Antifungal potential of some collagen-based nanocomposites Against Candida lusitaniae

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 immuneand 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].


Introduction
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.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., andAlternariaalternata 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 ofC.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].¯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 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, ZnTiO 3, RGO and Ag in different ratio combinations with Collagentill now, which provoked us to conduct this investigation.

Preparation and characterization of RGO
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).

Preparation of Collagen/ RGO composites
Type I fibril collagen gel with concentration of 2.64 wt.% was 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 4 0 C for 24 h and then lyophilized at -40 0 C 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].

Antifungal activity tests
In these study nanocomposites with compounds asRGO, Ag/RGO, Ag/SiO 2 /RGO, ZnTiO 3, ZnTiO 3 /SiO 2 /RGO and Ag/ ZnO/ZnTiO 3 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 4-6 0 C to afford diffusion of the nanoparticles and after that cultivated for 24 h at 36 0 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.

Results and discussion
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].

Antifungal potential of some collagen-based nanocomposites Against Candida lusitaniae
Copyright: © 2016 Ivanova, et al.The most pronounced results were obtained for the ratio 2:0,7and 2:0, 8.The antimicrobial effect of silver nanoparticles is well known [2, 7, 8, 13, 18, 30, 31, 33, 34, 35, 62, and 63].Our result is interesting with the ratio of two compounds -the distribution of the silver nanoparticles was more uniformly at a ratio of Collagen: Ag/RGO = 2:0.7,were the highest activity was obtained.The lower antifungal activity of higher concentration could be due to agglomeration of active nano-Ag.In the case of Coll: Ag/SiO 2 /RGOfor C. lusitaniaе the strongest effect was observed for the highest concentration and three time weaker effect for the lower ratios.Data are shown in Figure 3.As other authors mentioned earlier dual action of nanocomposites combined with antibiotic have stronger antifungal effect [64].The results of synergistic action of nanoAg, SiO 2 and RGO as a compounds of nanocomposite with collagen matrix were summarized and the data can be seen at Figure 3.The highest concentrations demonstrate significant effectiveness compared with other weight ratios 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  4).It is comparable to antifungal effect of Coll: Ag/ SiO 2 /RGO, but the effect is more stable and does not reduce so quickly as that of to the Coll: Ag/SiO 2 /RGO nanocomposite./SiO 2 /RGO nanocomposite had insignificant inhibition effect on test microorganismCandida lusitaniae,butthe other examined materials have significantly higher antifungal effect.The most pronounced results were obtained for the ratio 2:1 at the most of the tested materials but Coll: Ag/ZnO/ZnTiO 3 and Coll: Ag/RGOshow different results.The first is most effective at 2:0.8 and second nanocomposite at the ratio 2:0.7 respectively.If we compare effectiveness of the test nanomaterials, it can be concluded that the most effective against C. lusitaniaе was Chloramphenicol in the nutrient medium in combination withhold: RGO (2:1) followed by Coll: Ag/ SiO 2 /RGO (2:1)>Coll: ZnTiO3 (2:1)>Coll: Ag/ZnO/ZnTiO 3 (2:0.8)>Coll:Ag/RGO (2:0.7).Our results are comparable with these previously reported by other authors [61,62,63,64].Strongest effect of Coll: Ag/ SiO 2 /RGO nanocomposite without antibiotic in nutrient medium was possibly due to the role of SiO 2 as dispersing agent (vehiculum).It is important to mention that SiO 2 alone as micro size powder has no antibacterial effect till the concentration of 6 mg/ml (results are not presented).Noble metals have proved their activity to bacteria and fungi [65], but the supported materials are also important for the obtained antimicrobial effects.As [66] reported, the level of susceptibility of C. lusitaniae to amphotericin B did not differ much from those of C. albicansand C. glabrata, so it could be suggest similar antifungal activity of tested nanocomposites to the other fungal pathogens.

Figure 1 :
Figure 1: Antifungal effect of nanocomposite RGOin different weight ratio with collagen matrix -the size of the sterile zone around the disk.Antimicrobial effect of nanocomposites Ag/RGO in different ratios with collagen porous nanocompositescan is seen at Figure 2.Candida lusitaniaе growth was inhibited of the all four different concentrations.The most pronounced results were obtained for the ratio 2:0,7and 2:0, 8.The antimicrobial effect of silver nanoparticles is well known[2, 7, 8,  13, 18, 30, 31, 33, 34, 35, 62, and 63].Our result is interesting with the ratio of two compounds -the distribution of the silver nanoparticles was more uniformly at a ratio of Collagen: Ag/RGO = 2:0.7,were the highest activity was obtained.The lower antifungal activity of higher concentration could be due to agglomeration of active nano-Ag.

Figure 2 :
Figure 2: Antifungal effect of nanocompositeAg/RGO in different weight ratio with collagen matrix (the size of the disk is not included).In the case of Coll: Ag/SiO 2 /RGOfor C. lusitaniaе the strongest effect was observed for the highest concentration and three time weaker effect for the lower ratios.Data are shown in Figure3.As other authors mentioned earlier dual action of nanocomposites combined with antibiotic have stronger antifungal effect[64].The results of synergistic action of nanoAg, SiO 2 and RGO as a compounds of nanocomposite with collagen matrix were summarized and the data can be seen at Figure3.The highest concentrations demonstrate significant effectiveness compared with other weight ratios

Figure 3 :
Figure 3: Antifungal effect of nanocompositeAg/SiO 2 /RGOin different weight ratio with collagen matrix -the size of the sterile zone around the disk.Collagen/ZnTiO 3 nanocomposite has shown antifungal activity at higher tested concentrationsagainst Candida lusitaniae.ofCollagen: ZnTiO 3 = 2:1(Figure4).It is comparable to antifungal effect of Coll: Ag/ SiO 2 /RGO, but the effect is more stable and does not reduce so quickly as that of to the Coll: Ag/SiO 2 /RGO nanocomposite.

Figure 4 :
Figure 4: Antifungal effect of nanocomposite ZnTiO 3 in different weight ratio with collagen matrix (the size of the disk is not included).In the contrast the highest antifungal effect of nanocompositeColl: Ag/ZnO/ ZnTiO 3 was not at highest concentration of the active compounds to the collagen.The results may be due to agglomeration of nanoparticles.The results were illustrated at Figure 5.

Figure 5 : 3 /
Figure 5: Antifungal effect of nanocomposite Ag/ZnO/ZnTiO 3 in different weight ratio with collagen matrix (the size of the disk is not included).Antibacterial effect of nanocomposite ZnTiO3/SiO 2 /RGO on the tested fungal strain was the weakest by all tested nanomaterials as can be seen from Figure6.The result means possible gentle effect not only to the fungus, but also to the other eukaryotic cells and could be the aim of the further investigation.

Figure 6 : 3 /
Figure 6: Antifungal effect of nanocomposite ZnTiO 3 /SiO2/RGO in different weight ratio with collagen matrix-the size of the sterile zone around the disk.The Coll:ZnTiO