Understanding the Antifungal Mechanism of Ag@ZnO Core-shell Nanocomposites against Candida krusei

In the present paper, facile synthesis of Ag@ZnO core-shell nanocomposites is reported where zinc oxide is coated on biogenic silver nanoparticles synthesized using Andrographis paniculata and Aloe vera leaf extract. Structural features of as synthesized nanocomposites are characterized by UV-visible spectroscopy, XRD, and FTIR. Morphology of the above core-shell nanocomposites is investigated by electron microscopy. As synthesized nanocomposite material has shown antimicrobial activity against Candida krusei, which is an opportunistic pathogen known to cause candidemia. The possible mode of activity of the above material has been studied by in-vitro molecular techniques. Our investigations have shown that surface coating of biogenic silver nanoparticles by zinc oxide has increased its antimicrobial efficiency against Candida krusei, while decreasing its toxicity towards A431 human epidermoid carcinoma cell lines.

Fungal infections caused by Candida species have become one of the major causes of morbidity and mortality in immunocompromised situation, particularly for patients with hematologic malignancies and for transplant recipients [1][2][3][4] . Despite sincere efforts, treatment of candidemia remains a challenging issue due to frequent development of resistance to the antifungal drugs used in clinical settings 5 . For many years, most of the cases of candidemia were caused by Candida albicans. Recently, other Candida species have proved to be responsible for an increased proportion of candidemia. In particular, Candida krusei (C. krusei) has become one of the most frequent of these emerging pathogens since it is opportunistic in nature showing resistance to commonly used drugs 6,7 . It is most often found in patients who have had prior fluconazole exposure 8 . More recent studies have shown infections due to C. krusei among patients receiving amphotericin B 9 . While great effort is made to understand the biology, epidemiology and pathogenicity of this pathogen, little success has been achieved in developing new treatment strategies 10 . Thus, there is an urgent need to search for alternative antifungal agents. In this regard, nanomaterials based strategies have received much attention by the scientific community owing to their unique antimicrobial effectiveness caused by combination of their small size and high surface-to-volume ratio, which enable intimate interactions with microbial membranes 11,12 . Inorganic antibacterial agents such as metallic and metal oxide nanoparticles (NPs) are considered to be advantageous compared to organic compound due to their stability, chemical inertness, and potential biotechnological applications 13,14 . Out of various nano-structured materials under investigation, Ag NPs have shown interesting antifungal activity against Candida albicans. Since, there are several reports on acute toxic effects of Ag NPs against human cell lines; its practical application is hindered 15,16 . To overcome these limitations, researchers have been trying to develop nanocomposites (NC) which may preserve the antibacterial activity of the metallic NPs while increasing the biocompatibility of the resultant composite structure. NC materials containing metallic silver has been explored as antibacterial agents for a long time. Ag/SiO 2 NC are studied for their potential antibacterial effect against Escherichia coli and Staphylococcus aureus [17][18][19][20] . Zhang and co-workers have reported the antibacterial effect of Ag/TiO 2 NC powders against

Results and Discussion
UV-visible spectroscopy. Due to presence of free surface electrons, nobel metal NPs show strong surface Plasmon resonance (SPR) band in the UV-visible spectrum. Thus, bio-synthesis of Ag NPs has been followed by change in the SPR band of the aqueous dispersion. Surface coating with a metal oxide layer is expected to change the dielectric constant of the surrounding environment and hence can shift the SPR band of metal NPs. This concept has been extensively used to monitor the formation of metal@metal oxide core-shell NC 17,23 . UV-visible spectra of the aqueous dispersion of Ag NPs, Ag@ZnO core-shell NC, and only plant extract are shown in Fig. 1. Aqueous dispersion of biogenic Ag NPs showed a clear SPR band at 434 nm 17 . After the addition of aqueous sodium zincate sol, SPR band has shown