Tannic acid-modied silver nanoparticles enhance anti-Acanthamoeba activity without increasing cytotoxicity of the contact lens solutions

Background: Free living amoebae of Acanthamoeba genus are cosmopolitan, widely distributed protozoans causing severe, vision-threatening corneal infection known as Acanthamoeba keratitis (AK). Majority of the increasing number of AK cases are associated with contact lenses use. Due to lack of effective therapies against AK, proper eye hygiene and effective contact lenses disinfection are crucial in prevention of this infection. Currently available multipurpose contact lens disinfection systems are not fully effective against Acanthamoeba trophozoites and cysts. There is an urgent need to increase the disinfecting activity of these systems to prevent Acanthamoeba keratitis infections. Synthesized nanoparticles have been recently studied and proposed as a new generation of anti-microbial agents. It is also known that plant metabolites, including tannins, present anti-parasitic activity. The aim of this study was to evaluate the anti-amoebic activity and cytotoxicity of the tannic acid-modied silver nanoparticles (AgTANPs) conjugated with selected multipurpose contact lens solutions. Methods: The anti-amoebic activity of pure contact lens care solutions and nanoparticles conjugated with contact lens care solutions were examined in vitro by colorimetric assay, based on the oxido-reduction of AlamarBlue. The cytotoxicity assays were performed using a broblast HS-5 (ATCC CRL-11882) cell line. Results were statistically analyzed by ANOVA and Student-Newman-Keuls tests using the p<0.05 level of statistical signicance. Results: The obtained results showed that nanoparticles enhanced anti-Acanthamoeba activity of the tested contact lens solutions without increasing their cytotoxicity prole. The activity is enhanced within minimal disinfection time recommended by the manufacturer. Conclusions: The conjugation of selected contact lens solutions with AgTANPs might be a novel and promising approach as part of preventive actions of Acanthamoeba keratitis infections among contact lens users.


Background
Amoebae of the Acanthamoeba genus are free-living, cosmopolitan protozoans with broad occurrence spectrum and various degrees of pathogenicity. They are ubiquitous in both natural and man created environment. As facultative human parasites, when transmitted form the environment into the eye surface, may cause progressive, sight-threatening corneal infection known as Acanthamoeba keratitis [1]- [5]. Unproper use and disinfection of contact lenses, corneal damages and exposure of eyes to the polluted water are the primary risk factors of the AK. Lack of speci c symptoms in the early stage of infection, and co-infections with other microorganisms cause serious diagnostic di culties and delay the treatment. The number of AK infections have been increasing worldwide. There is no dedicated effective therapy against AK. Current therapeutic approaches are limited to prolonged application of diamides and biguanides. The treatment is often unsuccessful and very toxic to the eye [6]- [9]. Amoebae trophozoites may attach to the surface of both, contact lenses and contact lenses storage cases. Multipurpose contact lens disinfection systems are not effective against Acanthamoeba and need improvement on their antiamoebic activity [10]- [12]. Summarizing, the prevention, including proper eye hygiene and effective contact lenses disinfection seem to be the best approaches to limit the AK incidence.
In the recent years fast development of nanotechnology has been observed. Synthetized nanoparticles (NPs) are currently proposed as a new generation of anti-bacterial, anti-viral and anti-fungal agents [13], [14]. Moreover, nanoparticles activity against different protozoans such as Giardia intestinalis, Entamoeba histolytica, Cryptosporidium parvum and Leishmania spp. has been already con rmed [15]- [17]. Plant metabolites, including tannins, present anti-microbial activity. Tannins are polyphenolic plants metabolites with con rmed anti-obesity, anti-diabetes, anti-oxidant and anti-microbial activity. They are capable to form insoluble complexes with nucleic acids, carbohydrates, proteins and to chelate metal ions. Tannic acid (penta-m-digalloyl glucose) is the simplest, hydrolysable tannin with con rmed antibacterial, anti-cancer and anti-oxidant activity [18]- [21]. In our previous studies we demonstrated that tannic AgTANPs were well absorbed and showed anti-amoebic activity against Acanthamoeba strains belonging to T4 genotype [22]. Other authors con rmed that nanoparticles enhance anti-amoebic effect of biguanides such as chlorhexidine digluconate and other therapeutic compounds [23]- [25]. The aim of this study was to evaluate the activity and cytotoxicity of AgTANPs conjugated with selected multipurpose contact lens solutions against the trophozoite stage of a strain of Acanthamoeba castellanii T4 genotype. Methods 2.1. cultivation of the strain ATCC 30010 type A.castellanii Neff strain was cultured axenically in 25 cm 2 culture tissue asks (without shaking) at 27 °C in PYG medium [0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose] containing gentamicin 10 mg/mL in the Department of Medical Biology, Medical University of Warsaw, Poland. Amoebae were subcultured twice a month and observed for their growth under direct light microscope using a Bürker chamber (haemocytometer).
The long-term stability of the colloidal dispersions of al tested NPs (zeta potential) was measured and con rmed by the electrophoretic light-scattering method with a Zetasizer Nano ZS, model ZEN3500 (Malvern Instruments, Worcestershire, UK) [24], [26]. The size and shape of AgTANPs were determined by using the high-resolution scanning transmission electron microscopy (HR-STEM) technique (Fig. 1). Measurements were taken with a scanning electron microscope (Nova NanoSEM 450, FEI) using transmission mode (STEM II) at an accelerating voltage of 30 kV. Samples for HR-STEM investigations were prepared as follows: a drop of colloid was deposited onto carbon-coated copper grids (300 mesh) and left for 2 h for solvent evaporation. The well-dispersed nano uids were used as a stock solution and were appropriately diluted to various concentrations ranging between 0.25-2.5 ppm and used in subsequent activity and cytotoxicity assays.  Table 1. All multipurpose solutions used in the study were purchased from authorized agents.

