In Vitro Amoebicidal Activity of Titanium Dioxide/UV-A Combination Against Acanthamoeba

Immunology and Microbiology In Vitro Amoebicidal Activity of Titanium Dioxide/UV-A Combination Against Acanthamoeba

Gabrielle Gomart,1,2 Julie Denis,2 Tristan Bourcier,1,3 Anne Dory,4 Ahmed Abou-Bacar,2 Ermanno Candolﬁ,2 and Arnaud Sauer1,3 1Service d’Ophtalmologie, Hopitauxˆ Universitaires de Strasbourg, Nouvel Hopitalˆ Civil, Strasbourg, France 2Institut de Parasitologie et de Pathologie Tropicale de Strasbourg, Fed´ eration´ de Medecine´ Translationnelle de Strasbourg, Strasbourg, France 3Fed´ eration´ de Medecine´ Translationnelle de Strasbourg (FMTS), Universite´ de Strasbourg, Strasbourg, France 4Service de Pharmacie, Hopitauxˆ Universitaires de Strasbourg, Nouvel Hopitalˆ Civil, Strasbourg, France

Correspondence: Gabrielle Gomart, PURPOSE. To assess the amoebicidal effect of titanium dioxide (TiO2)/UV-A combination Service d’Ophtalmologie, Hopitauxˆ against Acanthamoeba sp trophozoites and cysts. Universitaires de Strasbourg, Nouvel ETHODS The amoebicidal effect of the TiO /UV-A combination was tested on trophozoites and Hopitalˆ Civil, 1 Place de L’Hopital,ˆ BP M . 2 426, Strasbourg 67091 Cedex, France; cysts of clinical isolates of Acanthamoeba hatchetti and Acanthamoeba sp genotype T4, [email protected]. obtained from two severe cases of ulcerative keratitis. Samples of cultured Acanthamoeba were transferred to a 96-well plate. We tested the effect of sterile water (blank control), TiO2 alone, EC and AS contributed equally to the UV-A alone, TiO2 and additional UV-A exposure, chlorhexidine 0.02% alone, chlorhexidine work presented here and should 0.02% and TiO2, chlorhexidine and UV-A, chlorhexidine 0.02% and TiO2, and additional UV-A therefore be regarded as equivalent exposure. Cell viability assessment was done using the trypan blue dye exclusion method. authors. RESULTS. The combination of TiO2 with UV-A demonstrated antitrophozoite and anticyst Submitted: June 8, 2018 activity (P < 0.05). This in vitro study showed a synergistic effect of the association of Accepted: July 30, 2018 chlorhexidine with TiO2 and UV-A on cysts (P < 0.001). Citation: Gomart G, Denis J, Bourcier CONCLUSIONS. Given the in vitro synergistic effectiveness of the association of chlorhexidine T, et al. In vitro amoebicidal activity of with TiO and UV-A against cysts, the treatment of Acanthamoeba keratitis could be titanium dioxide/UV-A combination 2 improved by this new therapeutic approach. against Acanthamoeba. Invest Oph- thalmol Vis Sci. 2018;59:4567–4571. Keywords: Acanthamoeba, cornea, infection, titanium dioxide, ultraviolet rays https://doi.org/10.1167/iovs.18-25003