a distinct blue shift of about 23 nm. This could be attributed to the formation of the oxide shell on the surface of Ag NPs. It has been previously demonstrated that the SPR band is highly sensitive to a variety of perturbations on the particle surface 17,23 . For ZnO (n ZnO = 1.92) with a refractive index remarkably higher than that of water (n H2O = 1.33), strong blue-shifts in the SPR band position is expected for Ag colloidal particles as they become encapsulated with zinc oxide shell. This interesting optical phenomenon is associated with the transfer of electronic charge between the core and shell. Ag NPs have negative charge due to excess surface electrons which causes the rapid aggregation of the colloid 17,24,25 . Formation of an oxide shell helps to accommodate the excess electrons present on the metal surface. Due to the large difference between the intrinsic Fermi level of the core and the conduction band energy of the (n-type) semiconductor shell, mobile electrons that diffuse within the shell will be trapped for long periods of time in the core 17 . This reduces the possibility of interaction between the adjacent metal NPs and hence may increase the stability of the resultant NC system. In the aqueous condition, zinc nitrate is expected to undergo rapid dissolution to produce Zn 2+ and NO 3ions. In presence of sodium hydroxide Zn 2+ may precipitate rapidly to form Zn(OH) 2 . In presence of high concentration of NaOH and water, the resultant Zn(OH) 2 would not simply undergo a thermal decomposition to produce ZnO and H 2 O rather it is expected to produce soluble Na 2 ZnO 2 . However in aqueous solutions principal zincate ion appears as [Zn(OH) 3 .H 2 O] − . Now under hydrothermal condition, solvated zincate ion converts to produce  X-ray diffraction studies. Phase and crystal structure of the Ag@ZnO core-shell NC are investigated by XRD and result is shown in Fig. 2 FTIR spectroscopy. The presence of adsorbed molecules and/or functional groups on surface of Ag@ZnO NC was analyzed with FTIR spectroscopy at room temperature in an acquired range of 500-4000 cm -1 . Figure 2(b) shows the FTIR spectra of as-synthesized Ag@ZnO core-shell NC. A broad band at 3200-3600 cm -1 may have originated from polyphenols that are present in the extract. The small vibration at 1620 could be attributed to the stretching peak of C = O group 26 present in the polyphenol (e.g. gallic acid) content of plant extract. The boarding band frequency of side chain of alkane groups (sp 3 C-H bending, 1460 cm -1 ) is obtained at 1450 cm -1 27,28 . The weak band of C-N group is shown at 1109 cm -1 which comes in the range of 1080-1360 cm -1 29 .
Total Phenolic content Detemination. Supplementary Figure 2 reveals that the phenolic content present in Ag@ZnO core-shell NCs is lesser than the phenolic content present in the extract solution. The decrement in the phenolic content (GAE (μ g/ml)) of Ag@ZnO NCs in comparison with the Andrographis paniculata and Aloe vera plant extract alone or together indicates possible explanation for the synthesis mechanism of Ag@ZnO core shell NCs, where components with phenolic groups (equivalent to galic acid) present in the plant extract solution may have acted as a reducing and/or stabilizing agent.

Transmission electron microscopy. Morphology of the Ag NPs and Ag@ZnO NC is investigated
by TEM and resultant images are shown in Fig. 3. TEM samples were prepared by dipping the TEM grid in aqueous dispersion of nanomaterials followed by freeze drying for 12 h. Well-dispersed Ag NPs are clearly observed. Approximate size of the Ag NP is 30∼ 35 nm. ZnO NPs are found in the broad size range of 40∼ 75 nm. Comparison of these results with XRD data suggests the formation of polycrystalline materials. Core-shell structure is observed for Ag@ZnO NCs. However, particles are obtained with multiple silver cores encapsulated inside single zinc oxide shell. This observation was in accordance with the data obtained from UV-visible spectrum of the core-shell NCs.

Investigation of size distribution and surface charge. Size distribution of the synthesized Ag@ZnO
NCs is investigated by differential light scattering (DLS) technique. As shown in Supplementary Figure 3a NC particles were found to be in the average size range of 42 nm. The surface charge of the NC particles was investigated by zeta-potential measurement and was found to be ≈ − 27 mV (Supplementary Figure 3b).
