Highly Potential Antifungal Activity of Quantum-Sized Silver Nanoparticles Against Candida albicans

Highly Potential Antifungal Activity of Quantum-SizedSilver 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-sizedsilver nanoparticles (SNPs) against the growth of Candida albicans has been demonstratedin the present study. C. albicans is a known opportunistic human pathogen causing superficialand systemic infections. Research data carried out on C. albicans so far have shown unequiv-ocally that it develops resistance against conventional antifungal drugs and that the infections itcauses are difficult to cure with conventional antifungal agents. Hence, it is urgent to findnewer materials for the treatment of infections caused by C. albicans that must be safe for thehost. PVP-capped SNPs were synthesized, and its surface plasmon band was observed at410 nm. The growth of C. albicanswas markedly inhibited when the cells were incubated withSNP. The minimum inhibitory concentration (MIC) of SNP was determined as 70 ng/ml, andthis value is relatively lower when compared with the conventionally used antifungal drugssuch as amphotericin B (0.5 μg/ml), fluconazole (0.5 μg/ml), and ketoconazole (8 μg/ml). Theviability of SNP-treated cells was checked by measuring the metabolic activity using XTTassay. Field emission scanning electron microscopic (FE-SEM) and transmission electronmicroscopic (TEM) analyses of the cells treated with SNP have lost the structural integrityto 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 asadvances in modern medicine prolong the lives of severely ill patients. These organisms causeinfection not only in those having HIV, cancer, organ transplant, and surgical operation andICU patients but also newborn infants. Fungi, being eukaryotic in nature and more complexthan bacteria, cause infections that are often difficult to diagnose and treat, resulting in

Appl Biochem BiotechnolDOI 10.1007/s12010-014-0782-9

M. Selvaraj : P. Pandurangan :N. Ramasami : S. B. Rajendran : S. N. Sangilimuthu : P. Perumal (*)University of Madras, Chennai, Tamil Nadu, Indiae-mail: [email protected]

P. Perumale-mail: [email protected]

unacceptably high mortality rates [1]. In recent years, a rapid increase in microbes that areresistant to conventionally used antibiotics has been observed [2]. Candida albicans is acommensal present on the mucosal surfaces of human oral and vaginal cavities and frequentlyturns into an opportunistic pathogen causing superficial and systemic infections if the hostimmune system is compromised due to immunosuppressive and broad-spectrum antibiotictherapy, HIV infection, and cancer chemotherapy [3]. Currently, most of the available anti-fungal agents are based on polyenes (amphotericin B), triazoles (fluconazole, itraconazole,voriconazole, and posaconazole), and echinocandins (caspofungin, micafungin, andanidulafungin). However, the administration of these antifungals is often accompanied byvarious complications such as amphotericin B toxicity and adverse effects of some azolesincluding 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 theinfections it causes are difficult to treat with conventional antifungal drugs. Therefore, it iscrucial to find newer molecules for the treatment of Candida infections without adverse effecton the host cells. It has been known, since the ancient times, that silver and its compounds areeffective antimicrobial agents [10, 11]. In particular, due to the recent advances in research onmetal nanoparticles, nano-Ag has received special attention as a possible antimicrobial agent[12–14]. Therefore, the preparation of uniformly sized silver nanoparticles with specificrequirements in terms of size, shape, physicochemical properties is of great interest in theformulation of newer pharmaceutical products [15, 16]. Though the biocidal effect and themode of action of silver ion are known, the antifungal effects and the mode of action of SNPagainst fungi have remained mostly unknown.

Therefore, an attempt has been made in the present investigation to synthesize polyvinyl-pyrrolidone (PVP)-stabilized quantum-sized silver nanoparticles (SNPs) and to evaluate theirantifungal activity against the growth of C. albicans. The growth of the organism wasmarkedly inhibited when the cells were incubated with SNP. The minimum inhibitoryconcentration (MIC) of SNP has been determined, and the cell viability was checked usingthe XTT assay by measuring the mitochondrial dehydrogenase activity of the live cells. FE-SEM and transmission electron microscopic (TEM) analyses have clearly indicated markedchanges in the integrity of the cells treated with SNP. For the first time, elemental analysis ofthe 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 offluconazole was purchased from CIPLA (India). Culture media such as yeast nitrogen base(YNB) and yeast extract peptone dextrose agar (YEPDA) were purchased from Difco Labo-ratories (Detroit, MI, USA) and HiMedia (India), respectively. The other solvents used in thestudy 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 theCentre for Advanced Studies in Botany, University of Madras. The organism was cultured onYNB medium and stored on YEPDA slants at 4 °C until further use.

