CHAPTER 4 IN VITRO CYTOTOXICITY ASSAY ON GOLD NANOPARTICLES WITH DIFFERENT STABILIZING AGENT

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1 81 CHAPTER 4 IN VITRO CYTOTOXICITY ASSAY ON GOLD NANOPARTICLES WITH DIFFERENT STABILIZING AGENT 4.1 INTRODUCTION The nanoparticles have been shown to adhere to cell membranes (Ghitescu and Fixman 1984) and be ingested by cells (Parak et al 2002). The breaching of the cell membrane and the intracellular storage may have a negative effect on the cells regardless of the toxicity of the particles and their subsequent functionality. In this study, gold nanoparticles stabilized with citrate, starch and gum arabic are used for cytotoxicity studies. The assays used are based on different modes of detection like (3-[4,5-dimethylthiazol-2- yl] -2,5-diphenyltetrazolium bromide reduction assay) (MTT), the neutral red cellular uptake assay and lactate dehydrogenase (LDH) release assay. We found that the gold nanoparticles stabilized with citrate, starch and gum arabic are viable to different cells through different assays with different concentrations and time of exposure of gold nanoparticles. It was found that the viability depends on the stabilizing agent and the types of cytotoxicity assay used. 4.2 MATERIALS All Chemicals were obtained from Sigma-Aldrich and used as received. Deionized water was used in all experiments. The prostate cancer cell lines (PC-3) and breast cancer cell lines (MC-7) were obtained from the

2 82 American Type Culture Collection (ATCC) through the Department of Microbology, PSG Institute of Medical Sciences and Research, Coimbatore, Tamilnadu, India. Gold nanoparticles stabilized with citrate, starch and gum arabic were prepared and characterization studies were performed before inducting into cytotoxicity studies as referred in chapter EXPERIMENTAL METHODS MTT Assay Gold Nanoparticles with Different Concentrations Cytotoxicity evaluation of citrate (CAuNPs), starch (SAuNPs) and gum Arabic (GAuNPs) stabilized gold nanoparticles were performed using MTT assay as described by Mosman (1983). About ml -1 cells (MCF-7 and PC-3) in their exponential growth phase were seeded in a flat-bottomed 96-well plate and were incubated for 24 hr at 37 0 C in 5% CO 2 incubator. Series of dilution (30, 60, 90, 120 and 150 g/ml) of gold nanoparticles stabilized with citrate, starch and gum arabic in medium were separately added to the plate. After 24 hr of incubation, 10 L of MTT reagent was added to each well and was further incubated for 4 hr. Formazan crystals formed after 4 hr in each well were dissolved in 150 l of detergent and the plates were read immediately in a microplate reader (BIO-RAD microplate reader-550) at 570nm. Untreated PC-3 and MCF-7 cells as well as the cell treated with (30, 60, 90, 120 and 150 g/ml) concentration of AuNPs for 24 hr were subjected to the MTT assay for cell viability determination Time of exposure assay Cytotoxicity was also assessed using MTT assay at different time period. About ml -1 cells lines in their exponential growth phase were seeded in a flat-bottomed 96-well polystyrene coated plate. Gold

3 83 nanoparticles stabilized with citrate, starch and gum arabic with concentration 50 g/ml were diluted in the growth medium and added to the plate. Incubations carried out for various times (12, 24, 36, 48, 60 and 72 hr) at 37 o C in an atmosphere of 5% CO 2 in air. At the end of incubation 10 µl of MTT reagent was added to each well and further incubated for 4 hr. Formazan crystals formed after 4 hr in each well were dissolved in 150 µl of detergent and the plates were read immediately in a micro plate reader (BIO-RAD micro plate reader-550) at 570nm. Untreated cell lines as well as the cell treated with gold nanoparticles at different time were subjected to the MTT assay for cell viability determination LDH Assay Gold nanoparticles with different concentrations Cytotoxicity was assessed using a LDH cytotoxicity detection kit (Roche applied sciences). This assay measures the release of cytoplasm enzyme lactate dehydrogenase (LDH) by damaged cells. Cells cultured in 96 plates were treated with increasing concentrations of gold nanoparticles (30, 60, 90, 120 and 150 g/ml) stabilized with citrate, starch and gum arabic. After 48 hr of treatment, culture supernatant was collected and incubated with reaction mixture. The LDH catalyzed conversion results in the reduction of the tetrazolium salt to formazan, that can be read absorbance 490nm. These data are measured in LDH activity as a percentage of the control. Any significant increase in LDH levels would indicate cellular disruption or death due to the treatment Time of exposure assay Cytotoxicity was assessed using an LDH detection kit (Roche Applied Science). This assay measures the release of cytoplasm enzyme lactate dehydrogenase (LDH) by damaged cells. Cells cultured in 96 plates

