Appl Biochem Biotechnol DOI 10.1007/s12010-014-0782-9
Highly Potential Antifungal Activity of Quantum-Sized Silver Nanoparticles Against Candida albicans Malathi Selvaraj & Prabhu Pandurangan & Nishanthi Ramasami & Suresh Babu Rajendran & Sriman Narayanan Sangilimuthu & Palani Perumal Received: 1 August 2013 / Accepted: 4 February 2014 # Springer Science+Business Media New York 2014
Abstract The antifungal activity of polyvinylpyrrolidone (PVP)-stabilized quantum-sized silver nanoparticles (SNPs) against the growth of Candida albicans has been demonstrated in the present study. C. albicans is a known opportunistic human pathogen causing superficial and systemic infections. Research data carried out on C. albicans so far have shown unequivocally that it develops resistance against conventional antifungal drugs and that the infections it causes are difficult to cure with conventional antifungal agents. Hence, it is urgent to find newer materials for the treatment of infections caused by C. albicans that must be safe for the host. PVP-capped SNPs were synthesized, and its surface plasmon band was observed at 410 nm. The growth of C. albicans was markedly inhibited when the cells were incubated with SNP. The minimum inhibitory concentration (MIC) of SNP was determined as 70 ng/ml, and this value is relatively lower when compared with the conventionally used antifungal drugs such as amphotericin B (0.5 μg/ml), fluconazole (0.5 μg/ml), and ketoconazole (8 μg/ml). The viability of SNP-treated cells was checked by measuring the metabolic activity using XTT assay. Field emission scanning electron microscopic (FE-SEM) and transmission electron microscopic (TEM) analyses of the cells treated with SNP have lost the structural integrity to a greater extent. Keywords Quantum-sized silver nanoparticles . Candida albicans . Antifungal activity . Minimum inhibitory concentration . Polyvinylpyrrolidone
Introduction Fungal infections caused by yeasts and molds represent an escalating problem in health care as advances in modern medicine prolong the lives of severely ill patients. These organisms cause infection not only in those having HIV, cancer, organ transplant, and surgical operation and ICU patients but also newborn infants. Fungi, being eukaryotic in nature and more complex than bacteria, cause infections that are often difficult to diagnose and treat, resulting in M. Selvaraj : P. Pandurangan : N. Ramasami : S. B. Rajendran : S. N. Sangilimuthu : P. Perumal (*) University of Madras, Chennai, Tamil Nadu, India e-mail: [email protected] P. Perumal e-mail: [email protected]
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unacceptably high mortality rates [1]. In recent years, a rapid increase in microbes that are resistant to conventionally used antibiotics has been observed [2]. Candida albicans is a commensal present on the mucosal surfaces of human oral and vaginal cavities and frequently turns into an opportunistic pathogen causing superficial and systemic infections if the host immune system is compromised due to immunosuppressive and broad-spectrum antibiotic therapy, HIV infection, and cancer chemotherapy [3]. Currently, most of the available antifungal agents are based on polyenes (amphotericin B), triazoles (fluconazole, itraconazole, voriconazole, and posaconazole), and echinocandins (caspofungin, micafungin, and anidulafungin). However, the administration of these antifungals is often accompanied by various complications such as amphotericin B toxicity and adverse effects of some azoles including toxicity and drug interactions [4–7] and yeast resistance to antifungal therapy [8, 9]. A remarkable property observed with C. albicans is that it develops resistance, and the infections it causes are difficult to treat with conventional antifungal drugs. Therefore, it is crucial to find newer molecules for the treatment of Candida infections without adverse effect on the host cells. It has been known, since the ancient times, that silver and its compounds are effective antimicrobial agents [10, 11]. In particular, due to the recent advances in research on metal nanoparticles, nano-Ag has received special attention as a possible antimicrobial agent [12–14]. Therefore, the preparation of uniformly sized silver nanoparticles with specific requirements in terms of size, shape, physicochemical properties is of great interest in the formulation of newer pharmaceutical products [15, 16]. Though the biocidal effect and the mode of action of silver ion are known, the antifungal effects and the mode of action of SNP against fungi have remained mostly unknown. Therefore, an attempt has been made in the present investigation to synthesize polyvinylpyrrolidone (PVP)-stabilized quantum-sized silver nanoparticles (SNPs) and to evaluate their antifungal activity against the growth of C. albicans. The growth of the organism was markedly inhibited when the cells were incubated with SNP. The minimum inhibitory concentration (MIC) of SNP has been determined, and the cell viability was checked using the XTT assay by measuring the mitochondrial dehydrogenase activity of the live cells. FESEM and transmission electron microscopic (TEM) analyses have clearly indicated marked changes in the integrity of the cells treated with SNP. For the first time, elemental analysis of the SNP-treated and nontreated cells has been carried out in this study.
Materials and Methods Chemicals Antifungal drugs such as amphotericin B and ketoconazole were purchased from HiMedia (India). Silver nitrate was purchased from Sigma Chemical Co. (USA). An injectable form of fluconazole was purchased from CIPLA (India). Culture media such as yeast nitrogen base (YNB) and yeast extract peptone dextrose agar (YEPDA) were purchased from Difco Laboratories (Detroit, MI, USA) and HiMedia (India), respectively. The other solvents used in the study were of analytical grade procured locally. Organisms and Growth Conditions C. albicans used in this study was obtained from the fungal culture collection facility at the Centre for Advanced Studies in Botany, University of Madras. The organism was cultured on YNB medium and stored on YEPDA slants at 4 °C until further use.
