Nanoscience & Technology Open Access

The possible mechanism of action of different nanomaterials on pathogenic fungi, significant for medical practice is summarizedin this article. Furthermore, the antifungal action of different collagen based nanocomposites against Candida lusitaniae was evaluated and compared. Collagen-based biomaterials with antimicrobial activity are attractive candidates for wound dressing, tissue engineering, components of implantable devices, etc. One of the easiest and most effective ways among the large variety of known approaches to add antimicrobial activity of biomaterials is development of composites including antimicrobial agents. In this study RGO, Ag/RGO, Ag/ SiO2/RGO, ZnTiO3, ZnTiO3/SiO2/RGO and Ag/ZnO/ZnTiO3were entrapped in a porous collagen matrix by sol-gel cryogen drying to preserve the native biological activity of the collagen. No chemical interactions were expected in the Collagen/antimicrobial compounds nanocomposites under these conditions.The nanocomposites were prepared in different ratios of the compounds.The formed sterile zones around the disc samples (diameter of 9.0 mm; thickness of 3 mm) were measured in mm (±0.5). The representative of eukaryotic organisms Candida lusitaniaе demonstrated high sensitivity with large sterile areas at some of the tested materials.

Key words: Pathogenic fungi; Collagen matrix; Nanoparticles; Zone of inhibition; RGO- Reduced Graphene oxide

Nanoscience has been emerged as a powerful tool to develop new approaches in the field of designing new antimicrobial drugs. Antifungal potential of nanoparticles should be considered in two directions connected to the seriousness and relevance of the issues. On the one hand is the clinical significance of fungal strains as C.albicans, T.asahii, T.mentagrophytes, A.niger, representing a direct threat to human health, causing skin, eye and otherdiseases. On the other handthe nanotechnology could be an alternative to deal with massive losses caused by phytopathogenic fungi such as Phomadestructiva, Curvularialunata, Alternaria alternate, Fusarium oxysporumetc. In striving to feed the increasing global population [1].

Candida albicans is the most common pathogenic fungus isolated in bloodstream infections in hospitalized patients, and candidiasis represents the fourth most common infection in United States hospitals, mostly due to the increasing numbers of immune- and medically-compromised patients. C. albicans has the ability to form biofilms and morphogenetic conversions between oval cells and hyphal morphologies contribute to biofilm development. Moreover, these attached communities of cells are surrounded by a protective exopolymeric matrix that effectively shelters Candida against the action of antifungals. Because of dismal outcomes, novel antifungal strategies, and in particular those targeting biofilms are urgently required. As fungi are eukaryotic, research and development of new antifungal agents is difficult due to the limited number of selective targets, also leading to toxicityto macro-organism [2].

Similar to Candida,Trichosporonasahii is an emerging fungal pathogen and exist in several growth forms in response to environmental conditions[3].T.asahii can invade the human body through implantable catheters, abraded skins, mucosa, or respiratory tract, causing systematic and fatal trichosporosis and also has shown drug resistance[4, 5,6].

Other important dermatophyte fungus is Trichophyton [7, 8]. Trichophyton rubrum (T. rubrum) is known to account for as many as 69.5% of all dermatophyte infections [9, 10, 11,12].The results of antifungal activity reveal that the growth of T. rubrum was inhibited atconcentration of 10 μg/ml Ag-NPsalone. In combination tests, fluconazole (20 μg/ml) together with Ag-NPs (2.5 μg/ml) and griseofulvin (0.4 μg/ml) with Ag-NPs (2.5 μg/ ml) demonstrated increasing antifungal effects on T. rubrum [13].

