Amoebicidal Activity of Poly-Epsilon-Lysine Functionalized Hydrogels

Purpose To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii. Methods A. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0–2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid–Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration. Results The toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL. Conclusions pɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.

A canthamoeba keratitis (AK) is a rare infection but may result in severe visual impairment or blindness. 1,2 Acanthamoeba are opportunistic protozoan, amoebic parasites that live naturally in soil, air, and aquatic habitats in one of two forms: trophozoites (mobile active form) or cysts (highly resilient, double-walled, dormant cysts). 3 Trophozoites and cysts are both able to infect the cornea, skin, and central nervous system. 4 The conversion of trophozoites into cysts results from adverse environmental conditions. 5,6 The corneal epithelium normally provides a barrier to invading pathogens, but Acanthamoeba invade the cornea through a breech in the corneal epithelium. Contact lens (CL) wear is a significant risk factor for the development of AK, and up to 80% of cases of AK are associated with the use of CLs or lens solutions contaminated with waterborne Acanthamoeba. In particular, there has been an increase in AK associated with the increased use of soft CLs. [7][8][9] In addition, a lack of compounds effective against Acanthamoeba cysts in CL solutions and tap water quality are also contributing risk factors of AK in CL wearers. [10][11][12][13] To initiate infection, trophozoites adhere to the corneal epithelium via mannose-decorated glycoproteins and invade the cornea as enzymes such as matrix metalloproteases, serine, and cysteine proteases degrade the corneal stroma. 14 Within the stroma, trophozoites may encyst, which may lead to a persistent, relapsing keratitis. 15 Successful treatment of AK requires early diagnosis, 16 but the diagnosis is challenging and may be mistaken for a bacterial, viral, or fungal keratitis. Treatment of established AK is difficult and currently reliant on intensive treatment with chlorohexidine, polyhexamethylene biguanide, brolene, or hexamide, with variable and often poor outcomes. 17 Poly-epsilon-lysine (pεK) is a cationic peptide with intrinsic antimicrobial properties and that reportedly disrupts the cell membrane and cell wall of microbes showing broadspectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts, and fungi. [18][19][20][21] Our previous studies have demonstrated pɛK hydrogels are effective against laboratory strains of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa while having no toxicity toward corneal epithelial cells. [22][23][24] Further functionalization of the free amine groups of the hydrogel with covalently bound pɛK (pɛK + ) increased pɛK levels with a 10fold increase in amine functionality due to additional pɛK molecules when compared with nonfunctionalized hydrogels. 22 Further studies associated this increase in pɛK with increased antimicrobial activity against P. aeruginosa, reducing the number of viable bacteria in in vitro and ex vivo corneal infection models.
The aim of this study was to evaluate whether pɛK offers amoebicidal activity against Acanthamoeba in both trophozoite and cyst forms. The effects of pɛK solution, pɛK hydrogel, and pɛK + hydrogel treatment upon both trophozoite and cyst forms of Acanthamoeba were investigated at 24 hours and 7 days in vitro, and significant toxicity was demonstrated with both forms. Using an ex vivo porcine cornea model of AK, we showed that no A. castellanii were detected in the stroma after application of pɛK + or pɛK hydrogels.

Cultivation of Acanthamoeba
A. castellanii (Douglas) Page (ATCC 30234) (ATCC, Manassas, VA, USA), originally isolated from a patient with corneal keratitis, was used in the study. Trophozoite cultures were maintained in axenic culture in ATCC Medium 712 Peptone-Yeast-Glucose (PYG) medium with additives in T-75 CELL-STAR tissue culture flasks (Greiner Bio-One, Stonehouse, UK) at 25°C, according to ATCC instructions. Trophozoites in exponential growth (72-96 hours) were centrifuged at 1000 × g for 10 minutes and resuspended in PYG media. Trophozoites were counted in a disposable Millicell, Neubauer hemocytometer (Millipore, Merck, UK) and adjusted to a final concentration of 2 × 10 4 amoeba/mL in PYG media.
Cysts were prepared following centrifugation of trophozoites and pellets washed in PBS by gentle pipetting and recentrifuged to remove traces of PYG media. Trophozoites were resuspended in PBS to a final concentration of 1 × 10 6 cysts/mL, and 1 mL of trophozoite suspension was added onto nonnutrient agar plates (3% w/v agar; Sigma-Aldrich, Dorset, UK) at 28°C for 7 to 10 days until 100% encystment was observed by microscopic analysis. Cysts were harvested using a cell scraper and resuspended in PBS containing sodium dodecyl sulfate 0.5% (w/v) (Sigma-Aldrich) to solubilize any remining trophozoites, followed by centrifugation at 1000 × g for 10 minutes and resuspended at 2 × 10 4 cysts/mL in PBS.