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Synthesis and Characterization of Quantum-Sized SNP The PVP-stabilized quantum-sized SNPs were synthesized as follows. Fifty milligrams of silver nitrate (AgNO3) was dissolved in 11.25 ml of ethylene glycol to which 1.5 g of PVP was added. The solution was thoroughly mixed using a magnetic stirrer. The agitation of the solution was continued for 20 min while increasing the temperature by 5 °C after every 5 min. The formation of a colloidal, wine-brown-colored solution confirms the formation of SNP. The SNP prepared as above was diluted to 10−4 and used for further investigations. The SNP was characterized using UV–Vis spectral analysis in a spectrophotometer (Lamda-650, Perkin Elmer, USA) and high-resolution transmission electron microscope (HR-TEM, FEI, Tecnai G2 model T 30 S-twin, 300 kV, Japan). Determination of MIC of SNP The determination of MIC of the PVP-stabilized SNP against C. albicans was performed as per the recommendations of the Clinical Laboratory Standard Institute (CLSI) [17]. The yeast cells of C. albicans were grown planktonically under aerobic conditions at 37 °C for 24 h in YNB broth. The cell count was made with hemocytometer, and a standardized cell suspension (1 × 105 cells/ml) was prepared. One hundred microliters of the cell suspension was dispensed in triplicate microtiter wells to which 100-μl suspension containing 1.380 to 0.092 μg of nanoparticles in YNB medium was added and mixed well. The following conventionally used antifungal agents such as amphotericin B [18], fluconazole, and ketoconazole were used in order to compare the antifungal potential of SNP. Amphotericin B and ketoconazole were dissolved in dimethyl sulfoxide (DMSO) and diluted with sterile YNB medium to obtain drug concentrations ranging from 0.0313 to 1,024 μg/ml. The cells with either nanoparticles or conventionally used antifungals were incubated at room temperature for 24 h, and the growth inhibition was measured spectrophotometrically at 600 nm (Powerserve XS Biotech, USA). Wells with cells without SNP were used as the control. The MIC was calculated as the lowest amount of SNPs that inhibited 80 % growth of C. albicans cells under the experimental conditions. Cell Viability Assay The metabolic activity of SNP-treated cells were determined by the XTT [2, 3-bis(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] assay [19]. The XTT tetrazolium salt was dissolved in phosphate-buffered saline (10 mM, pH 7.4) at a concentration of 0.5 g/l and filter sterilized through a 0.2 μm filter, and the aliquots were stored at −80 °C. Just before use, an aliquot was thawed, and 10 mM menadione prepared in acetone was added to the XTT solution to a final concentration of 1 μM. One hundred microliters of culture (1×105 cells/ml) was dispensed into 96-well plates. Different concentrations of SNP (1.380 to 0.092 μg) were added to the wells and incubated for 24 h at 37 °C. After 24 h of incubation, 100 μl of the XTT/menadione solution was added to each well and mixed thoroughly. The plates were incubated in dark at 37 °C for 2–3 h. The reduced formazan-colored product was measured at 490 nm using a microtiter plate reader (Powerserve XS Biotech, USA). The concentration of SNP showing 80 % reduction in cell viability was then calculated.
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Field Emission Scanning and Transmission Electron Microscopic Studies One milliliter of 1×105 cells/ml suspension was incubated with SNPs at their MIC values for 24 h and observed in a FE-SEM (H-7650, Hitachi, Japan) coupled with Energy Dispersive X-ray Analysis (EDAX) spectrum analysis, and the morphological changes were observed. The cells grown on YNB medium without SNP served as the control. For TEM, the cells were fixed with glutaraldehyde (1 %) solution, postfixed with osmium tetroxide for 2 h, and washed with buffer twice. The cells were dehydrated with increasing concentration of acetone and embedded in Epon–Araldite resin. Ultrathin sections were made, stained with uranyl acetate and lead citrate, and observed under TEM (SU 6600 Hitachi, Japan).
Results Synthesis and Physical Characterization of SNP The solution containing silver nitrate (AgNO3), ethylene glycol, and PVP turned into a colloidal, wine-brown-colored solution which indicated the formation of SNP. The SNP prepared as above was diluted to 10−4 and used for further investigations. The synthesized SNP showed an absorption peak at 410 nm (Fig. 1a), and this characteristic peak confirmed the formation of colloidal SNP. The TEM analysis revealed the formation of spherical SNP measuring about 2 nm (Fig. 1b). The HR-TEM analysis also revealed the formation of a thin outer layer measuring 0.28 nm (Fig. 1b). This thin layer was made by PVP encompassing SNP which appeared like core-shell morphology. Antifungal Activity of SNP The PVP-stabilized SNP showed antifungal activity against the tested organism. There was a marked reduction in the growth of the cells when incubated with SNP in a concentrationdependent manner (Fig. 2a). The MIC of the SNP was determined at 70 ng/ml. The MIC
Fig. 1 a UV–Vis spectra and b HR-TEM image of SNP
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Fig. 2 MIC values of a SNP and b amphotericin B, fluconazole, and ketoconazole against C. albicans. Each value represents the mean of three independent experiments
values for the known antifungals such as amphotericin B and fluconazole were observed at 0.5 μg/ml and for ketoconazole at 8 μg/ml (Fig. 2b). Cell Viability Assay To verify if the synthesized SNPs have the ability to kill the Candida cells, the cell viability assay was performed with XTT assay as described in the “Materials and Methods” section. The metabolic reduction of XTT sodium salt by mitochondrial dehydrogenase forms a colored water-soluble formazan product, and this was measured spectrophotometrically at 490 nm. The cells treated with PVP-stabilized SNP showed decreased metabolic activity in a dosedependent manner (Fig. 3) and confirmed that the SNPs have the ability to kill or inactivate the organism. Further, the MIC value of SNP as determined by the XTT assay coincided with the MIC value obtained through spectrophotometric measurement.
Fig. 3 Effect of PVP-stabilized silver nanoparticles on the cell viability. Each value represents the mean of three independent experiments
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Field Emission Scanning Electron Microscopic Studies The cells treated with SNP exhibited noticeable structural changes (Fig. 4c, d) when compared with the control cells (Fig. 4a, b). The SNP-treated cells that appeared deformed exhibited surface shrinkages when compared with the control. The SNP-treated cells aggregated into clumps, remained attached to the adjacent cells (Fig. 4c, d), forming EPS (extracellular polysaccharide)-rich biofilms. The SNP adhered onto the biofilm and cellular surfaces, besides being concentrated at the cellular interjections in the biofilm. The EDAX spectrum of SNPtreated cells showed the presence of Ag peak (Fig. 5b) along with other inorganic elements present in the medium, but in the case of untreated cells, the Ag peak was absent (Fig. 5a). The elemental analysis clearly indicated adherence of the SNP on the cell surface leading to cell damage and arrest of growth. Transmission Electron Microscopic Studies The cells treated with SNP were highly deformed (Fig. 6b, c, e, f), and the cells had shrunken to a greater extent (Fig. 6b, c, e, f). Alteration in the cell wall and cell membrane was also observed. Pronounced increase in the number and enlargement of vacuole was evident with reduction in the cytoplasm, and the cells started to disorient from its original shape. A significant portion of cells showed accumulation of granules in the cytoplasm and vacuoles. C. albicans cells treated with SNP also showed signs of pseudohypha and hypha morphogenesis.
