Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 64–71

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Green synthesis of pullulan stabilized gold nanoparticles for cancer targeted drug delivery Moorthy Ganeshkumar a, Thangavel Ponrasu a, Modhugoor Devendiran Raja b, Muthaiya Kannappan Subamekala c, Lonchin Suguna a,⇑ a b c

Department of Biochemistry, CSIR-Central Leather Research Institute, Council of Scientific and Industrial Research, Adyar, Chennai 600020, India Bio-Products Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600020, India Department of Biopharmaceutics, Anna University, Chennai, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Green chemistry based pullulan

stabilized gold nanoparticles (PAuNPs) were prepared by microwave irradiation method.  PAuNPs thus prepared were physiologically stable.  These PAuNPs could be used as carrier because of their biocompatibility studied in zebrafish model.  Functionalized PAuNPs could be used as targeted drug delivery system for liver cancer.

a r t i c l e

i n f o

Article history: Received 30 August 2013 Received in revised form 21 March 2014 Accepted 23 March 2014 Available online 5 April 2014 Keywords: Gold nanoparticles Drug delivery Biopolymer Liver cancer cell target Zebrafish embryo

a b s t r a c t The aim of this study was to synthesize green chemistry based gold nanoparticles using liver specific biopolymer and to develop a liver cancer targeted drug delivery system with enhanced efficacy and minimal side effects. Pullulan stabilized gold nanoparticles (PAuNPs) were coupled with 5-Fluorouracil (5-Fu) and folic acid (Fa) which could be used as a tool for targeted drug delivery and imaging of cancer. The toxicity of 5-Fu, 5-Fu adsorbed gold nanoparticles (5-Fu@AuNPs), Fa-coupled 5-Fu adsorbed gold nanoparticles (5-Fu@AuNPs-Fa), was studied using zebrafish embryo as an in vivo model. The in vitro cytotoxicity of free 5-Fu, 5-Fu@AuNPs, 5-Fu@AuNPs-Fa against HepG2 cells was studied and found that the amount of 5-Fu required to achieve 50% of growth of inhibition (Ic50) was much lower in 5-Fu@AuNP-Fa than in free 5-Fu, 5-Fu@AuNPs. The in vivo biodistribution of PAuNPs showed that higher amount of gold had been accumulated in liver (54.42 ± 5.96 lg) than in other organs. Ó 2014 Elsevier B.V. All rights reserved.

Introduction The role of plants and biopolymers for the production of nanoparticles is directly related to the symbiosis between nanotechnology and green chemistry. It is also vital to develop connections ⇑ Corresponding author. Tel.: +91 44 24911386; fax: +91 44 24911589. E-mail address: [email protected] (L. Suguna). http://dx.doi.org/10.1016/j.saa.2014.03.097 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

between nanotechnology and nature. Green chemistry based nanoparticles production plays a major role to address growing concerns on the overall toxicity of nanoparticles for biomedical applications [1]. The main focus of nanotechnology is to synthesize nanoparticles with predictable shape, size, polymeric coating and fine distribution for potential biomedical applications. Various techniques are involved in nanoparticles preparation like chemical and physical

M. Ganeshkumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 64–71

methods which may successfully produce pure, well-defined nanoparticles, but these are quite expensive and potentially dangerous to the environment [2]. Green chemistry based eco-friendly methods for synthesis of nanoparticles could be an alternative for chemical synthesis. Numerous natural derived materials and biopolymers have been successfully used for well-organized and speedy green synthesis of silver [3], copper oxide [4], zinc oxide [5], selenium [6] and platinum [7], gold nanoparticles (AuNPs) [8–13]. Natural Arabic gum reduced and stabilized AuNPs could be used as an X-ray contrast agent [14]. Gellulan gum reduced AuNPs could be utilized as a carrier of doxorubicin for cancer cell target therapy [15], AuNPs functionalized by folic acid coupled thiolated poly ethylene glycol-(FA-PEG-SH) were used to carry cisplatin [16]. Cetuximab functionalized gemcitabine adsorbed AuNPs have been used to target pancreatic cancer cells [17]. Park et al. have used AuNPs functionalized by thiolated poly ethylene glycol (PEG-SH)-anti epidermal growth factor receptors to target the delivery of b-Lapachon to human breast cancer cells (MCF-7), which were found useful for in vitro breast cancer studies. They have also compared MCF 7 with Human lung adenocarcinoma epithelial cell line (A549) and observed more up take of gold nanoparticles by A549 than MCF-7 based on the glutathione concentration in the cells [18]. Peptide caped AuNPs were used as tool for doxorubicin delivery to human epithelial cervical adenocarcinoma cells (HeLa) which showed active inhibition of cancer cell growth when compared to free drug and also found biocompatible with mouse fibroblast (L929) cells [19], AuNPs coated with lysozyme have been observed to induce bovine serum albumin nanoaggregation, which were used as carrier for doxorubicin delivery to HeLa cells which showed more inhibition for cancer cell growth compared to free drug and the drug free nanocarriers were found to be nontoxic to cells [20]. Chitosan polymer reduced gold nanoparticles could serve as a carrier for insulin in mucoadhesive drug delivery system for diabetes treatment [21]. Natural polysaccharide, dextran reduced and stabilized gold nanoparticles, could be employed for cell imaging when conjugated with cell penetrating peptide along with fluorescent dyes [22]. Apart from the great excitement about the potential uses of gold nanoparticles for medical imaging diagnostics and drug delivery applications, nanoparticle toxicity must be investigated before using them for any in vivo applications [23]. Zebrafish embryos are suitable in vivo model for rapid screening of nanoparticle toxicity because of low cost, transparent nature and possessing a high degree of homology to human genome. Some of the nanoparticles were screened by zebrafish embryos to assess the toxicity of dendrimers, copper nanoparticles, carbon nanotubes and Bucky balls, gold and silver nanoparticles and metal oxide nanoparticles [24–26]. 5-Fu is a chemotherapeutic drug that is widely used for the treatment of malignant cancers, and is usually the first choice of drug for the treatment of hepatic cancer. High doses of this drug are currently administered, mostly by continuous infusion, for over 5 to 21 days [27], but its use has been limited, because of its systemic toxicities like severe gastrointestinal irritations, hematologic side effects and severe disturbances in bone marrow [28]. Moreover, 5-Fu has a serum half-life of only 15 min which further limits its usage [29]. In order to prolong the circulation time of 5-Fu and increase its efficacy, researchers are attempting to modify its delivery by using polymer conjugates or by incorporating 5-Fu into particulate carriers [30–36]. Fig. 1 shows structure of pullulan, a neutral glucan, consisting of liner polymer of maltotriosyl units, connected by a-1,6 linkages, is an extracellular microbial polysaccharide produced by an yeast like fungus, Aureobasidium pullulans. Pullulan is water-soluble, nontoxic, non-immunogenic, nonmutagenic and noncarcinogenic in

