar to the biomolecule annexin V.

We further used environmentally benign so called green chemicals i.e.,

polyphenols, flavonoids, catechins, and various phytochemicals present in tea, soybean,

and cinnamon and their synergistic reduction potentials to reduce the gold salts into

AuNps. Such nanoparticles also showed excellent affinity toward epidermal growth

factor receptors (EGFR) on prostate and breast cancer cells and proved to be non-

cytotoxic at as high as 150µM. These studies showed that gold nanoparticles can be

coated not only to exert specific molecular interactions in specific cell types but also to

be devoid of adverse effects.

1

CHAPTER 1

Introduction:

Nanotechnology is a tremendously powerful technology, which holds a huge

promise for the design and development of many types of novel products with its

potential medical applications on early disease detection, treatment, and prevention. Gold

nanoparticles represent a new class of biocompatible vectors capable of fulfilling this

promise by selective cell and nuclear targeting of which will provide new means for the

site- specific diagnosis and treatment of medical conditions. This work outlines the

methodology for conjugation of AuNps with target specific biomolecules (Bombesin,

Annexin V) and details the results of studies assessing the target specificity and

Cytotoxicity effects of thus conjugated gold nanoparticles.

1.1 Gold Nanoparticles and their properties:

Gold nanoparticles are defined as stable colloid solutions of clusters of gold atoms

with sizes ranging from 1-100 nm (Figure 1.1).

Gold Nanoparticles Gold Nanoparticles are a cluster of gold atoms with

sizes in nanometer range.

Gold nanoparticle 1 nm – 100 nm Cluster of Gold atoms

Example: 0.65 nm Colloidal Gold-nanoparticle solution contains approximately 9 gold atoms

Figure 1.1: Gold Nanoparticles and their electronic properties

2

At this nanoscale, AuNps possess different physicochemical characteristics when

compared to the bulk gold [1, 2], most obvious example being the color change from

yellow to ruby red when bulk gold is converted into nanoparticulate gold. This ruby red

color of AuNps is explained by a theory called “surface plasmonics”.

According to this theory, when the clusters of gold atoms are hit by the

electromagnetic field of the incoming light, the surface free electrons (6 electrons in case

of AuNps) present in the conduction band of AuNps oscillate back and forth thus,

creating a plasmon band which has an absorption peak in the visible region at 530-540

nm (Figure 1.2) [3]. The surface plasmon band (SPB) of AuNps is used as an indicator for

formation during the synthesis of AuNps from their precursor salts. The sensitivity of

plasmon band absorptivity is the basic detection mechanism involved in the AuNps based

bio-sensors [4, 5].

Figure 1.2: Theory of Surface Plasmonics.

3

Physical properties of AuNps in turn depend on the size, shape, particle-particle distance

and the nature of the stabilizer used to prevent the agglomeration of nanoparticles [1].

According to Mie theory, Surface Plasmon Band (SPB) is absent for AuNps less than

2nm and greater than 500nm [2]. Gold nanorods have two SPB’s, one longitudinal-

wavelength band at 550-600nm and one transverse-wavelength band at 520nm [6, 7].The

longitudinal-wavelength band is very sensitive and changing the aspect ratio of Gold

nanorods changes the absorption region from visible to Near-infra red (NIR) [8]. This

unique optical property of Gold nanorods is used in Near-infra red ray therapy [9]; and

enhanced Raman scattering of adsorbed biomolecules [10]. Therefore, by changing the

size and shape of AuNps, the SPB and scattering may be tuned for application in cellular

imaging, drug delivery and therapy.

The six free electrons present in the conduction band of nanoparticulate gold

makes them potential candidates to bind with thiols and amines [11]. Therefore, AuNps

may be easily tagged with various proteins and bio-molecules rich in amino acids leading

to important biomedical applications including targeted drug delivery [12, 13], cellular

imaging [14], and biosensing [15]. Further, the free electrons also render AuNps useful as

contrast enhancement agents [16]. Imaging studies are based on comparisons of contrast

produced by the variations in the electron densities in different tissues. With their high

electron densities, AuNps serve as excellent contrast enhancement agents in the detection

of tumors.

4

1.2 Synthesis of Gold Nanoparticles:

Gold Nanoparticles are traditionally synthesized by reducing metallic gold in +3

state to nanoparticulate gold in +1 state. There are a number of reducing agents reported

in the literature for the synthesis of AuNps. Two most important ones are: Tri sodium

citrate (Citrate synthesis) discovered by Turkevitch in 1973 [17] and Sodium borate

(Borate synthesis) introduced by Brust-Schiffrin in 1994 [18]. These two synthesis

protocols pose potential problems in case of size control, stability and most importantly

toxicity [19]. Therefore, we followed a novel protocol reported by Katti et. al. in 2003

that uses a phosphino-amino acid based reducing agent, tris hydroxyl phosphine alanine

(THPAL) to synthesize AuNps (Figure 1.3).

THPAL is a water-soluble non-toxic reducing agent. It is reported that swine models can

withstand up to 100 mg/kg of body weight of THPAL with out showing toxicity, the most

important of criteria in the use of nanoparticles for bio medical applications.

Due to the strong surface reactivity of free electrons present on AuNps, they

easily tend to agglomerate posing stability problems. Naturally occurring, FDA approved

non-toxic compounds such as starch; gum arabic and gelatin were used to stabilize

AuNps immediately after they are formed [20]. These stabilizers form weak covalent

bonds with AuNps so that they easily shed off in the presence of biomolecules with

strong electronegative groups with which the AuNps can then react.

5

The Reaction:

Au+3(aq) + Reducing Agent (aq) + Stabilizer ----- Au0 (solid)/Au+1

[HAuCl4; NaAuCl4] THPAL [Starch; Gum Arabic; Gelatin]

Gold nano particles

5 minutes

NaAuCl4

THPALTHPAL

PNH

NH

NH

COOH COOH

COOH

Novel nontoxic phosphino- amino acid based reducing agent

Heat at 90-100C

+THPAL

(Katti et. al., J. Am. Chem. Soc. 2003, 125, 6955.)

STARCH +

Figure 1.3: General Synthesis protocol of Gold Nanoparticles.

1.3 Gold nanoparticle - based targeted delivery systems:

Gold nanoparticles; due to their ease of synthesis, uniform size distribution, rich

surface chemistry and a lack of toxicity; have been proven to be excellent candidates for

conjugation with numerous biomolecules for site-specific delivery. Selective cell and

receptor targeting of AuNps are likely to provide new pathways for the targeted delivery

of diagnostic/ therapeutic agents [21, 22]. Tkachenko et al. showed specific nuclear

targeting of AuNps by conjugating them with bovine serum albumin (BSA) using

differential contrast microscopy [23, 24]. Gold nanoparticles are used as targeted contrast

6

agents in detecting cervical cancer by tagging them with monoclonal antibodies and

oncoproteins associated with human papilomavirus [25]. Mercaptoalkyl-oligonucleotide-

conjugated AuNps have been used in the detection of polynucleotides by plasmon band

interactions with the surrounding environment [26]. In particular, Gold nanoshells, when

coated with breast tumor marker Her-2, effectively localized in the microscopic tumors

present in the breast tissue [27]. Most of the work in the area of nanoimaging is also

focusing on fabricating detectors that can detect efficiently cells undergoing apoptosis, an

effect common to many chemotherapeutic regimens. These examples of the use of

AuNPs in medical diagnosis although demonstrate the potential of AuNPs even in drug

delivery, their target specificity by conjugation with biomolecules has yet to attain the

desired refinement.

The overall goal of this research is to conjugate non-toxic AuNps with

biomolecules like Bombesin and Annexin V which posses’ high affinity towards specific

receptors over-expressed on cancer cells and phosphatidylserine (PS), a phospholipid

exposed on apoptotic cells respectively and studying their target specificity.

7

CHAPTER 2

Bombesin Conjugated Gold Nanoparticles for Targeted Delivery

2.1 Introduction:

Thirty years ago, Erspamer and Anastasi isolated Bombesin (BBN) from the skin of

amphibians Bombina variegata and Bombina bombina [29]. Bombesin is a

tetradecapeptide neurohormone with exocrine and endocrine effects. Its C-terminal amino

acid sequence is very similar to that of Gastrin releasing peptide (GRP) which has high

affinity towards G-protein coupled receptors (GPCRs) (Figure 2.1).