moebae of the Acanthamoeba genus are free-living, Titanium dioxide (TiO2) is a ﬁne crystal powder. There are A aerobic, eukaryotic organisms and are widely dispersed in two forms of TiO2: micro particles, widely used in surface 1 nature and can inhabit terrestrial and aquatic environments. coatings and cosmetic materials, and nanoparticles. TiO2 Acanthamoeba species present two morphologic stages in nanoparticles have photocatalytic activity17: they produce their life cycle: a vegetative trophozoite stage, in which there is oxygenated free radicals under UV-A radiation. Oxygenated reproduction by binary ﬁssion, and a cyst stage that is resistant free radicals cause cell lysis by lipid peroxidation,18 release of to environmentally adverse conditions.2 Acanthamoeba proto- intracellular components,19 and nucleic acid20,21 and protein zoan are involved in keratitis and encephalitis.3 damage.22,23 4 Acanthamoeba keratitis is a medical emergency. The main TiO2 antimicrobial activity is known and commonly used as 5 17 risk factors are contact lens wearing and corneal trauma. The a self-disinfecting surface option. TiO2 is used in number of 24 pathogenesis is characterized by two events: (1) trophozoites materials, including health care items. adhere to the epithelial surface and invade then degrade the The antimicrobial properties of some types of nanoparticles, stromal extracellular matrix6 and (2) encystment of the mainly metallic ones, have been recently demonstrated in vitro, 25 protozoan, which promotes resistance and recurrence of notably against Gram-negative and Gram-positive bacteria. infection.7 Currently, the treatment involves topical antimicro- This study presents results of a series of in vitro experiments 8 bials, such as chlorhexidine, polyhexamethylene biguanide, evaluating the amoebicidal effects of TiO2 with UV-A on diamidines,9 and antifungal agents10 belonging to membrane- Acanthamoeba cysts and trophozoites in an effort to ultimately acting agents.11 Contrary to bacterial or fungal keratitis, failure expand the armamentarium of antimicrobial agents for the of topical therapy is common.6 Based on the severity of treatment of amoebic keratitis. Acanthamoeba keratitis and as a last resort, the only viable surgical option is penetrating keratoplasty.12 Among the novel therapeutic approaches, corneal cross-linking seems promising METHODS 13,14 in the management of Acanthamoeba keratitis. Miltefosine Acanthamoeba Strains (hexadecylphosphocholine), an alkylphosphocholine, showed interesting results too, in topical15 and systemic therapy.16 The Two Acanthamoeba strains were selected from a panel of development of antiacanthamoebic agents remains a challenge human clinical ocular amoebic pathogens isolated by corneal in order to improve the visual prognosis of the disease. scraping from patients with severe amoebic keratitis and

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FIGURE. Chemiluminescence (in related light unit, RLU) of different concentrations of TiO2 eye drop with UV-A light.