Fig. 4 a, b Structural changes due to interaction of SNP with C. albicans cells: control. c, d SNP-treated Candida cells
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Fig. 5 EDAX analysis of C. albicans cells treated a in the absence and b in the presence of SNP
Fig. 6 TEM analysis of C. albicans cells treated a, b in the absence and b, c, e, f in the presence of SNP
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Discussion Nanosized inorganic particles have increasingly become an important material in the development of novel nanodevices that are being used in numerous physical, biological, biomedical, and pharmaceutical applications [20, 21]. Among them, nano-Ag has proved to be highly toxic to a variety of microorganisms and potentially inhibited the growth of both bacteria and fungi [22, 23] excepting few strains that showed resistance [24]. An attempt has been made in the present investigation to synthesize quantum-sized SNPs which are stabilized with PVP and to evaluate their antifungal activity against C. albicans. The PVP had reduced the silver nitrate leading to the formation of colloidal SNP, and the formation was manifested as a change in color from colorless to wine brown. As is evident from Fig.1a, the SNP exhibited characteristic absorption peak at 410 nm and had confirmed the formation of SNPs. The reducing and stabilizing capabilities of PVP in the synthesis of SNP have been well documented. The coordination of Ag+ ion and O atom of the carbonyl group of PVP facilitates the reduction of Ag+ ion with the lone pair of electrons of N atom from pyrrolidone ring that resulted in the formation of PVP-stabilized SNP [25–27]. The nanoparticles appeared spherical, and as it is highly evident from Fig. 1b, there was a conspicuous presence of an ultrathin cap formed by PVP. The monodispersed SNP had a mean primary grain size of 2–3 nm and endured higher stability due to the ultrathin (0.28 nm) stabilizing cap of PVP that precluded aggregates. The SNPs were evaluated for its antifungal activity against the cells of C. albicans. There was a marked reduction in the growth of the cells when incubated with SNP and the reduction in growth occurred in a concentration-dependent manner (Fig. 2a). In the present investigation, the MIC value of the SNP was observed at 70 ng/ml which is comparatively lower than the MIC values obtained with conventional antifungal agents such as amphotericin B (0.5 μg/ml), fluconazole, and ketoconazole (8 μg/ml; Fig. 2b). The antifungal activity of SNP observed in the present study agrees with an earlier report wherein comparatively higher antimicrobial activity has been observed when the SNPs were stabilized with stabilizing agents. The MIC value of SNP observed in the present study has been much lower when compared with the MIC values of the study reported previously [28]. The results have clearly indicated that SNP has a potential as an antifungal agent in treating fungal infectious diseases. It has been reported that the clinical applications of several antimicrobial agents have been restricted as they bring about cytolysis of human erythrocytes. Nano-Ag showed low hemolytic activity, while amphotericin B shows a slightly higher hemolytic activity that could be fatal in patients who are treated with this agent for fungal infection [29]. As is evident in Fig. 2, the growth of the C. albicans was inhibited effectively by PVP-capped SNP even at low concentration. The efficacy of the SNP was better when compared with the conventionally used antifungal drugs. The C. albicans cells treated with SNP have lost their structural integrity and have presumably induced the production of extracellular polymeric substances in which the cells were interconnected thus giving a biofilm-like appearance. Biofilms are formed in response to a wide array of environmental clues that include mechanical perturbations, nutritional constraints, and exposure to harmful metabolites (antibiotics). In the present investigation, the SNPs have strikingly inhibited the test strain at very low concentration (70 ng/ml), and SEM images showed the accumulation of SNP at the cellular interjections in the biofilm and fungal cell wall. The SNPs have been reported to detrimentally interact with cell membrane resulting in breakdown of the membrane permeability barrier in prokaryotic and eukaryotic systems [29, 30]. It has been reported about the oxidative damage of the cell membranes due to the release of
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Ag+ from SNPs and their detrimental action on bacterial membrane-bound proteins resulting in loss of cellular integrity and osmoticum culminating in acute toxicity to the cells [29]. The similar conclusive results are lacking in fungal systems; however, the current understanding of membrane depolarization, dynamics, and permeability corroborates with the mode of action reported in bacteria. Quite recently, transcriptomic analysis of Saccharomyces cerevisiae exposed to SNP confirmed the potential damage to the cell wall and transmembrane proteins and upregulation of cell-wall-strengthening genes in surviving cells [31]. Such changes result in the release of glucose and trehalose [29] which, according to our hypothesis, could eventually be used up in biofilm formation, as enhanced biofilm formation had been reported in glucose-rich conditions [3]; nevertheless, the membrane damages caused by the SNPs were severe to mend and hence result in cell death. Investigations on the mode of SNP entry into the fungal cells are scarce, and it persists as an intriguing query like the organization of the fungal cell wall itself, to be probed in detail. The highly heterogeneous C. albicans cell wall has a central core composed of branched β-1,3glucan cross-linked with chitin which, in turn, covalently bound to β-1,6-glucan and α and βmannans [32, 33] interwoven into a fine matrix with ultrafine porosity, permeable to small macromolecules of Mr 620 and 0.81-nm hydrodynamic radius (RH) [34, 35]. In the present study, we hypothesize that the endosomal trafficking systems involved in the uptake and release of macromolecules and nutrients may be involved in the uptake of SNP. Of the various types of endocytosis, fluid phase endocytosis has been documented to be involved in assimilation of Lucifer yellow, an impermeable dye by C. albicans and found to be accumulated in the