High Antifungal Activity against Candida Species of Monometallic and

of Microbiology, Universidad Industrial de Santander, Cra. 32 # 29-31, Bucaramanga CP680002, Colombia. ACS Biomater. Sci. Eng. , Article ASAP. DOI...
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High antifungal activity against Candida species of monometallic and bimetallic nanoparticles synthesized in nanoreactors Jorge Andres Gutierrez, Silvia J. Caballero, Laura A. Diaz, M. Alejandra Guerrero, Jennifer Ruiz, and Claudia C. Ortiz ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00511 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 27, 2017

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High antifungal activity against Candida species of monometallic and bimetallic nanoparticles synthesized in nanoreactors

Jorge A. Gutiérreza*, Silvia Caballeroa, Laura A. Díaza, M. Alejandra Guerreroa, Jennifer Ruizb, Claudia C. Ortizb*

a

School of Chemistry, Universidad Industrial de Santander, Cra 27 # 9 (CP680002)

Bucaramanga, Colombia. b

School of Microbiology, Universidad Industrial de Santander, Cra. 32 # 29-31 (CP680002)

Bucaramanga, Colombia.

*

Corresponding authors: [email protected] / [email protected]

Abstract Among all novel challenges nowadays worldwide, the infectious diseases is probably one of the most important. It is well known that commonly treatments used include high doses of antibiotics, being very invasive therapies for patients. These treatments are more intensive when the infection is related with multi-drug resistant microorganisms. In this sense, in this work we report the use of reverse micelles to form less than 5 nm gold, silver and gold-silver nanoparticles (NPs) with biological activity against five opportunistic Candida strains responsible of several diseases in human beings. As results, we evaluate the interface properties and droplet-droplet interactions of micelles founding high fluidity in polar head of surfactant, necessary to form a flexible interaction

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channel in the “dimmer” micelle-micelle. In this condition, we form monodispersed, high reactive NPs with sizes less than 5 nm with high antifungal activity against C. parapsilosis, C. Krusei, C. glabrata, C. guillermondii, and C. albicans, with Minimum Inhibitory Concentrations (MIC50) less than 0.7 ppm in all cases, the lowest reported under our knowledge. These are very promising results to develop alternative therapies to treat fungal diseases on humans, animals, plants or to coat conventional surfaces on surgery rooms.

Keywords Nanoparticles; reverse micelles; gold, silver, platinum; Candidas, minimum inhibitory concentration.

Introduction It is well known the high interest in the physical properties of nano-sized materials. Among all species studied in nanoscience, metallic NPs have proved to be widely versatile and useful, they can be used in communications, catalysis, magneto-optics and optoelectronic devices,1-3 applications modulable by size and morphology.2,4,5 Among all metals used in nanotechnology, gold and silver are the most explored. Gold nanoparticles (AuNPs) can be functionalized with a wide variety of compounds such as antibodies, polymers, molecular probes, drugs, peptides, etc.6-12 AuNPs are used as no cytotoxic and no immunogenic materials in “smart” systems of drug release by internal or external stimuli,1316

being excellent alternatives for nanobiotechnology and nanomedicine.2,17-20 On the other

hand, it is well known the antimicrobial activity of silver nanoparticles (AgNPs) and some ionic compounds, applied in the last years in coatings, unguents for topical use on burns, water sanitization, etc.21-26 However, there are few studies with bimetallic nanoparticles

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applied in medicine; in this sense, it is very interesting take advantages of both metals and to form bimetallic species.27,28 But for any application, there are two key factors, size and polydispersity. In nanomedicine the small NPs can enhance cells communication,29 and this is very important to develop new functional materials such as biofilms, thermo-sensitive gels or food packaging,24,30-32 in all cases using new or “unusual” methodologies such as sono-chemical irradiation,26 green synthesis using leaves exacts