4 84 were treated with 50 g/ml concentrations of gold nanoparticles stabilized with citrate, starch and gum arabic. Incubations were carried out for various times (12, 24, 36, 48, 60 and 72 hr). After the treatment, culture supernatant was collected and incubated with reaction mixture. The LDH catalyzed conversion results in the reduction of the tetrazolium salt to formazan, which can be read at the absorbance of 490nm. Any significant increase in LDH levels would indicate cellular disruption or cell death due to the treatment Neutral Red Cell Uptake Assay Gold nanoparticles with different concentrations PC-3 and MC-7 cells were seeded at a population of cells per well in a 96 well plate. The cells were incubated for 24 hr and reached 80-90% confluence. The spent media was removed and the cells were washed with PBS (0.01 M phosphate buffer, M KCl and M NaCl) and 1 L fresh media was added. Gold nanoparticles stabilized with citrate, starch and gum arabic with different concentrations (30, 60, 90, 120 and 150 g/ml) were mixed with fresh media. The plates were then incubated for 24 hr at 37 0 in a humidified incubator with a 5% CO 2 environment. After the incubation, the cells were washed twice with PBS (0.01 M Phosphate buffer M KCl and 0.137M NaCl) thereafter 50 L of dye release agent (a solution of 1% acetic acid: 50% ethanol) added to each well and the plates were incubated for further 10 minutes. The plate was placed on a shaker (Vortex Genie) for 30 minutes after that the optical density at 540nm was determined on a multiwall spectrophotometer Time of exposure assay The cells were seeded at a population of cells per well in a 96 well plate. The cells were incubated for 24 hr and reached 80-90% confluence. The spent media were removed and the cells were washed with

5 85 PBS (0.01 M phosphate buffer, M KCl and M NaCl) and 1 L fresh media was added. The gold nanoparticles stabilized with citrate, starch and gum arabic with 50 g/ml concentration is taken and mixed with fresh media. The plates were then incubated for various times (12, 24, 36, 48, 60 and 72 hr) at 37 0 C in a humidifier incubator with 5% CO 2 environment. After the incubation, the cells were washed twice with PBS (0.01 M phosphate buffer, M KCl and M NaCl) thereafter 50 L of dye release agent (A solution of 1% acetic acid:50% ethanol) was added to each well and the plates were incubated for further 10 minutes. The plate was placed on a shaker (Vortex Genie) for 30 minutes after that the optical density was determined at 540nm on a multiwall spectrophotometer. 4.4 RESULTS The PC-3 cell lines in the exponential growth phase were exposed to different concentrations of citrate, starch and gum arabic stabilized gold nanoparticles. The cell viability was measured as described in the experimental section. Each result represents the mean viability + standard deviation (SD) of three independent experiments and each of these was performed in triplicate. Cell viability was calculated as the percentage of the viable cells compared to the untreated controls. In MTT assay, cells that are viable after 24 hr exposed to the sample were capable of metabolizing a dye (3-(4,-dimethylthiozol-2-yl)-2,5- disphenyl tetrazolium bromide) efficiently and the purple coloured precipitate which is dissolved in a detergent was analyzed spectro photometrically. After 24 hr of post treatment of PC-3 cell lines showed excellent viability even up to the concentration of 150 g of citrate, starch and gum arabic capped gold nanoparticles, Figure 4.1(a). These results clearly demonstrate that the stabilizing agents provide non-toxic coating on AuNPs and corroborate the results of the internalization studies discussed above. But the data shows that there is a marginal cytotoxic effect of citrate stabilized gold nanoparticles

