Micellear Gold Nanoparticles as Delivery Vehicles for Dual Tyrosine

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Micellear Gold nanoparticle as delivery vehicle for dual tyrosine kinase inhibitor ZD6474 for metastatic breast cancer treatment Siddik Sarkar, Suraj Konar, Nagaprasad Puvvada, Shashi Rajput, BN Prashanth Kumar, Raj R. Rao, Amita Pathak, Paul B. Fisher, and Mahitosh Mandal Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01072 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 17, 2017

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Micellear Gold nanoparticle as delivery vehicle for dual tyrosine kinase inhibitor ZD6474 for metastatic breast cancer treatment

Siddik Sarkar1,4,*, Suraj Konar1, Puvvada Naga Prasad2, Shashi Rajput1, B.N. Prashanth Kumar1, Raj R. Rao3, Amita Pathak2,*, Paul B. Fisher4, Mahitosh Mandal1,*

1

School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, INDIA; Department of Chemistry, Indian Institute of Technology, Kharagpur, INDIA; 3 Department of Chemical and Life Science Engineering, School of Engineering, Virginia commonwealth University, Richmond, VA, USA; 4 Department of Human and Molecular Genetics, VCU Institute of Molecular Medicine, VCU Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA; *To whom requests for reprints should be addressed: Drs. Mahitosh Mandal, Siddik Sarkar School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, West Bengal, PIN-721302, INDIA. Tel: +91-3222-283578, Fax: +91-3222-282221. E-mail: [email protected], [email protected] 2

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ABSTRACT Therapeutic index of poorly water soluble drugs are often hampered due to poor pharmokinetics, reduced blood retention and lack of effective drug concentrations in the tumor region. In order to overcome these issues, drugs are often delivered using delivery vehicles to provide an enhanced therapeutic index. Gold nanoparticles synthesized in micellar networks of amphiphilic block copolymer (AuNM) provide an efficient nanocarrier for tissue- and site-specific drug delivery owing to their low cytotoxicity and immunogenicity. AuNM is formed by exploiting the properties of both inorganic Au material and an amphiphillic polymer of polyethylene glycol-block-polypropylene glycol-block-polyethylene glycol-block (PEG-PPG-PEG). We further functionalized AuNM with a FDA approved dual tyrosine kinase inhibitor-ZD6474, i.e., ZD6474AuNM, and studied its physio-chemical properties. Both AuNM and ZD6474-AuNM, with a diameter of ~70 nm, were very stable at physiological pH. Conversely, at an acidic pH of 5.2, a slow sustained release profile of ZD6474 was evident from AuNM, which could provide a method of facilitating release of the drug in an acidic tumor environment. In vitro in triple negative breast cells, ZD6474-AuNM inhibited tumor cell proliferation, migration and invasion, and induced apoptosis. There was no detectable lysis of RBCs observed when they were treated with AuNM and ZD6474-AuNM, confirming hemocompatibility. To reinforce the possibility of AuNM serving as a delivery vehicle, AuNM was conjugated with the IR680 dye for tracking, and systemically delivered in female nude mice bearing MDA-MB-231 human breast cancer xenografts. Fluorescence signal was retained in the tumor region in a temporal manner as compared to other organs, indicating passive retention of AuNM in the tumor locale. Moreover, delivery of 2 ACS Paragon Plus Environment

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ZD6474-AuNM in nude mice bearing MDA-MB-231 xenografts led to decreased tumor size as compared to the control group. The promising safety, targeting and therapeutic results of systemic delivery of ZD6474 by AuNM provide an attractive alternative method for treating patients with metastatic breast cancer.

Key words: Micellar stabilized gold nanoparticle or

Gold nanomicelle (AuNM),

Vandetanib, ZD6474, ZD6474-AuNM, breast cancer; apoptosis.