identified using the routine procedure (PCR) of the parasitol- water and carbomer gel (Gel Larmes; Thea,´ Clermont-Ferrand, ogy laboratory. The pathogens were one isolate of Acantha- France) at a 0.8-mg/mL final concentration. This final moeba sp belonging to T4 genotype (Acanthamoeba T4) and concentration was chosen because of imperatives of fabrica- one of Acanthamoeba hatchetti. tion (it was the highest final concentration that can be 27 produced) and the potential efficacy of TiO2 eye drops with Acanthamoeba Axenization Assay UV-A. This potential efficacy was evaluated by oxygenated free radical production measured by chemiluminescence. Briefly, Briefly, the amoeba was isolated from corneal tissue by plate- luminescence is based on photon emission when an excited culture procedure onto an nonnutrient agar medium covered molecule returns to its lowest energy state.28 Luminol (97%; with an avirulent, plasmid-less, heat-inactivated Escherichia Sigma-Aldrich Chimie, Saint-Quentin-Fallavier, France) produc- coli strain without antibiotic resistance at 258C. A piece of agar es photons in attendance of oxygenated free radicals.29 The culture was picked up aseptically from nonnutrient agar photon production is measured by a luminometer (Glomax 96 medium and transferred to a tissue culture flask (Dutscher, Microplate Luminometer; Promega, Madison, WI, USA), and Brumath, France) containing 5 ml of peptone-yeast extract chemiluminescence is expressed in related light unit (RLU). We 26 glucose (PGY) broth medium with penicillin-streptomycin to tested the production of oxygenated free radicals by TiO2 eye proceed with the axenization process of primary isolation. drop at final concentrations of 0.2, 0.4, and 0.8 mg/mL with For trophozoites, after 48 hours of incubation at 358C, the and without irradiance of UV-A for 30 minutes. The experi- excystment process was observed followed by the growth of ment was repeated three times. There was minimal chemilu- trophozoites in monolayer. Trophozoites were harvested by minescence without UV-A. The result of the three draining the supernatant medium and placing the tissue concentrations with UV-A is represented in the Figure. culture flask on ice for five minutes. Trophozoites were pelleted and washed in diluted saline solution twice by In Vitro Testing centrifugation at 405g for 10 minutes. The washing process was followed by cell counting and quantitative standardization To begin with, we tested the in vitro effect of TiO2 with UV-A of both the total and viable trophozoites, determined with exposure on trophozoites (Acanthamoeba T4, then Acantha- trypan blue (Corning, New York, NY, USA) staining. The moeba hatchetti). The second step was the in vitro test on experimental viable trophozoites concentration was adjusted cysts (Acanthamoeba T4 then Acanthamoeba hatchetti). to 1 3 105 trophozoites/mL. Eight groups were tested as follows: sterile water (blank For cysts, after 48 hours of incubation at 358C, the control), TiO2 alone(T),UV-Aalone(UV-A),TiO2 and excystment process was observed followed by growth of additional UV-A exposure (TþUV-A), chlorhexidine 0.02% alone trophozoites in a monolayer. Cell supernatants were discarded, (C; Gilbert, Herouville-Saint-Clair,´ France), chlorhexidine 0.02% and the encystment of adherent trophozoites was performed in and TiO2 (CþT), chlorhexidine 0.02% and UV-A (CþUV-A), and fresh PGY medium culture. Viable cysts were harvested and chlorhexidine 0.02% and TiO2 with additional UV-A exposure washed in diluted saline solution twice by centrifugation at (Cþ TþUV-A). Each group was assayed in triplicate. 405g for 10 minutes. The washing process was followed by cell An aliquot of 30 lLofanAcanthamoeba-containing counting and quantitative standardization of both the total and solution was placed into each well of a sterile 96-well viable cysts, determined with trypan blue staining. The microplate (Dutscher). We used three wells for each group. experimental viable cyst concentration was adjusted to 1 3 For a blank control, 30lL of saline solution was added. For the 5 10 cysts/mL. For each trial, the count of the chambers was TiO2 groups (T and TþUV-A) 30 lL of the TiO2 eye drop was performed in triplicate. added (with a final concentration of 0.8 mg/mL). For the chlorhexidine groups (C and CþUV-A), 30 lL of chlorhexidine Titanium Dioxide was added (with a final concentration of 0.2 mg/mL, clinical concentration). For the chlorhexidine and TiO2 groups (CþT The TiO2 eye drop solution was prepared by the pharmacy and CþTþUV-A), 15 lL of the TiO2 eye drop (with a final department (Hopitauxˆ Universitaires de Strasbourg, France) by concentration of 0.8 mg/mL) and 15 lL of chlorhexidine (with dissolving titanium powder (Inresa, Bartenheim, France; a final concentration of 0.2 mg/mL) was added. Irradiance of conformed to European pharmacopeia standard 9.2) in saline UV-A light source with a 365-nm wavelength at a power

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TABLE 1. Percentage of Trophozoite Viability and Percentage of Encystment at 24 hours*

Acanthamoeba T4 Acanthamoeba hatchetti

Trophozoite Viability Encystment Trophozoite Viability Encystment

Treatment† Mean 6 SD, % P value Mean 6 SD, % P value Mean 6 SD, % P value Mean 6 SD, % P value

Control 48 6 20 47 6 18 54 6 11 42 6 11 UV-A 52 6 13 >0.05 44 6 14 >0.05 48 6 15 >0.05 47 6 15 >0.05 TiO2 45 6 16 >0.05 19 6 8 <0.001 52 6 16 >0.05 38 6 12 >0.05 TiO2 and UV-A 34 6 13 <0.05 17 6 17 <0.001 42 6 16 >0.05 25 6 16 <0.05 C06 0 <0.001 0 6 0 <0.001 18 6 13 <0.001 5 6 4 <0.001

