Silver nanoparticles biosynthesis and impregnation in cellulose acetate membrane for anti-yeast therapy

1 Parasitology Department, Federal University of Amazonas, Manaus, AM, Brazil. 2 Chemistry Institute, University of Campinas, 13083-970, Campinas, SP, Brazil. 3 Chemistry Department, Federal University of Amazonas, Manaus-AM, Brazil. 4 Department of Polymer Technology, University of Campinas, 13083-970, Campinas, SP, Brazil. 5 Brazilian Nanotechnology National Laboratory (LNNano-CNPEM), Campinas, SP, Brazil. 6 Laboratory of Nanomaterials Synthesis and Biological Interactions (NanoBioss), Chemistry Institute, University of Campinas, Campinas, SP, Brazil.


INTRODUCTION
Silver nanoparticles (AgNPs) are nanoforms that express higher antimicrobial potential due to their shape and reduced size.The characters of AgNPs promote a better interaction with biological receptors, they intensify the interaction with cell surface and, consequently, they increase the bioactive efficiency (Tarafdar and Raliya, 2013;Pereira et al., 2014).Due to their excellent antimicrobial action, AgNPs are widely used in medical, technological and agricultural areas (Ingale and Chaudhari, 2013;Pereira et al., 2014;Moghaddam et al., 2015;Siddiqi and Husen, 2016).
The world market of nanotechnological products have been in evolution.The annual arrecadation is around 25% and the prevision is an increase to 3 billions until 2020.Among these products, the ones from AgNPs stand out.The antimicrobial properties of AgNPs make possible their use in a wide diversity of products as plastics, soaps, pastes, food and textile products (Tran et al., 2013).
The colloidal solution of AgNPs can be combined with many polymeric matrices to extend the release of antimicrobial and increase its biocompatibility.The electrospinning nanofibers, for example, are polymers that have high potential of application in pharmaceutical and cosmetic industries for regeneration of skin and organs, therapies of drugs administration and others (Nista et al., 2015;Segala et al., 2015).
The search for nanostructured polymers with antimicrobial action are increasing due to their great potential of use in devices that require antiseptic characters as applications in skin wounds (Abdelgawad et al., 2013;Segala et al., 2015).For that reason, AgNPs are the nanoforms most used in polymers due to their electronic, optical, catalytic and antimicrobial properties (Segala et al., 2015).
The use of nanomembranes for the treatment of skin wounds provides better comfort to the patient because it reduces the number of antibiotic applications and doses, decreasing the side effects.In these kinds of infections, the nanomembranes also can act like a sponge absorbing all the fluids from the lesion and insure the oxygenation through the porous net of nanofibers (Nista et al., 2013).The cellulose acetate is a polymer that presents good tenacity, high biocompatibility and relative low cost, besides, easily forms pellicles with porous structure, the characters that make it advantageous to build an antiseptic nanocomposite to wounds treatment (Nista et al., 2012;Segala et al., 2015).
The combination of AgNPs with polymers for application in many areas is very versatile.However, the method of chemical synthesis generates toxic residues that contaminate the environment and affect the ecosystem (Zhang et al., 2011;Dhanasekaran et al., 2011;Durán and Marcato, 2011;Kulkarni and Muddapur, 2014;Meva et al., 2016).In the last years, there is an increasing demand to for the development of processes mediated by microbial sources due to their low cost and the production of nontoxic molecules and for being ecofriendly (Mishra and Singh, 2015;Moghaddam et al., 2015;Siddiqi and Husen, 2016).
Among the microbial sources, the filamentous fungi are potential biologic factories to the production of metallic nanoparticles due to their fast growth, large scale cultivation, secretion of proteins with higher reducer properties and the production of molecules that stabilize nanoparticles (Durán et al., 2011;Tarafdar and Raliya, 2013;Phanjom and Ahmed, 2015).
Some species of fungi are alternative safe systems as intermediate for the AgNPs synthesis in function of their high tolerance and capacity of metallic absorption.Aspergillus, Fusarium, Trichoderma, Cladosporium, Pleurotus, Penicillium and Verticillium are examples of fungi that mediate the extracellular synthesis of AgNPs.The extracellular process is more desirable than the intracellular because is less expensive and difficult (Sastry et al., 2003;Roy et al., 2013;Siddiqi and Husen, 2016).
There are many reports in the literature showing the synthesis of AgNPs mediated by Aspergillus species but are rare studies showing the potential of amazonic fungi in this process.The interaction of biological AgNPs with cellulose acetate nanomembranes is also an inedited investigation because most of the researches present the development of antiseptic polymers using AgNPs obtained from chemical synthesis.
This investigation shows the capacity of Aspergillus oryzae DPUA 1624 to mediate AgNPs synthesis with antimicrobial potential and the anti-yeast action with the impregnation of these particles in cellulose acetate nanomembranes (NanoMAC).
The molecular authentication was made from partial sequence of rDNA.The colony mycelium (200 mg) was used for DNA extraction by phenol chloroform method.The Internal Transcribed Spacer Region (ITS2) and part of 5.8S and 28S subunits from rDNA were amplified by PCR using ITS-3 (5´-GCA TCG ATG AAG AAC GCA GC-3') developed by White et al. (1990) and UNI-R (5'-GGT CCG TGT TTC AAG ACG-3') developed by Haynes et al. (1995).The rDNA sequences were obtained by automated sequencing (3500 ABI Applied Biosystem) and the species confirmation by comparison of DNA sequences from GeneBank using Basic Local Alignment Search Tool (BLASTn) from National Center for Biotechnology Information (NCBI).
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 0.3% (w/v) yeast extract; and 0.5% (w/v) peptone].Fermentation was conducted at 25°C, 180 rpm, for 96 h.The biomass was separated by filtration using Whatman paper no. 1, washed three times with ultrapure water, transferred to Erlenmayer flasks containing 200 ml of ultrapure water and maintained at 28°C, 200 rpm, for 96 h.The biomass was filtrated again and the aqueous extract recovered was used for AgNPs biosynthesis (Sastry et al., 2003;Durán et al., 2007;Vigneshwaran et al., 2007).