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nature. An attempt has been made to investigate the role of this polysaccharide for various biomedical applications including tissue engineering, targeted drug and gene delivery [37,38]. Pullulan is known for its specificity for liver and this property has been exploited for liver targeting [37–42]. In order to improve the therapeutic efficacy of 5-Fu and patients’ compliance, it is desirable to formulate sustained release and targeted drug delivery compositions so as to maximize the therapeutic benefits and to reduce unwanted side effects. In this study, we have used a microwave irradiation method to prepare 5-Fu-adsorbed PAuNPs and evaluated their physical characteristics using UV, IR, TEM and Photon correlation spectroscopy. In vitro release behavior, toxicity in zebrafish embryo, in vitro efficacy in Hep G2 cell line and in vivo biodistribution in albino rat model have also been studied. Experimental section Materials 5-Fu obtained from SRL Limited, Mumbai (India). Hydrochloroauric acid (HAuCl4), Folic acid, 1-(3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) was obtained from Sigma–Aldrich Chemicals (USA). Hep G2 cell lines were obtained from National Centre for Cell Science (NCCS), Pune, (India). The Millipore milli Q water was used for all the experiments. All other chemicals and reagents used were of analytical grade. Synthesis of gold nanoparticles (PAuNPs) The formation of AuNPs is through reduction of HAuCl4 by microwave irradiation in the presence of pullulan. 1  104 M gold solution (1.35 mL) was added to Millipore water (30 mL). 1 g of pullulan was dissolved in 20 mL of water. From this, 15 mL was transferred to gold solution (30 mL) and the reaction mixture was placed in domestic microwave oven (Godrej). The parameters such as microwaving power and time required to reduce HAuCl4 was optimized by conducting the reactions at three different power levels i.e. 140, 280 and 420 W for 1, 2 and 3 min. HAuCl4 on reduction gives wine red color indicating the formation of AuNPs. Photographs were taken at 1 min interval to note the color changes during microwaving. Similarly, the optimum rpm and time of run for ultracentrifugation was found to be 60,000 rpm and 30 min respectively. Characterization of gold nanoparticles and drug adsorbed gold nanoparticles To study the formation of PAuNPs, the UV–visible absorption spectra of the prepared PAuNPs were recorded using a Tecan plate reader – infinite M200 model, from 450 to 650 nm. The FTIR spectra of samples were measured using a Fourier transform infrared spectrometer (Horizon instrument). Briefly, a small quantity of sample was mixed with 200 mg of KBr and compressed to form pellets. These pellets were scanned in the spectral region of 4000–400 cm1, using a resolution of 4 cm1 and 20 co-added scans. TEM analysis was carried out by dispersing 2–3 drops of PAuNPs solution on a copper grid and dried at room temperature after removing the excess solution with filter paper. TEM analysis was carried out using Techni-20 Philips Transmission Electron Microscope operated at 80 keV. Particle size analysis of the PAuNPs was carried out at 90° using photon correlation spectrometer (PCS) of Malvern Instruments – Zetasizer 3000 HSA equipped with a digital autocorrelator. The CONTIN method was used for data process-

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Fig. 1. Structure of pullulan.

ing. Differential Scanning Calorimetry was performed by using DSC-60 Schimadzu Corporation, Japan. The samples were placed in aluminium pans and were crimped, followed by heating under nitrogen flow at a scanning rate of 5 °C/min from 25 to 200 °C. Stability testing of PAuNPs In vitro stability studies 1 mL of PAuNPs was incubated with 0.5 mL each of 5% NaCl, 0.5% cysteine, 0.2 M histidine and 0.5% BSA solutions respectively at 37 ± 1 °C for 1 week and samples were withdrawn at specific time intervals like 1, 4, 24 and 48 h and analyzed for Surface Plasmon resonance (SPR) bands by UV-spectroscopy. Similarly, stability studies were performed in phosphate buffered saline (PBS) of pH 6.0, 7.4 and 8.0 in both RPMI and E3 medium. Stability of PAuNPs before and after autoclaving PAuNPs (45 mL) was taken in a conical flask, corked with cotton wool and autoclaved at 121 °C for 15 min. The samples withdrawn before and after autoclaving were analyzed for SPR by UV–vis spectroscopy. Temperature dependent stability PAuNPs were incubated at 4°, 25° and 40 °C for a period of 3 months to confirm the stability of PAuNPs. SPR bands of PAuNPs were measured by UV–vis spectroscopy. Preparation of 5Fu@PAuNPs and adsorption efficiency A calculated amount of 5-Fu was added to PAuNPs, obtained as described above, resulting in a final 5-Fu concentration of 2 mg/mL and kept under stirring for 24 h. The drug adsorbed nanoparticles were recovered by ultracentrifugation at 60,000 rpm for 30 min. The pellet obtained was redispersed in distilled water for further characterization. The free 5-Fu present in supernatant was determined by UV absorbance at 266 nm. Adsorption efficiency is calculated using the following formula:

Loading efficiency ¼

hydroxide solution (600 ll) was added and stirred for 24 h. The aminated PAuNPs was collected by centrifugation at 60,000 rpm for 30 min. To this solution, a calculated amount of 5-Fu (10 mg) was added and stirred for 24 h. The pellet was collected by centrifugation at 60,000 rpm for 30 min. Then folic acid was conjugated to 5Fu@PAuNPs by adding 1 mL of folic acid (carboxylic group activated) solution (5 mg each of folic acid and EDC dissolved in 2 mL of 0.1 M NaOH before 30 min of mixing) followed by stirring at 4 °C for 10 h. The 5Fu@AuNP-Fa formed was separated by centrifugation at 60,000 rpm for 30 min. In vitro drug release characteristics The in vitro drug release profile of 5Fu@PAuNPs was determined by dialyzing the nanoparticles against sodium phosphate buffer. Briefly, 5Fu@PAuNPs (15 mg) was placed in a dialysis bag (MWCO 12 kDa). This bag was dialyzed against 100 mL of sodium phosphate buffer; pH 7.4, at 37 °C with continuous stirring at 100 rpm. 1 mL of sample was withdrawn at specific time intervals and analyzed by UV absorbance at 266 nm. Sink condition was maintained by replacing equal volume of phosphate buffer, pH 7.4. The release studies were performed in triplicate and average was taken. In vivo toxicity study of PAuNPs, 5-Fu, 5-Fu@PAuNPs in zebrafish embryo model [26] Fertilized eggs of zebrafish were obtained from natural mating of adult zebrafish and embryos were collected within 2 h of spawning. 6 healthy embryos from newly fertilized eggs, approximately 2 h post fertilization (hpf), were transferred to each well of a 24 well plate containing 1 mL of E3 medium. The embryos were exposed to different concentrations of PAuNPs (124.17, 186.26, 248.35, 496.7, 745.05 lg), 5-Fu (325, 650, 975, 1300, 1350, 1625 lg/mL), 5-Fu@PAuNPs (130, 260, 650, 975, 1300, 1625, 3250 lg/mL), for 4 days. Duplicates were maintained for each experiment and the medium without the compounds served as control. Toxicity was assessed by studying the hatching rate, percentage survival rate and morphology of embryos.

Amount of drug added during preparation  Amount of drug in the supernatant  100 Amount of drug added during preparation

Preparation of 5Fu@PAuNPs-Fa To prepare 5Fu@PAuNPs-Fa, 250 ll of epichlorohydrin was added to 10 mL of PAuNPs, which was stirred for 24 h and centrifuged at 60,000 rpm for 30 min. The pellet obtained was redispersed in distilled water. To this solution, 6% ammonium

Cytotoxicity of free 5-Fu, 5-Fu@PAuNPs, 5-Fu@PAuNPs-Fa in HepG2 cell line 50,000 cells/well of HepG2 cells were seeded in a 96 well plate and incubated at 37 °C, 5% CO2. After 24 h, the cells were treated with known concentrations of 5-Fu, 5-Fu@PAuNPs, 5-Fu@PAuNP-Fa

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(3.2, 6.5, 13, 26, 52, 104, 208 lg/mL). After 72 h, MTT (5 mg/mL) was added to the cells and incubated for 4 h at 37 °C, 5% CO2. The purple colored formazan crystals were solubilized and measured at 570 nm using microplate reader. In vivo biodistribution studies of PAuNP [43] The in vivo liver targeting properties of PAuNPs (3380 lg) in PBS were assessed in male Wistar rats weighing between 150 and 200 g. All procedures were carried out according to the recommendations of Institutional Animal ethical committee (IAEC). The PAuNPs were administered to the rats intraperitoneally. Rats were euthanized after 2 h of administration; vital organs were collected and used to analyze the organ distribution data. PAuNPs concentration in liver, lung, kidney and spleen was determined using Inductively Coupled Plasma – Optical Emission Sprectroscopy (ICP-OES). Statistics All statistical analyses were performed using Graph pad Prism (version 5.0; Graph Pad software Inc. San Diego CA, California, USA). Results and discussion Synthesis and characterization of gold nanoparticles In the present study, we have exploited the reducing, stabilizing and biocompatible nature of a biopolymer, pullulan, for the synthesis of gold nanoparticles. PAuNPs allowed the attachment of a large number of small molecules which will exclusively target liver cancer. AuNPs were formed during microwaving at 420W power only. The solution turned to dark blue, purple and finally to wine red within 3 min and remained constant indicating the formation of stable PAuNPs and no significant change in color was seen in the 4th and 5th min (Fig. 2A insert). Formation of PAuNPs had been studied using visible spectroscopy by taking SPR (Fig. 2A). TEM images showed the external morphology of PAuNPs. They were found to be 19.67 ± 10.53 nm sizes and most of the particles were