The problem with ocular pathogenic fungi, which causes keratomycosis increase because they are responsible for vision loss in the developing world like China [14]. Clinical studies indicate that keratomycosis constitutes about 46.7% to 61.9% of all cases of suppurative keratitis inpatients. Filamentous fungi, mainly Fusarium spp. or Aspergillus spp., are the most frequently isolated fungi in keratomycosis and the most common ocular pathogenic fungi. Fluconazole has high bioavailability against Candida spp., but Fusarium spp. and Aspergillus spp. are resistant to it [14, 15, and 16]. Other antifungal is Natamycin, but it penetrates the cornea and conjunctiva poorly and effective drug levels are not achieved in corneas with intact epithelium. The penetrationfor amphotericin B was negligibleafter topical application because it was poorly soluble in water [16]. According to Gao et al [17]. The activity of nano-silver against Fusarium spp.,Aspergillus spp., and Alternariaalternata was significantly superior to those ofnatamycin and fluconazole against ocular pathogenic fungi in vitro.Previous studies demonstrate significant antifungal activity of Nano-Ag, in an IC80 range of 1-7 μg/ml against T. mentagrophytes and Candida species[18].

Candida spp. represent one of the most common fungal pathogens often causing hospital-acquired sepsis with an associated mortality rate of up to 40% [19].Little is known about the epidemiology of infection with C. lusitaniae[20, 21]. Candida lusitaniae was originally isolated from the intestinal contents of warm-blooded animals [22]. In humans, C. lusitaniae rarely causes opportunistic infections, although 13 cases involving various sites, including the kidneys, peritoneum, and blood stream, have been described [20, 21, 22, 23, and 24]. This species of Candida is of special interest because of its innate resistance to amphotericin B and its ability to develop resistance to amphotericin during therapy [23, 25, and 26].A literaturereview [24] on C. lusitaniae infections update and better characterize the illness in the era of azole availability and standardized methodologies for antifungal susceptibility testing. In this review C. lusitaniae infection occurred in relatively young patients in a 55 cases (median age, 44 years). Fungemia was found in 80% of patients. Other infection syndromes, including peritonitis, meningitis, and urinary tract infection, were much less common [24].

Antimicrobial effect of silver nanoparticles (Ag-NPs) were investigated on many bacteria and yeast more than 60 yearsalready, butthe mechanisms of action are still not fully understood [27]. The nanoparticles attack the respiratory chain, cell division and finally lead to cell death [28]. Silver also forms complexes with various DNA bases (adenine, guanine, cytosine and thymine) and is a potent inhibitor of fungal DNA ases and in the same time it could form complexes with electron donors containing Sulphur, oxygen or nitrogen (thiols, carboxylates, phosphates, hydroxyl, amines, imidazoles, indoles). The inhibition action of silver ions may include displacing of native metal cations from their usual binding sites in enzymes. The possible mechanism of action of Ag NPs has been suggested according to the morphological and structural changes in microbial cells. The Ag NPs show efficient antimicrobial properties that could be explained with their extremely large surface area, which provides better contact with microorganisms [29]. The antifungal activity of Ag NPs also includes damages of fungalsporesin nanocomposits with silica or organic material as pullulan [30, 31]. Silver nanoparticles may inhibit the growth of T. asahii by permeating the fungal cell and damaging the cell wall and cellular components, in concentration of 4μg/ml. SEM Images have shown the deformed, distorted and shrunk and fracturedmycelium, with damaged surface, leading to the outflow of intracellular components and shrinkage of mycelium [31].

Zamperini et al [32]reporteda membrane depolarization of Candida albicans, an arrest of the fungal cell cycle by flow cytometry, release of intracellular glucose and trehalose, a decrease in plasma membrane fluorescence with increasing concentrations of Ag nanoparticles,pits in the cell wall, and pores in the plasma membrane on the TEM images. Other findings demonstrate that Ag nanoparticles destruct the fungal membrane integrity,dissipating the electrical potential of the membrane and inhibiting the normal budding process[33, 34].AgNPs inhibited the growth of A. flavus by affecting cellular functions which caused deformation in fungal hyphae, reduction in spores number, malformation and hypertrophy, leading to destruction and damage of spores [35].

C. albicans and C. tropicalis showed high sensitivity to AgNPs, Kim et al. shows the inhibition of C. albicans at 2 μg/ ml concentration of Ag-NPs[36].Other authors reports ten to 400 times higher concentrations, depend on their size and ways to obtainAg-NPs[18, 19, 37].The combination betweenfluconazoleand AgNp(32.5 nm average size spherical AgNp obtained by extracellular biosynthesis by the fungus Alternaria)showed the maximum inhibition against C. albicans, followed by Phomaglomerata and Trichoderma spp., whereas no significant enhancement of activity was found against Pleosporaherbarum and F. semitectum [38].