Silver nanoparticles biosynthesis
The aqueous extract (200 ml) was mixed with a silver nitrate solution (1 mol L -1 ) until final concentration of 1 mmol L -1 and incubated at 28°C, 200 rpm for 96 h, in the absence of light.Simultaneously, two controls: one of the aqueous extract and other of nitrate solution were maintained under same conditions.The synthesis of silver nanoparticles was confirmed by the color change of the solution and the presence of plasmonic band in 400 nm (Vigneshwaran et al., 2007;Zomorodian et al., 2016).Ultravioletvisible (UV-Vis) spectral analysis was done by using Cary 60 Agilent Spectrophotometer in the range of 200 to 800 nm.

Zeta potential and size of AgNPs (DLS)
The AgNPs diameter, size distribution and superficial charge obtained were measured in Nano Zetasizer (ZS) (Malvern Instruments, Worcestershire, UK), at 25°C.

Transmission electron microscopy (TEM)
Size distribution and morphological variation of AgNPs were determined from high resolution images in TEM (Carl Zeiss CEM 902) operating at 80 KeV.A drop of nanoparticles solution (diluted ten times) was deposited on carbon-coated copper grid and the images were processed in image J software and the equation used to calculate de AgNPs diameter from the obtained area was .The statistical analysis was carried out using the software origin (version 8.5.1.315).

Fourier-transform infrared spectroscopy (FTIR)
The data of IR spectra were acquired using an Agilent cary 630 FTIR spectrometer, with diamond attenuated total reflectance (ATR) attachment.In ATR mode, the spectral variation was from 400 to 4000 cm -1 .The AgNPs were analyzed depositing a drop of the colloidal suspension on ZnSe (zinc selenide) window.