33

and bio-reduction with

Fungus.34 Thus, the applications of large NPs have been limited because they are less stable than smaller, mainly due to electrostatic repulsion and steric effects on surface by ligands and linker molecules.35,36 Thus, when surface area/volume ratio is high the NPs remain stable in the presence of few repulsive molecules linked on the surface.37 However, the use of reverse micelles (RMs) stand up as an interesting method to form and ensure small metal nanocomposites.38,39 RMs are supramolecular assemblies formed when surfactants molecules are dissolved in nonpolar organic solvents, their polar area is located in the core while their hydrocarbon tails extend into the nonpolar media.40 One of the most important properties of RMs is the ability to dissolve polar solvents such as metal precursors, acting these systems as nanoreactors (less than 10 nm).38,41-43 On the other hand, it is well know that fungal infections is a challenge in medicine nowadays, making necessary the development of new therapeutic treatments,44,45 this is because the abuse of antibiotics agents has induced resistance of several microorganisms.46 Among all fungal infections, Candida species stand up by its frequency and high impact on human health.46,47 Candidemia is a bloodstream infection with 50-60% of all nosocomial Candida infections in the world, being reported high rates of mortality.48,49

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Herein, we report the importance of small monometallic and bimetallic NPs in order to develop a biomedical application. We show physicochemical characterization and excellent fungal activities against five pathogenic Candida species.

Experimental Materials Hexane HPLC grade were purchased from Sigma and were used without further purification. Sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT) (Sigma >99% purity) was used as received, to minimize water absorption, it was kept under vacuum with P2O5. Ultrapure water was obtained from Labconco equipment model 90901-1. Tetrachloroauric acid (HAuCl4, Sigma-Aldrich) and Silver nitrate (AgNO3, Merck Millipore) as metal precursor, and hydrazine (N2H4, Sigma- Aldrich) as reducing agent. The reagents for gold, silver and gold-silver NPs synthesis were used as received. Candida albicans strains were clinically isolated and were kept under ideal conditions in our lab until analysis.

Methods Reverse micelles as nanoreactors The AOT RMs in hexane were formed by mass and volumetric dilution until obtain optically clear solution. The amount of solute dissolved in water into RMs is expressed through Ws as follow: Ws = [solute in water]/[AOT]. All experimental points were measured three times with different samples. In all the cases, the experimental temperature was kept at 25 °C ± 0.2 °C.

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The synthesis of monometallic50 and bimetallic NPs in RMs is described in supporting information section. This methodology is based in Brownian motion of RMs and in micelle-micelle interactions.

Antifungal activity of NPs The antifungal activity of AuNPs, AgNPs and AuAgNPs against C. parapsilosis ATCC 22019, C. krusei A2, C. glabrata A2, C. guilermondii A2, and C. albicans ATCC 10231 were tested according to Clinical and Laboratory Standards Institute guidelines,51 and the minimal inhibitory concentrations (MICs) of the NPs were determined using a broth microdilution method by triplicate. Details about methodology performed can be seen in supporting information section.

Instrumentation UV/vis spectra were recorded using a spectrophotometer Shimadzu UV-1800. Photon Technology International QM-40 fluorometer dotted with a xenon lamp was used for fluorescent emission measurements. FTIR spectra were obtained using a Bruker Tensor 27 spectrometer, the absorption spectra were obtained by co-adding 100 measures with 0.5 cm-1 of resolution, using pure hexane as blank and Platinum ATR support. All DLS experiments were carried out at fixed AOT concentration of 0.1 M. We introduce an apparent hydrodynamic diameter (dapp) in order compare all our systems.43 The dapp of NPs were determined by dynamic light scattering (DLS) Malvern ZetaSizer NanoZS90 with a He-Ne laser of 633 nm. Thirty independent size measurements were made for each individual sample at scattering angle of 90°; the polydispersity index was always below 0.2.

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The TEM micrographs were obtained in a Transmission Electron Microscopy (TEM) FEI TECNAI G2 STWIN at 20–200 kV with a camera Gatan ES100W and software Gatan Digital Micrograph. For TEM studies, the samples were placed into a formvarcovered copper grid.