It was also determined that AgNp were more effective in reducing biofilm biomass when applied to adhered cells (2 h) than to pre-formed biofilms (48 h), with the exception of C. glabrata, which in both cases showed a reduction of ˜90%. AgNp were highly effective on adhered C. globate and respective biofilms. On C. albicans the effect was evident as a reduction in the number of viable biofilm cells. These results suggest that AgNp could be an effective alternative to conventional antifungal agents for therapies in Candida associated denture stomatitis [39].

AgNp at ultralow doses of 0.001% was effective against C. albicans, C. globate and M. sympodialis, yeast species often found in atopic dermatitis patients. Despite its 3.4-times lower silver content, the AgNp preparation exhibits an antimicrobial activity against the bacteria yeasts and dermatophytes tested, comparable to that of silver sulfadiazine at concentrations of 0.1% [40].

Li et al[41].evaluate the surface morphology change of the native and treated C. albicans and C. tropicalis with the prepared GO nanoparticles.After treatment with pure GO for 24 h, the yeast cells were covered by a single layer wrinkled GO. After contact with carbon nanoscrolls/AgNPs, the yeast morphology significantly changed, with a major damage in cytoplasmic membrane of C. albicans, the content of the yeast cell leaked completely.The similar phenomena also can be seen in C. tropical is cells, as the ionic silver released, and a distinct concave could be observed on the cell membrane. When the C. tropical is cell was covered by a non-AgNPs content GO nanosheets the cell membrane kept relative integrity, but with GO/AgNPsthe membrane of the fungal cells collapsed apparently and the cytoplasm released partially [41].

The antimicrobial activity of zinc oxide nanoparticles (ZnONPs) is mainly due to generation of highly reactive species like OH^¯, H₂O₂, O₂ 2¯. H₂O₂ penetrates the cell and OH¯ and O₂ ^2¯damages the cell wall and cell membrane from outside [42]. The positive charged ZnONps were considered to have close physical interaction with fungal cells by direct electrostatic adsorption. As a result, cell membrane damage and cellular internalization were promoted [43]. The penetration of ZnONPs could cause more ZnO-induced oxidative damage intracellular than outside the cells.A lot of studies have shown that ZnONPs induced the generation of ROS which cause oxidative stress [42, 43, 44, 45].Though excessive ROS-generation imposed unacceptable oxidative stress to cells that result in cell damage, small amount ROS can be tolerated by most cell types [44].ZnO NPs has a very good antifungal activity against different types of yeast including Candida spp, Trichosporonspp, Geotrichumsppand S. cerevisiae. The ZnO NPs were also effective against fluconazole resistant Candida isolates [46, 47, and 48].Candidalusitaniae is important nosocomial pathogen; it is acquired by indirect contact transmission between patients with transplantations and other immunocompromised patients in medical intensive care units [21, 49].

Sponge-like matrices show significant potential for the regeneration and repair of a broad range of damaged anisotropic tissues.The manipulation of the structure of collagen scaffolds using a freeze-drying technique is an intrinsically biocompatible way of tailoring the inner architecture of the scaffold[50]. The Collagen matrix impregnated with antibacterial agents is attractive candidates for wound dressing, tissue engineering, components of implantable devices, etc [51].

No reports about sensitivity of Candidalusitaniae to the nanocomposites of ZnO, ZnTiO₃, RGO and Ag in different ratio combinations with Collagentill now, which provoked us to conduct this investigation.

The used in this study RGO (with less that 2-3 w % impurities of graphite materials) was prepared by commonly used chemical exfoliation method starting from purified natural graphite powder (99.9 %, Alfa Aesar Co.) and employing sodium borohydride as a reducing agent, as it was described in [52].