6 86 with different cell lines used. Also we had a similar result with LDH and Neutral red cell assay for the gold nanoparticles stabilized with citrate, starch and gum arabic. In addition, the cell viability is appreciable in the case of starch and gum arabic stabilized gold nanoparticles, but feeble toxicity is experienced in the case of citrate stabilized gold nanoparticles as shown in Figure 4.1(b) and Figure 4.1(c). Figure 4.1 The PC-3 cell lines in the exponential growth phase were exposed to different concentrations of citrate (CAuNps), starch (SAuNps) and gum Arabic (GAuNps) stabilized gold nanoparticles. The cell viability was measured by (a) MTT assay (b) LDH assay and (c) Neutral Red Cell uptake assay

7 87 We have investigated the cell viability using the cell line MCF-7. The cell lines in the exponential growth phase were exposed to different concentrations of gold nanoparticles. The cell viability was measured by MTT assay, LDH assay and Neutral Red assay and the results are shown in Figure 4.2 (a), Figure 4.2 (b) and Figure 4.2 (c) respectively. In the cell viability assay experiments like MTT, LDH and Neutral red cell, the nanoparticles stabilized with starch and gum arabic were showing more viable to cell lines. The citrate stabilized gold nanoparticles exerts little toxic effect on the cell lines used. Cytotoxicity was also assessed using MTT assay, LDH assay and Neutral red cell assay at different time period (12, 24, 36, 48, 60 and 72 hr) along with PC-3 and MCF-3. The PC-3 cell line in the exponential growth phase was exposed to 50 g/ml concentration of gold nanoparticles for 12h, 24h, 36h, 48h, 60h and 72h. The cell viability was measured by MTT assay, LDH assay and Neutral Red assay as described in the experimental section. Cell viability was calculated as the percentage of the viable cells compared to the untreated controls. The result shows, after 12 hour exposure to gold nanoparticles, the viability started to decline. Figure 4.3(a), Figure 4.3 (b) and Figure 4.3 (c) shows the result of MTT, LDH and Neutral Red cell assays respectively. It was observed that the cell viability was marginally lower in citrate stabilized gold nanoparticles. The gold nanoparticles stabilized with starch and gum arabic were shows more than 90% of viability even after 72 hr of exposure.

8 88 Figure 4.2 The MCF-7 cell lines in the exponential growth phase were exposed to different concentrations of citrate (CAuNps), starch (SAuNps) and gum Arabic (GAuNps) stabilized gold nanoparticles. The cell viability was measured by (a) MTT assay (b) LDH assay and (c) Neutral Red assay

9 89 Figure 4.3 The PC-3 cell lines in the exponential growth phase were exposed to citrate (CAuNps), starch (SAuNps) and gum Arabic (GAuNps) stabilized gold nanoparticles for 12h, 24h, 36h, 48h, 60h and 72h. The cell viability was measured by (a) MTTT assay (b) LDH assay and (c) Neutral Red assay. The MCF-7 cell line in the exponential growth phase was exposed to 50 g/mll concentration of gold nanoparticles for 12h, 24h, 36h, 48h, 60h and 72h. The cell viability was measured by (a) MTT assay (b) LDH assay

10 90 and (c) Neutral Red Assay as described in the experimental section. Figure 4.4(a), Figure 4.4(b) and Figure 4.4(c) shows the results of MTT, LDH and Neutral Red cell assays using MCF-7 cell line. We found that the cell viability was slightly lower in the case of citrate stabilized gold nanoparticles and the gold nanoparticles stabilized with starch and gum arabic were three to four fold viable to cell line used. Table 4.1 Average size, plasmon wavelength, plasmon width and IC 50 of gold nanoparticles stabilized with citrate (CAuNps), starch (SAuNps) and gum Arabic (GAuNps) Sample name Average size (nm) Plasmon wavelength ( max ) nm Plasmon width ( ) nm IC 50 values (µg/ml) CAuNp 21± SAuNp 21± GAuNp 20± We found that the gold nanoparticles stabilized with citrate, starch, and gum arabic are viable to different cells through different assays with different concentrations and time of exposure of gold nanoparticles. The viability of the cell lines are depending on the stabilizing agent and the types of cytotoxicity assay used. The cell viability test shows distinguishable cytotoxic effect for citrate stabilized gold nanoparticles at a higher concentration and this is may be the surface coating is acidic in nature compared to starch and gum arabic. The IC 50 values for citrate, starch and gum arabic stabilized gold nanoparticles were 63 g/ml, 220 g/ml and 239 g/ml depending on the particle stabilizer used. The possibility of size effect is ignored since we have used the same size of gold nanoparticles. Interestingly the gold nanoparticles stabilized with starch and gum arabic are three-to-four fold viable than citrate at higher concentrations and in long time