CþTiO2 0 6 0 <0.001 0 6 0 <0.001 6 6 16 <0.001 11 6 5 <0.001 CþUV-A 0 6 0 <0.001 0 6 0 <0.001 11 6 18 <0.001 8 6 8 <0.001 CþTiO2þUV-A 0 6 0 <0.001 0 6 0 <0.001 8 6 22 <0.001 9 6 7 <0.001 * P values are comparisons between control and each condition. Significant P values are in bold. Means and SD from triplicate experiments are shown. 2 † UV-A, 30 minutes of initial exposure, 365-nm wavelength, at a power density of 2.2 mW/cm ;TiO2 eye drop at 0.8 mg/ml final concentration; C, Chlorhexidine eye drop at 0.2 mg/ml final concentration.

density of 2.2 mW/cm2 was dispensed on the four groups with significant differences. A P < 0.05 was considered statistically UV-A exposure for 30 minutes. For cysts, based on the classical significant. clinical treatment regimen, we retreated after 24 hours, following the same steps. RESULTS Analysis of Tests Regarding Acanthamoeba T4 trophozoites (Table 1), after 24 After the 24-hour incubation period for trophozoites and the hours of incubation, the combination of TiO2 and UV-A 48-hour incubation period for cysts, the cell viability of each demonstrated antitrophozoite activity: the difference between assay was measured with the trypan blue dye exclusion the control group and the TiO2þUV-A group was statistically method in a counting chamber (Dutscher) under microscope significant (P < 0.001). Each group with chlorhexidine (versus trypan blue stain. Trypan blue is a vital stain that colors only control) was an amebicide (P > 0.001). All the trophozoites dead cells. Living amoebae have a refringent appearance under died in the presence of chlorhexidine. light microscopy, whereas dead amoebae exhibit a blue color Regarding Acanthamoeba hatchetti trophozoites (Table 1), (see Supplementary Fig. 1 for photographs of live and dead after 24 hours of incubation, the in vitro experiment showed trophozoites and cysts). no difference among the groups UV-A, TiO2 alone, and TiO2þUV-A: UV-A versus control (P > 0.05), TiO2 versus Statistical Analysis control (P > 0.05), and TiO2 þUV-A versus control (P > 0.05). The encystment was statistically lower in the TiO2þUV-A group The analysis focuses on the following Acanthamoeba out- than in the control group (P < 0.05). Each group with comes: trophozoites persist, die, or encyst; and cysts persist or chlorhexidine (versus control) was an amebicide (P > 0.001). die. Descriptive statistics were expressed as the mean and Regarding Acanthamoeba T4 (Table 2) and Acanthamoeba standard deviation (SD) of the triplicate of the three repeats. hatchetti (Table 2) cysts, after 48 hours of incubation, the in The 2-way ANOVA test was used for the analysis, assuming a vitro experiment showed no difference among the groups UV- theoretical normal population. Multiple comparisons were A and TiO2 alone: UV-A versus control (P > 0.05) and TiO2 made post hoc between the different groups in order to find versus control (P > 0.05). TiO2þUV-A had the following an

TABLE 2. Percentage of Cyst Viability of Acanthamoeba T4 and Acanthamoeba hatchetti at 48 hours*

Cyst Viability‡

Acanthamoeba T4 Acanthamoeba hatchetti

Treatment† Mean 6 SD, % P value Mean 6 SD, % P value

Control 94 6 6976 5 UV-A 95 6 5 >0.05 100 6 0 >0.05

TiO2 85 6 5 >0.05 88 6 8 >0.05 TiO2 and UV-A 51 6 15 <0.001 69 6 15 <0.001 C276 12 <0.001 39 6 9 <0.001 CþTiO2 22 6 16 <0.001 36 6 12 <0.001 CþUV-A 21 6 19 <0.001 38 6 9 <0.001 CþTiO2þUV-A 11 6 16 <0.001 25 6 6 <0.001 * Retreatment at 24 hours. 2 † UV-A, 30 minutes of initial exposure, 365-nm wavelength, at a power density of 2.2 mW/cm ;TiO2 eye drop at 0.8 mg/ml final concentration; C, Chlorhexidine eye drop at 0.2 mg/ml final concentration. ‡ P values are comparisons between control and each condition. Significant P values are in bold. Means and SD from triplicate experiments are shown.