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spherical in shape, few prisms were also observed, as shown in Fig. 2B. In vitro stability study of gold nanoparticles The kmax of PAuNPs remained at 525 nm before and after autoclaving indicating that PAuNPs were stable even after autoclaving, as shown in Fig. S1 (ESI ). The stability of PAuNPs at various temperatures like 4 °C, 25 °C and 40 °C/75% RH was checked and found to be stable at all the temperatures, which was also confirmed by visible spectroscopy by taking SPR. The peak appeared at 525 nm shown in Fig. S1(b) (ESI ) confirmed the formation of gold nanoparticles. The stability of PAuNPs in various medium was also studied by visible spectroscopy and a maximum of only 5 nm shift was observed and it was found to be stable for a period of 48 h as shown in Fig. S2(a–d) (ESI ). The results from these in vitro stability studies confirmed that the PAuNPs were intact and demonstrated excellent in vitro stability in biological fluids at various pH. Particles size analysis of gold nanoparticles The zetapotential of PAuNPs, 5-Fu@AuNPs were measured by dynamic light scattering method, Fig. S3(a) and (b) (ESI ) showed, 27.6 ± 4.05 mV respectively. PCS method was used to confirm the mean particle size of PAuNPs and it was observed to be 71 ± 3 nm. PCS measurements yield a significantly larger average gold nanoparticles diameter (71 nm) than the TEM analysis (20 nm), TEM visualizes the dried gold nanoparticles core of the particles, whereas PCS measures their hydrodynamic diameter in liquid medium, which can be enhanced by hydrophilic pullulan coated on the gold nanoparticles surface. These similar results also obtained by heparin [44], soya phytochemical [45] and lysozyme–albumin [20], bombesin [46] coated gold nanoparticles. The size of drug adsorbed gold nanoparticles was increased up to 166 ± 2 nm as depicted in Fig. S3(c and d) (ESI ). The increase in the diameter of the particles was not only due to the adsorption of the drug on the surface of the particles, but also because of the centrifugation process followed to remove the unadsorbed drug. We had used simple manual mixing method to redisperse

Fig. 2. A UV–visible spectrum of PAuNPs at various time intervals from 250 s to 300 s it represent by (a) 250 (b) 260 (c) 270 (d) 280 (e) 290 (f) 300 s. (A) Figure shows formation of PAuNPs from 0 min to 5 min (INSERTS). (B) TEM image of PAuNPs.

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the gold nanoparticles after centrifugation, which would not produce the particles with the same size as before drug adsorption. DSC analysis of gold nanoparticles The DSC thermograms corresponding to pullulan, 5-Fu, pullulan-5-Fu physical mixture and 5-Fu@AuNPs are shown in Fig. S4(a–d) (ESI ). The pullulan thermogram displayed an endothermic peak at 100.11 °C (Fig. S4(a)) (ESI ). The DSC curve of 5Fu showed a single melting peak at 283.96 °C (Fig. S4(b)) (ESI ). For physical mixture, the peak was detected at the melting point of 5-Fu (Fig. S4(c)) (ESI ). DSC of 5-Fu@AUNPs shows broadening of the characteristic endothermic peak corresponding to 5-Fu (271.2 °C), while the pullulan peak shifted from 110 °C to 92.45 °C, suggesting a decrease in its Tg (glass transition temperature). The negative deviation observed in Fig. S4(c) (ESI ) might be due to physical interaction between the polymer and drug, which could modify the Tg, melting temperature (Tm), shape and enthalpy of the thermogram of both pullulan and 5-Fu. The Tg value of polymer can be decreased by blending it with small amount of substance that can act as a plasticizer as observed in olanzapine loaded PLGA nanoparticles [47]. FTIR analysis of gold nanoparticles The strong absorption at 3406 cm1 indicated that pullulan had some repeating units of AOH as in sugars. The other strong absorption at 2930 cm1 indicated a sp3 CAH bond of alkane compounds existed in the samples (Fig. 3a). 5-Fu showed a characteristic peak at 3135 cm1 due to NAH stretching, at 1725, 1666 cm1 due to C@O stretching, at 1247 and 1497 cm1 due to CH in plane stretching and at 816 cm1 due to CH out of plane deformation (Fig. 3b). The same peak observed in 5-Fu@AuNPs indicated that the drug was adsorbed on PAuNPs (Fig. 3d) [45]. In pure Fa (Fig. 3c), the characteristic absorption peaks at 1411 cm1 due to OH deformation of phenyl skeleton, 1484 cm1 due to phenyl ring, 1605 cm1 due to NH2 bending vibration, 1693 cm1 due to stretching vibration from ACOOH, 2928, 2833 cm1 due to symmetric stretching vibration of ACH2, 3413, 3324 cm1 due to stretching vibration of NHAH, 3540 cm1 due to stretching vibration of OH were observed [48,49]. The FTIR (Fig. 3e) spectrum of 5-Fu@AuNPs-Fa displayed an absorption band