Appl Biochem Biotechnol

Synthesis and Characterization of Quantum-Sized SNP

The PVP-stabilized quantum-sized SNPs were synthesized as follows. Fifty milligramsof silver nitrate (AgNO3) was dissolved in 11.25 ml of ethylene glycol to which 1.5 gof PVP was added. The solution was thoroughly mixed using a magnetic stirrer. Theagitation of the solution was continued for 20 min while increasing the temperatureby 5 °C after every 5 min. The formation of a colloidal, wine-brown-colored solutionconfirms the formation of SNP. The SNP prepared as above was diluted to 10−4 andused for further investigations. The SNP was characterized using UV–Vis spectralanalysis in a spectrophotometer (Lamda-650, Perkin Elmer, USA) and high-resolutiontransmission 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 wasperformed as per the recommendations of the Clinical Laboratory Standard Institute(CLSI) [17]. The yeast cells of C. albicans were grown planktonically under aerobicconditions at 37 °C for 24 h in YNB broth. The cell count was made with hemocy-tometer, and a standardized cell suspension (1×105 cells/ml) was prepared. Onehundred microliters of the cell suspension was dispensed in triplicate microtiter wellsto which 100-μl suspension containing 1.380 to 0.092 μg of nanoparticles in YNBmedium was added and mixed well. The following conventionally used antifungalagents such as amphotericin B [18], fluconazole, and ketoconazole were used in orderto compare the antifungal potential of SNP. Amphotericin B and ketoconazole weredissolved in dimethyl sulfoxide (DMSO) and diluted with sterile YNB medium toobtain drug concentrations ranging from 0.0313 to 1,024 μg/ml. The cells with eithernanoparticles or conventionally used antifungals were incubated at room temperaturefor 24 h, and the growth inhibition was measured spectrophotometrically at 600 nm(Powerserve XS Biotech, USA). Wells with cells without SNP were used as thecontrol. 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(2-methoxy-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, and10 mM menadione prepared in acetone was added to the XTT solution to a finalconcentration of 1 μM. One hundred microliters of culture (1×105 cells/ml) wasdispensed 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-coloredproduct was measured at 490 nm using a microtiter plate reader (Powerserve XSBiotech, USA). The concentration of SNP showing 80 % reduction in cell viabilitywas then calculated.


Field Emission Scanning and Transmission Electron Microscopic Studies

One milliliter of 1×105 cells/ml suspension was incubated with SNPs at their MIC values for24 h and observed in a FE-SEM (H-7650, Hitachi, Japan) coupled with Energy DispersiveX-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 werefixed with glutaraldehyde (1 %) solution, postfixed with osmium tetroxide for 2 h, and washedwith buffer twice. The cells were dehydrated with increasing concentration of acetone andembedded in Epon–Araldite resin. Ultrathin sections were made, stained with uranyl acetateand 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 acolloidal, wine-brown-colored solution which indicated the formation of SNP. The SNPprepared as above was diluted to 10−4 and used for further investigations. The synthesizedSNP showed an absorption peak at 410 nm (Fig. 1a), and this characteristic peak confirmed theformation of colloidal SNP. The TEM analysis revealed the formation of spherical SNPmeasuring about 2 nm (Fig. 1b). The HR-TEM analysis also revealed the formation of a thinouter layer measuring 0.28 nm (Fig. 1b). This thin layer was made by PVP encompassing SNPwhich appeared like core-shell morphology.

