Colloids and Surfaces B: Biointerfaces 123 (2014) 549–556
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Apoptosis in liver cancer (HepG2) cells induced by functionalized gold nanoparticles Thirunavukkarasu Ashokkumar a , Durai Prabhu c , Ravi Geetha a , Kasivelu Govindaraju b , Ramar Manikandan c , Chinnasamy Arulvasu c , Ganesan Singaravelu a,∗ a
Nanoscience Division, Department of Zoology, Thiruvalluvar University, Vellore 632 115, Tamilnadu, India Centre for Ocean Research, Sathyabama University, Chennai 600119, Tamilnadu, India c Department of Zoology, University of Madras, Guindy Campus, Chennai 600 025, Tamilnadu, India b
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Article history: Received 22 May 2014 Received in revised form 20 September 2014 Accepted 25 September 2014 Available online 5 October 2014 Keywords: Cajanus cajan Gold nanoparticles Liver cancer in vitro Apoptosis
a b s t r a c t An ethnopharmacological approach for biosynthesis of gold nanoparticles is being demonstrated using seed coat of Cajanus cajan. Medicinal value of capping molecule investigated for anticancer activity and results disclose its greater potential. The active principle of the seed coat [3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate] is elucidated. Rapid one-step synthesis yields highly stable, monodisperse (spherical) gold nanoparticles in the size ranging from 9 to 41 nm. Anticancer activity has been studied using liver cancer cells and cytotoxic mechanism has been evaluated using MTT, Annexin-V/PI Double-Staining Assay, Cell cycle, Comet assay and Flow cytometric analysis for apoptosis. The present investigation will open up a new possibility of functionalizing gold nanoparticles for apoptosis studies in liver cancer cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Green chemistry approach for synthesis of nanomaterials has gained considerable attention due to their potential application in life science. Nanoscience offers the ability to explore again the properties of materials by manipulating their sizes [1]. It is well known that the unique natures of nanomaterials are determined by their size, shape, chemistry and controlled dispersities [2]. Metal nanoparticles have attracted increasing attention for their promising applications in a variety of areas of life science such as biosensors, labels for cells, biomolecules and cancer treatment [3–5]. Nanoscience is now creating a growing sense of excitement in the life science, especially in target drug delivery [6]. Medical world and material science are continually strengthened by ever widening spectrum of applications using gold nanomaterials [7]. Gold nanoparticles have emerged as a promising scaffold for drug and gene delivery that provide a useful complement to more drug delivery vehicles. Combination of low inherent toxicity, high surface to volume ratio and tunable stability provides them with unique attributes that should enable new delivery strategies [8].
∗ Corresponding author. E-mail address: [email protected] (G. Singaravelu). http://dx.doi.org/10.1016/j.colsurfb.2014.09.051 0927-7765/© 2014 Elsevier B.V. All rights reserved.
Cancer nanotechnology is expected to change the very foundations of cancer treatment, diagnosis and detection. Nanomaterials, especially gold nanoparticles have unique physico-chemical properties, such as ultra small size, large surface area to mass ratio and high surface reactivity, presence of Surface Plasmon Resonance (SPR) bands, biocompatibility and ease of surface functionalization [9]. Functionalized gold nanoparticles as nanocarriers have tremendous growth in the pharmaceutical field for intracellular drug and gene delivery, mainly due to their large interacting surface, offering indispensable advantages to enhance potency, high specificity and low toxicity [10,11]. Liver cancer (Hepatocellular carcinoma) is the third most common cause of death in cancer. The death rate is increasing day by day and 85% people are affected in developing countries, more commonly men [12]. Chemotherapy with single or combinatorial treatments have suffered from non-specific mode of action, high intrinsic toxicity and or low survival profile as well as inappropriate patient selection and/or different patient geno and phenotype [13] and research towards new drugs with better efficacy against cancer is imperative. Many therapeutic and chemopreventive agents eliminate cancer cells by apoptosis. Apoptosis is a mechanism by which cells undergo death to control cell proliferation or in response to DNA damage [14]. Apoptotic cells are phagocytosed and digested by nearby resident cells without inducing any associated inflammation [15].
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Nanomedicine holds great promise to conquer several limitations of conventional drug delivery mechanism such as nonspecific biodistribution, lack of targeting, lack of aqueous solubility, poor oral bioavailability and low therapeutic indices [16–18]. Nanocarriers originated from various molecules having different chemical compositions are tuned to deliver anticancer drugs and other therapeutic ligands in a controlled and targeted manner to make them momentous drug delivery vehicles [19–21]. Cajanus cajan leaf material possess several medicinal properties like antioxidant [22], antihelminthic [23], hepatoprotecive [24], glycemic [25], neuroactive [26], hypocholesterolemic [27], antimicrobial activity [28] and the seed extract possess antisickling function [29]. Indeed, it is expected that gold nanoparticles synthesized using the seed coat of C. cajan might have medicinal properties. On the basis, efforts are made to identify the medicinal value of C. cajan phytochemical functionalized gold nanoparticles. As a result, their anti liver cancer activity has been explored. In the present investigation, synthesis of gold nanoparticles using a phytochemical is demonstrated. Interestingly, the capping of phytochemical molecule of gold nanoparticles is acquainted of its functionality as anticancer nanomaterial. 2. Materials and methods 2.1. Isolation of compound C. cajan dried seed coat was ground using commercial blender. 1 kg of powder was soaked with methanol for 24 h at room temperature. The extract was filtered and evaporated under reduced pressure to give 100 g. It was partitioned between n-hexane and water. The aqueous part was further partitioned between acetone, ethyl acetate and water. Finally the aqueous part was again partitioned with methanol. Methanol fraction was further subjected to silica gel column chromatography using MeOH: Acetone (98:2 v/v) as an elutent to yield compound. 2.2. GC-MS (Gas Chromatography-Mass Spectroscopy) Analysis The filtrate was analyzed for secondary metabolites by using GCMATE II GC-MS (Agilent). 1 L of the extract was injected through HP-5 capillary column, maintained at the temperature at 220 ◦ C and Helium is used as carrier gas. After analysis, the compound was identified by matching with structural library. 2.3. Gold nanoparticles synthesis The active principle 3-butoxy-2-hydroxypropyl 2-(2,4dihydroxyphenyl) acetate is isolated from the seed coat of red gram was subjected to confirm the property of synthesizing gold nanoparticles. 50 mg of isolated active principle was dissolved in 50 ml of distilled water, which was added in to 50 ml of 1 mM HAuCl4 solution (1:1 ratio). The gold nanoparticles solution, thus obtained was centrifuged at 15,000 rpm for 15 min in order to isolate gold nanoparticles. 2.4. Characterization UV–Vis spectra were recorded as a function of reaction time using Techomp 2300 UV-Vis spectrophotometer operated at a resolution of 1 nm. To find out the functional molecule which responsible for the formation of gold nanoparticles, Fourier Transform Infrared (FTIR) analysis was undertaken for the newly formed gold nanoparticles and the active principle of C. cajan. The crystal nature of the newly formed nanoparticles and to ensure the bio-precursor capping the nanoparticles were determined using Xray diffraction measurements (Bruker, Germany). The size, shape