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INTRODUCTION Cancer therapy frequently involves the administration of increasing doses of chemotherapeutic drugs, which often results in nonspecific toxicities. Drugs, when locally administered in a soluble form, are easily absorbed through capillaries into the circulatory system and distributed nonspecifically in the body, which may be the reason for therapeutic failure and systemic toxicities. Although conventional radio- and chemotherapy have been the gold standard of cancer therapy for decades, these approaches are not optimal and can lead to resistance to these and other therapies. Effectiveness of cancer therapy depends on fine tuning of eradication of cancer cells with minimal or ideally no toxic effect on normal cells

1, 2, 3

. The development of molecular-targeted

therapy or delivery systems that can preferentially localize anticancer agents at the site of action have potential to enhance therapeutic outcomes. Nanoparticles (NPs) are appealing drug carriers based on their high tissue permeability4, high colloidal stability and small size in the nanometer nm range. The enhanced permeability and retention (EPR) 5 effect of nanoparticles permit accumulation at the tumor site. Different types of inorganic materials are used to prepare NPs , and the use of Au as inorganic materials to prepare NPs or colloidal solution has been studied over decades6, 7. The primary rationale for selecting inorganic material Au in preparion of nanoparticle is its biocompatibility 8, high surface to volume ratio (large amount of drug can be loaded), aqueous solubility, ease of characterization and surface modification (i.e., organic molecules such as drugs, peptides, antibodies, etc., can be easily attached to gold nanoparticles) 9, 10. The absorption of AuNP can be tuned at near-infra-red regions, where


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tissue penetration is optimal and tissue absorption is minimal, providing a sharp contrasting agent for imaging and diagnostic purposes 11, 12. Numerous approaches were previously reported for preparing AuNPs by the reduction of Au(III) to Au(0) in presence of common reducing agents like sodium borohydride (NaBH4) or Sodium citrate

10

. There were also few reports of using

biocompatible amphiphillic polymers as a reducing and stabilizing agents for preparing AuNPs

13, 14

. It was also observed that the size of AuNPs could be varied or tuned based

on application and purposes by changing the molar ratios of Au and polymers

15

. Three

types of block polymers were also used as reducing agents for the synthesis of AuNPs and there morphological variations were also reported by Khullar et al.16. Deng et al. reported the synthesis of biocompatible miceller AuNPs and studied their diagnostic and therapeutic

functions17.

The

Triblock

non-ionic

copolymer

PEO-PPO-PEO

[Poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO)] was also used for synthesizing the gold nanoparticles previously

18

. Continuing this idea, herein, we have

successfully synthesized the AuNP by using non-toxic triblock non-ionic copolymer PEG-PPG-PEG as a reducing and stabilizing agent. Since AuNPs is located in the core or hydrophobic region of the micellar PEG-PPG-PEG, we referred this structure as Gold nanomicelle (AuNM). The use of amphiphillic polymer will assist in further achieving high amount of colloidal AuNPs by preventing agglomeration, increased retention time in blood, and possibly targeting tumor vasculature via passive EPR effect targeting by using specific ligands

20, 21

5, 19

or active

. Keeping this consideration AuNM will be an

attractive delivery vehicle for chemotherapeutic drugs with low solubility. In this paper,


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we have used AuNM as vehicle to deliver the molecular targeted drug ZD6474 (Vandatanib). ZD6474, a dual tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR), is associated with cell growth inhibition of non-small cell lung, head and neck, thyroid and breast cancers 22, 23, 24. It reverses multidrug-resistance (MDR) by directly inhibiting the activity of P-glycoprotein

25

. It also inhibits angiogenesis, and tumor growth, progression and

metastasis. Currently ZD6474 is an FDA approved small molecule used for treating EGFR overexpressing metastatic thyroid cancer preclinical