Antifungal Activity of SNP

The PVP-stabilized SNP showed antifungal activity against the tested organism. There was amarked reduction in the growth of the cells when incubated with SNP in a concentration-dependent 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


values for the known antifungals such as amphotericin B and fluconazole were observed at0.5 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 viabilityassay was performed with XTTassay as described in the “Materials andMethods” section. Themetabolic reduction of XTT sodium salt by mitochondrial dehydrogenase forms a coloredwater-soluble formazan product, and this was measured spectrophotometrically at 490 nm. Thecells treated with PVP-stabilized SNP showed decreased metabolic activity in a dose-dependent manner (Fig. 3) and confirmed that the SNPs have the ability to kill or inactivatethe organism. Further, the MIC value of SNP as determined by the XTT assay coincided withthe MIC value obtained through spectrophotometric measurement.

Fig. 2 MIC values of a SNP and b amphotericin B, fluconazole, and ketoconazole against C. albicans. Eachvalue represents the mean of three independent experiments

Fig. 3 Effect of PVP-stabilized silver nanoparticles on the cell viability. Each value represents the mean of threeindependent experiments


Field Emission Scanning Electron Microscopic Studies

The cells treated with SNP exhibited noticeable structural changes (Fig. 4c, d) when comparedwith the control cells (Fig. 4a, b). The SNP-treated cells that appeared deformed exhibitedsurface shrinkages when compared with the control. The SNP-treated cells aggregated intoclumps, remained attached to the adjacent cells (Fig. 4c, d), forming EPS (extracellularpolysaccharide)-rich biofilms. The SNP adhered onto the biofilm and cellular surfaces, besidesbeing concentrated at the cellular interjections in the biofilm. The EDAX spectrum of SNP-treated cells showed the presence of Ag peak (Fig. 5b) along with other inorganic elementspresent in the medium, but in the case of untreated cells, the Ag peak was absent (Fig. 5a). Theelemental analysis clearly indicated adherence of the SNP on the cell surface leading to celldamage 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 agreater 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 thecytoplasm, and the cells started to disorient from its original shape. A significant portion of cellsshowed accumulation of granules in the cytoplasm and vacuoles. C. albicans cells treated withSNP 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-treatedCandida cells


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


Discussion

Nanosized inorganic particles have increasingly become an important material in the devel-opment 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 toxicto 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 thepresent investigation to synthesize quantum-sized SNPs which are stabilized with PVPand to evaluate their antifungal activity against C. albicans. The PVP had reduced thesilver nitrate leading to the formation of colloidal SNP, and the formation wasmanifested as a change in color from colorless to wine brown. As is evident fromFig.1a, the SNP exhibited characteristic absorption peak at 410 nm and had confirmedthe formation of SNPs. The reducing and stabilizing capabilities of PVP in thesynthesis of SNP have been well documented. The coordination of Ag+ ion and Oatom of the carbonyl group of PVP facilitates the reduction of Ag+ ion with the lonepair of electrons of N atom from pyrrolidone ring that resulted in the formation ofPVP-stabilized SNP [25–27]. The nanoparticles appeared spherical, and as it is highlyevident from Fig. 1b, there was a conspicuous presence of an ultrathin cap formed byPVP. The monodispersed SNP had a mean primary grain size of 2–3 nm and endured higherstability 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. Therewas a marked reduction in the growth of the cells when incubated with SNP and the reductionin 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 theMIC 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 inthe present study agrees with an earlier report wherein comparatively higher antimicrobialactivity has been observed when the SNPs were stabilized with stabilizing agents. The MICvalue of SNP observed in the present study has been much lower when compared with theMIC values of the study reported previously [28]. The results have clearly indicated thatSNP has a potential as an antifungal agent in treating fungal infectious diseases. It hasbeen reported that the clinical applications of several antimicrobial agents have beenrestricted as they bring about cytolysis of human erythrocytes. Nano-Ag showed lowhemolytic activity, while amphotericin B shows a slightly higher hemolytic activitythat 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 byPVP-capped SNP even at low concentration. The efficacy of the SNP was better whencompared with the conventionally used antifungal drugs.