and morphology of gold nanoparticles were analyzed by High Resolution Transmission Electron Microscopy (HRTEM-PHILIPS) and Field Emission Scanning Electron Microscopy (FESEM-Carl Zeiss, Germany). The height profile of the newly synthesized gold nanoparticles was carried out using Atomic Force Microscopy (AFM-NTMDT, Ireland). The effect of pH on stability of newly synthesized gold nanoparticles was studied. 2.5. Biocompatibility assay Vero cell line was purchased from National Centre for Cell Sciences (NCCS), Pune, India. Biocompatibility effect of 3-butoxy-2hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate synthesized gold nanoparticles on the proliferation of cells were determined by MTT assay [30]. 2.6. MTT assay HepG2 cell line was purchased from National Centre for Cell Sciences (NCCS), Pune, India. The cell line was grown in Minimum Essential Medium (MEM) supplemented with 2 mM lglutamine, 100 U/ml penicillin, 100 g/ml gentamicin and 10% fetal bovine serum (FBS). Cells were cultured in T25 cm2 cell culture flasks at 37 ◦ C in a 5% CO2 atmosphere. HepG2 cells were cultured and seeded into 96 well plates approximately as 1 × 104 cells in each well and incubated for 48 h. Cells were treated with different concentrations of 3-butoxy-2-hydroxypropyl 2-(2,4dihydroxyphenyl) acetate and newly synthesized gold nanoparticles (AuNPs) (2, 4, 6, 8, 10 g/ml) respectively. After treatment, the plates were incubated for 24 and 48 h in order to perform cytotoxic analysis using MTT assay. MTT (3-(4,5-dimethylthiazol-2-yl) -2,5diphenyltetrazolium bromide, a yellow tetrazole) was prepared at a concentration of 5 mg/ml and 10 l of MTT was added to each well and incubated for 4 h. Purple color formazone crystals formed were then dissolved in 100 l of dimethyl sulphoxide (DMSO). These crystals were observed at 570 nm in a multi well ELISA plate reader. Optical density value was subjected to percentage of viability [30]. 2.7. Cell morphological observation The morphological observation was followed by the method [31] and the cells were seeded at 1 × 105 cells/well into a six well plate and cells were incubated 24 h. Later, the medium was replaced with MEM which containing FBS and of 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate synthesized gold nanoparticles (6 g/mL) and control counterparts were also maintained for 24 h. The cell morphology was examined under phase contrast inverted microscope (RADICAL). 2.8. Cell cycle analysis Cell cycle dissemination and percentage of apoptotic cells were performed by flow cytometry using propidium iodide (PI) [31]. After treatment, floating cells in the medium were combined with attached cells collected by trypsinization. Cells were washed with cold PBS and fixed using 80% ethanol in PBS at −20 ◦ C. Fixed cells were pelleted and stained with PI (50 g/mL) in the presence of RNase A (20 g/mL) for 30 min at 37 ◦ C. About 104 cells were analyzed in a Becton Dickinson FAC scan flow cytometer. Cell cycle histograms were analyzed using cell Quest Software. 2.9. Single cell gel electrophoresis (Comet assay) Comet assay was performed to determine the degree of oxidative DNA damage [32]. Cells were exposed to AuNPs for 48 h and
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washed with PBS. The cell suspension was mixed with 75 l of 0.5% Low Melting Agarose (LMA) at 39 ◦ C and spread on a fully frosted microscopic slide pre-coated with 200 l of 1% normal melting agarose. After the solidification of agarose, slides were covered with another 75 l of 0.5% LMA and then immersed in lysis solution (2.5 M NaCl, 10 mM Na-EDTA, 10 mM Tris, 1% Trion × 100 and 10% DMSO, pH 10) for 1 h at 4 ◦ C. The slides were then placed in a gel electrophoresis apparatus (containing 300 mM NaOH and 10 mM Na-EDTA, pH 13) for 40 min to allow unwinding of DNA and the alkali labile damage. Next, an electrical field (3000 mA, 25 V) was applied for 20 min at 4◦ C to draw the negatively charged DNA toward an anode. After electrophoresis, slides were washed thrice for 5 min at 4◦ C in a neutralizing buffer (0.4 M Tris, pH 7.5), followed by staining with 75 l of propidium iodide (40 g/ml) and then the slides were observed using fluorescence microscopy. 2.10. Flow cytometric analysis Apoptotic cells were assessed by measuring phosphatidylserine using an annexin V- Fluorescein isothiocynate (FITC) apoptosis detection kit (SIGMA product) as per manufacturer’s protocol. Briefly, cells (1 × 106 /well) were plated in six-well plates and allowed to attach overnight, treated with an IC50 concentration of gold nanoparticles and control alone for 24 h, cells were harvested and washed twice with PBS, resuspended the cells in 500 L of 1x binding buffer, stained with 5 L of annexin V-FITC conjugate and 10 L of PI solution to each sample cell suspension and incubated for 15 min in the dark condition at room temperature. Stained cells were analyzed by FAC scan flow cytometer (Becton Dickinson) to find out the viability (annexin V and PI negative), early apoptotic (annexin V positive and PI negative), or late apoptotic (annexin V and PI positive). The degree of apoptosis was quantified as a percentage of the annexin V positive cells. 2.11. Annexin-V/PI Double-Staining Assay HepG2 cells were plated at 1 × 106 cells/well into a six well chamber plate. At >90% confluence, the cells were treated with an IC50 concentration of gold nanoparticles and control alone for 24 h. The cells were washed with PBS, fixed in methanol: acetic acid (3:1, v/v) for 10 min and stained with 5 l of annexin V-FITC conjugate and 10 l of PI for 20 min. Morphology of apoptotic cells was examined under Zeiss Axio observer fluorescent microscope. 3. Results and discussion 3.1. Synthesis and characterization of gold nanoparticles Synthesis of nanoparticles using biological resources such as microorganisms (bacteria, fungi and virus), yeast and plants is relatively a recent phenomenon. An amalgamation of curiosity, environmental compulsions and understanding that nature has evolved the process for synthesis of inorganic materials as nano and micro-length scale have created great interest among material scientists. The rational of this hypothesis would result in a versatile green nanotechnology with consequent application of gold nanoparticles in a myriad of applications in nanomedicine. Rapid biological synthesis of gold nanoparticles was achieved using the active principle of seed coat of C. cajan. Extensive screening was made, as a result it was found that 3-butoxy-2hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate (1a inset) is the active principle of the C. cajan responsible for the production of gold nanoparticles (Fig. 1a). Surface plasmon resonance of the newly synthesized gold nanoparticles is centered at 535 nm and the reduction of AuCl4 − ions reached saturation within 10 min of the reaction.
Fig. 1. UV–vis spectra of (a) 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate isolated from Cajanus cajan. (b) Gold nanoparticles synthesized using 3butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate.