pɛK Solution Toxicity Assay Against Trophozoites and Cysts
Trophozoites and cysts were used at 2 × 10 3 amoebae per well (100 μL) in a 96-well plate. Then, 100 μL of pɛK solutions at 2× concentration was added to each well to a final working volume of 200 μL, with a final pɛK concentration range of 0 to 4.34 mM. Chlorohexidine (CHX) (Sigma-Aldrich) at 0.02% was used as a positive control for toxicity of trophozoites and cysts. Toxicity of pɛK against A. castellanii was assessed using propidium iodide (PI) (Thermo Fisher Scientific, Loughborough, UK) added to wells at a final concentration of 1 μg/mL. Dead trophozoites/cysts imaged using a fluorescent microscope and the number of dead red-labeled A. castellanii were compared with the number of nonstained trophozoites and cysts per field of view expressed as percentage dead (%) compared with total live and dead.

pɛK Hydrogel Toxicity Assay Against Trophozoites and Cysts
Under sterile conditions, 10-mm discs of pɛK and pɛK + hydrogels or commercial hydrophilic cast-molded CL (Hydrogel CL; Filcon II 2, 77% water content; Ultravision, Leighton Buzzard, UK) were added to 48-well plates using sterile forceps. Trophozoites or cysts were used at 1 × 10 4 amoebae per well in 500 μL of PBS (nutrient deplete) or PYG media (nutrient replete) and incubated at 28°C for either 24 hours or 7 days. Toxicity of pɛK hydrogels was measured as described above following the addition of 1 μg/mL propidium iodide to label dead trophozoites and cysts.

Ex Vivo Porcine Cornea Keratitis Model
Porcine eyes were obtained from a local abattoir within 6 hours of slaughter, and corneas were processed with agarose supports to establish an air-liquid interface ex vivo organ culture model as previously described by Kennedy et al. 23,24 and Supplementary 1. Trophozoites were suspended in 10 μL PYG media containing 1 × 10 5 amoebae and pipetted onto an air-dried region of the central cornea and allowed to absorb for 30 minutes at room temperature (assessed by visual analysis). Once the droplet was no longer visible, 10-mm discs of commercial CL, pɛK, or pɛK + hydrogels were applied to corneas. Control infected and noninfected corneas were run in parallel. DMEM (+10% FBS) was added up to the scleral boundary of the cornea (∼3 mL) and incubated for 7 days at 37°C, 5% CO 2 .
Following infection, pɛK + and pɛK hydrogel lenses and commercial hydrogel CLs were removed from corneas, resuspended in PYG media, and vortexed for 30 seconds to remove A. castellanii from lenses, and the solution was transferred to a 6-well plate to monitor regrowth. Quantification of A. castellanii infection of corneas was achieved by trephining (Blink Medical Ltd, Solihull, UK) 10-mm discs from the central cornea. To isolate trophozoites, corneal discs were cut into quadrants and further quartered, resuspended in 3 mL PBS, and briefly vortexed, followed by homogenization using a Qiagen TissueRuptor (Qiagen, Manchester, UK) for 15 seconds. The homogenate was centrifuged at 1000 × g for 10 minutes, resuspended in PYG media (+ glucose 1.5% (w/v)), transferred to a well of a 6-well plate, and cultured for 7 days to monitor regrowth. A. castellanii from each experimental condition were imaged at 7 days using a Nikon (Nikon Europe BV, Netherlands) Ti-E microscope and quantified following resuspension of A. castellanii in PYG media using a hemocytometer.

Histology
A separate set of corneas from each experimental condition was fixed in 10% (v/v) neutral buffered formalin (Sigma-Aldrich) for 18 hours. Corneas were processed using a Leica ASP300 (Nussloch, Germany) tissue processor. Paraffinembedded tissue was sectioned at a thickness of 5 μm and stained with periodic acid-Schiff (PAS) (Abcam, Cambridge, UK) following the manufacturer's protocol for positive detection of chitin in the cell wall of A. castellanii. Tissue sections were imaged using a Nikon CI upright microscope using a 20× and 40× objective.

Statistical Analysis
Experiments were performed in triplicate (n = 3), with three wells per experiment and five fields of view in each well. One-way ANOVA was performed with a post hoc Tukey's analysis, and P < 0.05 was considered significant. Statistical analysis was performed using Prism software version 8.02.263 (GraphPad Software, La Jolla, CA, USA). Error bars are shown as standard deviations of biological replicates.