Energy dispersive X-ray (EDX) and inductively coupled plasma optical emission spectrometry (ICP-OES)
The EDX was carried out in a Micro fluorescence x-ray spectrometer (microEDX1300, Shimadzu) in five points of the sample using spectrums of 15 and 50 kV and courting time of 200 s.
The inductively coupled plasma optical emission spectrometry (ICP-OES) was used for quantification of silver in the solution of AgNPs.The analysis was carried out in plasma emission spectrometer (PerkinElmer, Optima 8000).The calibration curve was constructed using 9 silver nitrate solutions with concentrations from 0.1 to 50 mg/L.The AgNPs solution was diluted 10 times for reading in the equipment.
CA membranes were prepared by electrospinning the cellulose acetate solution at room temperature (25°C) and 50% humidity, using a 20 ml glass syringe fitted with a 4 cm long, 0.8 mm diameter metallic needle.The positive pole of a high voltage power supply was connected to the metallic needle of the syringe, while the ground electrode was used to ground the copper plate collector, which had dimensions of 30 × 40 cm.The feed stream was controlled by a KD Scientific pump, Model 100 (Campinas, Brazil) connected to a syringe.The distance from the needle to the collector was 10 cm; the applied voltage was 15 kV, and the flow rate was 1 ml h -1 .Membrane samples were collected in aluminium foil used to coat the copper plate during the experiments (Nista et al., 2012;Segala et al., 2015).
The morphological structure and uniformity of nanoMAC were observed in scanning electron microscope (JEOL JSM-6360LV), by vacuum, operated at 10 kV.The images were obtained after sample metallization with a layer of gold/palladium and the diameter was calculated in Image J software.
The nanoMAC coating was performed by slow dripping of the AgNPs solution in discs (5 mm) of the nanostructured membrane until the final concentration of 6.73 μg Ag on each nanoMAC disc.

AgNPs suspension
The agar diffusion method was used for the antimicrobial activity against Escherichia coli CBAM 0001, Staphylococcus aureus ATCC 25923 and Candida albicans DPUA 1706 (Ostrosky et al., 2008).The bacterial cultures (10 8 CFU/ml) were inoculated on Müller Hinton agar (MHA) and the yeast culture (10 6 CFU/ml) on Sabouraud agar (SAB) and incubated at 37°C.In each plate, 100 μl of biosynthesized AgNPs (134.5 mg/L) were transferred to wells (8 mm) made on agar surface.The plates were incubated at 37°C for 24 h.The positive controls [streptomycin (50 μg/ml) to bacteria and itraconazole (50 μg/ml) to the yeast] were maintained at the same conditions.After incubation, the plates were examined for the presence of an inhibition zone.

Morphological and molecular authentication of A. oryzae
The macro and microscopic characteristics of the culture confirmed that the fungus is A. oryzae (Raper and Fennel, 1977;Klick and Pitt, 1988).For the molecular authentication, the rDNA sequences presented 99% of similarity (Score, 1554) (Evalue 0) with sequences at the same region from A. oryzae (Access number KT964480.1).The amplified sequence of A. oryzae DPUA 1624 was deposited at GenBank (Access number KY655350).

Silver nanoparticles biosynthesis
The biosynthesis of silver nanoparticles (AgNPs) was confirmed by the color change of the mycelial aqueous extract from yellow to brown (Figure 1A).There was no formation of aggregate particles and the extract did not present color intensification after 96 h.These results indicate that the nanoparticles were dispersed in the solution.
The color change occurs due to the effect of superficial plasmon resonance (SPR) and the reduction of AgNO 3 (Li et al., 2012).SPR refers to the collective oscillation of free electrons in the metallic nanoparticles when they interact with electromagnetic radiation (Cao et al., 2011;González et al., 2014;Al Juraifani and Ghazwani, 2015).
The absorption spectrum of the AgNPs solution (Figure 1B) shows a SPR band with peak of absorbance in 446 nm confirming the presence of AgNPs after 96 h of incubation.Similar results were reported for AgNPs from Aspergillus tubingensis in the same wavelength (Rodrigues, 2013).