at 1650 cm1 due to the formation of amide bond between folic acid and aminated pullulan. Similar results were reported in folic acid coupled chitosan polymer [50–52]. The spectrum showed a peak at 1433 cm1 due to OH deformation of phenyl skeleton in folic acid, and also showed a characteristic peak of 5-Fu at 1725, 1666 cm1, 1247 and 1497 cm1 and at 816 cm1 confirmed the prescence of drug and folic acid in 5-Fu@AuNPs-Fa. 10 mL of PAuNPs was aminated and the percentage of aminated polymer was found to be 3.28% [53]. Adsorption efficiency and in vitro drug release study of gold nanoparticles The% adsorption of drug on gold nanoparticles (5-Fu@PAuNPs) was found to be 87.6 ± 2.70. The in vitro release profile of 5-Fu adsorbed PAuNPs up to 48 h is illustrated in Fig. 4a. The initial burst release was prominent for the first 1 h (19%), after this burst release, a constant and slow release was observed at 48 h (45.88%). When PAuNPs were conjugated with Fa, a red shift (SPR from 525 to 535 nm) was observed and also exhibited a peak at 260 nm, 280 nm, 365 nm which confirmed that Fa was coupled on PAuNPs Fig. S5(a) (ESI ) [54,55]. The interaction between PAuNPs and 5-Fu (Fig. S5(b)) (ESI ) was confirmed by quenched fluorescence intensity of 5-Fu due to the attachment of 5-Fu on the surface of PAuNPs with close proximity [56,57]. Toxicity study of gold nanoparticles in zebrafish embryo model The toxicity of PAuNPs (124.17, 186.26, 248.35, 496.7, 745.05 lg) on zebrafish embryos is shown in Fig. 4b. The PAuNPs with a concentration of 496.7 lg was observed to be less toxic as compared to that of control which was confirmed by the hatching rate, percentage survival rate and changes in the morphology of the embryos. The hatching rate did not change up to a maximum concentration of PAuNPs (745.05 lg). As for as the survival rate of the larvae was concerned, it was constant up to 248.35 lg of PAuNPs and later decreased. The LD50 value i.e. the concentration of PAuNPs required to kill 50% of zebra fish embryos after 72 h of exposure) was found to be 745.05 lg as shown in Fig. 4c. The changes in the morphology of zebrafish embryos were observed using stereomicroscope. The difference in total length of larvae exposed to various concentrations of PAuNPs was found

Fig. 3. (a) FTIR spectrum of pullulan (b) FTIR spectrum of 5-Fu (c) FTIR spectrum of folic acid (d) FTIR spectrum of 5-Fu@PAuNPs (e) FTIR spectrum of 5-Fu@PAuNPs-Fa.

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Fig. 4. (a) In vitro drug release of 5-Fu@PAuNPs in PBS at pH 7.4 (b) Morphologic analysis of PAuNPs toxicity (c) graph representing the toxicity of PAuNPs in terms of survival rate of larvae. Each bar represents the mean ± SD of triplicate experiments. Significant differences compared to controls are indicated by ***p < 0.001 and were calculated with one-way ANOVA and Dunnett’s post-test.

to be insignificant. But, the length of the tail was reduced in size; both the tail and body found bent and pericardial edema was observed when the concentration of PAuNPs was at 745.05 lg. Over all body shape, heart, pericardial sacs of the larvae were normal in all PAuNPs concentrations, but poor pigmentation was observed when the concentration of PAuNPs was increased from

496.7, 745.05 lg. Facial edema was observed only at the concentration of 745.05 lg. The toxic effect of free 5-Fu and 5-Fu@PAuNPs, on zebrafish embryo is shown in Fig. 5a and b respectively. The free 5-Fu, 5Fu@PAuNPs did not show any hatching delay and morphological changes. The % of survival was found to be decreased when the

Fig. 5. (a) Morphological analysis of 5-Fu toxicity. (b) Morphological analysis of 5-Fu@PAuNPs toxicity.