The brownish reaction mixture changed to ruby red, the reaction indicating the formation of gold nanoparticles. The color change is attributed to the collective oscillation of free electrons induced by an interacting electromagnetic field in metallic nanoparticles [33]. The color change arises because of the reduction of chloroauric acid (Au3+ ) into gold nanoparticles (Au0 ). UV–vis absorption spectrum of gold nanoparticles solution as a function of time of reaction with compound is shown in Fig. 1b. The active principle 3-butoxy-2-hydroxypropyl 2-(2,4dihydroxyphenyl) acetate of C. cajan synthesized gold nanoparticles shows intense peaks at 3433 cm−1 (O H Stretch), 2924 cm−1 (C H stretch), 2161 cm−1 ( C C stretch), 1614 cm−1 ( C O stretch), 1415 cm−1 (C H bend), 1089 cm−1 (C O bend), 1031 cm−1 , 947 cm−1 , 903 cm−1 and 819 cm−1 (ester groups) relative shift in position and intensity distribution were confirmed with FTIR (supplementary Fig. 1 a & b) recorded for dry powder of the compound 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate were the strong bands observed at 3434 cm−1 (O H Stretch), 2924 cm−1 (C H stretch), 2142 cm−1 ( C C stretch), 1629 cm−1 ( C O stretch) and 1419 cm−1 (C H bend), compared the both FTIR spectra it is identified that the changes have occurred
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(111)
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2θ Fig. 2. XRD pattern of crystalline gold nanoparticles.
in the peak 2142 cm−1 (alkynes group) and 1629 cm−1 (carbonyl group). The peak that appeared at 2142 cm−1 (alkynes group) in the compound peak shifted to higher wavelength 2161 cm−1 after reducing the choloroauric acid which indicates the alkynes group capping with gold nanoparticles. The peak shift from higher wavelength 1629 cm−1 (carbonyl group) to lower wavelength 1614 cm−1 and 1031 cm−1 , 947 cm−1 , and 903 cm−1 , ester groups was disappeared, which indicates the reduction of Au+ to Au0 . GC–MS spectra of isolated compound obtained were compared with NIST Mass spectra library. The compound observed at the retention time of 9.39 min with a concentration 97% was matched with 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate (Supplementary Fig. 2a) and the molecular mass of the compound was found to be 298.4095 Da. (Supplementary Fig. 2b). The crystalline nature of the newly synthesized Au nanoparticles was further confirmed from X-Ray diffraction (XRD) analysis. Fig. 2 shows the XRD pattern of the dried nanoparticles obtained using compound of 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate. The diffraction peaks were observed at 38.2◦ , 44.3◦ , 64.7◦ , and 77.8◦ in the 2 range 30◦ to 80◦ which can be indexed to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections of face centered cubic structure (FCC) of metallic gold, respectively (JCPDS on 04-0784) revealing that the synthesized gold nanoparticles are composed of pure crystalline gold. FCC metallic crystalline gold nanoparticles’ XRD pattern has additional as yet unassigned peaks are also observed, suggesting that the crystallization of bioorganic phase occurs on the surface of the gold nanoparticles, which is confirmed by XRD pattern of purified compound of 3-butoxy-2-hydroxypropyl 2-(2,4dihydroxyphenyl) acetate, single peak was observed at 29◦ in the 2 range 30◦ –80◦ (Supplementary Fig. 3). FE-SEM image provided further insight into the morphology and size of the nanoparticles. FE-SEM image (Supplementary Fig. 4a) shows well dispersed spherical nanoparticles. The purity of the biosynthesized gold nanoparticles was examined by EDAX combined with FE-SEM. The EDAX Spectrum revealed a strong signal for gold (Supplementary Fig. 4b). Other peaks corresponding to O, C were also observed, most likely because of the borosilicate glass on which the sample was coated for FE-SEM. Transmission electron microscopy was used to analyze the size and shape of the nanoparticles. TEM images (Fig. 3a and b) show clearly the newly formed gold nanoparticles were predominantly spherical in shape with diameters ranging from 9 to 41 nm where maximum number of gold nanoparticles were of average size around 29 nm. Gold nanoparticles were crystalline in nature, as can be seen from the selected area diffraction pattern can be indexed as (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections of fcc gold
Fig. 3. (a) TEM images of newly biosynthesized gold nanoparticles. (b) Inset SAED pattern of single gold nanoparticles.
nanoparticles (Fig. 3b Inset). The newly synthesized gold nanoparticles’ crystalline nature is in fairly good agreement with X- ray diffraction analysis. AFM study was used to identify the surface topology, height and shape of the biosynthesized nanoparticles. Supplementary Fig. 5a & b shows 3D and lateral AFM images which clearly reveals that particles are spherical in shape, and is in fairly good agreement with TEM and FE-SEM images. The average height of the gold nanoparticles is found to be 1.7 m. Effect of pH on stability of gold nanoparticles solution was measured after 120 h using UV–vis spectra that the absorption maxima are uniform in the pH range from 4 to 9 in acidic and basic conditions. The results indicating that the gold nanoparticles are extreme stability in different of physiological solutions (Supplementary Fig. 6). Biosynthesis of nanomaterials demonstrated herein is a kind of bottom up approach where the main reaction occurring is reduction or oxidation. The phytochemicals have been reducing molecules which are responsible for the reduction of metal into their respective nanomaterials [34]. In this study gold nanoparticles were synthesized using a plant compound 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate as a novel reducing and stabilizing agent. The newly synthesized gold nanoparticles SPR band is centered at 535 nm. Similarly, it is reported that the gold nanoparticles produced by C. amboinicus leaf extract show a strong Surface Plasmon Resonance, refers to the collective oscillations of the conduction electrons in resonance with the light field. Further, it has been reported that the surface plasmon mode arises from the electron confinement in the
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nanoparticles [35]. XRD pattern reveals that the newly synthesized gold nanoparticles are crystalline in nature as reported earlier [36]. 3.2. In vitro anticancer studies Biomedical application of nanoparticles necessitates their biocompatibility which is the important criteria to be assessed. On the basis, vero cells (monkey cell line) were treated with newly synthesized gold nanoparticles at various concentrations (10, 20, 40, 80, 160 and 320 g/ml). After 24 h of treatment with gold nanoparticles, IC50 value was noted at the concentration of 246 g/ml (Supplementary Fig. 7). Pharmacological nature of the plant C. cajan identified as a potential candidate on synthesizing gold nanoparticles lead to work on the anticancer activity. Investigation made by examining the antiproliferation effects of newly synthesized gold nanoparticles using HepG2 liver cancer cells. MTT assay was made with newly synthesized gold nanoparticles at concentrations ranging from 2 g/ml to 10 g/ml to determine the IC50 value and the value was 6 g/ml for 24 h exposure (Fig. 4i) HepG2 cells were treated with the IC50 concentration of newly synthesized gold nanoparticles. Morphological assessment of treated cells clearly indicates the play of apoptotic mechanisms leading to cell death which is evident by changes in membrane integrity, inhibition of cell growth, cytoplasmic condensation and cell shrinkage were observed [Fig. 4(iia and b)]. Cell cycle assays were performed on a control sample and on cells treated with IC50 concentrations of newly synthesized gold nanoparticles. Fig. 5a and b shows that incubation of gold nanoparticles with HepG2 cells for 24 h significantly reduced the DNA content and make them appear in the sub-G0 /G1 region which indicative of apoptosis, with consequent loss of cells in the G1 phase, 63.86% of cells were gated in sub-G0 /G1 phase. As cell cycle comprises four distinct phases (G1 phase, S phase, G2 phase and Mitosis) and two checkpoints (G0 /G1 and G2 /M checkpoints) which assure that no DNA damage is transmitted to daughter cells [37]. Interestingly, it was noticed that gold nanoparticles arrest all stages of the cell cycle and found to damage the DNA. The percentage of cell cycle arrest varying from one phase to another phase. Indeed, gold nanoparticles strongly arrest cell cycle in G0 –G1 phase compared to control. Analysis of DNA fragmentation effect of newly synthesized AuNPs on HepG2 cells was assessed by comet assay. Comet assay is a rapid and sensitive method for the detection of DNA damage in individual cells induced by a variety of genotoxic agents [38]. The length of comet tail was four and two times than that of control while, in 48 h treated HepG2 cells ten and six times of span of tail
Fig. 4. (i) In vitro cytotoxicity of newly synthesized gold nanoparticles (SD, n = 3) (ii) Morphology of control and treated HepG2 liver cancer cells (40x magnifications) (a) control (b) gold nanoparticles (IC50 ).