Antifungal activity against C. krusei. The comparative antifungal activity of Ag NPs, ZnO NPs and Ag@ZnO NC has been shown in Fig. 4. It can be observed that Ag NPs at a concentration of 500 and 1000 μ g/mL have completely inhibited the growth of C. krusei following 15 h and 6 h of incubation respectively. The MIC value for biosyn- thesized Ag NPs is taken as 500 μ g/mL. Although lower concentrations of the metal NPs (50, 100 and 250 μ g/mL) have shown antifungal activity, but complete growth inhibition has not been observed. Similar results have been obtained with lower concentrations of chemically synthesized ZnO NPs. 500 μ g/mL of ZnO NPs completely inhibited the growth of the organism and hence this concentration is taken as the MIC value. However the efficiency as compared to biosynthesized Ag NPs is less because the time taken to completely suppress the growth of the microbe is quite high (24 h) with ZnO NPs compared to Ag NPs. Ag@ZnO NC have shown the highest antifungal efficiency against C. krusei. 250, 500 and 1000 μ g/mL of the NC have completely inhibited the growth of the fungus within 18, 9 and 6 h of incubation respectively. Hence 250 μ g/mL of the core shell NC has been taken as the MIC value for completely killing the microbe. Lower concentrations (50 and 100 μ g/mL) have suppressed the growth of the fungus by more than 50% compared to untreated cells. Earlier reports with chemically synthesized Ag-ZnO NC have shown similar results against GFP expressing antibiotic resistant E. coli 30 . The MIC value with their material is observed to be 550 μ g/mL. Lu et al. have previously reported that the MIC value for E. coli and S. aures is 600 and 400 μ g/mL respectively with Ag-ZnO NC system 31 . In both the studies the MIC values obtained using Ag-ZnO NC is higher as compared to the core-shell NC used in the present study against the fungus C. krusei.
To confirm the cell viability, fluorescence based live dead staining assay has been done ( Fig. 5a-d). Figure 5a represents untreated cells which are used as control experiment. From Fig. 5b, it is observed that the live fungal cells have considerably decreased in number within 6 h of treatment with 250 μ g/mL of core-shell structure NC material. Following 12 h of treatment, the percentage of dead cells has considerably increased. By the end of 24 h, the live fungal count observed is almost negligible indicating very high cell death. With increase in the incubation time there is a corresponding increase in the percentage of dead cells. This might be due to the fact that increased incubation time favored more interaction of the fungal cells with the as synthesized NC material. Hence the cell death has occurred in time dependant manner. FE-SEM images of the untreated and treated cells have been shown in Fig. 5e-h respectively. From Fig. 5e, we can observe that the cells of C. krusei are intact having smooth surface. However in treated cells (Fig. 5f), within 6 h of treatment partial change in cell morphology has been noticed. 12 h of incubation has indicated clustering of the cells with breaks in the cell membranes (Fig. 5g). Formation of hollow pits has also been observed in the images. Cell shrinkage has been observed within 24 h of incubation with core-shell NC material (Fig. 5h). This might be due to damage of the cell membrane resulting in leakage of intracellular compounds leading to complete cell disruption. There are similar reports with ZnO NPs, Ag NPs and chemically synthesized Ag-ZnO NC against different microbes 32,33 . Interestingly, as compared to other Candida sps, C. krusei has multilayered cell wall and trilaminar cell membrane and hence it is very difficult to penetrate the cell by damaging the outer protective layer 34 . In the present study the effectiveness of Ag@ZnO NC against this microbe is very high which could be useful for future application purpose. Moreover there is hardly any report available on the mode of antifungal action of nanomaterial based core-shell structures against this pathogen.