Results and Discussion Physicochemical properties of AuNPs, AgNPs and AuAgNPs in hexane/AOT/water at Ws= 7 Nanomaterials based on metal NPs are very attractive on several fields for more than three decades.52 However, the synthesis method is a key factor on final reactivity and applicability of products.5,53 It is well known that AOTRMs in aliphatic hydrocarbons such as hexane or heptane have effective micelle-micelle interactions,43,50 these studies let us to know the ideal conditions to synthesize metal NPs inside RMs, but the internal phenomena in the polar core are still poorly investigated. The first experience performed was to evaluate the maximum amount of precursors (WsMax) that hexane/AOT RMs can dissolve, funding that with gold and silver precursors the WsMax exceeds 25, enough quantity to form a concentrated NP solution.

Table 1. dapp values obtained in hexane/AOT RMs at different Ws. [AOT] = 0.1 M.

dapp (nm) Ws HAuCl4 AgNO3

HAuCl4- AgNO3

5

3.3

3.1

3.3

10

4.1

3.9

4.2

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15

4.9

4.4

4.8

20

6.1

5

6.2

25

6.7

6.1

6.5

Table 1 clearly shows that size of RMs increases (proportional) with Ws. It is very important since linear tendency suggest discrete spherical shape of aggregates until Ws= 25. According to recent reports, this is possible in aliphatic hydrocarbons such as hexane or heptane.43,54 Based in our results and tacking into account that it is necessary small but fluid nanoreactors, we chose Ws= 7 to form NPs. Thus, in order to understand the surface nature, the optical properties of monometallic and bimetallic NPs were determined analyzing the form, intensity and absorption wavelength of Surface Plasmon Resonance (SPR), also the possible molecular interactions between metal surface and surfactants by FTIR of polar head of AOT.

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Figure 1: Optical properties of AuNPs, AgNPs and AuAgNPs in hexane/AOT/water at Ws= 7. A) Absorption spectra. Empty RMs was used as blank. B) Infrared absorption of asymmetric S=O stretching.

The Figure 1A show absorption spectra of three metal NPs, the SPR spectrum of AuNPs have an absorption maximum λabsmax= 518 nm, typical energy of less than 15 nm NPs in this type of systems.55,56 At high energy (from 450 to 300 nm) the spectra has no significant changes in absorbance, corresponding to no contributions of medium and atomic gold clusters. AgNPs have the λabsmax= 405 nm with high intense and gaussian band; similarly to gold, at high energy was no found changes in the spectrum, corresponding only to monodispersed colloidal AgNPs. Finally, bimetallic NPs were obtained with an SPR band at 490 nm. It is interesting to note some facts in bimetallic NPs, AgNPs SPR is more intense than AuNPs, but the intensity and absorption wavelength of AuAgNPs are more similar to gold than silver, also the band is more gaussian. This could be product of more gold atoms on NP surface because of plasmon resonance is a phenomenon of free electrons in a collective oscillation of materials,57 it is not necessary a silver core with a gold shell, or

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silver nucleus doped with gold atoms, could be a unorganized mixture of metals, but mainly gold. On the other hand, at high energy (around 300 nm) small changes of absorbance were observed, and these changes increases with time. This could be product of atomic clusters released from the surface, this is very interesting due to atomic clusters have properties between NPs and molecules. The IR stretching spectra of different NPs in AOT RMs suggest interactions of all metal surface with polar head of surfactant, however, it is clear to note that silver interactions with sulfonate group of AOT are stronger than gold, given by a notorious changes in form and frequency of spectra in the 1350-1100 cm-1 region, suggesting strong interaction between SO3- and metal atoms, displacing the Na+ counter-ion given by a loss of splitting located from 1216 to 1241cm-1.58 According with Uv-vis behavior, this could be product of less reduction potential of silver in comparison with gold. Thus, in this macromolecular system the optical spectra suggest the formation of mixed nanoparticle, with silver and gold atoms on surface, this has implications in microbial activity and others applications due to the union of reactivity of two different noble metals. Others sites of AOT molecule such as carbonyl and symmetric sulfonate were evaluated, but minor changes were found (see supporting information), suggesting that molecular interactions are mainly by the sulfonate group. The size of monometallic and bimetallic NPs was determined in solution by DLS with a polydispersity index below to 0.2 in all cases, giving similar values in both techniques: AuNPs 4 nm, AgNPs 3 nm, and AuAgNPs 5 nm. It is clear to note that these values are an average, because between TEM and DLS the sizes may vary. More TEM micrographs are showed in supporting information section.