vacuole. Assimilation of polystyrene beads (40 nm) and CdSe/ZnS quantum dots (20 nm) by sycamore plants’ protoplasts and cells in cell cultures further substantiates the involvement of fluid phase endocytosis in transport of macromolecules and synthetic nanomaterials across the cell wall [36]. Moreover, in the present study, the TEM image (Fig. 6b) showed a cluster of endocytic vesicles with SNP accumulated at the margins of the enlarged central vacuole. We extrapolate these ideas and resolve to the involvement of fluid phase endocytosis involvement in assimilation of quantum-sized SNP (2–3 nm). Our TEM results support the previous reports claiming significant changes in the membrane structures along with the dramatic enlargement and increase in the number of vacuoles on SNP treatment. Intense vacuolation has occurred in response to environmental cues and cellular stress in C. albicans; however, it has demonstrated the vitality of vacuoles in filamentous growth which may aid in survival and host tissue invasion through mutational studies [37]. In the present study, SNP treatment induced severe environmental stress that had resulted in vivid morphological changes like enlargement of existing vacuoles and formation of numerous vacuoles in the cytoplasm (Fig. 6b, c, e, f). The emergence of polarized growing points in SNP-treated cells, which is considered as one of the survival strategies under stress conditions, is evidence of the antifungal activity of SNP. A delay or arrest of cell cycle progression in C. albicans often results in a terminal phenotype, different from pseudohyphae and hyphae in the ability to divide, and hence eventually dies [38]. We observed very few highly elongated pseudohypha-like cells (Fig. 6e) and highly distorted hyphae with multiple vacuoles (Fig. 6f). In addition, the vast majority of the examined cells showed emergence of small evagination structures but failed to progress further. It could be inferred that the SNP treatments have impacted the cell cycle, and these observations also corroborate with the reported fungistatic, fungicidal, and cell cycle impedance activity of SNPs against C. albicans [29, 39]. In this study, we have evaluated the viability of the cells treated with SNP by measuring the mitochondrial dehydrogenase activity with XTT sodium salt. The
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amount of formazan product was directly correlated with the number of live cells. As the SNP concentration increased, there was a decrease in the reduced formazan product implying that the SNPs have prevented the enzymatic respiration. It has been reported that SNP disrupts the mitochondrial integrity and induces cytochrome c release and promotes apoptosis through phosphatidylserine exposure and the activation of metacaspases. The mechanism of SNP in mitochondria-dependent apoptosis in C. albicans has not been fully elucidated; however, it has been envisaged that the SNP induces programed cell death through ROS accumulation especially OH·[39]. The outstanding activity of PVP-coated SNP has also been proved against bacteria and viruses. It has been reported that the PVP-coated SNP ranging from 1 to 10 nm is inhibiting the HIV-1 virus from attaching to the host cells preferably via binding to gp120 glycoprotein knobs [40]. In a similar study, PVP-coated SNP was shown to reduce the respiratory syncytial virus infection by 44 % [41]. The mechanism of SNP uptake and accumulation has been reported for the bacteria such as Vibrio cholera, Pseudomonas aeruginosa, and Salmonella typhi. It has been observed that the attachment of the 10-nm-sized SNP to the bacterial cell membranes and inside the cells occurs [42]. The inactivation of lactate dehydrogenase (LDH) activity and increased protein leakage was observed with SNP-treated Staphylococcus aureus and Escherichia coli [43]. Irregular pit generation on E. coli cell surfaces was observed in cells treated with differentially shaped SNP [44]. As far as the cytotoxic effects of the PVP-stabilized SNP are concerned, there is no conclusive recorded evidence of cytotoxicity on human fibroblast and Paramecium caudatum at recommended MIC values against pathogenic microbes [45, 46]. Ruparelia et al. [47] also reported the strain-specific antimicrobial activity of 3-nm-sized SNP against E. coli and S. aureus. The inhibitory potential was observed 40–50 % greater than the copper nanoparticles. Reports on reduced efficacy of PVP-stabilized SNPs over nonstabilized or surfactantstabilized SNPs stated that PVP coating results in slower or reduced release of Ag+ [44], while other reports claimed that the bonding of Ag to PVP is accountable for the reduced antimicrobial activity over free Ag encapped within foamy carbon [40]. Interestingly, our results contradict the above claims and exhibited a superior antifungal activity because of the quantum size and high stability of the PVP-stabilized SNP. Assimilation of nanoparticles is inversely proportional to its size, quantum-sized particles finding greater entry than microdimensional particles, and directly correlates to the level of toxicity [42, 48]. Also, PVP encapsulation provides sustained release of Ag from SNPs that could ensure prolonged antifungal activity.
Conclusion The present study offers an unequivocal proof that PVP-stabilized quantum-sized SNPs work as a potent antifungal agent at very low concentration. The MIC values obtained were comparatively lower when compared with the MIC values of known antifungals. Furthermore, the viability of the yeast cell treated with PVP-stabilized SNP has been evaluated, and the results very well coincide with the MIC values. While SEM images confirmed the interaction of SNP onto the cell wall and biofilm, TEM analysis gives a picture of the loss of cellular integrity, vacuolation, and cell cycle arrest resulting in deformation when incubated with the PVP-stabilized SNP. It could turn out to be an effective and safe alternative to conventional oral and topical antifungal agents with due investigations for clinical applications.
Appl Biochem Biotechnol Acknowledgments The authors thank the National Centre for Nanoscience and Nanotechnology, MHRD and DST-INSPIRE for financial support in the form of a research grant and junior research fellowships. Thanks is also due to Mr S. Prathap Augustine, technician, NCNSNT, for assisting us with FE-SEM and TEM imaging.