Antifungal mechanism. Formation of ROS in microorganisms occurs due to metabolic activity of the cells. Particularly the accumulation of ROS plays an important role in inducing apoptosis in metazoans and yeasts 35 . Therefore determination of intracellular ROS plays a significant role in underlying the probable antimicrobial mechanism of a particular compound. Figure 6a Figure 4). However, the highest level of ROS has been generated with Ag@ZnO NC. We have qualitatively determined the ROS generation by using DCF-DA which can easily detect intracellular hydrogen peroxide in intact cells. DCF-DA is permeable to plasma membrane and is hydrolyzed into the cytoplasm to form DCFH-carboxylate anion 36,37 . From this observation, we may expect that the antimicrobial mechanism of as synthesized Ag NPs, ZnO NPs and Ag@ZnO NC against the tested pathogen is due to generation of ROS. It is well known that ROS targets biological cellular compounds and particularly lipid molecules which are highly complex in nature. In the present study we have tried to determine the effect of all the three nanomaterials on the cell membrane of the targeted pathogen by measuring MDA concentration which is one of the most important degradation products of lipid perooxidation 38 . The concentration of MDA has been evaluated by using TBARS, which is an indicator of MDA. It is observed from Fig. 6b that the concentration of MDA has significantly increased in cells treated with Ag@ZnO NC as compared to untreated sample with increase in incubation time of up to 20 h. The MDA level increased progressively and is found to be highest at 12 h of incubation (0.010 nmole/μ L). However at 24 h incubation, the concentration of MDA is observed to be lesser signifying that MDA might be degraded. However, the concentration of MDA is much less in cells treated with Ag and ZnO NPs (Supplementary Figure 4).
To further elucidate the antifungal mechanism, we tried to investigate the rate of DNA damage in C. krusei by using different concentration of Ag NPs, ZnO NPs and Ag@ZnO NC. From Fig. 6c represents the DNA degradation of the fungal cell with Ag@ZnO NC. It can be observed that following 24 h of incubation, there is appearance of a distinct DNA band in the control sample. With 50 μ g/mL of NC a slightly less intense band can be observed compared to the untreated sample. However with further increase in concentration of the NC, there is no appearance of DNA band suggesting complete DNA degradation. Ag and ZnO NP treated cells have shown DNA damage in doze dependant manner (Supplementary Figure 4). 500 μ g/mL and 1000 μ g/mL of the metal and metal oxide NPs have not shown any DNA band suggesting complete DNA degradation. However, Ag@ZnO NC have shown similar results with lower concentration (100 and 250 μ g/mL) indicating superior antifungal activity. This might be due to the fact that when fungal cells are exposed to the NC, the later can rupture the cell membrane thereby inducing significant oxidative stress and generating ROS which ultimately lead to DNA degradation. Previous reports on antimicrobial activity of NPs also suggest that oxidative stress in microbial cells could play an important role in inducing DNA damage through generation of ROS 39 . The later attacks DNA thereby producing Cytotoxicity and Genotoxicity of Ag@ZnO core-shell NC against A431 human epidermoid carcinoma cell line. For future application of the as synthesized core-shell NC, it is important to understand their biocompatibility factor. This will ensure that the antimicrobial material will target the pathogenic cells without causing any toxic effect towards the normal cells. Hence, we have investigated the cytotoxicity of the synthesized Ag@ZnO core-shell NC against normal A431 human epidermoid carcinoma cell lines. From Fig. 7(a), it is seen that after 24 h of incubation, as compared to the untreated cells, the percentage of viable cells in the treated samples is very high. Treatment with the optimized concentration of the NC material used in the present study (i.e. 250 μ g/mL), has shown 89% cell viability after 48 h of incubation indicating very less toxicity. Under similar experimental condition (250 μ g/mL treated for 48 h), Ag and ZnO NPs have shown 79% and 81% cell viability respectively (Fig. 7b,c). Both the NPs have shown very less toxicity towards the cell line used in the study. The reason might be due to the biocompatibility of the as synthesized Ag NPs. However, previous studies with chemically synthesized Ag NPs and ZnO NPs have shown considerable toxicity towards the above mentioned cell line at lower concentrations 42,43 . In the present study, least cytoxicity has been observed with Ag@ZnO NC which might be due to the core-shell morphology which may not have allowed the leaching of the silver ion, thereby maintaining its antimicrobial efficiency. Das et al. have observed similar results with chemically synthesized Ag@ZnO core-shell NCs 44 .
Since core shell NC are used as topical antifungal agents, the cytotoxicity of the as synthesized Ag@ZnO NC has been observed against the endothelial cell line A570. From the MTT data (Supplementary Figure 6) it has been observed that the above mentioned material has shown very less toxicity towards A570 cell line. The cell viability has been observed to be 91% following 48 h of incubation.