23, 27

26

. It had shown promising results in

models in breast cancer. But the results were not satisfactory or

promising in clinical trials as compared to preclinical results 28. It is highly possible that the lower effective drug concentration of ZD6474 available to cancer cells in patients is responsible for its reduced efficacy1, 2, 3. In order to enhance the effective dose of ZD6474 in the tumor region, we have conjugated it with micellar gold nanoparticles or Gold nanomicelle (AuNM). In this manuscript we address the preparation of AuNM using micellar PEGPPG-PEG as both reducing as well as stabilizing agent, and studied its physiochemical properties. We have also shown that chemotherapeutic ZD6474 can also be conjugated with micellar AuNP (ZD6474-AuNM) for effective delivery. Few studies have been conducted regarding the potential of AuNP in drug delivery in cancer cells

29, 30

. We

hypothesized that ZD6474 conjugated with AuNM could improve the therapeutic index of ZD6474 in treating breast cancer. We have tested this hypothesis using triple negative breast cancer (most aggressive of all breast cancer subtypes) cell lines MDA-MB-468 and 6 ACS Paragon Plus Environment

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MDA-MB-231. Our in vitro and in vivo studies provide impetus and rationale for potentially using AuNM for drug delivery, which can be further improved by conjugating AuNM with ligands binding with receptors that are up-regulated in cancers or their vasculature, thus, enhancing therapeutic index of chemotherapeutic agents in cancer patients with minimal side effects.

EXPERIMENTAL SECTION Reagents: Stock solutions of 100 mM ZD6474 (AstraZeneca Pharmaceuticals, Macclesfield, United Kingdom) were prepared in DMSO (Sigma-Aldrich, St. Louis, MO, USA), stored at -20°C, and diluted in fresh medium or solvent prior to use. Stock solutions of 20 mM Gold (III) chloride trihydrate (HAuCl4.3H2O) (Sigma-Aldrich) and 10 mM of Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), i.e., PEG-PPG-PEG (Sigma-Aldrich) and 10 mM of O,O′-Bis(2-aminopropyl) poly (propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol), i.e., PPGPEG-PPG were prepared in distilled water. 1 mg/mL of 3-(4,5-dimethylazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT) (Sigma Aldrich) was prepared in cell culture medium just prior to use. All the reagents used in this study were of analytical grade and were used without further purification. Cell Lines: Human breast cancer cell lines MDA-MB-231 and MDA-MB-468 were cultured in Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (Ham) (D-MEM/F-12) with 15 mM HEPES buffer, L-glutamine, pyridoxine hydrochloride, supplemented with 1.2 g Sodium bicarbonate (Gibco® Invitrogen™, Auckland, NZ), antibiotics (10,000 U/L penicillin and 10 mg/L streptomycin) (Himedia, Mumbai, India)


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and 10% FBS (Gibco® Invitrogen™, Auckland, NZ). Cells were incubated at 37°C in a 5% CO2 and 95% humidified incubator (Heraeus® Hera Cell, USA). Preparation of Micelles: In order to prepare the micelle solution, PEG-PPG-PEG (0.5 g) was dissolved in 10 mL of milli Q water and stirred vigorously for 20 min at room temperature in a sealed container and stored overnight under refrigeration to stabilize the micelle solution. Synthesis of micellar stabilized Au nanoparticle (AuNM): 2 mL of micelle solution were mixed with 20 µl of HAuCl4.3H2O. 20 mM of HAuCl4.3H2O solution was added drop wise (1 µL at a time for a total volume 20 µL) to 2 mL of micelle solution of PEG-PPG-PEG in a container kept on a magnetic stirrer at 25˚C. After addition of 20 µL of gold solution to the micelle solution the stirring of the mixture was stopped and the mixture was left for an additional 1 h at 25˚C. During the 1 h incubation time (ageing) without stirring the solution color changes to colorless and further changed to reddish pink. Synthesis of ZD6474 loaded AuNM (ZD6474-AuNM): 1 mg of ZD6474 was dissolved in 200 µL of ethanol and dried in a vacuum oven until dried forming a white layer in RB Flask. To this layer we added 1 mL of Au nanoparticle-loaded micelles and the solution was shaken in a roller apparatus for 3 h, which resulted in the formation of ZD6474-loaded AuNM. Characterization: UV-Visible spectrophotometer (Lamda 45, Perkin Elmer Instruments, California, USA) was used to measure formation of micelles after gold nanocolloid formation and ZD6474 loading. The morphology and particle size of nanoconjugates were measured using High Resolution Transmission Electron