Amoebicidal, Both Trophozoicidal and Cysticidal, Activity of pɛK Solution Against A. castellanii
Trophozoicidal and cysticidal effects of pɛK solution (0-4.34 mM) on A. castellanii were assessed following incubation for 24 hours and 7 days. Toxicity was assessed by the number of dead, red PI-stained A. castellanii compared to nonstained live A. castellanii and expressed as percentage death at each dose.
After 24-hour pɛK treatment of trophozoites, 80% (SD ± 6.14%) toxicity was observed at 0.54 mM pɛK (P < 0.0001) compared to untreated trophozoites, and no further increase in toxicity above 80% was achieved at any higher concentrations (Fig. 1Ai). After 24 hours, trophozoites remained attached to the tissue culture plastic surface (TCPS) surface showing characteristic morphology, while treatment with CHX showed 100% toxicity. An increased dose of pɛK increased the number of dead A. castellanii, while live A. castellanii detached from the surface and did not show characteristic trophozoite morphology. At 7 days posttreatment, amoebicidal effects of pɛK upon trophozoites were observed at lower pɛK doses (Fig. 1Aii). After 7-day treatment with lower concentrations of pɛK (0.016 mM), the number of dead trophozoites increased to 99% (SD ± 1.04%) (P < 0.0001).
Effects of pɛK solution upon cysts after 24-hour treatment showed 76% (SD ± 1.50%) cyst death at 0.54 mM and above (P < 0.0001) (Fig. 1Bi). At 7 days (Fig. 1Bii), treatment with pɛK solution led to increased number of dead cysts at lower pɛK concentrations. Toxicity against cysts reached 77% (SD ± 2.14%) at 0.004 mM (P < 0.0001), and cyst death did not significantly increase (P < 0.05) further with increased pɛK concentrations. At 24 hours and 7 days, only live cysts were visible in control images, while in comparison, CHX showed red PI-stained cysts in the field of view.

Toxicity of pɛK Solution Against hCE-T Cells
Maximum toxicity of pɛK treatment toward trophozoites and cysts was observed at 0.54 mM at 24 hours and at 0.016 mM and 0.004 mM at 7 days for trophozoites and cysts, respectively. Toxicity of pɛK solution at different concentrations was determined upon confluent monolayers of hCE-T cells.
After 24-hour treatment with pɛK solution, confluent monolayers of live hCE-T cells (stained green with calcein- AM dye) were observed after application up to 0.54 mM pɛK, with comparable dead hCE-T cells (stained red with PI) to untreated controls (P > 0.05) (Figs. 2Ai, Aii). At 1.09 mM pɛK and above, live cells were reduced, with increased dead cells per image compared to untreated controls (P < 0.05). At 7 days, treatment with pɛK 0 to 0.067 mM was not toxic to hCE-T cells (P > 0.05) and, at 0.135 mM and above, proved toxic to hCE-T cells with a reduction in live hCE-T cells (P < 0.05) (Figs. 2Bi, Bii).
At 7 days, growth on pɛK + hydrogels showed 83.31% (SD ± 7.96%) toxicity against trophozoites compared to untreated controls (P < 0.0001). The total number of live and dead trophozoites decreased by 7 days compared to 24 hours, with dead trophozoites being degraded within the media, accounting for the lower percentage reduction. In comparison, trophozoites cultured on nonfunctionalized pɛK hydrogels, commercial hydrogel CL, and TCPS all showed growth of trophozoites with negligible toxicity <1% in the total population (Figs. 3Bi, Bii), with no significant difference (P < 0.05) compared to untreated controls. Treatment with CHX showed 96.01% (SD ± 4.12%) dead trophozoites and was slightly more effective compared to pɛK + hydrogels (P = 0.024).

Toxicity of pɛK Hydrogels Against A. castellanii in Ex Vivo Porcine Corneas
Porcine corneas were infected with trophozoites for 7 days in the presence and absence of commercial hydrogel CL, pɛK hydrogel, or pɛK + hydrogel (Fig. 6). Histologic analysis of fixed corneas using PAS to label A. castellanii showed there were no A. castellanii within the corneal stroma in unin-fected corneas. Infected corneas and infected in the presence of commercial hydrogel CL both showed cysts below the corneal surface. Infected corneas incubated with pɛK or pɛK + hydrogels showed an absence of A. castellanii within tissue sections.