Gastrin-releasing peptide –Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2

Bombesin –Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2

Figure 2.1: Comparison of Amino acid sequences in Gastrin releasing peptide and Bombesin

Bombesin is proved to have numerous pharmacological effects, such as control of

gastrin release, modulation of gastrointestinal secretions, smooth muscle motility and the

amount of food intake [30-34]. Recently, it has been shown to play a major role in cell

proliferation, tumor growth and inflammation [35, 36]. There is substantial evidence

indicating increased BBN/GRP (Gastric releasing peptide) receptor expression in

prostate, small-cell lung, ovarian, breast, pancreatic, gastric, renal cell, and thyroid

8

cancers, suggesting their use as specific markers for targeting [37-45]. Since BBN and

GRP have high affinity towards GRP receptors, conjugating AuNps with these peptides

should allow for greater binding of these particles to the tumor sites, thus making them

suitable for early detection of tumors by imaging techniques.

By modifying the structure of BBN several derivatives of BBN with high affinity

towards GRP receptors (agonists) and some with low affinity towards receptors

(antagonists) have been synthesized [46, 47, 48]. In this research, 7-amino acid truncated

BBN (Figure 2.2) derivative was conjugated to AuNps that has been proven to have high

agonistic action towards prostate, small-cell lung and breast cancer cell lines. A Disulfide

bond (-S-S) present in thioctic acid (TA), an antioxidant, was used as a Linker to bind

truncated BBN with AuNps. Starch was used as a stabilizer.

2.2 Materials and Methods:

Sodium tetrachloroaurate (NaAuCl4) was obtained from Alfa-Aesar (Ward Hill,

MA). Tris hydroxyl phosphino alanine (THPAL) was synthesized according to the

standard protocols described in the literature [49, 50]. Sephadex G -100 gel was obtained

from Pharmcia (Newyork). 7-amino acid Bombesin (BBN) was obtained from Anaspec

(San Jose, CA). De-ionised water was used as a solvent to synthesize gold nanoparticles.

Minimum essential medium (MEM with nonessential amino acids, powdered),

HEPES, insulin, streptomycin sulfate, penicillin-G, were obtained from Sigma Chemical

Company (St. Louis, MO). Bovine calf serum, phenol red (sodium salt), and lyophilized

trypsin were obtained from Gibco BRL (Grand Island, NY).

9

Cell Culture:

MCF-7 breast cancer cells and PC-3 prostate cancer cells were obtained from V.

Craig Jordan (University of Wisconsin-Madison) and ATCC (Manassas, VA)

respectively. MCF-7 cells were maintained in MEM with nonessential amino acids, 10

pgml-1 phenol red, 10 mM HEPES, 6 ngml-1 insulin, 100 units ml-1 penicillin, 100 pgml-1

streptomycin, and 5% charcoal-stripped calf serum (maintenance medium) [51,52,53].

PC-3 cells were maintained in RPMI medium supplemented with 4.5 gL-1 D-glucose, 25

mM HEPES, 0.11 gL-1 sodium pyruvate, 1.5 gL-1 sodium bicarbonate, 2 mM L-glutamine

and 10 % FBS and100 units ml-1 penicillin and 100 pgml-1 streptomycin.

2.3 Synthesis of Bombesin conjugated Gold nanoparticles

(BBN-AuNps):

2.3.1. Step-1: Synthesis of Starch stabilized Gold Nanoparticles (S-AuNPs):

To a 10 ml vial, 0.0225 g of starch was added and dissolved in 6 ml of double-

distilled and deionized water. This starch solution was heated up to 900C -1000C and

stirred continuously until dissolved. To the dissolved starch solution, 100 μl of 0.1 M

NaAuCl4 solution was added resulting in a pale yellow color solution. To this solution, 20

μl of 0.1 M THPAL solution was added with continuous stirring. The resulting solution

slowly turned into purplish-wine color representing the formation AuNps. The mixture

was cooled to room temperature and S-AuNPs were characterized by UV-Vis absorption

Spectroscopy and Transmission Electron Microscopy (TEM) measurements [54].

10

2.3.2 STEP: 2 – Filtration:

Thus synthesized S-AuNps were filtered using Sephadex columns to remove any

unreacted starch from the mixture [54]. The absorbance peak of filtered S-AuNps was

adjusted to 0.7 by diluting the resulting solution to suit further conjugation with thioctic

acid-BBN conjugate.

2.3.3 STEP: 3-Conjugation of Thioctic acid-BBN (TA-BBN) conjugate to S-AuNps:

Based on the studies done by Kattumuri et. al [54] 0.885 μM of Thioctic acid-

BBN conjugate (1 mg in 1 ml of methanol) was added to 1 ml of filtered S-AuNps and

stirred for 60 hrs. The product, thus formed was further filtered and purified by washing

several times with mixture a of methanol and water to remove all traces of unconjugated

reactants. The purified Bombesin-conjugated gold nanoparticles (BBN-AuNPs) solution

was vacuum dried and re-dissolved in PBS (without Ca2+ and Mg2+). This conjugate was

used to study its target specificity towards GRP receptors present on PC-3 and MCF-7

cell lines.

2.4. Cell internalization procedure:

About 16,000 cells (PC-3/MCF-7) were plated into each well in a 6 well plate

were incubated at 370C for 20.0 hrs to allow the cells to recover. The medium from each

well was aspirated and 4 ml of fresh growth medium was added per each well. Cells were

allowed to grow until they reached confluence by changing the medium every alternate

day. To the confluent cell layer, 150 μl of BBN-AuNps solution made up in PBS was

added and further incubated for 4 hrs at 370C. The medium was then aspirated from each

11

well and the cell layer was rinsed 3 times with complete growth medium to remove any

traces of uninternalized BBN-AuNps. Then the cell layer was washed with CMFH-EDTA

(Calcium-Magnesium-Free-Hank’s +HEPES - EDTA) solution to remove all traces of

serum, a trypsin inhibitor. About 0.5 ml of 0.1 M Trypsin-EDTA solution was added to

each well to detach the cell layer from the plastic. Detached cells were dispersed in 4 ml

of complete growth medium and gently pipetted out of the well. The cell suspension was

transferred into a centrifuge tube and centrifuged at approximately 125 x g for 5 minutes.

Supernatant was discarded and cell pellet was fixed with 0.1 M Na-Cacodylate buffer

containing 2% glutaraldehyde and 2% paraformaldehyde. The pellets were post-fixed

with 1 % osmium tetraoxide, dehydrated and embedded in Epon/ Spurr’s resin and 80 nm

sections were collected and placed on TEM grids followed by sequential counterstaining

with uranyl acetate and lead citrate. TEM grids were observed under TEM (Joel 1400)

and images were recorded at different magnifications. As a control, similar incubations

were performed with either 25 μM unconjugated AuNps and/or with the same

concentration BBN-AuNps in the presence of 0.035mg of BBN, (since this is the amount

of BBN used to conjugate S-AuNps).

12

2.5 Results and Discussion:

2.5.1. Synthesis of Bombesin conjugated Gold nanoparticles (BBN-AuNps):

7-amino acid truncated BBN, a derivative of BBN, has been proven to have high

agonistic action towards GRP receptors [55, 56]. According to literature, AuNps show

high affinity towards sulfur [11]. Indeed, sulfur bonds present in various biomolecules

could be used as linkers to conjugate them with AuNps. In fact, disulfide bond (S-S)

present in thioctic acid serves as a linker to conjugate AuNps selectively with BBN even

in the presence of thiol groups in other biomolecules. Thioctic acid is conjugated to BBN

by first activating its carboxyl group using “2-(1H-benzotriazol-1-yl)-1, 1, 3, 3,-

tetramethyluronium hexafluorophosphate” (HBTU) followed by an addition reaction with

BBN (Figure 2.2). The product thus formed TA-BBN, was then conjugated with AuNps.

Gold Nanoparticles were synthesized using NaAuCl4 as a gold precursor, THPAL

as a reducing agent and starch solution as a stabilizer. Starch stabilizes AuNps by

forming weak coordination bonds between its –OH groups and gold atoms. These weak

coordination bonds are broken down in the presence of powerful -S-S- bond present in

Thioctic acid-BBN conjugates to remove starch coating on AuNps. Filtered S-AuNps

showed a uniform size distribution of 15±5 nm and an absorption peak at 530 nm.

Absorption studies and TEM analysis of the S-AuNps shows excellent uniformity and

desired size distribution of S-AuNps necessary for further conjugation with TA-BBN

(Figure 2.3).

For the synthesis of BBN-AuNps 1 ml of stable S-AuNps were stirred with 0.855

μM of TA-BBN for 60 hrs. The purified BBN-TA-AuNps when analyzed by UV-visible

spectroscopy and TEM showed a uniform size distribution of 20nm and has an absorption

13

peak at 530 nm (Figure 2.4). These values are similar to the values reported indicated by

Petersen.et.al for AuNps conjugated to CTAB and by Katti.et.al for AuNps conjugated to

cysteine [57].