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Infection and Immunity, 77(6), 2343–2355. 38. Berman, J. (2006). Morphogenesis and cell cycle progression in Candida albicans. Current Opinion in Microbiology, 9, 595–601. 39. Hwang, I. S., Lee, J., Hwang, J. H., Kim, K. J., & Lee, D. G. (2012). Silver nanoparticles induce apoptotic cell death in Candida albicans through the increase of hydroxyl radicals. FEBS Journal, 279(7), 1327–1338. 40. Elechiguerra, J. L., Burt, J. L., Morones, J. R., Bragado, A. C., Gao, X., Lara, H. H., et al. (2005). Interaction of silver nanoparticles with HIV-1. Journal of Nanobiotechnology, 3, 6. 41. Sun, L., Singh, A. K., Vig, K., Pillai, S. R., & Singh, S. R. (2008). Silver nanoparticles inhibit replication of respiratory syncytial virus. Journal of Biomedical Nanotechnology, 4, 149–158. 42. Morones, J. R., Elechiguerra, J. L., Camacho, A., Holt, K., Kouri, J. B., Ramirez, J. T., et al. (2005). The bactericidal effect of silver nanoparticles. Journal of Nanotechnology, 16(10), 2346–2353. 43. Kim, S. H., Lee, H. S., Ryu, D. S., Choi, S. J., & Lee, D. S. (2011). Antibacterial activity of silvernanoparticles against Staphylococcus aureus and Escherichia coli. Korean Journal of Microbiology and Biotechnology, 39(1), 77–85. 44. Pal, S., Tak, Y. K., & Song, J. M. (2007). Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli. Applied and Environmental Microbiology, 73(6), 1712–1720. 45. Panacek, A., Kolar, M., Vecerova, R., Prucek, R., Soukupova, J., Krystof, V., et al. (2009). Antifungal activity of silver nanoparticles against Candida spp. Biomaterials, 30(31), 6333–6340. 46. Kvitek, L., Vanickova, M., Panacek, A., Soukupova, J., Dittrich, M., Valentova, E., et al. (2009). Initial study on the toxicity of silver nanoparticles (NPs) against Paramecium caudatum. Journal of Physical Chemistry C, 113, 4296–4300. 47. Lee, H. J., Lee, S. G., Oh, E. J., Chung, H. Y., Han, S. I., Kim, E. J., et al. (2011). Antimicrobial polyethyleneimine silver nanoparticles in a stable colloidal dispersion. Colloids and Surfaces B: Biointerfaces, 88(1), 505–511. 48. Choi, O., & Hu, Z. (2008). Size dependent and reactive oxygen species related nanosilver toxicity to nitrifying bacteria. Environmental Science Technology, 42, 4583–4588.
Highly Potential Antifungal Activity of Quantum-Sized Silver Nanoparticles Against Candida albicans Malathi Selvaraj & Prabhu Pandurangan & Nishanthi Ramasami & Suresh Babu Rajendran & Sriman Narayanan Sangilimuthu & Palani Perumal Received: 1 August 2013 / Accepted: 4 February 2014 # Springer Science+Business Media New York 2014
Abstract The antifungal activity of polyvinylpyrrolidone (PVP)-stabilized quantum-sized silver nanoparticles (SNPs) against the growth of Candida albicans has been demonstrated in the present study. C. albicans is a known opportunistic human pathogen causing superficial and systemic infections. Research data carried out on C. albicans so far have shown unequivocally that it develops resistance against conventional antifungal drugs and that the infections it causes are difficult to cure with conventional antifungal agents. Hence, it is urgent to find newer materials for the treatment of infections caused by C. albicans that must be safe for the host. PVP-capped SNPs were synthesized, and its surface plasmon band was observed at 410 nm. The growth of C. albicans was markedly inhibited when the cells were incubated with SNP. The minimum inhibitory concentration (MIC) of SNP was determined as 70 ng/ml, and this value is relatively lower when compared with the conventionally used antifungal drugs such as amphotericin B (0.5 μg/ml), fluconazole (0.5 μg/ml), and ketoconazole (8 μg/ml). The viability of SNP-treated cells was checked by measuring the metabolic activity using XTT assay. Field emission scanning electron microscopic (FE-SEM) and transmission electron microscopic (TEM) analyses of the cells treated with SNP have lost the structural integrity to a greater extent. Keywords Quantum-sized silver nanoparticles . Candida albicans . Antifungal activity . Minimum inhibitory concentration . Polyvinylpyrrolidone
Introduction Fungal infections caused by yeasts and molds represent an escalating problem in health care as advances in modern medicine prolong the lives of severely ill patients. These organisms cause infection not only in those having HIV, cancer, organ transplant, and surgical operation and ICU patients but also newborn infants. Fungi, being eukaryotic in nature and more complex than bacteria, cause infections that are often difficult to diagnose and treat, resulting in M. Selvaraj : P. Pandurangan : N. Ramasami : S. B. Rajendran : S. N. Sangilimuthu : P. Perumal (*) University of Madras, Chennai, Tamil Nadu, India e-mail: [email protected] P. Perumal e-mail: [email protected]
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unacceptably high mortality rates [1]. In recent years, a rapid increase in microbes that are resistant to conventionally used antibiotics has been observed [2]. Candida albicans is a commensal present on the mucosal surfaces of human oral and vaginal cavities and frequently turns into an opportunistic pathogen causing superficial and systemic infections if the host immune system is compromised due to immunosuppressive and broad-spectrum antibiotic therapy, HIV infection, and cancer chemotherapy [3]. Currently, most of the available antifungal agents are based on polyenes (amphotericin B), triazoles (fluconazole, itraconazole, voriconazole, and posaconazole), and echinocandins (caspofungin, micafungin, and anidulafungin). However, the administration of these antifungals is often accompanied by various complications such as amphotericin B toxicity and adverse effects of some azoles including toxicity and drug interactions [4–7] and yeast resistance to antifungal therapy [8, 9]. A remarkable property observed with C. albicans is that it develops resistance, and the infections it causes are difficult to treat with conventional antifungal drugs. Therefore, it is crucial to find newer molecules for the treatment of Candida infections without adverse effect on the host cells. It has been known, since the ancient times, that silver and its compounds are effective antimicrobial agents [10, 11]. In particular, due to the recent advances in research on metal nanoparticles, nano-Ag has received special attention as a possible antimicrobial agent [12–14]. Therefore, the preparation of uniformly sized silver nanoparticles with specific requirements in terms of size, shape, physicochemical properties is of great interest in the formulation of newer pharmaceutical products [15, 16]. Though the biocidal effect and the mode of action of silver ion are known, the antifungal effects and the mode of action of SNP against fungi have remained mostly unknown. Therefore, an attempt has been made in the present investigation to synthesize polyvinylpyrrolidone (PVP)-stabilized quantum-sized silver nanoparticles (SNPs) and to evaluate their antifungal activity against the growth of C. albicans. The growth of the organism was markedly inhibited when the cells were incubated with SNP. The minimum inhibitory concentration (MIC) of SNP has been determined, and the cell viability was checked using the XTT assay by measuring the mitochondrial dehydrogenase activity of the live cells. FESEM and transmission electron microscopic (TEM) analyses have clearly indicated marked changes in the integrity of the cells treated with SNP. For the first time, elemental analysis of the SNP-treated and nontreated cells has been carried out in this study.