compared to control. Comet assay also discloses that the DNA has not been damaged in control HepG2 cells. DNA damage was seen in AuNPs treated cells significantly increased number of tail DNA, tail length, tail moment, olive tail moment in HepG2 cells (Fig. 6a and b). Cytotoxicity mechanism of newly synthesized gold nanoparticles in HepG2 cells were analyzed using annexin-V flow cytometry. Fig. 7a and b shows 24 h of exposure to gold nanoparticles the HepG2 cell death rate was 69.99% with 63.85% early apoptotic cells, 6.14% late apoptotic cells and that there was no necrotic noticed. Phosphatidylserine (PS) signal in this assay is associated with apoptosis, which is an indicator of nonviable cells. This result is also corroborated with the morphological investigation on HepG2 cells [Fig. 8(ia–c)] and gold nanoparticles treated HepG2 cells which discloses broken up cells leads to apoptotic bodies [Fig. 8(iia-c)].
Fig. 5. Cell Cycle arrest in HepG2 cell line a) Control b) gold nanoparticles (IC50 ).
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Fig. 6. Effect of AuNPs on DNA damage in HepG2 cells by comet assay (a) Control (b) gold nanoparticles (IC50 ).
Fig. 7. Flow cytometric analysis of apoptotic cells (a) Control (b) gold nanoparticles (IC50 ).
Phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane of the viable cells. But, in apoptotic cells PS is translocated from the inner to the outer leaflet of the plasma membrane, thus exposing PS to the external cellular environment [39].
According to the phenomenon that the annexin V is a 35–36 KDa Ca2+ dependent phospholipid-binding protein that has high affinity for PS [40]. Further, annexin V FITC and PI is not able to bind to normal live cells since the molecule is not able to penetrate the phospholipid bilayer. Gold nanoparticles induced HepG2 cells apoptosis was confirmed by using annexin V FITC and PI, which is easily penetrate to the apoptotic cells and, it may be due to the features of apoptotic cells such as membrane blebbing, shrinkage of the cytoplasm and DNA fragmentation of the nuclear chromatin [41]. The number of cells undergone apoptosis was quantified and the result reveals that 63.5% early apoptotic cells induced by gold nanoparticles and the untreated cells was not shown the apoptotic cells. Nanomaterials are at the cutting edge of the exciting area of cancer nanotechnology. The potential of nanomaterials in cancer drug delivery is infinite with novel new applications constantly being explored. Marangoni et al. [42] reported jacalin-conjugated goldnanoparticles a nanoconjugate for cancer diagnosis. Thevenot et al. [43] suggest that surface functional groups of TiO2
Fig. 8. Morphology of apoptotic cells (ia–c) Control (iia–c) gold nanoparticles (IC50 ).
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nanoparticles influence the degree of toxicity against cancer cells based on the cancer cell’s membrane. Further, it has been demonstrated that NH2 and OH groups showed significantly higher toxicity than COOH. Mohanty et al. [19] observed resveratrol stabilized gold nanoparticles turn to be a potential vehicle for cancer drug delivery. Joshi et al. [39] investigated chloroquineconjugated gold nanoparticles’ anticancer property and observed that autophagy is likely the cause of necrotic cell death. Cytotoxicity of molybdenum trioxide (MoO3 ) nanoplates toward invasive breast cancer iMCF-7 cells was made by analyzing morphological changes and performing Western blot and flow cytometry analyses. The findings suggested that MoO3 exposure induces apoptosis and generates reactive oxygen species (ROS) in iMCF-7 cells. Nevertheless, despite recent advances in this area, the biomedical applications of nanomaterials are still limited by the low efficiency of functionalization, low stability and high toxicity [44]. Interestingly, the newly synthesized gold nanoparticles are found to be biocompatible and the anticancer activity was achieved neither with dopping nor conjugation of molecule. The stabilized molecule (3-butoxy2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate) provides the functionality to the newly formed gold nanoparticles as anticancer nanomaterial. Results of the present investigation reveals that excessive reactive oxygen species (ROS) generation may play a dominant role in the newly synthesized gold nanoparticles induced cancer cells’ apoptosis. It might be due to the generated ROS favours the DNA damage leading to apoptosis. The targeted delivery of a drug should result in enhanced therapeutic efficacy with low to minimal side effects. This is wide accepted concept, but limited in application due to lack of available technologies and process of validation. Results of the present investigation demonstrate the prospects of nanoscience to overcome the limitations of conventional technologies. Acknowledgement The authors G.S and T.A are thankful to Indian Council of Medical Research (ICMR), New Delhi for their grant. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.09.051. References [1] H. Bonnemann, R. Richards, Nanoscopic metal particles synthetic methods and potential applications, Eur. J. Inorg. Chem. 10 (2001) 2455. [2] A. Ahmad, S. Senapati, M.I. Khan, R. Kumar, M. Sastry, Extracellular biosynthesis of monodisperse gold nanoparticles by a novel extremophilic actinomycete, thermomonospora sp, Langmuir 19 (8) (2003) 3550. [3] J.M. Nam, C.S. Thaxton, C.A. Mirkin, Nanoparticle-based-bar codes for the ultrasensitive detection of proteins, Science 301 (2003) 1884. [4] A.G. Tkachenko, H. Xie, W. Coelmm, J. Ryan, M.F. Anderson, S. Franzen, D.L. Feldheim, Multifunctional gold nanoparticle-peptide complexes for nuclear targeting, J. Am. Chem. Soc. 125 (2003) 4700. [5] L.R. Hirsch, R.J. Stafford, J.A. Bankson, S.R. Sershen, B. Rivera, R.E. Price, J.D. Hazle, N.J. Halas, J.L. West, Nanoshell-mediated near –infrared thermal therapy of tumors under magnetic resonance guidance, Proc. Natl. Acad. Sci. USA 100 (2003) 13549. [6] N. Prabhu, T.R. Divya, G. Yamuna, Synthesis of silver phyto nanoparticles and their antibacterial efficacy, Digest. J. Nanomater. Biostruct. 5 (2010) 185. [7] X. Chen, H.J. Schluesener, Nanosilver: a nanoproduct in medicinal application, Toxicol. Lett. 176 (2008) 1. [8] P. Ghosh, G. Han, M. De, C.K. Kim, V.M. Rotello, Gold nanoparticles in delivery applications, Adv. Drug Del. Rev. 60 (2008) 1307. [9] C.R. Parta, R. Bhattacharya, D. Mukhopadhyay, P. Mukherjee, Fabrication of gold nanoparticles for targeted therapy in pancreatic cancer, Adv. Drug Del. Rev. 62 (2010) 346.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Apoptosis in liver cancer (HepG2) cells induced by functionalized gold nanoparticles Thirunavukkarasu Ashokkumar a , Durai Prabhu c , Ravi Geetha a , Kasivelu Govindaraju b , Ramar Manikandan c , Chinnasamy Arulvasu c , Ganesan Singaravelu a,∗ a
Nanoscience Division, Department of Zoology, Thiruvalluvar University, Vellore 632 115, Tamilnadu, India Centre for Ocean Research, Sathyabama University, Chennai 600119, Tamilnadu, India c Department of Zoology, University of Madras, Guindy Campus, Chennai 600 025, Tamilnadu, India b
a r t i c l e
i n f o