14

Reaction steps in the synthesis of BBN-AuNps

Figure 2.2: Synthesis protocol of BBN-AuNps

15

a) b)

c)

Size distribution of Starch-AuNps

0

5

10

15

20

25

30

35

1 3 5 7 9 11 13 15 17 19 21 23 25

Size of Starch-AuNps

Num

ber o

f AuN

ps

Figure 2.3: a) TEM Image, b) UV-Visible absorption spectrum, and c) size distribution of Starch Gold Nanoparticles.

16

a) b)

c)

Size distribution of BBN-AuNps

0

5

10

15

20

25

30

35

1 3 5 7 9 11 13 15 17 19 21 23 25

Size of BBN-AuNps

Num

ber o

f AuN

ps

Figure 2.4: a) TEM Image, b) UV-Visible absorption spectrum, and c) size distribution of Bombesin-Gold Nanoparticles (BBN-AuNps).

17

a) b)

c) d)

Figure 2.5a, b, c, d: Different stages of the cellular uptake process of BBN-AuNps. TEM images of PC-3 depicting the arrival of a BBN-AuNps at the cell membrane, binding of the nanoparticles to surface receptors, membrane wrapping of the Nps, and finally internalization into the cell nucleus, respectively.

18

a)

b)

Figure 2.6: TEM images of MCF-7 cells showing the internalization of BBN-AuNps into the nucleus.

19

a)

b)

Figure 2.7 a, b: TEM images of PC-3 cells showing no uptake of BBN-AuNps when the GRP-receptors are blocked by excess free BBN (0.035 mg).

20

Figure 2.8: TEM images of MCF-7 cells showing no uptake of BBN-AuNps when the GRP-receptors are blocked by excess free BBN (0.035 mg).

21

a)

b)

c)

Figure 2.9 a, b, c: TEM Images of three different PC-3 cells showing uptake of Unconjugated-AuNps in to the lysosomes.

22

a)

b)

c)

Figure 2.10 a, b, c: TEM Images of three different MCF-7 cells showing uptake of Unconjugated-AuNps in to the lysosomes.

23

2.5.2. Cellular interactions of Bombesin conjugated Gold nanoparticles:

To determine whether BBN-AuNps can be used to label BBN/GRP receptors over

expressed in prostate and breast cancer cells, cultures of confluent PC-3 and MCF-7 were

exposed for 4 hrs to BBN-AuNps. These incubated samples are analyzed under TEM to

verify whether the BBN-AuNps are bound to the GRP receptors present in PC-3 and

MCF-7 cells. Figures 2.5a,b,c,d clearly show binding of BBN-AuNps to the plasma

membrane of PC-3 cells by forming clathrin pits and then those pits delivering the

nanoparticles into the nucleus via endosomes. Figures 2.6 show a similar uptake of BBN-

AuNps into the nucleus of MCF-7 cells. A control experiment, in which cells were

exposed to the same concentration of BBN-AuNps in the presence of 0.035 mg of BBN,

showed no uptake of BBN-AuNps Figures 2.7 a, b and 2.8. These results suggest that the

uptake of BBN-AuNps occurs likely by the same mechanism as that of Bombesin. These

results are also in agreement with the results of similar studies showing the uptake of

Bombesin coated Qdots in Swiss 3T3 cells, which over-express GRP receptors [58].

To further substantiate the results, we also incubated unconjugated gold

nanoparticles with PC-3 and MCF-7 cells. TEM analysis (Figures 2.9 a, b, c and 2.10 a,

b, c) demonstrated that unconjugated gold nanoparticles were packed inside intact single

membrane vesicles, similar to lysosomes or phagosomes, and showed no specific uptake

process. Therefore, it may be concluded that unconjugated AuNps were taken up by the

cells via non-specific phagocytosis pathway since they contained no targeting peptide and

BBN-AuNps likely being taken up by a GRP-receptor mediated endocytosis. Based on

these results, it appears feasible that Bombesin conjugated AuNps could be used to

24

specifically target and bind to the endogenously expressed GRP receptors on PC-3 and

MCF-7 cells and could be effectively used for targeted delivery and/or early detection of

tumors by various imaging techniques.

25

CHAPTER 3

Detection of Apoptosis using Annexin-Gold Nanoparticles

3.1 Introduction:

Apoptosis is an extremely organized, energy dependent form of programmed cell

death. It plays a crucial role in the maintenance of normal cellular homeostasis by

removing the damaged cells. Apoptosis is a fundamental feature reported in the

pathology of wide range of diseases including Alzheimer’s disease, various lung diseases,

in cardiac ischemia and in progressive heart failure [59, 60, 61]. The process of apoptosis

is characterized by distinct morphological features like loss of plasma membrane

integrity, nucleus and cytoplasm condensation, membrane blebbing, formation of

apoptotic bodies. Early apoptotic phase is marked by the exposure of a phospholipid

molecule, phosphatidylserine present on the cytoplasm side of the cell on to the outer cell

surface [62, 63]. This key feature may be effectively utilized in fabricating various

apoptosis detection probes. Therefore, Annexin V a 35 kDa phospholipid-binding protein

that has high affinity towards PS exposed on apoptotic cells has been chosen to

conjugated with gold nanoparticles to specifically target AuNps on to the apoptotic cells

[64, 65, 66]. Annexin V conjugated to various fluorescence and magnetic probes such as

FITC and Iron oxide are reported to show high affinity to apoptotic cells [67, 68, 69, 70].

26

3.2 Materials and Methods:

Annexin-V and an Annexin V-FITC Apoptosis Detection Kit was procured from

BioVision Research Products, (CA, USA). Thioctic acid, N-hydroxy succinimide (NHS),

dicylcohexyl carbodiamide (DCC), phosphate buffer (pH 7) and methanol are obtained

from Sigma Aldrich Chemicals, (St. Louis, MO).

Cell Culture:

Jurkat T cells obtained from ATCC (ATCC TIB-152) were used to induce apoptosis.

The cells were grown in ATCC complete growth medium, which contains RPMI 1640

medium, adjusted with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose,

10 mM HEPES, 1.0 mM sodium pyruvate, 90% and 10% FBS.

3.2.1 Synthesis of Annexin V-Gold Nanoparticles (Anx V-AuNps):

STEP: 1

3.2.1a Synthesis of Thioctic acid analogue of Annexin V:

Sixty micro grams of Annexin V was dissolved in 9 ml of 2:1 solution of

phosphate buffer (pH 7) and methanol by stirring continuously. To this solution, 400 µL

of Thioctic acid (26.89 mg of TA in 10 mL MeOH), 400 µL of H-hydroxy succinimide

(NHS) (15.1 mg of NHS in 10 mL MeOH) and 400 µL of dicyclohexyl carbodiamide

(DCC) (26.89 mg of DCC in 10 mL MeOH) were added. The reaction mixture was

stirred for 16 hrs and the small amount of urea (reaction by-product) that is formed at the

end of the reaction was filtered using 1.2 micron filter. The remaining Annexin V-

Thioctic acid analogue solution was used insitu further in the conjugation reaction with

S-AuNps.

27

Figure: 3.1 Synthesis of Thioctic acid analogue of Annexin-V

STEP: 2

3.2.1b Starch stabilized AuNps:

Starch stabilized AuNps are synthesized as described in the section 2.3.1.

STEP: 3

3.2.1c Conjugation reaction of Annexin V-TA with S-AuNps:

Annexin V-TA analogue and filtered S-AuNps obtained in steps 1 and 2 respectively are

used for further conjugation reactions. To 1 ml of Annexin V-TA in 2:1 mixture of

phosphate buffer/methanol 1 ml of S-AuNps solution was added drop-by-drop, stirring

continuously. This mixture was stirred for 16 hrs to obtain the gold conjugate of

Annexin-V. The Annexin V-AuNps conjugate thus obtained was purified to remove all

traces of unconjugated Annexin V-TA by repeated centrifugation and washing, with 2:1

phosphate buffer/ methanol solution. The pure Anx V-AuNps conjugate formed was

characterized using UV-Vis spectroscopy and TEM analysis.