The C. albicans cells treated with SNP have lost their structural integrity and havepresumably induced the production of extracellular polymeric substances in which thecells were interconnected thus giving a biofilm-like appearance. Biofilms are formedin response to a wide array of environmental clues that include mechanical perturba-tions, nutritional constraints, and exposure to harmful metabolites (antibiotics). In thepresent investigation, the SNPs have strikingly inhibited the test strain at very lowconcentration (70 ng/ml), and SEM images showed the accumulation of SNP at thecellular interjections in the biofilm and fungal cell wall. The SNPs have been reportedto detrimentally interact with cell membrane resulting in breakdown of the membranepermeability barrier in prokaryotic and eukaryotic systems [29, 30]. It has beenreported about the oxidative damage of the cell membranes due to the release of


Ag+ from SNPs and their detrimental action on bacterial membrane-bound proteinsresulting in loss of cellular integrity and osmoticum culminating in acute toxicity tothe cells [29]. The similar conclusive results are lacking in fungal systems; however,the current understanding of membrane depolarization, dynamics, and permeabilitycorroborates with the mode of action reported in bacteria. Quite recently,transcriptomic analysis of Saccharomyces cerevisiae exposed to SNP confirmed thepotential damage to the cell wall and transmembrane proteins and upregulation ofcell-wall-strengthening genes in surviving cells [31]. Such changes result in therelease of glucose and trehalose [29] which, according to our hypothesis, couldeventually be used up in biofilm formation, as enhanced biofilm formation had beenreported in glucose-rich conditions [3]; nevertheless, the membrane damages causedby 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 anintriguing query like the organization of the fungal cell wall itself, to be probed in detail. Thehighly heterogeneous C. albicans cell wall has a central core composed of branched β-1,3-glucan cross-linked with chitin which, in turn, covalently bound to β-1,6-glucan and α and β-mannans [32, 33] interwoven into a fine matrix with ultrafine porosity, permeable to smallmacromolecules of Mr 620 and 0.81-nm hydrodynamic radius (RH) [34, 35]. In the presentstudy, we hypothesize that the endosomal trafficking systems involved in the uptake andrelease of macromolecules and nutrients may be involved in the uptake of SNP. Of the varioustypes of endocytosis, fluid phase endocytosis has been documented to be involved in assim-ilation of Lucifer yellow, an impermeable dye by C. albicans and found to be accumulated inthe 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 involvementof fluid phase endocytosis in transport of macromolecules and synthetic nanomaterials acrossthe cell wall [36]. Moreover, in the present study, the TEM image (Fig. 6b) showed a cluster ofendocytic vesicles with SNP accumulated at the margins of the enlarged central vacuole. Weextrapolate these ideas and resolve to the involvement of fluid phase endocytosis involvementin assimilation of quantum-sized SNP (2–3 nm).

Our TEM results support the previous reports claiming significant changes in the mem-brane structures along with the dramatic enlargement and increase in the number of vacuoleson SNP treatment. Intense vacuolation has occurred in response to environmental cues andcellular stress in C. albicans; however, it has demonstrated the vitality of vacuoles infilamentous growth which may aid in survival and host tissue invasion through mutationalstudies [37]. In the present study, SNP treatment induced severe environmental stress that hadresulted in vivid morphological changes like enlargement of existing vacuoles and formationof numerous vacuoles in the cytoplasm (Fig. 6b, c, e, f). The emergence of polarized growingpoints in SNP-treated cells, which is considered as one of the survival strategies under stressconditions, is evidence of the antifungal activity of SNP. A delay or arrest of cell cycleprogression in C. albicans often results in a terminal phenotype, different frompseudohyphae and hyphae in the ability to divide, and hence eventually dies [38].We observed very few highly elongated pseudohypha-like cells (Fig. 6e) and highlydistorted hyphae with multiple vacuoles (Fig. 6f). In addition, the vast majority of theexamined cells showed emergence of small evagination structures but failed toprogress further. It could be inferred that the SNP treatments have impacted the cellcycle, 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 bymeasuring the mitochondrial dehydrogenase activity with XTT sodium salt. The


amount of formazan product was directly correlated with the number of live cells. Asthe SNP concentration increased, there was a decrease in the reduced formazanproduct implying that the SNPs have prevented the enzymatic respiration. It has beenreported that SNP disrupts the mitochondrial integrity and induces cytochrome crelease and promotes apoptosis through phosphatidylserine exposure and the activationof metacaspases. The mechanism of SNP in mitochondria-dependent apoptosis inC. albicans has not been fully elucidated; however, it has been envisaged that theSNP induces programed cell death through ROS accumulation especially OH·[39].