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Microscopy (HR-TEM). For this study, the sample was dropped on a copper mesh coated with an amorphous carbon film for HR-TEM ((JEOL, JEM-2010, Tokyo, Japan) operated at 200 kV to obtain the images of micelle-reduced AuNP and ZD6474-loaded micellereduced AuNP. Drug entrapment: ZD6474-loaded micellar AuNP (ZD6474-AuNM) were separated from untrapped ZD6474 by centrifuging (5000g, 20 min) the drug-loaded solution through Amicon® Ultra Centrifugal Filter Devices (cut off 10 kD) (Millipore, Carrigtwohill, Ireland) and the absorbance of free drug in the filtrate was measured using UV-VIS spectrophotometer. The percentage of entrapment efficiency was calculated by the formula: Drug released (%) =

× 100

where, E % = Entrapment efficiency of ZD6474-loaded AuNM Atotal drug = Optical density of ZD6474 used for preparation of ZD6474-AuNM Aunbound drug = Optical density of filtrate containing unbound ZD6474. Drug releasing studies: The in vitro drug release profile of the ZD-6474 loaded miceller AuNPs (i.e., ZD6474-AuNM) was determined at two different pH values of 7.4 and 5.2 by dialysis membrane method and measuring the absorbance using a UV-Visible spectrophotometer. Briefly, the ZD6474 loaded nanoparticles placed in a dialysis bag with a molecular cut off of 14 kDa. The bag was then suspended in ethanol (maintained at pH 7.4 and pH 5.2) at 37 °C as the release medium. Ethanol was used instead of water as the release medium to provide sink conditions as ZD6474 is poorly soluble in water, whereas pH values of 7.4 and 5.2 were maintained to mimic the physiological pH of blood and the acidic intracellular environment in tumor cells respectively. At appropriate


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time intervals, requisite amount of aliquots was taken from the released medium and analyzed by a spectrophotometer at λmax value of 248 nm. The amount of drug released (%) was calculated using the following equation31, 32.

Drug released (%) =

× 100

Zeta Potential (ζ): Photon correlation spectroscopy (DLS) used to determine the resultant sample hydrodynamic particle size distribution and the corresponding zeta potential was measured by Nano Zetasizer (Zetasizer nano, Malvern Instruments, UK) after diluting the synthesized nanocarriers with PBS (pH 7.2) solution. Cell proliferation by MTT assay: Cell suspensions of actively growing MDAMB-231 and MDA-MB-468 were dispensed (200 µL) in quadruplicate into 96-well tissue culture plates at an optimized concentration of 1 x 104 cells/well in complete medium. Twenty-four h after seeding, cells were untreated or treated with 5 and 10 µM of ZD6474-AuNM (functionalized AuNM), AuNM or ZD6474 (drug only) and incubated for 72 h. Cell growth was assessed by 3-(4,5-dimethylazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay 33. The MTT assay provides growth assessment by measuring the enzymatic reduction of yellow tetrazolium MTT to a purple formazan, as measured at 540 nm using microplate reader (Bio-Rad, Hercules, CA, USA). All experiments were performed 3 times and the average of all of the experiments has been shown as cellgrowth percentage in comparison with the control experiment, where untreated controls were considered as 100% proliferation. Boyden chamber assay. To test the invasive and migratory behavior of treated cells Boyden chamber assay was performed as mentioned previously

23

with slight

modifications. Cells (1 x 105) that were treated with non-toxic dose of 1 µM ZD6474 and 10 ACS Paragon Plus Environment