Genotoxicity of Ag NPs, ZnO NPs and Ag@ZnO NC against the above mentioned cell line has been evaluated through fluorescence microscopy after staining with DAPI. Figure 8f clearly shows that the nuclear morphology of A431 cells is unaltered in the presence of 250 μ g/mL NC up to 48 h of incubation. This confirms that the synthesized NC used in the study does not have any genotoxicity towards the human epidermoid carcinoma cell line. Similar results have been obtained with 250 μ g/mL of Ag and ZnO NPs (Fig. 8i,l respectively).
To confirm the cytotoxicity induced in treated A431 cell lines by incorporation of Ag@ZnO NC, propidium iodide (PI) uptake assay has been performed and quantified through FACS. From the results shown in Fig. 9, it is evident that the population of cells in the lower right quadrants, which is stained by PI, corresponds to the percentage of nonviable cells. It has increased to 2.12% and 4.10% after treatment with 250 μ g/mL of Ag@ZnO NC for 24 and 48 h respectively (Fig. 9c,d). However, uptake of PI is very less in control or untreated cells after 24 and 48 h incubation (Fig. 9a,b). Similarly we have also performed the FACS of Ag and ZnO NPs with the above mentioned cell line. The obtained data suggests that the toxicity of Ag NPs and ZnO NPs is marginally higher than Ag@ZnO (Supplementary Figure 5). Considering these results it is expected the as synthesized Ag@ZnO core-shell NCs show effective antibacterial activity against C. krusei while it does not offer severe toxicity towards normal A431 cells.

Conclusion
In summary, antifungal activity of Ag@ZnO NC with core-shell morphology is investigated against opportunistic pathogen C. krusei. From experimental observations, present report communicates a promising consequence. Core-shell NC is obtained by coating of zinc oxide on biogenic silver NPs synthesized using Andrographis paniculata and Aloe vera. Comparative antifungal data indicates that NC material could inhibit the growth of the targeted pathogen at a lower dose size (250 μ g/mL) than that of pure Ag and ZnO NPs. Our investigation suggests that the antifungal mechanism of core-shell NC system could be attributed to the generation of ROS which may degrade cellular compounds. Additionally the core-shell NC was found to be less toxic towards normal A431 human epidermoid carcinoma cell lines. These findings justify further studies for possible biomedical application of the proposed Ag@ZnO core-shell NC material.

Materials and Methods
Bio-synthesis of Ag NPs. Andrographis paniculata (AP) and Aloe vera (AV) were collected from the local market of Bhubaneswar. 10 gm of clean and washed leaves of AP and AV each were taken in 100 mL of de-ionized (DI) water. They were macerated thoroughly by motor pestle for 15 minutes and boiled at 60 °C for 30 minutes than filtered using Whatman filter paper 1. The clear filtrate was collected for biosynthesis of NPs. 80 ml of Silver perchlorate hydrate (Sigma-Aldrich, 99%) solution was taken, kept under stirring condition on hot plate. At 90 °C, 10 mL of clear filtrate aqueous solution obtained from the plant extract mixture (10 times diluted) was added under stirring condition. The color of the solution was changed to yellowish-brown which indicated the formation of metallic Ag NPs. Four parameters were monitored for optimizing the process of synthesis i.e. temperature, reaction pH, plant extract dilution factor and concentration of silver salt. pH of the Silver perchlorate hydrate solution was adjusted by adding 1 M NaOH and 0.5 N HCl. The process was first optimised for the reaction pH and temperature. Four different pH (pH 4.0, 7.0, 10.0, and the natural pH = 6.34) of the silver salt was taken and synthesis was observed at four different reaction temperatures (4 °C, 50 °C, 70 °C, 90 °C and room temperature i.e. 28 ± 2 °C). Similarly four different dilutions of the plant extract were made (1× , 10× , 100× , 1000× ). Each dilution was used for biosynthesis at four different concentration of silver salt solution (0 mM, 1 mM, 2 mM, 3 mM, and 4 mM). Spectroscopic results were used to obtain the optimized synthesis condition. These two parameters were further used to optimize results for dilution rate and concentration of Ag NPs.

Synthesis of Ag@ZnO NC.