Materials and Methods Chemicals Antifungal drugs such as amphotericin B and ketoconazole were purchased from HiMedia (India). Silver nitrate was purchased from Sigma Chemical Co. (USA). An injectable form of fluconazole was purchased from CIPLA (India). Culture media such as yeast nitrogen base (YNB) and yeast extract peptone dextrose agar (YEPDA) were purchased from Difco Laboratories (Detroit, MI, USA) and HiMedia (India), respectively. The other solvents used in the study were of analytical grade procured locally. Organisms and Growth Conditions C. albicans used in this study was obtained from the fungal culture collection facility at the Centre for Advanced Studies in Botany, University of Madras. The organism was cultured on YNB medium and stored on YEPDA slants at 4 °C until further use.
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Synthesis and Characterization of Quantum-Sized SNP The PVP-stabilized quantum-sized SNPs were synthesized as follows. Fifty milligrams of silver nitrate (AgNO3) was dissolved in 11.25 ml of ethylene glycol to which 1.5 g of PVP was added. The solution was thoroughly mixed using a magnetic stirrer. The agitation of the solution was continued for 20 min while increasing the temperature by 5 °C after every 5 min. The formation of a colloidal, wine-brown-colored solution confirms the formation of SNP. The SNP prepared as above was diluted to 10−4 and used for further investigations. The SNP was characterized using UV–Vis spectral analysis in a spectrophotometer (Lamda-650, Perkin Elmer, USA) and high-resolution transmission electron microscope (HR-TEM, FEI, Tecnai G2 model T 30 S-twin, 300 kV, Japan). Determination of MIC of SNP The determination of MIC of the PVP-stabilized SNP against C. albicans was performed as per the recommendations of the Clinical Laboratory Standard Institute (CLSI) [17]. The yeast cells of C. albicans were grown planktonically under aerobic conditions at 37 °C for 24 h in YNB broth. The cell count was made with hemocytometer, and a standardized cell suspension (1 × 105 cells/ml) was prepared. One hundred microliters of the cell suspension was dispensed in triplicate microtiter wells to which 100-μl suspension containing 1.380 to 0.092 μg of nanoparticles in YNB medium was added and mixed well. The following conventionally used antifungal agents such as amphotericin B [18], fluconazole, and ketoconazole were used in order to compare the antifungal potential of SNP. Amphotericin B and ketoconazole were dissolved in dimethyl sulfoxide (DMSO) and diluted with sterile YNB medium to obtain drug concentrations ranging from 0.0313 to 1,024 μg/ml. The cells with either nanoparticles or conventionally used antifungals were incubated at room temperature for 24 h, and the growth inhibition was measured spectrophotometrically at 600 nm (Powerserve XS Biotech, USA). Wells with cells without SNP were used as the control. The MIC was calculated as the lowest amount of SNPs that inhibited 80 % growth of C. albicans cells under the experimental conditions. Cell Viability Assay The metabolic activity of SNP-treated cells were determined by the XTT [2, 3-bis(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] assay [19]. The XTT tetrazolium salt was dissolved in phosphate-buffered saline (10 mM, pH 7.4) at a concentration of 0.5 g/l and filter sterilized through a 0.2 μm filter, and the aliquots were stored at −80 °C. Just before use, an aliquot was thawed, and 10 mM menadione prepared in acetone was added to the XTT solution to a final concentration of 1 μM. One hundred microliters of culture (1×105 cells/ml) was dispensed into 96-well plates. Different concentrations of SNP (1.380 to 0.092 μg) were added to the wells and incubated for 24 h at 37 °C. After 24 h of incubation, 100 μl of the XTT/menadione solution was added to each well and mixed thoroughly. The plates were incubated in dark at 37 °C for 2–3 h. The reduced formazan-colored product was measured at 490 nm using a microtiter plate reader (Powerserve XS Biotech, USA). The concentration of SNP showing 80 % reduction in cell viability was then calculated.
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Field Emission Scanning and Transmission Electron Microscopic Studies One milliliter of 1×105 cells/ml suspension was incubated with SNPs at their MIC values for 24 h and observed in a FE-SEM (H-7650, Hitachi, Japan) coupled with Energy Dispersive X-ray Analysis (EDAX) spectrum analysis, and the morphological changes were observed. The cells grown on YNB medium without SNP served as the control. For TEM, the cells were fixed with glutaraldehyde (1 %) solution, postfixed with osmium tetroxide for 2 h, and washed with buffer twice. The cells were dehydrated with increasing concentration of acetone and embedded in Epon–Araldite resin. Ultrathin sections were made, stained with uranyl acetate and lead citrate, and observed under TEM (SU 6600 Hitachi, Japan).
Results Synthesis and Physical Characterization of SNP The solution containing silver nitrate (AgNO3), ethylene glycol, and PVP turned into a colloidal, wine-brown-colored solution which indicated the formation of SNP. The SNP prepared as above was diluted to 10−4 and used for further investigations. The synthesized SNP showed an absorption peak at 410 nm (Fig. 1a), and this characteristic peak confirmed the formation of colloidal SNP. The TEM analysis revealed the formation of spherical SNP measuring about 2 nm (Fig. 1b). The HR-TEM analysis also revealed the formation of a thin outer layer measuring 0.28 nm (Fig. 1b). This thin layer was made by PVP encompassing SNP which appeared like core-shell morphology. Antifungal Activity of SNP The PVP-stabilized SNP showed antifungal activity against the tested organism. There was a marked reduction in the growth of the cells when incubated with SNP in a concentrationdependent manner (Fig. 2a). The MIC of the SNP was determined at 70 ng/ml. The MIC
Fig. 1 a UV–Vis spectra and b HR-TEM image of SNP
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Fig. 2 MIC values of a SNP and b amphotericin B, fluconazole, and ketoconazole against C. albicans. Each value represents the mean of three independent experiments
values for the known antifungals such as amphotericin B and fluconazole were observed at 0.5 μg/ml and for ketoconazole at 8 μg/ml (Fig. 2b). Cell Viability Assay To verify if the synthesized SNPs have the ability to kill the Candida cells, the cell viability assay was performed with XTT assay as described in the “Materials and Methods” section. The metabolic reduction of XTT sodium salt by mitochondrial dehydrogenase forms a colored water-soluble formazan product, and this was measured spectrophotometrically at 490 nm. The cells treated with PVP-stabilized SNP showed decreased metabolic activity in a dosedependent manner (Fig. 3) and confirmed that the SNPs have the ability to kill or inactivate the organism. Further, the MIC value of SNP as determined by the XTT assay coincided with the MIC value obtained through spectrophotometric measurement.