28

Figure 3.2: Conjugation reaction of Annexin-V-TA with S-AuNps

3.2.2 Interaction of Annexin-V-AuNps with Apoptotic Cells:

To induce apoptosis, 7 µL of Camptothecin (1 mM in DMSO) per ml of culture

medium was added to approximately 2 million Jurkat T cells, followed by incubation at

370C for 7hrs [71] . Apoptosis induced by camptothecin was verified by staining the

samples with FITC-Annexin V and Propidium iodide (PI). PI stains the necrotic cells

(more specifically a DNA stain) and FITC-Annexin V stains the apoptotic cells present in

the sample. Increasing concentrations of Anx V-AuNps were added to a series of tubes

containing similar amounts of apoptotic Jurkat T cells in a binding buffer (from apoptosis

detection kit) followed by incubation for 30 min at room temperature in dark. Five micro

liters of FITC-Annexin V (fluorescent probe) and 5 µL of PI were added simultaneously

to each and every tube and allowed to incubate for 30 min. Then all the samples were

analyzed by FACS. As a reference, a positive control with no apoptosis (live cells) and a

negative control with 100% apoptosis were used.

29

3.2.3 Scanning Electron Microscopy:

Yuri/Jensen top-on SEM method:

Apoptotic Jurkat T cells were incubated for 3 hrs with 278.4 µM Anx V-AuNps, washed

3 times with chilled PBS buffer and fixed with 0.1 M Na-Cacodylate buffer containing

2% glutaraldehyde and 2% paraformaldehyde. A small amount of the fixed sample was

placed onto a 0.22 µM nucleopore membrane (shiny side up) sitting on top of a slightly

moistened piece of filter paper in a small petri dish. For each of the following steps listed

below enough buffer solution was added to the filter paper to keep the membrane

floating. As long as the whattman paper stays wet, it will pull ever-increasing amounts of

solution through the nucleopore filter.

After placing the fixed sample onto the membrane, series of five buffer rinses

with 0.1 M cacodylate buffer followed by ultra pure water were done. Samples were then

dehydrated with a series of 10%, 20%, 35%, & 50% ethanol dilutions. At this point

another 0.22 µM membrane was gently placed on top of the membrane with sample (top-

on). Dehydration was continued with 70%, 90%, 95% and 3x100% ethanol dilutions.

Following dehydration, samples were critical point dried immediately after which, the

sandwiched nucleopore membranes were separated and samples were mounted onto SEM

stubs coated with a thin carbon layer.

30

3.3 Results and Discussion:

3.3.1 Synthesis of Annexin V-Gold Nanoparticles (Anx V-AuNps):

We have successfully generated Annexin V-Thioctic acid analogue and

conjugated it with S-AuNps via activated carboxyl group present in Thioctic acid (TA)

and the amino groups on Annexin-V. N-hydroxy succinimide (NHS) was used to activate

the C-terminal carboxyl group in thioctic acid and dicylcohexyl carbodiamide (DCC), a

dehydrating agent was used to facilitate the coupling reaction between annexin V and

thioctic acid.

Figures 3.3 a and b show the results of UV-Vis spectroscopy and TEM images of

Anx V-AuNps respectively. Anx V-AuNps show a sharp absorption peak at 530 nm and

a uniform size distribution of 12±5 nm, which agree with the earlier reported values of

CLIO-nanoparticles conjugated to annexin V [72].

a) b)

Figure 3.3: a) UV-Visible absorption spectrum, b) TEM image of Annexin V-Gold Nanoparticles.

Annexin-V-TA-Au

0.09

0.1

0.11

0.12

0.13

0.14

0.15

0.16

400 450 500 550 600 650 700 750 800

Wavelength (nm)

Abs

31

3.3.2. Indirect Binding Assay:

Since, Anx V-AuNps do not posses any fluorescent probe on them, it makes it

difficult to analyze their binding capacity towards apoptotic cells using flowcytometry

(detects the fluorescence marker attached to apoptotic cells only). Therefore, an indirect

binding assay was designed to analyze the ability of Anx V-AuNps to detect the apoptotic

cells. According to this assay, increasing concentrations of Anx V-AuNps were added to

a series of tubes containing similar amounts of apoptotic Jurkat T cells in a binding buffer

followed by addition of equal amounts of FITC-Annexin V (fluorescent probe) and PI to

each and every tube. The strength of Anx V-Gold Nanoparticles to detect apoptotic Jurkat

–T cells was examined by using a dual wavelength FACS. As shown in Figures 3.4 a, b,

c, d, e, f as the concentration of Anx V –AuNps was increased from 58 µM to 278.4 µM;

signal given by FITC-Annexin V (fluorescent dye) was decreased from 99.13 to 5.55%

(Table 3.1). These results indicate that at higher concentrations Anx V-AuNps saturate all

the available apoptotic cells and are not competitively displaced by FITC–Annexin V.

Therefore, it may be concluded that Anx V-AuNps show high specificity to bind with

phosphatidylserine exposed on surface of the apoptotic Jurkat T cells.

32

3.4 a. Live Cells (No Apoptosis):

3.4 b. Anx-AuNps (0 µM) + FITC- Annexin V (5 µl) + PI (5 µl)

3.4 c. Anx-AuNps (58 µM) + FITC- Annexin V (5 µl) + PI (5ul)

33

3.4 d. Anx-AuNps (116 µM) + FITC- Annexin V (5 µl) + PI(5 µl)

3.4 e. Anx-AuNps (232 µM) + FITC- Annexin V (5 µl) + PI (5 µl)

3.4 f. Anx-AuNps (278.4 µM) + FITC- Annexin V (5 µl) + PI (5 µl)

Figures 3.4: a, b, c, d, e, f: Effect of increasing concentrations of Anx V-AuNps on FITC-Anx fluorescence signal in apoptotic Jurkat T-cells.

34

Indirect Binding Assay:

Figure 3.5: Dose response curve for Anx V- AuNps in displacement of FITC-Anx from apoptotic Jurkat-T cells.

Treatment % of Apoptotic cells

detected in FACS

Live Cells (No Apoptosis) 0

Anx-AuNps (0 µM) + FITC-Annexin V (5 µl) + PI (5 µl) 75.89

Anx-AuNps (58 µM) + FITC- Annexin V (5 µl) + PI (5ul) 50.9

Anx-AuNps (116 µM) + FITC- Annexin V (5 µl) + PI(5 µl) 39.14

Anx-AuNps (232 µM) + FITC- Annexin V (5 µl) + PI (5 µl) 15.99

Anx-AuNps (278.4 µM) + FITC- Annexin V (5 µl) + PI (5 µl) 5.55

Figure 3.6: Table showing the effect of increasing concentrations of Anx V-AuNps on FITC-Anx fluorescence signal in apoptotic Jurkat T-cells.

In-direct Binding Assay

0

20

40

60

80

100

0 58 116 232 278.4

Concentration of Anx V-Gold nanoparicles (uM)

% o

f apo

ptot

ic c

ells

de

tect

ed in

FA

CS

35

3.3.3 Detection of Binding of Anx V-Gold Nanoparticles to Apoptotic Cells through

scanning electron microscopy:

To further validate our results we examined the ability of Anx V-AuNps to bind

with apoptotic cells via Scanning electron microscopy. Confluent cultures of Jurkat-T

cells are apoptocized using 7 µl of Camptothecin and then incubated with 278.4 µM Anx

V-AuNps, washed 3 times with chilled PBS buffer and fixed with 0.1 M Na-cacodylate

buffer containing 2% glutaraldehyde and 2% paraformaldehyde. Analysis of these

samples under dark field back scattering mode in SEM clearly show the Anx V-AuNps

attached on the rough surfaces of apoptotic Jurkat T cells as shinning little spots (Figures

3.8 a, b, c, d). As a control similar incubations are made with unconjugated AuNps

.These controls show absolutely no binding of unconjugated AuNps with the apoptotic

cells. Figure 3.7 shows an Apoptotic cell with rough, disrupted cell surface when viewed

under normal SEM mode (no back scattering applied).

Figure 3.7: Apoptotic Jurkat-T cell showing rough surface.

36

Figu

res 3

.8: a

, b, c

, d: B

ack

scat

ter S

EM im

ages

show

ing

surf

ace

bind

ing

of A

nx V

-AuN

ps to

apo

ptot

ic Ju

rkat

-T c

ells

incu

bate

d w

ith 2

78.4

µM

Anx

V-A

uNps

.

a b

c d

37

CHAPTER 4

Green Gold Nanoparticles

4.1 Introduction:

The use of phytochemicals in the synthesis of nanoparticles is an important

symbiosis between nanotechnology and green Chemistry [73, 74, 75]. As the

nanorevolution unfolds, it is imperative to develop ‘nano-naturo’ connections between

nanotechnology and green domains of the nature. Production of nanoparticles under

nontoxic green conditions is of vital importance to address growing concerns on the

overall toxicity of nanoparticles for medical and technological applications [76, 77, 78].