Article history: Received 22 May 2014 Received in revised form 20 September 2014 Accepted 25 September 2014 Available online 5 October 2014 Keywords: Cajanus cajan Gold nanoparticles Liver cancer in vitro Apoptosis
a b s t r a c t An ethnopharmacological approach for biosynthesis of gold nanoparticles is being demonstrated using seed coat of Cajanus cajan. Medicinal value of capping molecule investigated for anticancer activity and results disclose its greater potential. The active principle of the seed coat [3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate] is elucidated. Rapid one-step synthesis yields highly stable, monodisperse (spherical) gold nanoparticles in the size ranging from 9 to 41 nm. Anticancer activity has been studied using liver cancer cells and cytotoxic mechanism has been evaluated using MTT, Annexin-V/PI Double-Staining Assay, Cell cycle, Comet assay and Flow cytometric analysis for apoptosis. The present investigation will open up a new possibility of functionalizing gold nanoparticles for apoptosis studies in liver cancer cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Green chemistry approach for synthesis of nanomaterials has gained considerable attention due to their potential application in life science. Nanoscience offers the ability to explore again the properties of materials by manipulating their sizes [1]. It is well known that the unique natures of nanomaterials are determined by their size, shape, chemistry and controlled dispersities [2]. Metal nanoparticles have attracted increasing attention for their promising applications in a variety of areas of life science such as biosensors, labels for cells, biomolecules and cancer treatment [3–5]. Nanoscience is now creating a growing sense of excitement in the life science, especially in target drug delivery [6]. Medical world and material science are continually strengthened by ever widening spectrum of applications using gold nanomaterials [7]. Gold nanoparticles have emerged as a promising scaffold for drug and gene delivery that provide a useful complement to more drug delivery vehicles. Combination of low inherent toxicity, high surface to volume ratio and tunable stability provides them with unique attributes that should enable new delivery strategies [8].
∗ Corresponding author. E-mail address: [email protected] (G. Singaravelu). http://dx.doi.org/10.1016/j.colsurfb.2014.09.051 0927-7765/© 2014 Elsevier B.V. All rights reserved.
Cancer nanotechnology is expected to change the very foundations of cancer treatment, diagnosis and detection. Nanomaterials, especially gold nanoparticles have unique physico-chemical properties, such as ultra small size, large surface area to mass ratio and high surface reactivity, presence of Surface Plasmon Resonance (SPR) bands, biocompatibility and ease of surface functionalization [9]. Functionalized gold nanoparticles as nanocarriers have tremendous growth in the pharmaceutical field for intracellular drug and gene delivery, mainly due to their large interacting surface, offering indispensable advantages to enhance potency, high specificity and low toxicity [10,11]. Liver cancer (Hepatocellular carcinoma) is the third most common cause of death in cancer. The death rate is increasing day by day and 85% people are affected in developing countries, more commonly men [12]. Chemotherapy with single or combinatorial treatments have suffered from non-specific mode of action, high intrinsic toxicity and or low survival profile as well as inappropriate patient selection and/or different patient geno and phenotype [13] and research towards new drugs with better efficacy against cancer is imperative. Many therapeutic and chemopreventive agents eliminate cancer cells by apoptosis. Apoptosis is a mechanism by which cells undergo death to control cell proliferation or in response to DNA damage [14]. Apoptotic cells are phagocytosed and digested by nearby resident cells without inducing any associated inflammation [15].
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Nanomedicine holds great promise to conquer several limitations of conventional drug delivery mechanism such as nonspecific biodistribution, lack of targeting, lack of aqueous solubility, poor oral bioavailability and low therapeutic indices [16–18]. Nanocarriers originated from various molecules having different chemical compositions are tuned to deliver anticancer drugs and other therapeutic ligands in a controlled and targeted manner to make them momentous drug delivery vehicles [19–21]. Cajanus cajan leaf material possess several medicinal properties like antioxidant [22], antihelminthic [23], hepatoprotecive [24], glycemic [25], neuroactive [26], hypocholesterolemic [27], antimicrobial activity [28] and the seed extract possess antisickling function [29]. Indeed, it is expected that gold nanoparticles synthesized using the seed coat of C. cajan might have medicinal properties. On the basis, efforts are made to identify the medicinal value of C. cajan phytochemical functionalized gold nanoparticles. As a result, their anti liver cancer activity has been explored. In the present investigation, synthesis of gold nanoparticles using a phytochemical is demonstrated. Interestingly, the capping of phytochemical molecule of gold nanoparticles is acquainted of its functionality as anticancer nanomaterial. 2. Materials and methods 2.1. Isolation of compound C. cajan dried seed coat was ground using commercial blender. 1 kg of powder was soaked with methanol for 24 h at room temperature. The extract was filtered and evaporated under reduced pressure to give 100 g. It was partitioned between n-hexane and water. The aqueous part was further partitioned between acetone, ethyl acetate and water. Finally the aqueous part was again partitioned with methanol. Methanol fraction was further subjected to silica gel column chromatography using MeOH: Acetone (98:2 v/v) as an elutent to yield compound. 2.2. GC-MS (Gas Chromatography-Mass Spectroscopy) Analysis The filtrate was analyzed for secondary metabolites by using GCMATE II GC-MS (Agilent). 1 L of the extract was injected through HP-5 capillary column, maintained at the temperature at 220 ◦ C and Helium is used as carrier gas. After analysis, the compound was identified by matching with structural library. 2.3. Gold nanoparticles synthesis The active principle 3-butoxy-2-hydroxypropyl 2-(2,4dihydroxyphenyl) acetate is isolated from the seed coat of red gram was subjected to confirm the property of synthesizing gold nanoparticles. 50 mg of isolated active principle was dissolved in 50 ml of distilled water, which was added in to 50 ml of 1 mM HAuCl4 solution (1:1 ratio). The gold nanoparticles solution, thus obtained was centrifuged at 15,000 rpm for 15 min in order to isolate gold nanoparticles. 2.4. Characterization UV–Vis spectra were recorded as a function of reaction time using Techomp 2300 UV-Vis spectrophotometer operated at a resolution of 1 nm. To find out the functional molecule which responsible for the formation of gold nanoparticles, Fourier Transform Infrared (FTIR) analysis was undertaken for the newly formed gold nanoparticles and the active principle of C. cajan. The crystal nature of the newly formed nanoparticles and to ensure the bio-precursor capping the nanoparticles were determined using Xray diffraction measurements (Bruker, Germany). The size, shape