ZnO NPs were coated on the surface of biogenic Ag NPs via colloidal technique. 2 mL of 2 mM sodium hydroxide solution was added to 10 mL of 10 mM zinc nitrate hexahydrate (Sigma Aldrich, 98%) to obtain a white precipitate of zinc hydroxide. The obtained precipitate was dissolved by the addition of excess amount of sodium hydroxide solution to form sodium zincate. Then 1 mL sodium zincate solution was diluted to 5 mL with DI water and added to 100 mL of biologically synthesized Ag NPs at 95 ± 2 °C under vigorous stirring. It was stirred for 30 minutes to prepare Ag@ZnO NC. Change in color from yellowish brown to black indicated the formation of Ag@ZnO core-shell NC.
Characterization of the materials. Synthesis of Ag NPs and Ag@ZnO NC was followed by UV-visible spectroscopy (Shimadzu UV-1800). Bio-synthesized and purified NPs were characterized by using various instrumental techniques. The morphology of the materials was investigated by transmission electron microscopy (TEM, JEOL-JEM-2010). The crystal structure of the material was investigated by X-ray diffraction technique (D/Max 2005, Rigaku). Fourier Transform infrared spectroscopy (FTIR) (Shimadzu 8201PC, Japan) was used to investigate the presence and nature of surface functional groups. Determination of total phenolic contents. The total phenolic content of Andrographis paniculata, Aloe vera plant extract as well as Ag@ZnO core shell NCs has been assayed by Folin-Ciocalteau's method. Being one of the member of phenolic family, galic acid is chosen as a standard for determining the phenolic content. 0.1 ml of Andrographis paniculata, Aloe vera plant extract, mixture of both the above mentioned plant extracts (1:1 ratio) and Ag@ZnO samples were taken in conical flasks and final volume was adjusted to 46 ml by adding 45.9 ml distilled water. Then 1 ml Folin-Ciocalteau reactive solution (1 N) was added and incubated at room temperature for 3 min. Finally, 3 ml (2%) sodium carbonate solution was added and mixtures were put on shaker for 2 h at room temperature followed by measurement of OD at 760 nm in spectrophotometer. The phenolic compound content was expressed as gallic acid equivalent (GAE, μ g).
Antifungal assay. Comparative antimicrobial activity of Ag NPs, ZnO NPs and Ag@ZnO NC was investigated against Candida krusei. Fungal growth inhibition was performed by inoculating 1 × 10 6 CFUs of C. Krusei cells into 20 mL of PDB with different concentrations (0, 50, 100, 250, 500, 1000 μ g/mL) of nanomaterials and incubated in shaking incubator at 30 °C in 200 rpm for 24 h. Then 100 μ L of sample was taken at 3 h interval and serially diluted (10 −1 to 10 −3 ) in normal saline solution (0.9% NaCl). Samples were taken from 10 −3 dilution and plating was done on PDA by drop plate method to determine inhibition of cell growth by CFU assay. The Minimum inhibitory concentration (MIC) for each of the above mentioned nanomateirals was determined by considering the lowest concentration of the materials which could completely inhibit the growth of C. krusei.
Live/dead staining. To check viability of C. krusei after treatment with 250 μ g/mL of Ag@ZnO NC, live/dead staining was performed by using fluorescein diacetate (FDA) (Sigma-Aldrich) and propidium iodide (PI) (MP Biomedicals). The protocol followed is same as reported by Ehgartner et al. 45 . Initially 1 × 10 6 cells of C. krusei were incubated with 250 μ g/mL of Ag@ZnO NC for 0, 12 and 24 h. It was then centrifuged to remove culture media and washed with 1x PBS. Then pellets were again resuspended in 1x PBS. Subsequently 10 μ g/mL FDA and 20 μ g/ml PI was added into untreated and treated samples and incubated in the dark at room temperature for 15 minutes. Cell viability was analyzed under fluorescence microscope (Floid Cell Imaging Station, Life Technology).

FE-SEM of treated fungal cell. Samples of treated and untreated C. krusei cells for Field Emission Scanning
Electron Microscope (FE-SEM) were prepared according to previously reported protocol 46 . Initially 1 × 10 6 cells of C. Krusei were treated with 250 μ g/mL of Ag@ZnO NC for 12 and 24 h. Then treated and untreated cells were fixed with 4% glutaraldehyde (Sigma-Aldrich) in 0.2 M sodium cacodylate buffer (pH 5.5 to 6.0; Sigma-Aldrich). The cells were washed with cacodylate buffer, and dehydrated using ethanol. The samples (untreated and treated cells) were further sputter coated with gold and analyzed under FE-SEM.