Zeta potential and size of AgNPs
Zeta potential (ζ) indicates the repulsion between the disperse particles with the same charge by electrophoretic mobility.Its value can be related to the stability of colloidal dispersions (Phanjom and Ahmed, 2015).In this study, the zeta potential of AgNPs mediated by A. oryzae was 28.7 mv.This result suggests that the AgNPs have an elevated electrostatic repulsion which contributes to their stabilization.According to Roy et al. (2013), particles in suspension with high negative or positive zeta potential tend to repel themselves and do not form aggregates.
Dynamic light analysis (DLS) is a technique that determines particles sizes by measurement of aleatory changes in the intensity of light from a solution or suspension (Fatima et al., 2015).The average hydrodynamic diameter of A. oryzae AgNPs by DLS was 57.88 nm (Figure 2) and the polydispersity index was Silva et al. 1493 0.217, confirming the formation of particles in nano scale dimension (1-100 nm) and the definition of the solution as monodisperse due to the interval defined for this type of particles from 0.01 to 0.7 (Honary et al., 2013;Tarafdar and Raliya, 2013).

Transmission eletron microscopy (TEM)
The results of TEM images (Figure 3A) show that AgNPs are predominately rounded and, in less quantity, some are triangle with average size of 61.15±11.45nm.AgNPs with variable shapes and sizes are common in the biological systems; however, the ones with triangle shapes have more antimicrobial efficiency than the rounded AgNPs (Pal et al., 2007;Rai et al., 2009;Roy et al., 2013).
The size distribution of AgNPs is as shown in Figure 3B.Most of the AgNPs are from 50 to 60 nm in size.Some studies report nanoparticles from Aspergillus with sizes from 1 to 140 nm (Sagar and Ashok, 2012;Khalil, 2013;Roy and Das, 2015).Binupriya et al. (2010) and Pereira et al. (2014) reported AgNPs synthesis by A. oryzae species with sizes of 5 to 50 nm and 25 to 76 nm, respectively.

X-Ray diffraction (XRD)
The XRD patterns of the AgNPs showed the crystalline nature of the sample and confirmed the presence of pure silver in the nanoparticles solution analyzed.Figure 4 shows that it is possible to observe five distinct bands in 37, 97°, 44,29°, 64,66°, 77,46° and 81,44° corresponding to (111), ( 200), ( 220), ( 311) and ( 222) planes.These planes correspond to the Ag° nanoparticles face-centered cubic (fcc) (Crystal Structure Database-ICSD/Code 64,994) with a lattice parameter of a = 4.077 Å.They confirm the formation of metallic silver in the suspension of AgNPs.

Fourier-transform infrared spectroscopy (FTIR)
The spectra presented four distinct bands: 3288, 2101, 1994 and 1633 cm -1 (Figure 5).The 3288 cm -1 band is  related to the vibration energy of primary and secondary amides from proteins and the 1633 cm -1 band is related to the vibration energy of carbonyl group and is assigned to the primary amide bond of the protein (El-Aziz et al., 2012).The presence of carbonyl and primary amide groups indicate that the proteins present in the aqueous extract of A. oryzae biomass are connected to the AgNPs.Many reports testify that proteins can connect to silver nanoparticles by the free amine groups turn them more stable particles (Phanjom and Ahmed, 2015).

Energy dispersive X-ray spectroscopy (EDX) and inductively coupled plasma optical emission spectrometry (ICP-OES)
The energy dispersive X-ray spectroscopy (EDX) revealed strong sign in the region next to 3 KeV that correspond to the peak of silver (Pereira et al., 2014;Elgorban et al., 2016).In EDX spectra, a peak was observed in 2.98 keV and others in 3.17 keV that correspond to bonds energy of AgLa and AgLb1, respectively (Figure 6).Both peaks composed of the optical absorption profile of silver element.These results indicate that the AgNP mediated by A. oryzae are high pure.
The inductively coupled plasma optical emission spectrometry analysis showed that the concentration of the AgNPs solution was 134.5 µg/ml.