activity assays
Pure contact lens solutions and nanoparticles at concentrations of 0.25, 0.5, 1.25, and 2.5 ppm conjugated with contact lens care solutions were examined in vitro and assessed for their anti-amoebic activity. To determine the anti-amoebic e cacy on trophozoites (log growth phase after 6 days following sub-culturing), a previously described colorimetric 96-well microtitre plate assay, based on the oxidoreduction of AlamarBlue was used [27]. Subsequently, the plates were analysed over a period of 6 h, 24

cytotoxicity
Brie y, the cytotoxicity assays were performed using a broblast HS-5 (ATCC CRL-11882) cell line as described in our previous studies [22]. A commercial kit for the evaluation of drug-induced cytotoxic effects based on the measurement of lactate dehydrogenase (LDH) activity released to the media (Pierce LDH cytotoxicity assay kit 88953, 88954) was used as per protocol. Fibroblasts were incubated with each of contact lens solution separately and contact lens solution + nanoparticles added in the same concentration as in the activity assays. To calculate the % of cytotoxicity, absorbance was measured at 490 nm and 680 nm.

statistical analysis
All the experiments were performed three times in triplicate. For all activity and cytotoxicity detailed results standard deviation (SD) and mean values were calculated. Results were statistically analyzed by ANOVA and Student-Newman-Keuls tests using the p < 0.05 level of statistical signi cance. For the not statistically signi cant results, "no activity" comment was added in the Tables 2 and 3.

activity
Obtained results con rmed insu cient anti-amoebic effect of the tested contact lens solutions against Acanthamoeba trophozoites. Anti-amoebic activity was revealed for SCA and reached 32% of inhibition after 6 h of incubation. ReNu and O-F did not show anti-amoebic effect on the tested Acanthamoeba strain within the rst 24 h of incubation. The detailed data are shown in the Table 2.
AgTANPs signi cantly enhanced anti-Acanthamoeba activity of the tested contact lens solutions. Speci cally, AgTANPs conjugated with SCA, after the minimal disinfection time recommended by the manufacturers (6 h), showed the most promising dose dependent increase of the amoebae inhibition (Fig. 2). Similar anti-amoebic effect was achieved for AgTANPs conjugated with ReNu (Fig. 3). The enhanced anti-amoebic effect of both conjugates last up to 96 h of incubation. O-F conjugated with the nanoparticles did not show any enhanced effect during the rst 24 h of incubation (Fig. 4). The antiamoebic effect was revealed just after 48 h of incubation. The detailed results are shown in the Table 3.