The power of phytochemicals, which initiate varieties of chemical transformations within

biological systems, is well known [77, 79, 80, 81]. For example, a high level of genistein

found in Soybeans is both a phytoestrogen and antioxidant, and has been extensively used

to treat conditions affected by estrogen levels in the body [82, 83]. Polyphenolic

flavonoids in tea, of which epigallocatechin gallate (EGCG) is the major constituent, has

anticarcinogenic activity [84, 85]. Cinnamon a common household spice is known to

have potential properties to treat diabetes mellitus [86, 87]. While the tremendous health

benefits of chemical cocktails present within tea, soya, cinnamon is beyond doubt, the

actual applications of the chemical reduction power of the myriad of chemicals present in

herbs and spices is still in infancy. Therefore we investigated the synergistic potentials of

polyphenols, flavonoids, catechins, and various phytochemicals present tea, soya,

cinnamon for the reduction reactions of gold salts to produce AuNps which have

potential applications in the diagnosis and therapy of various deadly diseases including

cancer.

38

4.2 Materials and Methods:

4.2.1 Synthesis of Soybean Gold Nanoparticles (Soy-AuNps):

Step 1: Soybean Extract Preparation

Intact soybeans (8 g) were washed with DI water to remove any traces of contaminants.

Soybeans were then soaked in 50 ml of DI water at room temperature for 72 hrs.The

supernatant was decanted, and centrifuged at 8000 rpm for 10 min at room temperature

and was stored at 40C and for use within 3 days.

Step 2: Four ml of soybean supernatant were diluted to 8 ml in DI water and was heated

to simmer for 1 min.

Step 3: To this solution, 100 µl of NaAuCl4 (0.1 M) were added and further heated to

simmering with constant stirring. Within 20minutes, the color of the solution turned to

ruby red indicating the formation of gold nanoparticles (Soy-AuNP).

4.2.2 Synthesis of Tea Gold Nanoparticles (T-AuNps):

Step 1: 100mg of Tea leaves (Darjeeling Tea) were added to 6 ml of DI water and the

reaction mixture was stirred continuously at 25 °C for 15 min.

Step 3: To the stirring mixture, 100 μl of 0.1 M NaAuCl4 solution (in DI water) were

added. The color of the mixture slowly turned purple-red from pale yellow within 10

minutes, which indicates the formation of gold nanoparticles (T-AuNps).

Step 4: The reaction mixture was stirred for an additional 15 minutes and the gold

nanoparticles thus formed were separated from residual tea leaves immediately using a

5µ filter and analyzed using UV-Visible spectroscopy and TEM.

39

4.2.3 Synthesis of Cinnamon gold nanoparticles (Cin-AuNps):

100 μl of 0.1 M NaAuCl4 was added to a stirring solution of 6ml DI water

containing 30 mg of cinnamon powder. Within 15 minutes, the color of the solution

turned to ruby red indicating the formation of gold nanoparticles (Cin-AuNps).

4.3 Cell Culture and Cellular Interactions:

MCF-7 breast cancer cells and PC-3 prostate cancer cells were cultured and the cellular

interactions were studied as described earlier for AuNps in Chapter 2.

4.4 Cytotoxicity Studies (MTT assay):

Cytotoxicity evaluation of soy-, tea- and Cin-AuNps was performed using MTT

assay as described by Mosman [88]. Approximately 1 × 105 ml-1 cells (MCF-7 and PC-3)

in their exponential growth phase were seeded in a flat-bottomed 96-well polystyrene-

coated plate and were incubated for 24 hrs at 37 °C in a 5% CO2 incubator. Series of

dilutions (10, 30, 50, 70, 90, 110, and 150 µM) of AuNps in the medium was added to the

plate in hexaplets. After 24 hrs of incubation, 10 μl of MTT reagent was added to each

well and was further incubated for 4 hrs. Formazan crystals formed after 4 hrs in each

well were dissolved in 150 μl of detergent and the plates were read immediately in a

microplate reader (Spectramex, 190 Molecular Devices Inc., USA) at 570 nm. Wells with

complete medium, nanoparticles, and MTT reagent, without cells were used as blanks. A

control experiment with series of dilutions of NaAuCl4 was performed using the same

MTT kit to validate the assay.

40

4.5 Results and Discussion:

4.5.1 Synthesis of Green gold Nanoparticles:

Our new green process for the production of gold nanoparticles uses direct

interaction of sodium tetrachlroaurate (NaAuCl4) with black Darjeeling tea, soyabean

supernatant and cinnamon powder in the absence of man-made chemicals and thus,

satisfies all the principles of a 100% green chemical process. Various phytochemicals

present in tea, soy and cinnamon are presumably responsible for making a robust coating

on gold nanoparticles and thus, rendering stability against agglomerations. Absorption

measurements indicated that the plasmon resonance wavelength, λmax of T-AuNPs, Soya-

AuNps and Cin-AuNps are 535 nm, 540 nm and 540 nm respectively. The sizes of T-

AuNPs, Soya-AuNps and Cin-AuNps are in the range of 12±4 nm; 16±5 nm and 15±5

nm respectively as measured from TEM techniques (Figures 4.1, 4.2, 4.3).

41

a) b)

c)

Size Distribution of TGAAuNP-H

0

5

10

15

20

25

6 8 10 12 14 16

Size (nm)

No.

of P

artic

les

Figure 4.1: a) UV-Visible absorption spectrum, b) TEM Image c) size distribution of Tea Gold Nanoparticles.

42

a) b)

c)

Figure 4.2: a) UV-Visible absorption spectrum, b) TEM Image, c) size distribution of Soy Gold Nanoparticles

0

2

4

6

8

10

12

6 8 10 12 14 16 18 20 22More

S ize (n m )

No.

of P

artic

les

43

a) b)

c)

Figure 4.3: a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Cinnamon Gold Nanoparticles

Size distribution of Cinnamon stabilized AuNP

05

1015202530

7 9 11 13 15 17 19

Size (nm)

No.

of P

artic

les

44

4.5.2 Cellular Internalization studies:

Results of cellular internalization studies of AuNps solutions are key to providing

insights into their use in biomedicine. Their selective cell and nuclear targeting will

provide new pathways for their site-specific delivery as diagnostic/ therapeutic agents. A

number of studies have demonstrated that phytochemicals in tea, soya and cinnamon

have the ability to penetrate the cell membrane and internalize within the cellular matrix

[89, 90]. Cancer cells are highly metabolic and porous in nature and are known to

internalize solutes rapidly compared to normal cells [90]. Therefore, we hypothesized

that tea, soya and cinnamon derived phytochemicals, if coated on AuNps, will show

internalization within cancer cells.

TEM images of prostate (PC-3) and breast tumor (MCF-7) cells treated with

AuNPs unequivocally validated our hypothesis. Significant internalization of

nanoparticles via endocytosis within the MCF-7 and PC-3 cells was observed (Figures

4.4a, b, c; 4.5a, b, c; 4.6a, b, c). The internalization of nanoparticles within cells could

occur via processes including phagocytosis, fluid-phase endocytosis, and receptor-

mediated endocytosis. The viability of both PC-3 and MCF-7 cells post-internalization

suggests that the phytochemical coating renders the nanoparticles non-toxic to cells. Such

a harmless internalization of AuNps will provide new opportunities for probing cellular

processes via nanoparticulate-mediated imaging.

45

a) b)

c)

Figures 4.4a, b, c: TEM Images of different MCF-7 cells showing uptake of Tea-AuNps in to the lysosomes.

46

a)

b)

Figures 4.5a, b: TEM Images of different MCF-7 cells showing uptake of Soya-AuNps in to the lysosomes.

47

a)

b)

Figures 4.6 a, b: TEM images of MCF-7 cell showing uptake of Cin-AuNps in to the lysosomes.

48

4.5.3 Cytotoxicity Studies:

Untreated PC-3 and MCF-7 cells as well as cells treated with 10, 30, 50, 70, 90,

110 and 150 μM concentrations of various AuNps for 24 hrs were subjected to the MTT

assay for cell-viability determination. In this assay, only cells that are viable after 24 h

exposure to the sample are capable of metabolizing a dye (3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide) efficiently and produce a purple colored precipitate

which is dissolved in a detergent and analyzed sphectrophotometrically. After 24 hrs

post-treatment, PC-3, MCF-7 cells showed excellent viability even up to 150 μM

concentrations of tea, soy and cinnamon-AuNps (Figures 4.7 a, b; 4.8 a, b;4.9 a, b). These

results clearly demonstrate that the phytochemicals within these herbs provide a non-

toxic coating on AuNps and corroborate the results of the internalization studies

discussed above. It is also important to recognize that a vast majority of Gold (I) and

Gold (III) compounds exhibit varying degrees of cytotoxicity to a variety of cells (Figure

4.10). The lack of any noticeable toxicity of tea, soy and cinnamon-AuNps provides new

opportunities for the safe application in molecular imaging and therapy.