and morphology of gold nanoparticles were analyzed by High Resolution Transmission Electron Microscopy (HRTEM-PHILIPS) and Field Emission Scanning Electron Microscopy (FESEM-Carl Zeiss, Germany). The height profile of the newly synthesized gold nanoparticles was carried out using Atomic Force Microscopy (AFM-NTMDT, Ireland). The effect of pH on stability of newly synthesized gold nanoparticles was studied. 2.5. Biocompatibility assay Vero cell line was purchased from National Centre for Cell Sciences (NCCS), Pune, India. Biocompatibility effect of 3-butoxy-2hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate synthesized gold nanoparticles on the proliferation of cells were determined by MTT assay [30]. 2.6. MTT assay HepG2 cell line was purchased from National Centre for Cell Sciences (NCCS), Pune, India. The cell line was grown in Minimum Essential Medium (MEM) supplemented with 2 mM lglutamine, 100 U/ml penicillin, 100 g/ml gentamicin and 10% fetal bovine serum (FBS). Cells were cultured in T25 cm2 cell culture flasks at 37 ◦ C in a 5% CO2 atmosphere. HepG2 cells were cultured and seeded into 96 well plates approximately as 1 × 104 cells in each well and incubated for 48 h. Cells were treated with different concentrations of 3-butoxy-2-hydroxypropyl 2-(2,4dihydroxyphenyl) acetate and newly synthesized gold nanoparticles (AuNPs) (2, 4, 6, 8, 10 g/ml) respectively. After treatment, the plates were incubated for 24 and 48 h in order to perform cytotoxic analysis using MTT assay. MTT (3-(4,5-dimethylthiazol-2-yl) -2,5diphenyltetrazolium bromide, a yellow tetrazole) was prepared at a concentration of 5 mg/ml and 10 l of MTT was added to each well and incubated for 4 h. Purple color formazone crystals formed were then dissolved in 100 l of dimethyl sulphoxide (DMSO). These crystals were observed at 570 nm in a multi well ELISA plate reader. Optical density value was subjected to percentage of viability [30]. 2.7. Cell morphological observation The morphological observation was followed by the method [31] and the cells were seeded at 1 × 105 cells/well into a six well plate and cells were incubated 24 h. Later, the medium was replaced with MEM which containing FBS and of 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate synthesized gold nanoparticles (6 g/mL) and control counterparts were also maintained for 24 h. The cell morphology was examined under phase contrast inverted microscope (RADICAL). 2.8. Cell cycle analysis Cell cycle dissemination and percentage of apoptotic cells were performed by flow cytometry using propidium iodide (PI) [31]. After treatment, floating cells in the medium were combined with attached cells collected by trypsinization. Cells were washed with cold PBS and fixed using 80% ethanol in PBS at −20 ◦ C. Fixed cells were pelleted and stained with PI (50 g/mL) in the presence of RNase A (20 g/mL) for 30 min at 37 ◦ C. About 104 cells were analyzed in a Becton Dickinson FAC scan flow cytometer. Cell cycle histograms were analyzed using cell Quest Software. 2.9. Single cell gel electrophoresis (Comet assay) Comet assay was performed to determine the degree of oxidative DNA damage [32]. Cells were exposed to AuNPs for 48 h and
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washed with PBS. The cell suspension was mixed with 75 l of 0.5% Low Melting Agarose (LMA) at 39 ◦ C and spread on a fully frosted microscopic slide pre-coated with 200 l of 1% normal melting agarose. After the solidification of agarose, slides were covered with another 75 l of 0.5% LMA and then immersed in lysis solution (2.5 M NaCl, 10 mM Na-EDTA, 10 mM Tris, 1% Trion × 100 and 10% DMSO, pH 10) for 1 h at 4 ◦ C. The slides were then placed in a gel electrophoresis apparatus (containing 300 mM NaOH and 10 mM Na-EDTA, pH 13) for 40 min to allow unwinding of DNA and the alkali labile damage. Next, an electrical field (3000 mA, 25 V) was applied for 20 min at 4◦ C to draw the negatively charged DNA toward an anode. After electrophoresis, slides were washed thrice for 5 min at 4◦ C in a neutralizing buffer (0.4 M Tris, pH 7.5), followed by staining with 75 l of propidium iodide (40 g/ml) and then the slides were observed using fluorescence microscopy. 2.10. Flow cytometric analysis Apoptotic cells were assessed by measuring phosphatidylserine using an annexin V- Fluorescein isothiocynate (FITC) apoptosis detection kit (SIGMA product) as per manufacturer’s protocol. Briefly, cells (1 × 106 /well) were plated in six-well plates and allowed to attach overnight, treated with an IC50 concentration of gold nanoparticles and control alone for 24 h, cells were harvested and washed twice with PBS, resuspended the cells in 500 L of 1x binding buffer, stained with 5 L of annexin V-FITC conjugate and 10 L of PI solution to each sample cell suspension and incubated for 15 min in the dark condition at room temperature. Stained cells were analyzed by FAC scan flow cytometer (Becton Dickinson) to find out the viability (annexin V and PI negative), early apoptotic (annexin V positive and PI negative), or late apoptotic (annexin V and PI positive). The degree of apoptosis was quantified as a percentage of the annexin V positive cells. 2.11. Annexin-V/PI Double-Staining Assay HepG2 cells were plated at 1 × 106 cells/well into a six well chamber plate. At >90% confluence, the cells were treated with an IC50 concentration of gold nanoparticles and control alone for 24 h. The cells were washed with PBS, fixed in methanol: acetic acid (3:1, v/v) for 10 min and stained with 5 l of annexin V-FITC conjugate and 10 l of PI for 20 min. Morphology of apoptotic cells was examined under Zeiss Axio observer fluorescent microscope. 3. Results and discussion 3.1. Synthesis and characterization of gold nanoparticles Synthesis of nanoparticles using biological resources such as microorganisms (bacteria, fungi and virus), yeast and plants is relatively a recent phenomenon. An amalgamation of curiosity, environmental compulsions and understanding that nature has evolved the process for synthesis of inorganic materials as nano and micro-length scale have created great interest among material scientists. The rational of this hypothesis would result in a versatile green nanotechnology with consequent application of gold nanoparticles in a myriad of applications in nanomedicine. Rapid biological synthesis of gold nanoparticles was achieved using the active principle of seed coat of C. cajan. Extensive screening was made, as a result it was found that 3-butoxy-2hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate (1a inset) is the active principle of the C. cajan responsible for the production of gold nanoparticles (Fig. 1a). Surface plasmon resonance of the newly synthesized gold nanoparticles is centered at 535 nm and the reduction of AuCl4 − ions reached saturation within 10 min of the reaction.
Fig. 1. UV–vis spectra of (a) 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate isolated from Cajanus cajan. (b) Gold nanoparticles synthesized using 3butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate.