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Figure 2: DLS histogram and TEM micrographs of colloidal NPs, A) AuNPs, B) AgNPs and C) AuAgNPs. Nanoreactor hexane/AOT Ws= 7.

As can be seen, the NPs obtained are spherical, monodispersed and have sizes less than 10 nm. This is considered a monodispersed system due to the small difference in sizes (2-3 nm) of all colloidal solutions, something very difficult to obtain with other synthesis methodologies. The use of RMs allow us the control of this kind of issues, due to a fast reduction process in confined environments. But it is clear to note that depending of surfactant and organic phase used, the NPs could be tunable in form and size. Once

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obtained the NPs, the organic media can be changed by aqueous by solvent evaporation, followed by washings with 50:50 of ethanol:water, and centrifugation at 9000 rpm at 4°C. According with spectroscopy, microscopic and DLS results, these could be very good solutions for our antifungal applications, this is mainly due to a scale factor, and this resides in more and effective interactions over the microorganisms, avoiding phagocytes and other biological species that can affect the action of NPs in specific sites.

Antibacterial activity of AuNPs, AgNPs and AuAgNPs against Candida parapsilosis, C. Krusei, C. glabrata, C. guillermondii, and C. albicans The fungicidal activity was performed through MIC evaluating the growth kinetics at 490 nm during 48 hours. All results of fungal growth kinetics in presence of several concentrations of monometallic and bimetallic NPs as well the MIC90 are shown in supporting information. In this document only are showed the graphics corresponding to C. glabrata, and the MIC50 values in Table 2 Initially, the fungicidal activity of AOT residues were tested in all Candida species in order to discard surfactant effects on growth inhibition (Figure 3).

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Figure 3: Growth kinetics of C. glabrata in presence and absence of AOT surfactant. The other four kinetics are showed in supporting information section.

As can be seen, AOT molecules have no effect on normal growth of C. glabrata, suggesting that any possible inhibition in NPs solutions will be product of metals. In all others strains of fugal cultures: C. parapsilosis, C. krusei, C. guillermondii, and C. albicans, the growth kinetics were similar, and AOT have no effect at all. When NPs solution were evaluated at different concentrations, there was a strong change in the normal Candida behavior (Figure 4).

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Figure 4: Growth kinetics of C. glabrata A2 in the presence of A) AuNPs, B) AgNPs, and C) AuAgNPs synthesized into reverse micelles.

The Figure 4 shows growth inhibition at the first NPs concentration (0.33 ppm), then a slight increase in the optical density after 25 hours, also as the NPs concentration increases, the colonies growth decreases, where at between 1 and 0.5 ppm all Candida species are practically inhibited, suggesting cell death in stationary state. In all Candida spp, several NPs concentrations were tested, but the inhibition was achieved at the lowest concentration, suggesting high impact and reactivity of these NPs in comparison with similar studies.59,60 If we look all strains with each NP system, it is possible to determine the importance of the first stage of inhibition (Figure 5).

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Figure 5: Growth inhibition percent of Candida spp by several concentrations of metal NPs. A) AuNPs, B) AgNPs, and C) AuAgNPs.