49

a)

b)

4.7 a, b: Dose dependent cytotoxicity of Tea-AuNPs in cultured PC-3 and MCF-cells after 24 hrs of

exposure using MTT assay.

MTT Assay of Tea-AuNps in PC-3 cell line

0

20

40

60

80

100

120

140

160

10 30 50 70 90 110 150Concentration (um )

% C

ell V

iabi

lity

PC-3 cells-Tea AuNps

MTT Assay of Tea-AuNps in MCF-7 cell line

0

20

40

60

80

100

120

140

10 30 50 70 90 110 150Concentration (um )

% C

ell V

iabi

lity

MCF-7cells-Tea-AuNps

50

4.8 a, b: Dose dependent cytotoxicity of Soy-AuNPs in cultured PC-3 and MCF-cells after 24 hrs of

exposure using MTT assay.

MTT Assay of Soy-AuNps in PC-3 cell line

0

20

40

60

80

100

120

140

160

10 30 50 70 90 110 150Concentration (um )

% C

ell V

iabi

lity

PC-3 cells-Soy-AuNps

M TT Assay of Soy-AuNps in M CF-7 ce ll line

0

20

40

60

80

100

120

140

10 30 50 70 90 110 150Concentration (um )

% C

ell V

iabi

lity

MCF-7 cells-Soy-AuNps

51

a)

b)

4.9 a, b: Dose dependent cytotoxicity of Cin-AuNPs in cultured PC-3 and MCF-cells after 24 hrs of

exposure using MTT assay.

M TT Assay of Cin-AuNps in PC-3 ce lls

0

20

40

60

80

100

120

140

10 30 50 70 90 110 150Concentration (um )

% C

ell V

iabi

lity

PC-3 cells-Cin-AuNps

M TT Assay of Cin-AuNps in M CF-7 ce ll line

0

20

40

60

80

100

120

140

10 30 50 70 90 110 150Concentration (uM )

% C

ell V

iabi

lity

MCF-7 cells-Cin-AuNps

52

MTT Assay of natural constructs in MCF-7and PC-3 cell lines.

0

20

40

60

80

100

120

0.01 0.1 1 10 10

01,0

00

Concentration (M)

% C

ell V

iabi

lity NaAuCl4 in MCF-

7 cellsNaAuCl4 in PC-3cells

4.10: Dose dependent cytotoxicity of NaAuCl4 in cultured PC-3 and MCF-cells after 24 hrs of exposure

using MTT assay

53

CHAPTER 5

SUMMARY:

Functionalized and biomolecule conjugated gold nanoparticles will play an

indispensable role in the overall design and development of AuNps-based nano

pharmaceuticals. For molecular imaging and therapy, it will be of pivotal importance to

generate nanoparticles in bio-friendly media including, in the presence of chemically

sensitive proteins, peptides and receptor specific bio vectors. This research resulted in the

synthesis of non-toxic, biologically friendly gold nanoparticles that are conjugated with

highly specific biomolecules such as Bombesin and Annexin V. In addition, the

conjugated AuNps were tested for their target specificity towards cancerous cells and

apoptotic cells.

Our results show that Bombesin conjugated gold nanoparticles can enter prostate

and breast cancer cells via BBN/GRP receptor-mediated mechanism. These results show

a proof of concept for the in vivo delivery of AuNps to the tumor regions. Their use as

contrast enhancement agents to visualize tumors in vivo, using Computer Tomography

(CT) imaging technique depends on whether their uptake is selective in such cells as

opposed to surrounding non-tumerous tissue at non-toxic doses.

Conjugates of AuNps with Annexin V to detect apoptosis may be of use: in the

detection of apoptosis by CT-imaging, for cell sorting, and for better appreciation of

interactions between various chemotherapeutic agents and cells. Therefore, the synthesis

of biomolecule/protein conjugated gold nanoparticles outlined opens the way for the

54

fabrication of a wide range of biological probes, which can specifically target wide range

of cells.

Future Work

The present work may be continued by working towards the following directions

1. Evaluating the in vivo uptake profile of Bombesin gold nanoparticles and

Annexin V gold nanoparticles in tumors and apoptotic cells.

2. Determining the apt amount of conjugated AuNps to use them as contrast

enhancement agents.

3. Investigating the target specificity of Anx V-AuNps by inducing apoptosis via

different mechanisms.

4. Fabricating gold nanoparticles with more than one targeting agent such as

conjugating a receptor specific biomolecule and a florescent probe.

55

BIBLIOGRAPHY

1. Alivisatos AP. Semiconductor Clusters, Nanocrystals, and Quantum Dots. Science

271: 933-937, 1996.

2. Feldheim DL, Colby AF, Marcel D. Metal Nanoparticles: Synthesis,

Characterization and Applications, J. Phys. Chem 300: 11202-11208, 2002.

3. Kreibig U, Vollmer M. Optical Properties of Metal Clusters. Springer 25: 532-538,

1995.

4. Chandrasekharan N, Kamat PV, Hu J, Jones G. Dye- Capped Gold Nanoclusters:

Photoinduced Morphological Changes in Gold/Rhodamine 6G Nanoassemblies. J.

Phys. Chem 104:11103-11109, 2000.

5. Nath N, and Chilkoti A. Interfacial Phase Transition of an Environmentally

Responsive Elastin Biopolymer Adsorbed on Functionalized Gold Nanoparticles

Studied by Colloidal Surface Plasmon Resonance. J. Am. Chem. Soc 123:8197-8202,

2001.

6. Link S, El-Sayed MA. Shape and size dependence of radiative, non-radiative and

photothermal properties of gold nanocrystals. Int. ReV. Phys. Chem 19 (3): 409-453,

2000.

7. Link S, El-Sayed MA. Optical properties and ultrafast dynamics of metallic

nanocrystals. Annu. ReV. Phys. Chem 54:331-366, 2003.

8. Link S, Mohamed MB, El-Sayed MA. Simulation of the optical absorption spectra

of gold nanorods as a function of their aspect ratio and the effect of the medium

dielectric constant. J. Phys. Chem. B. 103:3073-3077.

9. Link S, El-Sayed MA. Additions and corrections to “Simulation of the optical

absorption spectra of gold nanorods as a function of their aspect ratio and the effect of

the medium dielectric constant”. J. Phys. Chem. B 109:10531-10532, 2005.

10. Nikoobakht B, Wang J, El-Sayed MA. Surface enhanced Raman scattering of

molecules adsorbed on gold nanorods: off-surface plasmon resonance condition.

Chem. Phys. Lett 366: 17-23, 2002.

56

11. Leff DV, Brandt L, and Heath JR. Synthesis and Characterization of Hydrophobic,

Organically-Soluble Gold Nanocrystals Functionalized with Primary Amines.

Langmuir 12:4723, 1996.

12. Niemeyer CM, Angew. Nanoparticles, Proteins, and Nucleic Acids: Biotechnology

Meets Materials. Science Chem. Int. Ed 40:4128-41582001, 2001.

13. Sanford JC, Smith FD, and Russell JA. Optimizing the biolistic process for

different biological applications. Methods Enzymol 217: 483-509, 1993.

14. Bielinska A, Eichman JD, Lee I, Baker JR, Balogh LJ. Imaging {Au0-PAMAM}

Gold-dendrimer Nanocomposites in Cells. Nanopart. Res 4: 395-403, 2002.

15. Olofsson L, Rindzevicius T, Pfeiffer I, Kall M, and Hook F. Surface-Based Gold-

Nanoparticle Sensor for Specific and Quantitative DNA Hybridization Detection.

Langmuir 19: 10414-10419, 2003.

16. Anderson SA, Rader RK, Westlin WF, Null C, Jackson D, Lanza GM, Wickline

SA, Kotyk JJ. Magnetic resonance contrast enhancement of neovasculature with

alpha(v)beta(3)-targeted nanoparticles. Magn Reson Med 44:433–439, 2000.

17. Turkevitch J, Stevenson PC, Hillier J. Nucleation and Growth Process in the

Synthesis of Colloidal Gold. Faraday Soc 11:55-75, 1951.

18. Brust M, Walker M, Bethell D, Schiffrin DJ, and Whyman RJ. Synthesis of

Thiol-Derivatized Gold Nanoparticles in a Twophase Liquid-Liquid System. J. Chem.

Soc., Chem. Commun 801-802, 1994.

19. Guo S, Wang E. Synthesis and electrochemical applications of gold

nanoparticles. Analytica Chimica Acta 598: 181-192, 2007.