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amoebicidal effects: on Acanthamoeba T4, there were 49% of cystidal potency, with minimal associated host-cell cytotoxic- 10,34 dead cysts with TiO2 þUV-A against 6% with control (P < ity. 0.001); and on Acanthamoeba hatchetti, there were 31% of Results obtained in vitro do not always correlate with in dead cysts with TiO2 þUV-A against 3% with control (P < vivo efficacy; therefore, further tissue culture models and 0.001). TiO2þUV-A was better than TiO2 alone (P < 0.001). animal studies are under way to test the efficacy of this Each group with chlorhexidine was better than the control (P treatment for infectious keratitis. < 0.001). There was a synergistic effect of chlorhexidine with Furthermore, it was important to determine the cytotoxic- 35 TiO2þUV-A: after 48 hours, the percentage of dead cysts was ity of TiO2.Eometal. evaluated the effect of TiO2 higher with the combination of CþTiO2þUV-A than with nanoparticle exposure on the ocular surface in vivo on 40 chlorhexidine alone and on Acanthamoeba T4 cysts (P < rabbits. Of the five toxicity criteria, two increased after TiO2 0.05) as on Acanthamoeba hatchetti cysts (P < 0.01). exposure. Given that we were able to demonstrate in vitro activity of the TiO2/UV-A against Acanthamoeba,itis necessary to establish safety with other in vivo tests on corneal DISCUSSION epithelia cells and animal studies. In conclusion, the combination TiO2þ UV-A presents The work outlined here is directed to the development of TiO2 antitrophozoite and an adjunctive anticyst activity in vitro and UV-A as a new adjunctive method for the treatment of when applied with the parameters used in the present study. Acanthamoeba keratitis, which is a cause of significant morbidity worldwide and can cause rapid and devastating vision loss.6 Acanthamoeba keratitis continues to be difficult Acknowledgments to treat despite the use of topical agents and adjuvant surgery, The authors thank Nicolas Meyer (Laboratoire de Biostatistique, such as corneal transplantation.30 Many studies worked on the FacultedeM´ edecine´ de Strasbourg, Universite´ de Strasbourg), photocatalytic utility of TiO2/UV in a disinfection system, with Philippe Andre´ (Laboratoire de Biophotonique et Pharmacologie, efficacy on bacteria, fungi, and viruses. Sökmen et al.31 used Faculte´ de Pharmacie de Strasbourg, Universite´ de Strasbourg), and Nicolas Keller (Institut de Chimie et Proced´ es´ pour L’Energie, TiO2 for photocatalytic disinfection of Giardia intestinalis and Acanthamoeba castellanii cysts in water with UV-C expo- L’Environnement et la Sante,´ Universite´ de Strasbourg) for 31 32 technical assistance. sure. Imran et al. synthesized TiO2 nanoparticles and demonstrated their inhibitor effects on Acanthamoeba cas- Disclosure: G. Gomart, None; J. Denis, None; T. Bourcier, None; tellanii trophozoite growth and viability. The antimicrobial A. Dory, None; A. Abou-Bacar, None; E. Candolfi, None; A. Sauer, None effect of photocatalyse is a reason why we tested TiO2 with UV- A on cysts and trophozoites of Acanthamoeba hatchetti and T4 in vivo. References Acanthamoeba keratitis has been characterized as a painful and vision-threatening disease. The infection cascade starts 1. Visvesvara GS, Moura H, Schuster FL. 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