As was mentioned before, the slopes of inhibition versus NPs concentrations are higher in the first stage, under 0.5 ppm for Ag and AuAgNPs, and between 0.5 and 1.0 ppm for AuNPs (Figure 5). Although AgNPs are known as the best antimicrobials agents in this kind of studies, it is important to emphasize that also AuNPs and AuAgNPs have excellent activity. In this sense, it is clear as the new properties emerge when we mix two noble metals, inhibiting growth at very low concentration (Table 2). Table 2: MIC50 of metal NPs against Candida species. AuNPs

AgNPs

AuAgNPs

Candida Strains ppm C. parapsilosis ATCC 22019

0.033

0.125

0.25

C. krusei A2

0.33

0.062

0.125

C. glabrata A2

0.62

0.125

0.062

C. guillermondii A2

0.62

0.062

0.03

C. albicans ATCC 10231

0.25

0.03

0.03

The MIC50 values for all NPs systems were below 0.7 ppm, suggesting significant antifungal activity. If we compare all results, the C. albicans ATCC 10231 and C. guillermondii A2 were the species more inhibited with MIC50 of 0.033 ppm of AuAgNPs and 0.03 and 0.062 ppm respectively of AgNPs. In the most cases the best inhibition effect was caused by bimetallic NPs, something unusual, but this could be product of some factors. All fungi species have cell membrane and cell wall, the membrane is composed by

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phospholipids, and the wall contain mannoproteins, β-glucan-chitin, β-glucan and mannoprotein again. Thus, the NPs need to interact with all of these macromolecules before reach the phospholipids, the integral proteins, peripheral proteins and ionic channels. According to this, it is well known that silver is an excellent antimicrobial agent,21 gold is an excellent material to link molecules and functional groups,7 so, if we mix these atoms at NP surface, it is possible to take advantage of gold surface as “tool” reacting with biomolecules near and through the cell wall, and then use the silver to generate damage. Bimetallic NPs could allow double effect, fungistatic and fungicidal. The fungistatic effect is a result of inhibition of β-glucan synthase, and consequently of cell wall.61 While the fungicidal effect, is caused by changes in the wall integrity, losing its mechanic resistance, leading to cell destruction by osmotic pressure variations.62 We think that all of these effects on the fungi could be increased by gold atoms and its high reactivity with several functional groups of biomolecules. One important fact in the development of new antimicrobial agent is the concentration necessary to cause damage over the microorganism, due to must be low enough to have no side effects. Our results show different MIC50, but all data under 1 ppm. As it is well known, the NPs toxicity is size dependent, in AgNPs smaller than 20 nm the % of cell viability partially decreases, e.g. in A549 for lung, and SK-Mel28 and A375 for Skin, the viability decreases 50% at concentrations > 25 ppm. In the case of NPs of 5 nm, the concentration to decrease the 50% of cell viability in these cell lines is 12.5 ppm.63 Thus, in our case, where the activity was found under 1ppm, the cytotoxicity is not an issue. Among all species tested in this work, C. Krusei and C. albicans are the main responsible of fungal disease in humans. In this sense and taking into account our results,

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there are very promising possibilities to develop new antifungal treatments to affront several public health issues.

Conclusions We have shown the importance of reverse micelle as nanoreactors in the formation of metal NPs. According with our recent reports, we chose reverse microemulsions with effective micelle-micelle interaction to synthesize < 5 nm NPs. The colloidal solutions obtained were stables on time, making possible to evaporate the organic media, keep up NPs for several weeks, and re-suspends again for specific applications. Our experimental results show monometallic and bimetallic NPs stabilized by surfactant molecules with excellent antifungal activity against five Candida species. The smallest MIC50 values were found in C. parapsilosis ATCC 22019, C. guillermondii A2 and C. albicans ATCC 10231. It is interesting that several species of Candida were inhibited first by bimetallic NPs, standing out the importance of mix metals at nanoscale, where the properties of different materials converge and novel phenomena arise. These results open new approaches to develop alternative therapies such as ointments, fabrics or bandages to treat infections caused by fungi.

Supporting Information Supporting information file contain FTIR spectra of other sections of AOT with Au, Ag and AuAgNPs, more TEM micrographs. Also, contain MIC90 of Au, Ag and AuAgNPs in the five Candidas and all fungal growth kinetics in presence and absence of metal NPs.

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Acknowledgments We gratefully acknowledge the financial support for this work at “Estancias Postdoctorales” program of Universidad Industrial de Santander (UIS) and Departamento Administrativo de Ciencia, Tecnología e Innovación (Colciencias).

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