cytotoxicity
The overall cytotoxicity measured for SCA and O-F was similar and reached 36%. The cytotoxicity of ReNu reached 26%. Cytotoxicity values of nanoparticles conjugated with contact lens solutions comparing to pure contact lens solutions were not statistically signi cant. The cytotoxicity results are listed in Table 4.

Discussion
In the recent years, AK incidents have been increasingly recognized worldwide. Available anti-amoebic therapies are not fully effective and results in high cytotoxicity to the human eye. The main key predisposing factor for AK is contact lens use. Effective contact lens disinfection is the best approach to minimize the number of AK incidences. In this study, we tested multipurpose contact lens disinfecting systems containing different active ingredients but characterized by similar mode of action, resulting in cell membrane perturbation (Table 1) The obtained results con rmed lack of amoebicidal activity of all tested multipurpose contact lens solutions against Acanthamoeba strain. Our results are in accordance with other publications and showed that disinfecting capabilities of market available contact lens solutions are insu cient [11], [12], [28]- [31].
Fast development of nanotechnology showed signi cant anti-microbial potential of the nanoparticles, especially silver nanoparticles (AgNPs) [14], [32], [33]. Speci c mechanism of action of AgNPs is still not entirely understood, however recent studies conducted on bacteria, shed more light on this process. We know that nanoparticles cause damages leading to disruption of membrane permeability and alteration of the respiratory functions of the cell. This process eventually leads to disruption of the cell integrity. After crossing the cell membrane, nanoparticles can penetrate inside the cell and interact with DNA, RNA and proteins altering both transcription and translation processes. Presence of nanoparticles in the cell matrix raises the oxidative stress. The intracellular damages and disruption of enzymatic pathways are done by the free radicals. Altogether, nanoparticles cause cytotoxic effects and nally lead to the cell death. Cytotoxicity of AgNPs depends on their physico-chemical properties such as size and density. Typically, smaller nanoparticles have relatively increased stability and enhanced anti-microbial activity. Similarly, higher concentrations of nanoparticles show increased anti-microbial activity. However, this property is strictly correlated to the tested microbial species and type of nanoparticles used. Shape of the nanoparticles has not been proved to be crucial factor in uencing the anti-microbial activity. Some authors showed that truncated triangular or similar geometries such as hexagonal and octahedral shape of the AgNPs are more effective against bacteria while other authors reported that shape of AgNPs does not have any in uence on their activity [34]- [36]. Recent publications showed, that nanoparticles can prolong the ocular retention of some topical drugs, thus enabling treatment of eye diseases using reduced drug dosages [37], [38]. It was con rmed, that nanoparticles coated on the contact lenses caused signi cant reduction in microbial colonization on the contact lens surface [39]. Contact lenses impregnated with AgNPs, after 6 h of incubation, did not exhibit desirable anti-bacterial activity against Staphylococcus aureus while demonstrated excellent anti-bacterial effects against Pseudomonas aeruginosa [40]. Silver-impregnated lens cases showed lower proportion of microbial contamination compared to the control cases. Most microorganisms isolated from silver-impregnated cases were members of the normal skin ora [41].