Fig. 3 Effect of PVP-stabilized silver nanoparticles on the cell viability. Each value represents the mean of three independent experiments
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Field Emission Scanning Electron Microscopic Studies The cells treated with SNP exhibited noticeable structural changes (Fig. 4c, d) when compared with the control cells (Fig. 4a, b). The SNP-treated cells that appeared deformed exhibited surface shrinkages when compared with the control. The SNP-treated cells aggregated into clumps, remained attached to the adjacent cells (Fig. 4c, d), forming EPS (extracellular polysaccharide)-rich biofilms. The SNP adhered onto the biofilm and cellular surfaces, besides being concentrated at the cellular interjections in the biofilm. The EDAX spectrum of SNPtreated cells showed the presence of Ag peak (Fig. 5b) along with other inorganic elements present in the medium, but in the case of untreated cells, the Ag peak was absent (Fig. 5a). The elemental analysis clearly indicated adherence of the SNP on the cell surface leading to cell damage and arrest of growth. Transmission Electron Microscopic Studies The cells treated with SNP were highly deformed (Fig. 6b, c, e, f), and the cells had shrunken to a greater extent (Fig. 6b, c, e, f). Alteration in the cell wall and cell membrane was also observed. Pronounced increase in the number and enlargement of vacuole was evident with reduction in the cytoplasm, and the cells started to disorient from its original shape. A significant portion of cells showed accumulation of granules in the cytoplasm and vacuoles. C. albicans cells treated with SNP also showed signs of pseudohypha and hypha morphogenesis.
Fig. 4 a, b Structural changes due to interaction of SNP with C. albicans cells: control. c, d SNP-treated Candida cells
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Fig. 5 EDAX analysis of C. albicans cells treated a in the absence and b in the presence of SNP
Fig. 6 TEM analysis of C. albicans cells treated a, b in the absence and b, c, e, f in the presence of SNP
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Discussion Nanosized inorganic particles have increasingly become an important material in the development of novel nanodevices that are being used in numerous physical, biological, biomedical, and pharmaceutical applications [20, 21]. Among them, nano-Ag has proved to be highly toxic to a variety of microorganisms and potentially inhibited the growth of both bacteria and fungi [22, 23] excepting few strains that showed resistance [24]. An attempt has been made in the present investigation to synthesize quantum-sized SNPs which are stabilized with PVP and to evaluate their antifungal activity against C. albicans. The PVP had reduced the silver nitrate leading to the formation of colloidal SNP, and the formation was manifested as a change in color from colorless to wine brown. As is evident from Fig.1a, the SNP exhibited characteristic absorption peak at 410 nm and had confirmed the formation of SNPs. The reducing and stabilizing capabilities of PVP in the synthesis of SNP have been well documented. The coordination of Ag+ ion and O atom of the carbonyl group of PVP facilitates the reduction of Ag+ ion with the lone pair of electrons of N atom from pyrrolidone ring that resulted in the formation of PVP-stabilized SNP [25–27]. The nanoparticles appeared spherical, and as it is highly evident from Fig. 1b, there was a conspicuous presence of an ultrathin cap formed by PVP. The monodispersed SNP had a mean primary grain size of 2–3 nm and endured higher stability due to the ultrathin (0.28 nm) stabilizing cap of PVP that precluded aggregates. The SNPs were evaluated for its antifungal activity against the cells of C. albicans. There was a marked reduction in the growth of the cells when incubated with SNP and the reduction in growth occurred in a concentration-dependent manner (Fig. 2a). In the present investigation, the MIC value of the SNP was observed at 70 ng/ml which is comparatively lower than the MIC values obtained with conventional antifungal agents such as amphotericin B (0.5 μg/ml), fluconazole, and ketoconazole (8 μg/ml; Fig. 2b). The antifungal activity of SNP observed in the present study agrees with an earlier report wherein comparatively higher antimicrobial activity has been observed when the SNPs were stabilized with stabilizing agents. The MIC value of SNP observed in the present study has been much lower when compared with the MIC values of the study reported previously [28]. The results have clearly indicated that SNP has a potential as an antifungal agent in treating fungal infectious diseases. It has been reported that the clinical applications of several antimicrobial agents have been restricted as they bring about cytolysis of human erythrocytes. Nano-Ag showed low hemolytic activity, while amphotericin B shows a slightly higher hemolytic activity that could be fatal in patients who are treated with this agent for fungal infection [29]. As is evident in Fig. 2, the growth of the C. albicans was inhibited effectively by PVP-capped SNP even at low concentration. The efficacy of the SNP was better when compared with the conventionally used antifungal drugs. The C. albicans cells treated with SNP have lost their structural integrity and have presumably induced the production of extracellular polymeric substances in which the cells were interconnected thus giving a biofilm-like appearance. Biofilms are formed in response to a wide array of environmental clues that include mechanical perturbations, nutritional constraints, and exposure to harmful metabolites (antibiotics). In the present investigation, the SNPs have strikingly inhibited the test strain at very low concentration (70 ng/ml), and SEM images showed the accumulation of SNP at the cellular interjections in the biofilm and fungal cell wall. The SNPs have been reported to detrimentally interact with cell membrane resulting in breakdown of the membrane permeability barrier in prokaryotic and eukaryotic systems [29, 30]. It has been reported about the oxidative damage of the cell membranes due to the release of
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Ag+ from SNPs and their detrimental action on bacterial membrane-bound proteins resulting in loss of cellular integrity and osmoticum culminating in acute toxicity to the cells [29]. The similar conclusive results are lacking in fungal systems; however, the current understanding of membrane depolarization, dynamics, and permeability corroborates with the mode of action reported in bacteria. Quite recently, transcriptomic analysis of Saccharomyces cerevisiae exposed to SNP confirmed the potential damage to the cell wall and transmembrane proteins and upregulation of cell-wall-strengthening genes in surviving cells [31]. Such changes result in the release of glucose and trehalose [29] which, according to our hypothesis, could eventually be used up in biofilm formation, as enhanced biofilm formation had been reported in glucose-rich conditions [3]; nevertheless, the membrane damages caused by the SNPs were severe to mend and hence result in cell death. Investigations on the mode of SNP entry into the fungal cells are scarce, and it persists as an