The outstanding activity of PVP-coated SNP has also been proved against bacteriaand viruses. It has been reported that the PVP-coated SNP ranging from 1 to 10 nmis inhibiting the HIV-1 virus from attaching to the host cells preferably via binding togp120 glycoprotein knobs [40]. In a similar study, PVP-coated SNP was shown toreduce the respiratory syncytial virus infection by 44 % [41]. The mechanism of SNPuptake and accumulation has been reported for the bacteria such as Vibrio cholera,Pseudomonas aeruginosa, and Salmonella typhi. It has been observed that the attach-ment of the 10-nm-sized SNP to the bacterial cell membranes and inside the cellsoccurs [42]. The inactivation of lactate dehydrogenase (LDH) activity and increasedprotein leakage was observed with SNP-treated Staphylococcus aureus andEscherichia coli [43]. Irregular pit generation on E. coli cell surfaces was observedin cells treated with differentially shaped SNP [44]. As far as the cytotoxic effects ofthe PVP-stabilized SNP are concerned, there is no conclusive recorded evidence ofcytotoxicity on human fibroblast and Paramecium caudatum at recommended MICvalues against pathogenic microbes [45, 46]. Ruparelia et al. [47] also reported thestrain-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 surfactant-stabilized 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 reducedantimicrobial activity over free Ag encapped within foamy carbon [40]. Interestingly, ourresults contradict the above claims and exhibited a superior antifungal activity because of thequantum size and high stability of the PVP-stabilized SNP. Assimilation of nanoparticles isinversely proportional to its size, quantum-sized particles finding greater entry thanmicrodimensional particles, and directly correlates to the level of toxicity [42, 48].Also, PVP encapsulation provides sustained release of Ag from SNPs that couldensure prolonged antifungal activity.

Conclusion

The present study offers an unequivocal proof that PVP-stabilized quantum-sizedSNPs work as a potent antifungal agent at very low concentration. The MIC valuesobtained were comparatively lower when compared with the MIC values of knownantifungals. Furthermore, the viability of the yeast cell treated with PVP-stabilizedSNP 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 cellcycle arrest resulting in deformation when incubated with the PVP-stabilized SNP. Itcould turn out to be an effective and safe alternative to conventional oral and topicalantifungal agents with due investigations for clinical applications.


Acknowledgments The authors thank the National Centre for Nanoscience and Nanotechnology, MHRD andDST-INSPIRE for financial support in the form of a research grant and junior research fellowships. Thanks isalso due to Mr S. Prathap Augustine, technician, NCNSNT, for assisting us with FE-SEM and TEM imaging.

References

1. Perlroth, J., Choi, B., & Spellberg, B. (2007). Nosocomial fungal infections: epidemiology, diagnosis, andtreatment. Medical Mycology, 45, 321–346.

2. Goffeau, A. (2008). Drug resistance: the fight against fungi. Nature, 452, 541–542.3. Jin, Y., Samaranayake, L. P., Samaranayake, Y., & Yip, H. K. (2004). Biofilm formation of Candida albicans is

variably affected by saliva and dietary sugars. Archives of Oral Biology, 49(10), 789–798.4. Levin, M. D., Hollander, J. G. D., Van der Holt, B., Rijnders, B. J., Vliet, M. V., Sonneveld, P., et al. (2007).

Hepatotoxicity of oral and intravenous voriconazole in relation to cytochrome P450 polymorphisms. Journalof Antimicrobial Chemotherapy, 60(5), 1104–1107.

5. Taxvig, C., Hass, U., Axelstad, M., Dalgaard, M., Boberq, J., Andeasen, H. R., et al. (2007). Endocrine-disrupting activities in vivo of the fungicides tebuconazole and epoxiconazole. Toxicological Sciences,100(2), 464–473.

6. Worth, L. J., Blyth, C. C., Booth, D. L., Kong, D. C., Marriott, D., Cassumbhoy, M., et al. (2008).Optimising antifungal drug dosing and monitoring to avoid toxicity and improve outcomes in patients withhaematological disorders. Internal Medicine Journal, 38(6b), 521–537.