Determination of reactive oxygen species (ROS).
Generation of intracellular reactive oxygen species (ROS) in C. krusei cells were measured qualitatively by using 2′ ,7′ -dichlorofluorescin diacetate (DCF-DA) through flourescence microscope as described by Wojtala et al. 47 . DCF-DA is a specific dye for intracellular reactive oxygen species (ROS) which enters into the cells passively and reacts with ROS to form highly fluorescent dichlorofluorescein (DCF). 1 mL sample of Ag NPs, ZnO NPs and Ag@ZnO NC (250 μ g/mL) treated cells were taken and washed with 1x PBS by centrifugation at 8,000 rpm for 15 minutes. Then cell pellets were resuspended in 1x PBS buffer and 10 μ M of DCF-DA was added and incubated for 10 minutes at 37 °C under dark condition. The images were analysed under fluorescence microscope. Determination of lipid peroxidation by malondialdehyde (MDA) assay. MDA is a common end product of lipid peroxidation. Estimation of MDA through its reaction with thiobarbituric acid (TBA) forms a pink colored MDA-TBA complex which predicts the degree of damage in microbial cell membrane 48 . Lipid peroxidation assay kit (Sigma-Aldrich) was used to determine the concentration of MDA in the reaction system. DNA degradation. Candida krusei cells were treated with different concentrations (0, 50, 100, 250, 500, 1000 μ g/mL) of Ag NPs, ZnO NPs and Ag@ZnO for 24 h. DNA was isolated from treated and untreated cells followed by previously reported protocol 49 . The isolated DNA pellets were dissolved in Tris EDTA buffer and 5 μ L of the DNA samples were separated on 0.9% agarose gel prepared in Tris acetate EDTA buffer that was stained with ethidium bromide (1 μ g/mL). Image was taken in gel documentation system (Gel Doc TM EZ Imager, Bio-Rad).
Cytotoxicity assay. A431 human epidermoid carcinoma cell lines were obtained from American Type Culture Collection (ATCC), Manassas, USA. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with L-glutamine (4 mM), penicillin (100 units/mL), streptomycin (100 μ g/mL) and 10% (v/v) fetal bovine serum (growth medium). Firstly, 3 × 10 5 cells were seeded in 24 well plate and incubated for 48 h at 37 °C in presence of 5% CO 2 in humidified incubator. Then supernatants were aspirated from the wells and fresh growth medium was added, which was supplemented with desired concentrations of Ag NPs, ZnO NPs and Ag@ZnO NC in the range of 0, 50, 100, 250, 500, 1000 μ g/mL. Cells were incubated at 37 °C in humidified incubator in presence of 5% CO 2 for 24 and 48 h. The cytotoxicty of materials against A431 cell lines was performed followed by standard methylthiazole tetrazolium (MTT) assay 50  were covered with 300 nM DAPI stain and incubated for 10 minutes in dark at room temperature. Then cells were washed with 1x PBS to remove the stain solution. The nuclear morphology of the cells was analyzed under fluorescence microscope at 40x. Untreated cells were used as control.
Determination of cytotoxicity by FACS. Uptake of PI by cells was evaluated through FACS according to previously described protocol 51 . A431 cell lines (3 × 10 5 cells/well) were seeded in 24 well plate and incubated at 37 °C (in the presence of 5% CO 2 ) for 48 h. The supernatant was discarded with the help of aspirator and cells were incubated with 250 μ g/mL of Ag NPs, ZnO NPs and Ag@ZnO NC (suspended in culture medium) for 24 and 48 h. Treated and untreated cells were fixed using 70% ethanol at 4 °C for 1 h. Then cells were resuspended in 1x PBS and 50 μ g/mL of PI was added to each wells and incubated at 37 °C for 15 minutes. Samples were analyzed using a FACS calibur flow cytometer and CELL-Quest software (Becton-Dickinson, BD Biosciences, Franklin Lakes, NJ). In total, 10000 cells were analyzed per sample.