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Fig. 6. (a) Graph representing the toxicity of 5-Fu, in terms of survival rate of larvae. (b) Graph representing the toxicity of 5-Fu@PAuNPs, in terms of survival rate of larvae Fig. 5(a and b). Each bar represents the mean ± SD of triplicate experiments. Significant differences compared to controls are indicated by ***p < 0.001 and were calculated with one-way ANOVA and Dunnett’s post-test. (c) The cytotoxicity of free 5-Fu, 5-Fu@AuNPs, 5-Fu@PAuNPs-Fa against HepG2 cells. Each bar represents the mean ± SD of triplicate experiments. Significant differences compared to controls are indicated by ***p < 0.001 and were calculated with two way ANOVA followed by Bonferroni test.

concentration of free 5-Fu was increased from 975 lg to 1625 lg as shown in Fig. 6a. 5-Fu@PAuNPs did not affect the survival up to 260 lg, but as the concentration increased, 25% was found to be dead at the concentration of 3250 lg, as depicted in Fig. 6b. In vivo biodistribution of gold nanoparticles The biodistribution of PAuNPs (per gram of tissue) in different organs such as liver, kidney, spleen and lungs of rats after intraperitoneal injection, was quantified by ICP-OES (inductively coupled plasma optical emission spectroscopy). The accumulated gold concentration in lungs, kidney, liver and spleen was found to be 9.44 ± 1.07, 32.10 ± 1.80, 54.42 ± 5.96 and 45.49 ± 4.49 lg respectively. In vitro cytotoxicity study

nanoparticles that carry 5-Fu to target liver cancer cells. Zebrafish embryos were used to screen the 5-Fu and 5-Fu@PAuNPs toxicity. This type of functionalized gold nanoparticles could be used for both therapeutic applications and imaging of cancers. Tumor targeting of 5-Fu, 5-Fu@PAuNPs, 5-Fu@PAuNPs-Fa was carried out in Hep G2 cell line as they are known to over-express folate receptors. The results obtained by the in vitro drug release and cytotoxicity studies confirmed that 5-Fu@PAuNPs-Fa could be a promising alternative carrier for targeting liver cancer. Acknowledgements M. Ganeshkumar and T. Ponrasu would like to acknowledge the Council of Scientific and Industrial Research (CSIR), New Delhi, for Granting Senior Research fellowship and financial support. The authors thank Dr. M.D. Naresh (Biophysics Lab in CLRI) for help in the microscopic technique. We acknowledge SAIF, IITM, Chennai for providing ICP-OES. Pullulan was a gift sample from Hayashibara Ltd, Okayama, (Japan). The authors thank Central instrumentation lab, Tamilnadu Veterinary and Animal Science University for help in the TEM analysis.

Using MTT assay, the cytotoxicity of free 5-Fu, 5-Fu@PAuNPs, 5-Fu@AuNPs-Fa against HepG2 cells was compared and shown in Fig. 5c. The Ic50 values of free 5-Fu, 5-Fu@PAuNPs, 5-Fu@PAuNPsFa were 52 lg/mL (46% cell death), 52 lg/mL (49.15% cell death), 26 lg/mL (48.32% cell death) respectively. 5-Fu@PAuNPs-Fa showed significant cytotoxicity than other formulations after 24 h of incubation in HepG2 cell lines. The amount of 5-Fu required to achieve 50% of growth of inhibition (Ic50) was much lower in 5-Fu@PAuNPs-Fa than in free 5-Fu, 5-Fu@PAuNPs.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.03.097.

Conclusion

References

In Conclusion, we have reported a simple green chemistry based approach for the synthesis of gold nanoparticles using a natural, biocompatible, liver cell specific biopolymer. We have established a method to synthesize physiologically stable functionalized gold

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Green synthesis of pullulan stabilized gold nanoparticles for cancer targeted drug delivery.

The aim of this study was to synthesize green chemistry based gold nanoparticles using liver specific biopolymer and to develop a liver cancer targete...
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