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1 µM ZD6474-AuNM (AuNM containing equivalent dose of 1 µM of ZD6474), AuNM along with the control were added on the top chamber. Conditioned medium from mouse fibroblast NIH-3T3 cells was used as a source of chemoattractant and placed in the bottom compartment of the 6-well plate. After 12 h, noninvaded cells are scrapped off, and the cells that had migrated to the lower surface of the filter inserts were stained with 1 µM of cell labeling reagent Calcein-AM (Molecular Probes, Inc., Eugene, OR). The results are expressed as the

number of migrated cells as compared to the control

(untreated cells) observed under fluorescent microscope with 10X objective . Cell cycle and apoptosis: Cells were treated with 2.5 µM ZD6474, 2.5 µM ZD6474-AuNM, AuNM for 48 h and along with control (untreated) MDA-MB-268 and MDA-MB-231 after seeding in 60-mm tissue culture plates. After treatment, both attached and floating cells were collected and washed in phosphate-buffered saline (PBS) and incubated in 70% ethanol, kept at -20ºC overnight for fixation. Cells were centrifuged, washed and then incubated with PI solution (40 µg/mL PI, 100 µg/mL RNase A in PBS) at 37ºC for 1 h. Apoptotic cells were determined by their hypochromic sub-diploid staining profiles. The distribution of cells in the different cell-cycle phases was analyzed from the DNA histogram using Becton-Dickinson FACSCalibur flow cytometer and CellQuest software. In order to further study apoptosis in more detailed manner, we have performed an assay with FITC-Annexin V/ PI staining kit (Molecular Probes, Inc.) as par manufacturer's instructions. Hemocompatibility: Goat blood was collected and RBCs were separated using HISTOPAQUE®-1077 (Sigma-Aldrich). 5% v/v RBCs were prepared in PBS (pH 7.2) to which AuNM, ZD6474-AuNM or ZD6474 were added and the reading was recorded at


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570 nm in triplicate after 3 h using a microplate reader. A negative control (PBS) along with a positive control (0.1% Triton X-100) was also used. RBC lysis was calculated in comparison with 100% lysis in the positive control. In vivo tumor xenograft studies and antitumor efficacy of ZD6474-AuNM: Biodistribution and tumor delivery of AuNM, ZD6474-AuNM was studied using a nude mouse model of breast cancer. The animal research protocol was approved, and mice were maintained in accordance with the institutional (IACUC) guidelines of Virginia Commonwealth University, School of Medicine. Exponentially growing MDA-MB-231 cells were harvested and 2.5 x 106 cells in Matrigel were injected s.c. in 12–16 w-old female athymic BALB/c (nu+/nu+) mice. Tumor volume was calculated using the formula (A) (B2) π/6, where A was the length of the longest aspect of the tumor, and B was the length of the tumor perpendicular to A. All mice were randomly divided into two groups after 7 days of post injection of cells, AuNM control (n=5) and ZD6474-AuNM (n=5). ZD6474-AuNM was systemically injected twice a week by tail vein route for a period of 4-wk and tumor volume was monitored over the period of treatment. The dosage of ZD6474-AuNM used in this study was adjusted to contain equivalent amount of ZD6474 (30 mg/kg) dissolved in 200 µL PBS. Mice were sacrificed at the end of the experimental protocol. In vivo and ex vivo Fluorescence Imaging to study biodistribution and tumor delivery of IR680-AuNM: Mice were subjected to anesthesia prior to and during the imaging procedure. The animals were positioned within the scanner (IVIS Spectrum Preclinical in vivo Imaging System, Perkin Elmer, MA) of the imaging platform with IVIS Flow switched on, that regulate the flow of oxygen, isoflurane and remove the waste