DISCUSSION
This study demonstrated the antimicrobial peptide pɛK is effective at reducing viable A. castellanii trophozoites and cysts, both as a solution and when covalently bound to pɛK hydrogels using in vitro assays. pɛK was nontoxic to hCE-T cells at doses up to 0.54 mM at 24 hours and up to 0.067 mM at 7 days, incorporating doses that were toxic to A. castellanii. pɛK + hydrogels demonstrated toxicity toward both cysts and trophozoites compared to nonfunctionalized pɛK hydrogels and commercial hydrogel CLs. In our ex vivo porcine corneal infection model, pɛK and pɛK + hydrogels prevented infection of A. castellanii within the cornea stroma, and reduced numbers of A. castellanii adhered to hydrogels.
Current preventative treatments against AK are targeted at maintaining good hygiene regimes 15 and use of disinfectants within CL solutions. Therapeutic treatments for established AK involve the use of active reagents such as polyhexamethylene biguanide (0.02%) (or CHX 0.02%) and propamidine (0.1%) and hexamidine (0.1%), often in combination. 15,26,27 Increased resistance to many of these reagents has been reported in different strains of Acanthamoeba. 28,29 We have investigated only one particular strain of Acanthamoeba, but we have examined the effectiveness of pɛK + hydrogels at killing both trophozoites and cysts, the latter of which are particularly difficult to kill. MeniCare Pure CL solution (Menicon Ltd., Nagoya, Japan) contains pεK as an active ingredient, which is effective against Acanthamoeba. It is used as a disinfectant instead of polyhexamethylene biguanide, but this CL solution is only suitable for use with rigid gas-permeable CLs.
The effects of pɛK solution against trophozoites and cysts were investigated in both nutrient-rich and nutrient-deplete environments. Toxic effects of pɛK against A. castellanii occurred in a dose-and time-dependent manner against both cysts and trophozoites in nutrient-replete and nutrientdeplete environments. The effects of pεK were more effective against the trophozoites, which are easier to treat compared to the cysts. [30][31][32] Current treatments for prevention or treatment of an AK are typically harsh and damaging to the ocular surface, in particular the corneal epithelium. 33 We demonstrated that effective doses of pεK were toxic against trophozoites or cysts, respectively, but not to hCE-T cells. pεK is generally regarded as safe, is used in many applications, 18 and offers potential use as a treatment for AK.
There are currently no commercial CLs available that offer antimicrobial activity, and CLs themselves provide a potential route of infection into the cornea. Having established pεK solution was toxic to A. castellanii, we demonstrated pɛK + hydrogels showed toxicity at 24 hours and 7 days toward A. castellanii, compared to the commercial CLs tested. pɛK + hydrogels offer inherent antiamoebicidal (both trophozoicidal and cysticidal) activity without the potential need for topical application. CLs themselves provide a potential surface for Acanthamoeba to prolifer-  ate, and trophozoites and cysts are reported to adhere to all types of lenses. [34][35][36][37][38] Studies have shown Acanthamoeba possess an affinity for attachment to CLs (in particular soft CL) contributing toward development of AK. 9,39,40 Lee et al. 7,8 demonstrated that rigid gas-permeable CLs with a smoother, waxier, and more homogeneous surface showed decreased adhesion of Acanthamoeba. The water content and mobility of CLs are important factors in the adhesion of Acathamoeba. 41,42 Our study showed that A. castellanii can adhere and proliferate on the surface of the commercial CLs, whereas the pɛK + hydrogel reduced the number of viable A. castellanii.
In addition, the ex vivo infection model demonstrated pεK + hydrogels prevented infection by A. castellanii into the corneal stroma with no cyst formation, as was seen with commercial CLs. There was also a reduced recovery of A. castellanii from the pεK + hydrogels compared to commercial CLs or infected corneas. Teuchner et al., 43 using a porcine corneal ex vivo model, reported at 7 days there was deeper penetration of Acanthamoeba into stroma compared to 4 days with higher infection using PYG media compared to PBS. We used DMEM to ensure nutrients were supplied to the cornea during the experiment; it is not clear, therefore, if we would have observed higher rates of infection with PYG, supplying a nutrient-rich media for Acanthamoeba growth.
Antimicrobial effects of pεK are well documented, and our studies have reported antimicrobial effects of pεK + hydrogels toward Gram-positive and Gram-negative bacteria as well as fungi. 22,24,44,45 Acanthamoeba predate upon bacteria and fungi, which may be transported within Acanthamoeba, leading to a coinfection. [46][47][48] pεK hydrogels offer the ability to provide effective treatment against multiple microbes, due to functionalization of the free amine group on pεK providing innate antimicrobial properties. The current proposed mechanism of action for pεK involves the positively charged pεK peptide interacting with negatively charged cell membranes, leading to cell lysis. 18 AK is a rare but serious condition leading to visual loss, with an increase in number of cases associated with increased use of CLs. 10,49 Infection in non-CL wearers can also occur when the integrity of the ocular surface is compromised through surgery or trauma or if patients are immunocompromised. [49][50][51] CLs are important medical devices for visual and therapeutic purposes but pose a significant risk factor in the development and progression of AK. 7,13 Diagnosis and treatment of AK are difficult, and treatments that offer toxicity toward Acanthamoeba are limited, with many only effective against trophozoites and not cysts. 10 There is therefore a need for development of new therapies and approaches to prevent and treat AK and combat resistance to current Acanthamoeba treatments. 52 In summary, our data show pεK + hydrogels offer amoebicidal and cysticidal activity against A. castellanii, which could potentially lessen the risk of CL-associated AK.