There are just few studies that examined nanoparticles in uence against Acanthamoeba spp. Cobalt nanoparticles have been studied for their anti-amoebic potential and con rmed that hexagonal micro akes showed the most promising anti-Acanthamoeba effects compared to nano akes and granular cobalt nanoparticles. Apart from the concentration and size, also composition and morphology of the tested noncompounds determined their anti-amoebic activity [42], [43]. AgNPs are well absorbed by the Acanthamoeba trophozoites and integrate in the cell matrix. The nanoparticles decrease trophozoites viability and alter their metabolic activity on the dose dependent manner [44]. In our previous studies we con rmed, that AgNPs conjugated with contact lens solutions showed dose dependent enhanced antiamoebic activity [45]. Recently published studies con rmed enhanced anti-microbial effect of silver and gold (AuNPs) nanoparticles conjugated with commonly used drugs like chlorhexidine, uconazole or amphotericin B as well as with some disinfectants [25], [46]. Guanabenz, a drug already approved for hypertension that crosses the blood − brain barrier, conjugated with AuNPs and AgNPs showed signi cant anti-amoebic activity against both A.castellanii and Neagleria fowleri. Signi cant reduction in the host cell cytopathogenicity, especially for silver nanoconjugates, was revealed and associated with negligible cytotoxicity against human cells [47].
In the 21st century, eco-friendly and cost-effective bio-nanotechnology techniques are used to prepare anti-microbial active conjugates as potential candidates to eradicate infections and reduce microbial contaminations of a healthcare equipment including contact lenses. The integration and conjugation of bioactive agents into nanomaterials was tested mainly for their anti-bacterial activities. Green synthesis of AgNPs, AuNPs and platinum (PtNPs) nanoparticles showed enhanced anti-bacterial activity after combining with different classes of antibiotics [48]. Biosynthesis of AgNPs with the plant extract of Salvia spinosa resulted in increased bactericidal activity against Gram-positive and Gram-negative bacteria [49]. Novel conjugates using biogenic AgNPs from Convolvulus arvensi extract and chitosan showed anti-microbial, anti-bio lm, and anti-cancer potentialities [50]. Extract of Oscillatoria limnetica conjugated with silver nanoparticles exhibited strong anti-bacterial activity against multidrug-resistant bacteria as well as cytotoxic effects against both human breast cell line and human colon cancer cell line [51]. Synthesis of silver chloride nanoparticles (AgCl-NPs), using walnut green husk extract as well as silver nanoparticles with Peganum harmala L leaf extract resulted in signi cant inhibitory effects against Escherichia coli and S.aureus clinical isolates [52], [53]. Bio-nanotechnology has been not studied on protozoan species extensively. There are just a few published studies focusing on the in uence of nanoparticles conjugated with plants extracts on amoebae. Studies performed on the Jatropha curcas, Jatropha gossypifolia and Euphorbia milii extracts combined with nanoparticles exhibited that such combination caused signi cant reduction of the Acanthamoeba trophozoites with low cytotoxic effect to human cells [23]. In our previous studies we con rmed that tannic acid-modi ed silver nanoparticles showed increased anti-amoebic activity and less cytotoxicity to human cells in comparison to the pure silver nanoparticles [22]. In this study we revealed that tannic acid-modi ed silver nanoparticles conjugated with contact lens solutions exhibited even better anti-amoebic activity in relation to the cytotoxicity than in results obtained in our previous studies where we tested pure silver nanoparticles conjugates [45]. We conclude that differences in the anti-amoebic activity of the tested conjugates may be mainly driven by the anti-amoebic activity of the pure contact lens solutions. Nanoparticles in the tested concentration seem to enhance the already existing anti-amoebic potential of the selected contact lens solution.

Conclusions
In this study we showed dose dependent enhanced anti-amoebic effect of the tannic acid-modi ed silver nanoparticles (AgTANPs) conjugated with SCA and ReNu solutions against Acanthamoeba T4 strain. The promising results were obtained within the minimal disinfection time recommended by the manufacturers (6 h) and without increased toxicity to the human cells. Summarizing, conjugation of selected contact lens solutions with AgTANPs might be a promising approach to prevent Acanthamoeba keratitis infections among contact lens users. Nevertheless, further studies should be conducted to elucidate stability of the conjugation and activity against Acanthamoeba spp. cysts.  Figure 1 HR-STEM image of AgTANPs distribution and diameter. Anti-Acanthamoeba activity of AgTANPs conjugated with O-F contact lens solution after 6h of incubation in relation to cytotoxicity.