The brownish reaction mixture changed to ruby red, the reaction indicating the formation of gold nanoparticles. The color change is attributed to the collective oscillation of free electrons induced by an interacting electromagnetic field in metallic nanoparticles [33]. The color change arises because of the reduction of chloroauric acid (Au3+ ) into gold nanoparticles (Au0 ). UV–vis absorption spectrum of gold nanoparticles solution as a function of time of reaction with compound is shown in Fig. 1b. The active principle 3-butoxy-2-hydroxypropyl 2-(2,4dihydroxyphenyl) acetate of C. cajan synthesized gold nanoparticles shows intense peaks at 3433 cm−1 (O H Stretch), 2924 cm−1 (C H stretch), 2161 cm−1 ( C C stretch), 1614 cm−1 ( C O stretch), 1415 cm−1 (C H bend), 1089 cm−1 (C O bend), 1031 cm−1 , 947 cm−1 , 903 cm−1 and 819 cm−1 (ester groups) relative shift in position and intensity distribution were confirmed with FTIR (supplementary Fig. 1 a & b) recorded for dry powder of the compound 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate were the strong bands observed at 3434 cm−1 (O H Stretch), 2924 cm−1 (C H stretch), 2142 cm−1 ( C C stretch), 1629 cm−1 ( C O stretch) and 1419 cm−1 (C H bend), compared the both FTIR spectra it is identified that the changes have occurred
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(111)
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2θ Fig. 2. XRD pattern of crystalline gold nanoparticles.
in the peak 2142 cm−1 (alkynes group) and 1629 cm−1 (carbonyl group). The peak that appeared at 2142 cm−1 (alkynes group) in the compound peak shifted to higher wavelength 2161 cm−1 after reducing the choloroauric acid which indicates the alkynes group capping with gold nanoparticles. The peak shift from higher wavelength 1629 cm−1 (carbonyl group) to lower wavelength 1614 cm−1 and 1031 cm−1 , 947 cm−1 , and 903 cm−1 , ester groups was disappeared, which indicates the reduction of Au+ to Au0 . GC–MS spectra of isolated compound obtained were compared with NIST Mass spectra library. The compound observed at the retention time of 9.39 min with a concentration 97% was matched with 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate (Supplementary Fig. 2a) and the molecular mass of the compound was found to be 298.4095 Da. (Supplementary Fig. 2b). The crystalline nature of the newly synthesized Au nanoparticles was further confirmed from X-Ray diffraction (XRD) analysis. Fig. 2 shows the XRD pattern of the dried nanoparticles obtained using compound of 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate. The diffraction peaks were observed at 38.2◦ , 44.3◦ , 64.7◦ , and 77.8◦ in the 2 range 30◦ to 80◦ which can be indexed to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections of face centered cubic structure (FCC) of metallic gold, respectively (JCPDS on 04-0784) revealing that the synthesized gold nanoparticles are composed of pure crystalline gold. FCC metallic crystalline gold nanoparticles’ XRD pattern has additional as yet unassigned peaks are also observed, suggesting that the crystallization of bioorganic phase occurs on the surface of the gold nanoparticles, which is confirmed by XRD pattern of purified compound of 3-butoxy-2-hydroxypropyl 2-(2,4dihydroxyphenyl) acetate, single peak was observed at 29◦ in the 2 range 30◦ –80◦ (Supplementary Fig. 3). FE-SEM image provided further insight into the morphology and size of the nanoparticles. FE-SEM image (Supplementary Fig. 4a) shows well dispersed spherical nanoparticles. The purity of the biosynthesized gold nanoparticles was examined by EDAX combined with FE-SEM. The EDAX Spectrum revealed a strong signal for gold (Supplementary Fig. 4b). Other peaks corresponding to O, C were also observed, most likely because of the borosilicate glass on which the sample was coated for FE-SEM. Transmission electron microscopy was used to analyze the size and shape of the nanoparticles. TEM images (Fig. 3a and b) show clearly the newly formed gold nanoparticles were predominantly spherical in shape with diameters ranging from 9 to 41 nm where maximum number of gold nanoparticles were of average size around 29 nm. Gold nanoparticles were crystalline in nature, as can be seen from the selected area diffraction pattern can be indexed as (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections of fcc gold
Fig. 3. (a) TEM images of newly biosynthesized gold nanoparticles. (b) Inset SAED pattern of single gold nanoparticles.
nanoparticles (Fig. 3b Inset). The newly synthesized gold nanoparticles’ crystalline nature is in fairly good agreement with X- ray diffraction analysis. AFM study was used to identify the surface topology, height and shape of the biosynthesized nanoparticles. Supplementary Fig. 5a & b shows 3D and lateral AFM images which clearly reveals that particles are spherical in shape, and is in fairly good agreement with TEM and FE-SEM images. The average height of the gold nanoparticles is found to be 1.7 m. Effect of pH on stability of gold nanoparticles solution was measured after 120 h using UV–vis spectra that the absorption maxima are uniform in the pH range from 4 to 9 in acidic and basic conditions. The results indicating that the gold nanoparticles are extreme stability in different of physiological solutions (Supplementary Fig. 6). Biosynthesis of nanomaterials demonstrated herein is a kind of bottom up approach where the main reaction occurring is reduction or oxidation. The phytochemicals have been reducing molecules which are responsible for the reduction of metal into their respective nanomaterials [34]. In this study gold nanoparticles were synthesized using a plant compound 3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate as a novel reducing and stabilizing agent. The newly synthesized gold nanoparticles SPR band is centered at 535 nm. Similarly, it is reported that the gold nanoparticles produced by C. amboinicus leaf extract show a strong Surface Plasmon Resonance, refers to the collective oscillations of the conduction electrons in resonance with the light field. Further, it has been reported that the surface plasmon mode arises from the electron confinement in the
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nanoparticles [35]. XRD pattern reveals that the newly synthesized gold nanoparticles are crystalline in nature as reported earlier [36]. 3.2. In vitro anticancer studies Biomedical application of nanoparticles necessitates their biocompatibility which is the important criteria to be assessed. On the basis, vero cells (monkey cell line) were treated with newly synthesized gold nanoparticles at various concentrations (10, 20, 40, 80, 160 and 320 g/ml). After 24 h of treatment with gold nanoparticles, IC50 value was noted at the concentration of 246 g/ml (Supplementary Fig. 7). Pharmacological nature of the plant C. cajan identified as a potential candidate on synthesizing gold nanoparticles lead to work on the anticancer activity. Investigation made by examining the antiproliferation effects of newly synthesized gold nanoparticles using HepG2 liver cancer cells. MTT assay was made with newly synthesized gold nanoparticles at concentrations ranging from 2 g/ml to 10 g/ml to determine the IC50 value and the value was 6 g/ml for 24 h exposure (Fig. 4i) HepG2 cells were treated with the IC50 concentration of newly synthesized gold nanoparticles. Morphological assessment of treated cells clearly indicates the play of apoptotic mechanisms leading to cell death which is evident by changes in membrane integrity, inhibition of cell growth, cytoplasmic condensation and cell shrinkage were observed [Fig. 4(iia and b)]. Cell cycle assays were performed on a control sample and on cells treated with IC50 concentrations of newly synthesized gold nanoparticles. Fig. 5a and b shows that incubation of gold nanoparticles with HepG2 cells for 24 h significantly reduced the DNA content and make them appear in the sub-G0 /G1 region which indicative of apoptosis, with consequent loss of cells in the G1 phase, 63.86% of cells were gated in sub-G0 /G1 phase. As cell cycle comprises four distinct phases (G1 phase, S phase, G2 phase and Mitosis) and two checkpoints (G0 /G1 and G2 /M checkpoints) which assure that no DNA damage is transmitted to daughter cells [37]. Interestingly, it was noticed that gold nanoparticles arrest all stages of the cell cycle and found to damage the DNA. The percentage of cell cycle arrest varying from one phase to another phase. Indeed, gold nanoparticles strongly arrest cell cycle in G0 –G1 phase compared to control. Analysis of DNA fragmentation effect of newly synthesized AuNPs on HepG2 cells was assessed by comet assay. Comet assay is a rapid and sensitive method for the detection of DNA damage in individual cells induced by a variety of genotoxic agents [38]. The length of comet tail was four and two times than that of control while, in 48 h treated HepG2 cells ten and six times of span of tail
Fig. 4. (i) In vitro cytotoxicity of newly synthesized gold nanoparticles (SD, n = 3) (ii) Morphology of control and treated HepG2 liver cancer cells (40x magnifications) (a) control (b) gold nanoparticles (IC50 ).