7. Venkatakrishnan, K., VonMoltke, L. L., &Greenblatt, D. J. (2000). Effects of the antifungal agents on oxidativedrug metabolism: clinical relevance. Clinical Pharmacokinetics, 38(2), 111–180.

8. White, T. C., Holleman, S., Dy, F., Mirels, L. F., & Stevens, D. A. (2002). Resistance mechanisms in clinicalisolates of Candida albicans. Antimicrobial Agents and Chemotherapy, 46(6), 1704–1713.

9. Perea, S., Lopez-Ribot, J. L., Kirkpatrick, W. R., McAtee, R. K., Santillan, R. A., Martinez, M., et al. (2001).Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strainsdisplaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients.Antimicrobial Agents and Chemotherapy, 45(10), 2676–2684.

10. Silver, S. (2003). Bacterial silver resistance: molecular biology and uses and misuses of silver compounds.FEMS Microbiology Reviews, 27, 341–353.

11. Klasen, H. J. (2000). A historical review of the use of silver in the treatment of burns. II. Renewed interest forsilver. Burns, 26(2), 131–138.

12. Baker, C., Pradhan, A., Pakstis, L., Pochan, D. J., & Shah, S. I. (2005). Synthesis and antimicrobialproperties of silver nanoparticles. Journal of Nanoscience and Nanotechnology, 5(2), 244–249.

13. Melaiye, A., Sun, Z., Hindi, K., Milsted, A., Ely, D., Reneker, D. H., et al. (2005). Silver(I)-imidazolecyclophane gem-diol complexes encapsulated by electrospun tecophilic nanofibers: formation of nanosilverparticles and antimicrobial activity. Journal of the American Chemical Society, 127(7), 2285–2291.

14. Sondi, I., & Salopek-Sondi, B. (2004). Silver nanoparticles as antimicrobialagent: a case study on E. coli as amodel for Gram-negative bacteria. Journal of Colloid and Interface Science, 275(1), 177–182.

15. Liversidge, E. M., Liversidge, G. G., & Copper, E. R. (2003). Nanosizing: A formulation approach forpoorly- water- soluble compounds. European Journal of Pharmaceutical Sciences, 18(2), 113–120.

16. Brigger, I., Dubernet, C., & Couvreur, P. (2002). Nanoparticles in cancer therapy and diagnosis. AdvancedDrug Delivery Reviews, 54, 631–652.

17. CLSI (2008b). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi;approved standard CLSI document M38-A2. Wayne: Clinical and Laboratory Standards Institute.

18. Hartsel, S., & Bolard, J. (1996). Amphotericin B: new life for an old drug. Trends in PharmacologicalSciences, 17, 445–449.

19. Ramage, G., Vande Walle, K., Wickes, B. L., & Lopez-Rebot, J. L. (2001). Standardisation method for invitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrobial Agents andChemotheraphy, 45, 2475–2479.

20. Chan, W. C. W., Maxwell, D. J., Gao, X. H., Bailey, R. E., Han, M. Y., & Nie, S. M. (2002). Luminescentquantum dots for multiplexed biological detection and imaging. Current Opinion Biotechnology, 13(1), 40–46.

21. Sondi, I., Siiman, O., Koester, S., & Matijevic, E. (2000). Preparation of aminodextran- CdS nanoparticlecomplexes and biologically active antibody-amino dextran-CdS nanoparticle conjugates. Langmuir, 16,3107–3118.

22. Zhao, G. J., & Stevens, S. E. (1998). Multiple parameters for the comprehensive evaluation of thesusceptibility of Escherichia coli to the silver ion. Biometals, 11, 27–32.


23. Herrera, M., Carrion, P., Baca, P., Liebana, J., & Castillo, A. (2001). In vitro antibacterial activity of glass-ionomer cements. Microbios, 104, 141–148.

24. Klaus, T., Joerger, R., Olsson, E., & Granqvist, C. G. (1999). Silver-based crystalline nanoparticles,microbially fabricated. Proceedings of National Academy of Sciences, USA, 96, 13611–13614.

25. Wu, C., Mosher, B. P., Lyons, K., & Zeng, T. (2010). Reducing ability and mechanism forPolyvinylpyrrolidone (PVP) in silver nanoparticles synthesis. Journal of Nanoscience andNanotechnology, 10, 2342–2347.