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isoflurane gases by anesthesia gas filter unit (f/air) (A.M. Bickford Inc., NY). Acquisition of fluorescence image was done in spectral unmixing mode and digitally captured for further processing using Living Image 4.3.1 module. Following the imaging scan, the mice were allowed to recover from anesthesia and housed in the appropriate cage for repeated imaging studies. In order to study biodistribution and tumor delivery of AuNM, IR680 conjugated AuNM was prepared by using IR680dye Labelling kit (LI-COR, Lincoln, NE) with slight modifications. During the preparation of micelle, we have added PEG-PPG-PEG: PPGPEG-PPG at molar ratio of 9:1 in order to incorpoarate small amount of PPG-PEG-PPG polymer coating or stabilizing the core AuNP. This was done purposefully inorder to have reactive amine (NH2) group of PPG-PEG-PPG for conjugation with IR680 reactive NHS ester group as shown in Suppl Fig. S2. The conjugated IR680 in AuNM (IR680AuNM) was further washed to remove traces of unconjugated IR680 dye. Finally IR680AuNM was resuspended in PBS and systemically injected into female nude mice bearing MDA-MB-231 human breast cancer xenografts. Mice were imaged over time to detect and follow the accumulation and/or retention of IR680-AuNM. Mice were sacrificed 1 h or 18 h post-injection of IR680-AuNM, and tumor and organs were collected and ex vivo fluorescence imaging was done using an IVIS Spectrum imager. Statistical analysis: Statistical analyses were done using software GraphPad Prism 5 or OriginPro 9.0. The statistical analyses between different groups were computed by using 1-way ANOVA, whereas statistical analyses between two groups were computed by using the unpaired t-test.


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RESULTS Preparation and Characterization of micellar gold nanoparticles (AuNM): Homogeneous micelle-reduced gold nanocarriers were prepared through the reduction of HAuCl4 solution using PEG-PPG-PEG micelle as a reducing agent as shown in Fig. 1. During this process Au(III) was reduced to Au(0) to form nano-gold colloidal solution (Fig. 1). The color of the solution was ruby red, which was further mixed with ZD6474 overnight to obtain ZD6474-functionalized micellar AuNp (ZD6474-AuNM). The color of the resulting solution was violet (Fig. 2A). The solution was further centrifuged using Amicon® Ultra-4 centrifugal devices (10 kD cut off) (Millipore) to remove excess salts and un-bound ZD6474. This ZD6474-AuNM was used for subsequent experiments. For the optical characterization of AuNM, UV-VIS spectral measurements were performed. The presence of a characteristic surface Plasmon resonance band at 528 nm in the UVVIS spectrum (Fig. 2B) of uncomplexed AuNM solution can be ascribed to the formation of spherical shaped AuNP. The ZD6474-AuNM solution shows a surface Plasmon resonance band at 561 nm (Fig. 2B). The red shift of surface Plasmon resonance band from 528 to 561 nm may be due to the functionalization of AuNM with ZD6474. The resultant bands at 248 and 329 nm are due to presence ZD6474 moiety (Fig.2 and Suppl Fig. S1) and these values are consistent with those reported in the literature 34. Next, we investigated the size and shape of uncomplexed AuNM and ZD6474AuNM by using high-resolution transmission electron microscopy (HR-TEM). HR-TEM was done after drop coating the gold nanoparticles on a carbon-coated copper grid. From the HR-TEM images (Fig. 3), it is confirmed that uncomplexed AuNM is quasi-spherical in shape with rough surface and has an average size of ~60 nm. The TEM image of