compared to control. Comet assay also discloses that the DNA has not been damaged in control HepG2 cells. DNA damage was seen in AuNPs treated cells significantly increased number of tail DNA, tail length, tail moment, olive tail moment in HepG2 cells (Fig. 6a and b). Cytotoxicity mechanism of newly synthesized gold nanoparticles in HepG2 cells were analyzed using annexin-V flow cytometry. Fig. 7a and b shows 24 h of exposure to gold nanoparticles the HepG2 cell death rate was 69.99% with 63.85% early apoptotic cells, 6.14% late apoptotic cells and that there was no necrotic noticed. Phosphatidylserine (PS) signal in this assay is associated with apoptosis, which is an indicator of nonviable cells. This result is also corroborated with the morphological investigation on HepG2 cells [Fig. 8(ia–c)] and gold nanoparticles treated HepG2 cells which discloses broken up cells leads to apoptotic bodies [Fig. 8(iia-c)].
Fig. 5. Cell Cycle arrest in HepG2 cell line a) Control b) gold nanoparticles (IC50 ).
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Fig. 6. Effect of AuNPs on DNA damage in HepG2 cells by comet assay (a) Control (b) gold nanoparticles (IC50 ).
Fig. 7. Flow cytometric analysis of apoptotic cells (a) Control (b) gold nanoparticles (IC50 ).
Phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane of the viable cells. But, in apoptotic cells PS is translocated from the inner to the outer leaflet of the plasma membrane, thus exposing PS to the external cellular environment [39].
According to the phenomenon that the annexin V is a 35–36 KDa Ca2+ dependent phospholipid-binding protein that has high affinity for PS [40]. Further, annexin V FITC and PI is not able to bind to normal live cells since the molecule is not able to penetrate the phospholipid bilayer. Gold nanoparticles induced HepG2 cells apoptosis was confirmed by using annexin V FITC and PI, which is easily penetrate to the apoptotic cells and, it may be due to the features of apoptotic cells such as membrane blebbing, shrinkage of the cytoplasm and DNA fragmentation of the nuclear chromatin [41]. The number of cells undergone apoptosis was quantified and the result reveals that 63.5% early apoptotic cells induced by gold nanoparticles and the untreated cells was not shown the apoptotic cells. Nanomaterials are at the cutting edge of the exciting area of cancer nanotechnology. The potential of nanomaterials in cancer drug delivery is infinite with novel new applications constantly being explored. Marangoni et al. [42] reported jacalin-conjugated goldnanoparticles a nanoconjugate for cancer diagnosis. Thevenot et al. [43] suggest that surface functional groups of TiO2
Fig. 8. Morphology of apoptotic cells (ia–c) Control (iia–c) gold nanoparticles (IC50 ).
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nanoparticles influence the degree of toxicity against cancer cells based on the cancer cell’s membrane. Further, it has been demonstrated that NH2 and OH groups showed significantly higher toxicity than COOH. Mohanty et al. [19] observed resveratrol stabilized gold nanoparticles turn to be a potential vehicle for cancer drug delivery. Joshi et al. [39] investigated chloroquineconjugated gold nanoparticles’ anticancer property and observed that autophagy is likely the cause of necrotic cell death. Cytotoxicity of molybdenum trioxide (MoO3 ) nanoplates toward invasive breast cancer iMCF-7 cells was made by analyzing morphological changes and performing Western blot and flow cytometry analyses. The findings suggested that MoO3 exposure induces apoptosis and generates reactive oxygen species (ROS) in iMCF-7 cells. Nevertheless, despite recent advances in this area, the biomedical applications of nanomaterials are still limited by the low efficiency of functionalization, low stability and high toxicity [44]. Interestingly, the newly synthesized gold nanoparticles are found to be biocompatible and the anticancer activity was achieved neither with dopping nor conjugation of molecule. The stabilized molecule (3-butoxy2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate) provides the functionality to the newly formed gold nanoparticles as anticancer nanomaterial. Results of the present investigation reveals that excessive reactive oxygen species (ROS) generation may play a dominant role in the newly synthesized gold nanoparticles induced cancer cells’ apoptosis. It might be due to the generated ROS favours the DNA damage leading to apoptosis. The targeted delivery of a drug should result in enhanced therapeutic efficacy with low to minimal side effects. This is wide accepted concept, but limited in application due to lack of available technologies and process of validation. Results of the present investigation demonstrate the prospects of nanoscience to overcome the limitations of conventional technologies. Acknowledgement The authors G.S and T.A are thankful to Indian Council of Medical Research (ICMR), New Delhi for their grant. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.09.051. References [1] H. Bonnemann, R. Richards, Nanoscopic metal particles synthetic methods and potential applications, Eur. J. Inorg. Chem. 10 (2001) 2455. [2] A. Ahmad, S. Senapati, M.I. Khan, R. Kumar, M. Sastry, Extracellular biosynthesis of monodisperse gold nanoparticles by a novel extremophilic actinomycete, thermomonospora sp, Langmuir 19 (8) (2003) 3550. [3] J.M. Nam, C.S. Thaxton, C.A. Mirkin, Nanoparticle-based-bar codes for the ultrasensitive detection of proteins, Science 301 (2003) 1884. [4] A.G. Tkachenko, H. Xie, W. Coelmm, J. Ryan, M.F. Anderson, S. Franzen, D.L. Feldheim, Multifunctional gold nanoparticle-peptide complexes for nuclear targeting, J. Am. Chem. Soc. 125 (2003) 4700. [5] L.R. Hirsch, R.J. Stafford, J.A. Bankson, S.R. Sershen, B. Rivera, R.E. Price, J.D. Hazle, N.J. Halas, J.L. West, Nanoshell-mediated near –infrared thermal therapy of tumors under magnetic resonance guidance, Proc. Natl. Acad. Sci. USA 100 (2003) 13549. [6] N. Prabhu, T.R. Divya, G. Yamuna, Synthesis of silver phyto nanoparticles and their antibacterial efficacy, Digest. J. Nanomater. Biostruct. 5 (2010) 185. [7] X. Chen, H.J. Schluesener, Nanosilver: a nanoproduct in medicinal application, Toxicol. Lett. 176 (2008) 1. [8] P. Ghosh, G. Han, M. De, C.K. Kim, V.M. Rotello, Gold nanoparticles in delivery applications, Adv. Drug Del. Rev. 60 (2008) 1307. [9] C.R. Parta, R. Bhattacharya, D. Mukhopadhyay, P. Mukherjee, Fabrication of gold nanoparticles for targeted therapy in pancreatic cancer, Adv. Drug Del. Rev. 62 (2010) 346.
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