AgNPs solution and NanoMAC impregnated with AgNPs
The antimicrobial activity with the AgNPs solution  showed that E. coli CBAM 0001 and S. aureus ATCC 25923 were resistant to the AgNPs (134 µg/ml), while C. albicans DPUA 1706 was sensible.The yeast growing was completely inhibited by the AgNPs showing an inhibition zone of 23.6 cm (Figure 7).Xue et al. (2016) reported similar results with AgNPs mediated by Arthroderma fulvum.The particles presented antifungal activity against many fungi including C.

albicans.
AgNPs present efficient antimicrobial properties compared to other molecules due to their large superficial area that promotes a better contact with the microorganims (Rai et al., 2009;Kon and Rai, 2013).However, according to Segala et al. (2015), the antimicrobial activity of AgNPs depends on some factors such as crystallinity, geometry, size, superficial oxidation,  agglomeration in the biological environment, superficial charge, groups present in the nanoparticles surface, and others.For that reason, we can deduce that the properties of A. oryzae AgNPs enabled a better interaction with the yeast cells promoting the penetration of the nanoparticles through the plasmatic membrane of the micro-organism causing cell death.
The AgNPs antimicrobial effects are widely known, although their action mechanism in yeast cells not been completely described.Kim et al. (2009) observed that the AgNPs interact with C. albicans cells causing significant changes in their membranes resulting in the formation of "pits" and finally, the formation of pores and death.The authors also believe that the AgNPs break the membrane permeability barrier of C. albicans by the disarray of the lipid bilayer.It causes the scape of ions and other materials and the formation of pores that dissipate the membrane electrical potential.
Although many studies report the cytotoxic and genotoxic action of AgNPs in human and animal cells, there is still a lack of consistent and trustable data about them.Some of them are contradictory and, for that reason, there is no general consense about the action of these molecules in living organisms and in the environment (Rai et al., 2017).The cytotoxicity of AgNPs depends on many factors as size, shape, stability, given dose and time of exposition (Tran et al., 2013;Rai et al., 2017).
The AgNPs biosynthetized by the mycelial extract of A. oryzae DPUA 1624 can be evaluated as efficient agent for use in clinical application, especially in treatments of infections caused by C. albicans.
Yeasts highlight as main etiologic agent of fungal diseases primarily dermatomycosis, a skin infection that have been considerably increasing in the last decades.One of the causes that favor the incidence of this disease is due to the resistance of these fungi to the medications.For that reason, a considerable motivation occurs which is to find new anti-yeasts agents (Sidrim and Rocha, 2010;Xue et al., 2016).According to the necessity to develop new strategies against skin infections caused by yeasts, this study carried out the antimicrobial activity of AgNPs impregnated in cellulose acetate membrane (15%) and DMAc: acetone (1:2) (NanoMAC).The nanoMAC contains nanofibers with average diameter of 410.76 nm.They are random deposited forming a thinny and homogeneous tissue without beads formation (Figure 8A).
The nanoMAC impregnated with AgNPs (Table 1), allowed the increase of 24.22% in antifungal action against C. albicans when compared with the antimicrobial activity using the colloidal solution of AgNPs (Figure 8B).This result is relevant because it demonstrates that the AgNPs impregnation in cellulose acetate membrane can potentiate the action against yeasts of Candida genus.
NanoMAC impregnated with biological AgNPs present a large potential to be used in medical and pharmaceutical industries.They can be considered as sustainable nanocomposites with anti-yeast action.

Conclusion
The fungi A. oryzae DPUA 1624 produces molecules capable to reduce silver (Ag) ions and mediate stable silver nanoparticles synthesis.They are monodisperse with antimicrobial activity.This result provides a viable and eco-friendly alternative to antifungal production based on AgNPs without causing any polluting residues.The impregnation of AgNPs in cellulose acetate membrane increases the anti-yeast action of these particles resulting in the development of a nanocomposite with antifungal properties that can be used in medical and pharmaceutical industries.

Table 1 .
Antimicrobial activity of AgNPs from A. oryzae.