The phase formation and structural transformation were detected by X-ray phase analysis (Bruker D8 Advance, Germany; Cu Kα; Lynx Eye detector). extracted from calf hide using previously described technology [53]. The concentration of the collagen gel was adjusted at 1% and pH at 7.3 (that of the physiological medium) using 1M sodium hydroxide and antimicrobial agent (RGO powder) in 2:1, 2:0.8, 2:0.6, 2:0.4 or 2:0.2 ratios (wt/wt) was added. The in this way prepared collagen/antimicrobial agent composites were cross-linked with 0.5 % glutaraldehyde (to dry collagen) at 40C for 24 h and then lyophilized at -400C to obtain porous (sponge) material using a Martin Christ freeze-dryer for 48 hours, as it was previously described [54, 55]. Test samples with diameter of 9 mm were prepared after that from each composite.RGO aggregates with different dimensions are wrapped in the collagen matrix, some of them partially covered by matrix collagen.The compressive modulus at 10 % deformation was estimated of the studied Collagen/RGO composites, expecting that the presence of RGO could influence their mechanical strength [56].

In these study nanocomposites with compounds asRGO, Ag/RGO, Ag/SiO₂/RGO, ZnTiO₃, ZnTiO₃ /SiO₂/RGO and Ag/ ZnO/ZnTiO₃ were impregnated in collagen matrix in different ratios and were examined as antifungal agent against Candida lusitaniae. The test fungal strain was obtained by National Bank of Industrial Microorganisms and Cell Cultures, propagated in YGS at 36°C and 120 rpm. At 12 h [57]. Microbial density of 0.5-0, 8 were determined according to McFarland. The aliquots of 100 μL microbial suspension was randomly spread on solid medium (YGS agar) and discs of investigated material were put on them. The plates were left for 20 h at 4-60 C to afford diffusion of the nanoparticles and after that cultivated for 24 h at 360 C. The formed sterile zones around the disks samples were measured in mm (±0.5). Minimum two till 4 replicates for every composite were tested and mean values presented in the figures.

The antifungal activity, as a sterile zone in mm, of the studied antimicrobial agents loaded in collagen sponges is presented in Figures 1 to 6.

The used in this study RGO, consists of multilayer (up to 5) sheets with relatively large area (up to about 10 x 20 μm) that tend to aggregate. They were dispersed in a collagen gel to be formed porous collagen/RGO composite after a cryogen drying, the last one keeping the native biological activity of the collagen. No chemical interactions between RGO and the collagen matrix were expected under these conditions. Aggregates of RGO sheets were wrapped in the collagen matrix, some of them partially coated by matrix collagen, as depicted by SEM images.

Although that the mechanism of the antimicrobial activity of RGO is not fully understood, it is generally accepted that it includesan effect of the direct cell membrane contact with sharp RGO nanosheets [58, 59]. In addition, destructive extraction of large amount phospholipids from E. coli cell membrane by graphene nanosheets (due to strong dispersion interactions between the RGO and the lipid molecules) is shown as a reason for the antibacterial activity of the graphene nanosheets [60].

In result of the test the antibacterial effect of RGO on the representative eukaryotic organisms Candida lusitaniaе demonstrated high sensitivity with large sterile areas (data was shown in Figure 1) at themaximum tested concentration of RGO and small ones at the lowest of the tested concentrations. The high sensitivity of fungus is probably due to usage of nutrient medium with antibiotic Chloramphenicoland combination of

both antimicrobial agents: antibiotic and nanosheets. Such kind of interaction is reported by [61, 62, and 63].

As a result of the current study it can be concluded, that the inhibition effects of the studied collagen-based nanocompositeson the fungal growth of C. lusitaniaеdepends on the type of compoundsincluded in thenanocomposites and their concentration. The results from diffusion method in solid mediumindicate that some of the studied nanomaterials have strong pronounced effect on C.lusitaniaеwhich are a close relative of the resistant to antibiotics Candida albicans. Thus we can say, that the studied collagen-based nanocomposites are promising anti-infective biomaterials.

Bulgarian Scientific Fund is acknowledged for the financial support of this investigation (grand DCOST 01/14 /16.08.2016.) that is in the frame of COST Action TD1305 "Improved protection of medical devices against infections".

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