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ZD6474-functionalized AuNM shows the average particle size of ~70 nm. The size is similar without much difference due to interaction between amine moiety of ZD6474 with micelle reduced Au nanocarrier. The rough surface of AuNM can able to adsorb maximum amount of ZD6474 and after adequate adsorption of drug the size of the AuNM was going to be increased. The higher surface energy of rough surface leads to adsorb more amount of drug and after sufficient adsorption of drug, the surface energy of AuNM was minimized which helps to form more stable ZD6474 loaded AuNM with smooth surface. The interparticle distance between the nanoparticle decreases as compare to micelle-reduced AuNM. This decrease in interparticle distance between the nanomicelles is due to the functionalization of AuNM with ZD6474 from absorption spectral studies. Next, we studied the size, charge and stability of the nanocarrier through dynamic light scattering studies of zetasizer. There was >60% of the particles having sizes in the range of 60 to 75 nm in both AuNM and ZD6474-AuNM (Fig. 3A and 3B) and were in agreement with size distribution observed by TEM. The polydispersity index (PDI) values of AuNM and ZD6474-AuNM were found to be 0.09 and 0.15 respecetively. These values suggest that AuNM particles are more monodispersed than drug loaded AuNM. Interestingly, drug-loaded nanocarrier exhibited positive charge due to amine structural modification on the nanocarrier, in-contrast the free nanocarrier without ZD6474 exhibited negative charge (Fig. 4A). The +ve surface charge of ZD6474-AuNM in acidic medium will be beneficial for enhanced uptake by the cells of tumor due to its – ve surface charge. Fig. 4B showed the variation of hydrodynamic sizes of AuNM and ZD6474-AuNM at different values of pH. The results showed that the average


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hydrodynamic size of the polymeric gold species in absence and presence of ZD6474 decreases with increasing pH values. At low pH (pH < 7), the miceller AuNP was composed of stable and predominantly larger polymeric structures. Although at high pH (pH > 7) the hydrodynamic size of AuNM was decreased which could be attributed to the rupturing of larger polymers to smaller polymers. It was found that the stability of AuNM is unaltered even after 90 days as shown in Fig. 4C at physiological pH ~7. Efficiency of entrapment of ZD6474 in AuNM: AuNM was used as a delivery vehicle of ZD6474 and can be used as a transport approach for targeting tumors. We selected AuNM of ~60 nm diameter due to its simplicity of synthesis and purification, while retaining high surface area and size. The entrapment efficiency was calculated as described in Experimental Section. The entrapment of ZD6474 on the surface of AuNM was 94.66 ± 1.22%. The higher entrapment was due to large surface area of AuNM or the presence of more gold atoms. Drug release study: Regulated release of drug minimizes its side effects and prevents exposure to normal cells or tissues to avoid toxicity. Tumor in acidic environment favors controlled release of drug at target site. Here, the acidic media favors the regulated release of drug. This in turn fulfils to attain therapeutic index of drug at the site of the tumor. The release of ZD6474 from ZD6474-AuNM at physiological pH was 20 % after 45-h, but at pH 5.2 the release percentage raised to 82 % following 45 h incubation (Fig. 4D). These results attribute to the ZD6474-AuNM causing the control release of drug in acidic environment i.e., in tumor environment. ZD6474-AuNM decreases cell proliferation in breast cancer cells: In order to evaluate the effect of ZD6474-AuNM in vitro in cell culture, cell proliferation (MTT)


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assays were performed as described in Materials and Methods. We selected 5 and 10 µM as the concentration of ZD6474 as IC50 was found to be 5-10 µM ZD6474 in MDA-MB231 and other breast cancer cell lines 23. Cell proliferation was found to be 87.39 ± 1.59, 54.40 ± 3.475 and 39.80 ± 0.60 in MDA-MB-231 treated with AuNM, 5 µM ZD6474 and 5 µM ZD6474-AuNM (AuNM containing 5 µM equivalent concentration of ZD6474), respectively, as compared to 100% cell cell growth in control cultures. There was a further decrease in cell growth as the concentration of ZD6474 was increased. Cell proliferation in comparison with untreated controls was found to be 26.64 ± 2.097 and 19.49 ± 2.79 in MDA-MB-231 cells treated with 10 µM ZD6474 and 10 µM ZD6474AuNM (Fig. 5A). There was significant decrease in cell growth between the treated groups as compared to control (P

Micellear Gold Nanoparticles as Delivery Vehicles for Dual Tyrosine

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