20. Kattumuri V, M Chandrasekhar, S Guha, K Raghuraman, KV Katti, K Ghosh,

and RJ Patel. Agarose-stabilized gold nanoparticles for surfaceenhanced Raman

spectroscopic detection of DNA nucleosides. Applied Physics Letters 88(15), 2006.

21. Yang PH, Sun XS, Chiu JF, Sun HZ, He QY. Functionalized gold nanoparticles for

drug delivery. Bioconj. Chem. 16:494-496, 2005.

22. Chithrani BD, Chan WCW. Elucidating the Mechanism of Cellular Uptake and

Removal of Protein-Coated Gold Nanoparticles of Different Sizes and Shapes. Nano

Lett. 7:1542-1550, 2007.

57

23. Hong R, Han G, Fernandez JM, Kim BJ, Forbes NS, and Rotello VMJ.

Glutathione-Mediated Delivery and Release Using Monolayer Protected Nanoparticle

Carriers J. Am. Chem. Soc. 128:1078-1079, 2006.

24. Tkachenko AG, Xie H, Coleman D, Glomm W, Ryan J, Anderson MF, Franzen

S, Feldheim DL. Multifunctional gold nanoparticle-peptide complexes for nuclear

targeting. J. Am. Chem. Soc. 125:4700-4701, 2003.

25. Tkachenko AG, Xie H, Liu Y, Coleman D, Ryan J, Glomm WR, Shipton MK,

Franzen S, Feldheim DL. Cellular trajectories of peptide-modified gold particle

complexes: comparison of nuclear localization signals and peptide transduction

domains. Bioconjugate Chem. 15:482-490, 2004.

26. Sokolov K, Aaron J, Hsu B, Nida D, Gillenwater A, Follen M, MacAulay C,

Adler-Storthz K, Korgel B, Descour M, Pasqualini R, Arap W, Lam W,

Richards-Kortum R. Optical systems for in vivo molecular imaging of cancer.

Technol Cancer Res Treat. 2:491– 504, 2003.

27. Elghanian R, Storhoff JJ, Mucic RC, Letsinger RL, Mirkin CA. Selective

colorimetric detection of polynucleotides based on the distance-dependent optical

properties of gold nanoparticles. Science 277:1078–81, 1997.

28. Hirsch LR, Stafford RJ, Bankankson JA, Sershen SR, Rivera B, Price RE, Hazle

JD, Halas NJ, West JL. Nanoshell-mediated nearinfrared thermal therapy of tumors

under magnetic resonance guidance. Proc Natl Acad Sci 100:13549–13554, 2003.

29. Anastasi A, Erspamer V, Bucci M. Isolation and structure of bombesin and alytesin

2 analogous active peptides from the skin of the European amphibians Bombina and

Alytes. Experientia 27: 166–167, 1971.

30. Hampton LL, Ladenheim EE, Akeson M, Way JM, Weber HC, Sutliff VE,

Jensen RT, Wine LJ, Arnheiter H, Battey JF. Loss of bombesin-induced feeding

suppression in gastrin-releasing peptidereceptor-deficient mice. PNAS 95: 3188-3192,

1998.

31. Ladenheim EE, Emond M, Moran TH. Leptin enhances feeding suppression and

neural activation produced by systemically administered bombesin. Am J Physiol

Regulatory Integrative Comp Physiol 289: R473-R477, 2005.

58

32. Michaud D, Anisman H Merali Z. Capsaicin-sensitive fibers are required for the

anorexic action of systemic but not central bombesin. Am J Physiol Regulatory

Integrative Comp Physiol 276: R1617-R1622, 1999.

33. Stephen CW, Randy JS, Daniel P, Michael WS. Signals That Regulate Food

Intake and Energy Homeostasis. Science 280: 1378-1383, 1998.

34. McDonald TJ, Jornvall H, Nilsson J, Vagne M, Ghatei M, Bloom SR, Mutt V.

Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue.

Biochem Biophys Res Commun 90: p. 227, 1979.

35. Dembinski A, Konturek PK, Konturek SJ. Role of gastrin and cholecystokinin in

the growth-promoting action of bombesin on the gastroduodenal mucosa and the

pancreas. Regul Pept 27: p. 343, 1990.

36. Dembinski A, Warzecha Z, Konturek SJ, Banas M, Cai RZ, Schally AV. The

effect of antagonist of receptors for gastrin, cholecystokinin and bombesin on

growth of gastroduodenal mucosa and pancreas. J Physiol Pharmacol 42: 263-65,

1991.

37. Cuttitta F, Caney DN, Mulshine J, Moody TW, Fedorko J, Fishler A, Minna

JD. Bombesin-like peptides can function as autocrine growth factors in human

small-cell lung cancer. Nature 316: 823 – 826, 1985.

38. Reubi JC, Wenger S, Schmuckli-Maurer J, Schaer J.C, Gugger M. Bombesin

receptor subtypes in human cancers: detection with the universal radioligand (125)I-

[D-TYR(6), beta-ALA(11), PHE(13), NLE(14)] bombesin(6-14). Clin Cancer Res,

8:1139-1143, 2002.

39. Carroll RE, Matkowskyj KA, Chakrabarti S, McDonald TJ, Benya RV. Aberrant expression of gastrin-releasing peptide and its receptor by well-

differentiated colon cancers in humans. Am J Physiol 276: G655-65, 1999.

40. Siegfried JM, DeMichele MA, Hunt JD, Davis AG, Vohra KP, Pilewski JM. Expression of mRNA for gastrin-releasing peptide receptor by human bronchial

epithelial cells. Association with prolonged tobacco exposure and responsiveness to

bombesin-like peptides. Am J Respir Crit Care Med 156: 358-66, 1997.

41. Pansky A, De Weerth A, Fasler-Kan E, Boulay JL, Schulz M, Ketterer S, Selck

C, Beglinger C, Von Schrenck T, Hildebrand P. Gastrin releasing peptide-

59

preferring bombesin receptors mediates growth of human renal cell carcinoma. J Am

Soc Nephro 11:1409-1418, 2000.

42. Smith CJ, Volkert WA, Hoffman TJ. Gastrin releasing peptide (GRP) receptor

targeted radiopharmaceuticals: a concise update. Nucl Med Biol 30:861-868, 2003.

43. Sun B, Schally AV, Halmos G. The presence of receptors for bombesin/GRP and

mRNA for three receptor subtypes in human ovarian epithelial cancers. Regul Pept

30: 77-84, 2000

44. Burghardt B, Wenger C, Barabás K, Rácz G, Oláh A, Flautner L, Coy DH,

Gress TM, Varga G. GRP-receptor-mediated signal transduction, gene expression

and DNA synthesis in the human pancreatic adenocarcinoma cell line HPAF.

Peptides 22: 1119-1128, 2001.

45. Bartholdi MF, Wu JM, Pu H, Troncoso P, Eden PA, Feldman RI. In situ

hybridization for gastrin-releasing peptide receptor (GRP receptor) expression in

prostatic carcinoma. Int J Cancer 20:82-90, 1998.

46. Lin KS, Luu A, Baidoo KE, Hashemzadeh-Gargari H, Chen MK, Brenneman

K, Pili R, Pomper M, Carducci MA, Wagner HN Jr. A new high affinity

technetium-99m-bombesin analogue with low abdominal accumulation. Bioconjug

Chem 16: 43-50, 2005.

47. Kunstler JU, Veerendra B, Figueroa SD, Sieckman GL, Rold TL, Hoffman TJ,

Smith CJ, Pietzsch HJ. Organometallic 99mTc(III) '4 + 1' bombesin(7-14)

conjugates: synthesis, radiolabeling, and in vitro/in vivo studies. Bioconjug Chem

18: 1651-1661, 2007.

48. Hoffman TJ, Quinn TP, Volkert WA. Radiometallated receptor-avid peptide

conjugates for specific in vivo targeting of cancer cells. Nucl Med Biol 28:527-532,

2001.

49. Raghuraman K, Katti KK, Barbour LJ, Pillarsetty N, Barnes CL, Katt KV.

Characterization of supramolecular (H2O)(18) water morphology and water-

methanol (H2O)(15)(CH3OH)(3) clusters in a novel phosphorus functionalized

trimeric amino acid host. Journal of the American Chemical Society 125: 6955-6960,

2003.

60

50. Neves M, Gano L, Pereira N, Costa MC, Costa MR, Chandia M, Rosado M,

Fausto R. Synthesis, characterization and biodistribution of bisphosphonates Sm-

153 complexes: correlation with molecular modeling interaction studies. Nuclear

Medicine and Biology 29:329-335, 2002.