11 91 exposure. The averagee size, plasmon wavelength, plasmon width and IC 50 of gold nanoparticles stabilized with citrate, starch and gum arabic for the elucidation of stabilizer based cytotoxicity studies were summarized in Table 4.1. Figure.4.4 The MCF-7 cell line in the exponential growth phase were exposed to citrate (CAuNps), starch (SAuNps) and gum Arabic (GAuNps) stabilized gold nanoparticles for 12h, 24h, 36h,48h 60h and 72h. The cell viability was measured by (a) MTTT assay (b) LDH assay and (c) Neutral Red assay

12 DISCUSSION Comparison of cytotoxicity studies based on stabilizing agents revealed that citrate produced little toxic as compared to starch and gum arabic stabilized gold nanoparticles in different concentration and at different time. Even though the citrate has a feeble cytotoxicity, it was found to be viable because, it has more than 80% cell viability. Cell viability was also determined by a LDH release assay which employed to measure the cytotoxicity of the gold nanoparticles at different concentrations and time. The absorbance of the produced formazan at 490nm is proportional to the number of damaged or dying cells. The cytotoxicity of various cell lines exposed to increasing concentrations of nanoparticles stabilized with three different stabilizing agents were analyzed for 24 hr. At each concentration, there were no significant cytotoxicity effect is produced. The cell viability results indicate that gold nanoparticles are non-toxic to the array of cells tested. The incorporation of surface functionalities via citrate, starch and gum arabic renders these nanoparticles highly biocompatible. Noble metal particles, such as gold are generally non-toxic due to their inert nature. The cell survival at different concentrations of gold nanoparticles stabilized with different capping agent shows that there is a small variation in cell viability with the increase in concentration and at the long time exposure of nanoparticles with cell lines. Cell based cytotoxic assay with different concentrations of gold nanoparticles shows very small variation among citrate, starch and gum arabic. The gold nanoparticles used were having the same size, it differs only by its stabilizing agent. In comparison the citrate stabilized gold nanoparticles show less viability than starch and gum arabic. This is may be the citrate is acidic in nature because the size dependent cytotoxicity is ruled out since in all the three cases, the particles sizes were same.

13 93 These results are consistent with previous investigations performed with dermal fibroblasts (Pernodet et al 2006) that demonstrated the gold and citrate nanoparticles impaired the proliferation of dermal fibroblasts and induced an abnormal formation of actin filaments, causing a reduced cellular morphology. On the contrary (Connor et al 2005) reported that the gold and citrate nanoparticles impaired the proliferation of dermal fibroblasts and induced an abnormal formation of actin filaments, causing therefore a reduced cellular motility and influencing the cell morphology. In addition Connor (2005) reported that the citrate and biotinylated 18nm gold nanoparticles did not induce any toxicity in leukemia cells. 4.6 CONCLUSION In conclusion, we have found that the nanoparticles stabilized with citrate, starch and gum arabic are viable to different cells through different assays used. It was found that, the viability of the treated cell lines were depending on the stabilizing agent. The cell viability studies show that a feeble cytotoxic effect of citrate stabilized gold nanoparticles at higher concentrations and long time exposure. It was concluded that the surface coating is acidic in nature compared to starch and gum arabic. The IC 50 values for citrate, starch and gum arabic stabilized gold nanoparticles were 63 g/ml, 220 g/ml and 239 g/ml respectively depending on the particle stabilizer used. The possibility of size dependent cytotoxicity is ignored since we have used the same size of gold nanoparticles. Interestingly the gold nanoparticles stabilized with starch and gum arabic were three-to-four fold viable than citrate at higher concentrations and in long time exposure.