26. Sahoo, P. K., Kalyan Kamal, S. S., Jagadeesh Kumar, T., Sreedhar, B., Singh, A. K., & Srivastava, S. K.(2009). Synthesis of silver nanoparticles using facile wet chemical route. Defence Science Journal, 59(4),447–455.

27. Jiang, L. P., Wang, A. N., Zhao, Y., Zhang, J. R., & Zhu, J. J. (2004). A novel route for the preparation ofmonodisperse silvernanoparticles via a pulsed sonoelectrochemical technique. Inorganic ChemistryCommunications, 7(4), 506–509.

28. Kvitek, L., Panacek, A., Soukupova, J., Kol, M., Vecero, R., Prucek, R., et al. (2008). Effect of surfactant andpolymers on stability and antibacterial activity of silver Nanoparticles (NPs). Journal of Physical ChemistryC, 112, 5825–5834.

29. Kim, K. J., Sung,W. S., Suh, B. K., Moon, S. K., Choi, J. S., Kim, J. G., et al. (2009). Antifungal activity andmode of action of silver nano-particles on Candida albicans. Biometals, 22, 235–242.

30. Hwang, E. T., Lee, J. H., Chae, Y. J., Kim, Y. S., Kim, B. C., Sang, B. J., et al. (2008). Analysis of the toxicmode of action of silver nanoparticles using stress-specific bioluminescent bacteria. Small, 4, 746–750.

31. Niazi, J. H., Sang, B. I., Kim, Y. S., & Gu, M. B. (2011). Global Gene Response in Saccharomyces cerevisiaeexposed to silver nanoparticles. Applied Biochemistry and Biotechnology, 164(8), 1278–1291.

32. Klis, F. M., de Groot, P., & Hellingwerf, K. (2001). Molecular organization of the cell wall of Candidaalbicans. Medical Mycology, 39(1), 1–8.

33. Latgé, J. P. (2010). Tasting the fungal cell wall. Cellular Microbiology, 12(7), 863–872.34. Cope, J. E. (1980). The porosity of the cell wall of Candida albicans. Journal of General Microbiology, 119,

253–255.35. Basrai, M. A., Naider, F., & Becker, J. M. (1990). Internalization of lucifer yellow in Candida albicans by

fluid phase endocytosis. Journal of General Microbiology, 136(6), 1059–1065.36. Etxeberria, E., Gonzalez, P., Fernandez, E. B., & Romero, J. P. (2006). Fluid phase endocytic uptake of

artificial nano-spheres and fluorescent quantum dots by sycamore cultured cells. Plant Signaling andBehavior, 1(4), 196–200.

37. Johnston, D. A., Eberle, K. E., Sturtevant, J. E., & Palmer, G. E. (2009). Role for endosomal and vacuolarGTPases in Candida albicans pathogenesis. Infection and Immunity, 77(6), 2343–2355.

38. Berman, J. (2006). Morphogenesis and cell cycle progression in Candida albicans. Current Opinion inMicrobiology, 9, 595–601.

39. Hwang, I. S., Lee, J., Hwang, J. H., Kim, K. J., & Lee, D. G. (2012). Silver nanoparticles induce apoptoticcell 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). Interactionof 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 ofrespiratory 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). Thebactericidal 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 silver-nanoparticles against Staphylococcus aureus and Escherichia coli. Korean Journal of Microbiology andBiotechnology, 39(1), 77–85.

44. Pal, S., Tak, Y. K., & Song, J. M. (2007). Does the antibacterial activity of silver nanoparticles depend on theshape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli. Applied andEnvironmental Microbiology, 73(6), 1712–1720.

45. Panacek, A., Kolar, M., Vecerova, R., Prucek, R., Soukupova, J., Krystof, V., et al. (2009). Antifungalactivity 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 studyon the toxicity of silver nanoparticles (NPs) against Paramecium caudatum. Journal of Physical ChemistryC, 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). Antimicrobialpolyethyleneimine 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 tonitrifying bacteria. Environmental Science Technology, 42, 4583–4588.


Documents

Highly Potential Antifungal Activity of Quantum-Sized Silver Nanoparticles Against Candida albicans