51. Grady LH, Nonneman DJ, Rottinghaus GE,Welshons WV. PH-Dependent

cytotoxicity of contaminants of phenol red for MCF-7 breast cancer cells.

Endocrinology 129:3321-3330, 1991.

52. Welshons WV, Grady LH, Engler KS, Judy BM. Control of proliferation of

MCF-7 breast cancer cells in a commercial preparation of charcoalstripped adult

bovine serum. Breast Cancer Res Treat 23:97-104, 1992.

53. Read LD, Greene GL, Katzenellenbogen BS. Regulation of estrogen receptor

messenger ribonucleic acid and protein levels in human breast cancer cell lines by

sex steroid hormones, their antagonists, and growth factors. Mol Endocrinol 3:295-

304, 1989.

54. Kattumuri V. Gold Nanoparticles for Bio-medical applications: Synthesis,

Characterization, In Vitro and In Vivo studies. Thesis, Dept of Physics and

Astronomy, UMC.

55. Hoffman TJ, Sieckman GL, Volkert WA, Truman HS. Iodinated bombesin

analogues: Effect of N-terminal vs side chain iodine attachment on BBN/GRP

receptor binding. Journal of Nuclear Medicine 37: 850-855, 1996.

56. Hoffman TJ, Quinn TP, Volkert WA. Radiometallated receptor-avid peptide

conjugates for specific in vivo targeting of cancer cells. Nucl Med Biol 28 :527-532,

2001.

57. Narayanan R, Lipert RJ, Porter MD. Cetyltrimethylammonium bromide-modified

spherical and cube-like gold nanoparticles as extrinsic Raman labels in surface-

enhanced Raman spectroscopy based heterogeneous immunoassays. Anal Chem 80:

2265-71, 2008.

58. Steven H Young and Enrique Rozengurt. Qdot Nanocrystal Conjugates

conjugated to bombesin or ANG II label the cognate G protein-coupled receptor in

living cells. Am J Physiol Cell Physiol 290:C728–C732, 2006.

61

59. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science

267:1456-1462, 1995.

60. Peter ME, Heufelder AE, Hengartner MO. Advances in apoptosis research. Proc.

Natl. Acad. Sci. U.S.A. 94:12736-12737, 1997.

61. Bursch W, Oberhammer F, Schulte-Hermann R. Cell death by apoptosis and its

protective role against disease. Trends Pharmacol. Sci. 13:245-251, 1992.

62. Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, Schie RC, LaFace

DM. Early redistribution of plasma membrane phosphatidylserine is a general

feature of apoptosis regardless of the initiating stimulus: inhibition by

overexpression of Bcl-2 and Abl. J. Exp. Med. 182:1545-1556, 1995.

63. Schlegel RA, Williamson P. Phosphatidylserine, a death knell. Cell Death Differ

8:551-563, 2001.

64. Funakoshi T, Heimark RL, Hendrickson LE, McMullen BA, Fujikawa K.

Human placental anticoagulant protein: isolation and characterization. Biochemistry,

26:5572-5578, 1987.

65. Van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, and Reutelingsperger

CP. Annexin V-affinity assay: a review on an apoptosis detection system based on

phosphatidylserine exposure. Cytometry 31:1-9, 1998.

66. Hammill AK, Uhr JW, Scheuermann RH. Annexin V staining due to loss of

membrane asymmetry can be reversible and precede commitment to apoptotic death.

Exp. Cell Res 251:16-21, 1999.

67. Bossy-Wetzel E, Green DR. Detection of apoptosis by annexin V labeling. Methods

Enzymol 322:15-18, 2000.

68. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for

apoptosis. Flow cytometric detection of phosphatidylserine expression on early

apoptotic cells using fluorescein labeled Annexin V. J Immunol. Methods 184:39-51,

1995.

69. Schellenberger E, Bogdanov AJ, Petrovsky A, Ntziachristos V, Weissleder R,

and Josephson L. Optical imaging of apoptosis as a biomarker of tumor response to

chemotherapy. Neoplasia 5:187-192, 2003.

62

70. Petrovsky A, Schellenberger E, Josephson L, Weissleder R, and Bogdanov A Jr.

Near-infrared fluorescent imaging of tumor apoptosis. Cancer Res. 63:1936-42,

2003.

71. Sestier C, Da-Silva MF, Sabolovic D, Roger J, and Pons JN. Surface modification

of superparamagnetic nanoparticles (Ferrofluid) studied with particle

electrophoresis: application to the specific targeting of cells. Electrophoresis 19:

1220-1226, 1998.

72. Eyk A Schellenberger, David Sosnovik, Ralph Weissleder, and Lee Josephson.

Magneto/Optical Annexin V, a Multimodal Protein. Bioconjugate Chem. 15 (5),

1062 -1067, 2004.

73. Huang J, Li Q, Sun D, Lu Y, Su Y, Yang X, Wang H, Wang Y, Shao W, Hong

NJ, Chen C. Biosynthesis of silver and gold nanoparticles by novel sundried

Cinnamomum camphora leaf. Nanotechnol. 18:105104-105115, 2007.

74. Jorge L, Torresdey G, Gomez E, Peralta-Videa JR, Parsons JG, Troiani H,

Yacaman MJ. Phytoremediation of heavy metals and study of the metal

coordination by X-ray absorption spectroscopy. Langmuir 19: p.p1357, 2003.

75. Gardea-Torresdey JL, Tiemann KJ, Parsons JG, Gamez G, Herrera I, Jose

Yacaman M. Investigation into the Mechanism(s) of Au (III) Binding and

Reduction by Alfalfa Biomass. Microchemical Journal. 71:193-204, 2002.

76. Hardman R. Toxicologic Review of Quantum Dots: Toxicity Depends on

Physicochemical and Environmental Factors. Environ. Health. Perspect.114: p.p165,

2006.

77. Curtis J, Greenberg M, Kester J,Phillips S, Krieger G. Nanotechnology and

Nanotoxicology: A Primer for Clinicians. Toxicol. Rev. 25: p.p 245, 2006.

78. Lewinski N, Colvin V, Drezek R. Cytotoxicity of nanoparticles. Small 4:26-49,

2008.

79. Espín JC, García-Conesa MT, Tomás-Barberán FA. Nutraceuticals: Facts and

fiction. Phytochemistry 68: p.p 2986, 2007.

80. Rochfort S, Panozzo J. Class targeted metabolomics: ESI ion trap screening

methods for glucosinolates based on MSn fragmentation. J. Agric. Food. Chem. 55:

p.p7981 2007.

63

81. Setchell KD, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, Brashear

WT, Kirschner AS, Cassidy A, Heubi JE. Bioavailability of pure isoflavones in

healthy humans and analysis of commercial soy isoflavone supplements. J Nutr.

131:1362S-75S, 2001.

82. Magee PJ, Rowland IR. Phyto-oestrogens, their mechanism of action: current

evidence for a role in breast and prostate cancer. Br. J. Nutr 91:513-520, 2004.

83. Limer JL, Speirs V. Phyto-oestrogens and breast cancer chemoprevention.Breast

Cancer Res 6: 119-127, 2004.

84. Bandele OJ, Osheroff N. (-)-Epigallocatechin Gallate, A Major Constituent of

Green Tea, Poisons Human Type II Topoisomerases. Chem Res Toxicol. 21:936-43,

2008.

85. Shankar S, Ganapathy S, Srivastava RK. Green tea polyphenols: biology and

therapeutic implications in cancer. Front Biosci 12:4881-99, 2007.

86. Dannemann K, Hecker W, Haberland H, Herbst A, Galler A, Schäfer T,

Brähler E, Kiess W, Kapellen TM. Use of complementary and alternative

medicine in children with type 1 diabetes mellitus - prevalence, patterns of use, and

costs. Pediatr Diabetes, 2008.

87. Suppapitiporn S, Kanpaksi N. The effect of cinnamon cassia powder in type 2

diabetes mellitus. J Med Assoc Thai 89 Suppl 3:S200-5, 2006.

88. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application

to proliferation and cytotoxicity assays. J Immunol Methods 65:55-63, 1983.

89. Mizuno H, Cho YY, Zhu F, Ma WY, Bode AM,Yang CS,Ho CT, Dong ZG.

Theaflavin-3, 3′-Digallate Induces Epidermal Growth Factor Receptor Down-

Regulation Mol. Carcinog 45: 204-212, 2007.

90. Sun DJ, Liu Y, Lu DC, Kim W, Lee JH, Maynard J, Deisseroth A. Endothelin-3

growth factor levels decreased in cervical cancer compared with normal cervical

epithelial cells Human Pathology 38: 1047-1056, 2007.