intriguing query like the organization of the fungal cell wall itself, to be probed in detail. The highly heterogeneous C. albicans cell wall has a central core composed of branched β-1,3glucan cross-linked with chitin which, in turn, covalently bound to β-1,6-glucan and α and βmannans [32, 33] interwoven into a fine matrix with ultrafine porosity, permeable to small macromolecules of Mr 620 and 0.81-nm hydrodynamic radius (RH) [34, 35]. In the present study, we hypothesize that the endosomal trafficking systems involved in the uptake and release of macromolecules and nutrients may be involved in the uptake of SNP. Of the various types of endocytosis, fluid phase endocytosis has been documented to be involved in assimilation of Lucifer yellow, an impermeable dye by C. albicans and found to be accumulated in the vacuole. Assimilation of polystyrene beads (40 nm) and CdSe/ZnS quantum dots (20 nm) by sycamore plants’ protoplasts and cells in cell cultures further substantiates the involvement of fluid phase endocytosis in transport of macromolecules and synthetic nanomaterials across the cell wall [36]. Moreover, in the present study, the TEM image (Fig. 6b) showed a cluster of endocytic vesicles with SNP accumulated at the margins of the enlarged central vacuole. We extrapolate these ideas and resolve to the involvement of fluid phase endocytosis involvement in assimilation of quantum-sized SNP (2–3 nm). Our TEM results support the previous reports claiming significant changes in the membrane structures along with the dramatic enlargement and increase in the number of vacuoles on SNP treatment. Intense vacuolation has occurred in response to environmental cues and cellular stress in C. albicans; however, it has demonstrated the vitality of vacuoles in filamentous growth which may aid in survival and host tissue invasion through mutational studies [37]. In the present study, SNP treatment induced severe environmental stress that had resulted in vivid morphological changes like enlargement of existing vacuoles and formation of numerous vacuoles in the cytoplasm (Fig. 6b, c, e, f). The emergence of polarized growing points in SNP-treated cells, which is considered as one of the survival strategies under stress conditions, is evidence of the antifungal activity of SNP. A delay or arrest of cell cycle progression in C. albicans often results in a terminal phenotype, different from pseudohyphae and hyphae in the ability to divide, and hence eventually dies [38]. We observed very few highly elongated pseudohypha-like cells (Fig. 6e) and highly distorted hyphae with multiple vacuoles (Fig. 6f). In addition, the vast majority of the examined cells showed emergence of small evagination structures but failed to progress further. It could be inferred that the SNP treatments have impacted the cell cycle, and these observations also corroborate with the reported fungistatic, fungicidal, and cell cycle impedance activity of SNPs against C. albicans [29, 39]. In this study, we have evaluated the viability of the cells treated with SNP by measuring the mitochondrial dehydrogenase activity with XTT sodium salt. The
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amount of formazan product was directly correlated with the number of live cells. As the SNP concentration increased, there was a decrease in the reduced formazan product implying that the SNPs have prevented the enzymatic respiration. It has been reported that SNP disrupts the mitochondrial integrity and induces cytochrome c release and promotes apoptosis through phosphatidylserine exposure and the activation of metacaspases. The mechanism of SNP in mitochondria-dependent apoptosis in C. albicans has not been fully elucidated; however, it has been envisaged that the SNP induces programed cell death through ROS accumulation especially OH·[39]. The outstanding activity of PVP-coated SNP has also been proved against bacteria and viruses. It has been reported that the PVP-coated SNP ranging from 1 to 10 nm is inhibiting the HIV-1 virus from attaching to the host cells preferably via binding to gp120 glycoprotein knobs [40]. In a similar study, PVP-coated SNP was shown to reduce the respiratory syncytial virus infection by 44 % [41]. The mechanism of SNP uptake and accumulation has been reported for the bacteria such as Vibrio cholera, Pseudomonas aeruginosa, and Salmonella typhi. It has been observed that the attachment of the 10-nm-sized SNP to the bacterial cell membranes and inside the cells occurs [42]. The inactivation of lactate dehydrogenase (LDH) activity and increased protein leakage was observed with SNP-treated Staphylococcus aureus and Escherichia coli [43]. Irregular pit generation on E. coli cell surfaces was observed in cells treated with differentially shaped SNP [44]. As far as the cytotoxic effects of the PVP-stabilized SNP are concerned, there is no conclusive recorded evidence of cytotoxicity on human fibroblast and Paramecium caudatum at recommended MIC values against pathogenic microbes [45, 46]. Ruparelia et al. [47] also reported the strain-specific antimicrobial activity of 3-nm-sized SNP against E. coli and S. aureus. The inhibitory potential was observed 40–50 % greater than the copper nanoparticles. Reports on reduced efficacy of PVP-stabilized SNPs over nonstabilized or surfactantstabilized SNPs stated that PVP coating results in slower or reduced release of Ag+ [44], while other reports claimed that the bonding of Ag to PVP is accountable for the reduced antimicrobial activity over free Ag encapped within foamy carbon [40]. Interestingly, our results contradict the above claims and exhibited a superior antifungal activity because of the quantum size and high stability of the PVP-stabilized SNP. Assimilation of nanoparticles is inversely proportional to its size, quantum-sized particles finding greater entry than microdimensional particles, and directly correlates to the level of toxicity [42, 48]. Also, PVP encapsulation provides sustained release of Ag from SNPs that could ensure prolonged antifungal activity.
Conclusion The present study offers an unequivocal proof that PVP-stabilized quantum-sized SNPs work as a potent antifungal agent at very low concentration. The MIC values obtained were comparatively lower when compared with the MIC values of known antifungals. Furthermore, the viability of the yeast cell treated with PVP-stabilized SNP has been evaluated, and the results very well coincide with the MIC values. While SEM images confirmed the interaction of SNP onto the cell wall and biofilm, TEM analysis gives a picture of the loss of cellular integrity, vacuolation, and cell cycle arrest resulting in deformation when incubated with the PVP-stabilized SNP. It could turn out to be an effective and safe alternative to conventional oral and topical antifungal agents with due investigations for clinical applications.
Appl Biochem Biotechnol Acknowledgments The authors thank the National Centre for Nanoscience and Nanotechnology, MHRD and DST-INSPIRE for financial support in the form of a research grant and junior research fellowships. Thanks is also due to Mr S. Prathap Augustine, technician, NCNSNT, for assisting us with FE-SEM and TEM imaging.
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