In Vitro Evaluation of Combined Commercialized Ophthalmic Solutions Against Acanthamoeba Strains

Acanthamoeba is a free-living amoebae genus which is present worldwide in natural and artificial environments. These amoebae are clinically important as causative agents of diseases in humans and other animals such as a fatal encephalitis or a sight threatening Acanthamoeba keratitis (AK). Lately; studies have focused on the search of novel therapeutic options for AK but also to prevent infections. Furthermore; the evaluation of commercialized products seems to be an option for this case since not clinical assays would be required. Thus; we aimed to test the amoebicidal activity of different mixtures of two commercial ophthalmic solutions: Systane® Ultra; which has already shown anti-Acanthamoeba properties; and Naviblef® Daily Care. In addition, we tested their cytotoxic effect against murine macrophages. At the individual level; Naviblef® Daily Care showed to be the most active product against Acanthamoeba spp. Nevertheless; the combinations of Systane® Ultra and Naviblef® Daily Care; showed an improvement in the activity against trophozoites and cysts of Acanthamoeba castellanii Neff. Moreover; the concentration necessary to generate cytotoxic effect against murine macrophages (J774.1) was much higher than the required for the amoebicidal and cysticidal effect achieved in the most effective mixtures.


Introduction
Acanthamoeba is a widely distributed protozoa which has been isolated from many sources such as soil, water, contact lenses, air-conditioning units, clinical samples among others [1,2]. The main human pathologies caused by this genus are Acanthamoeba Keratitis (AK) and Granulomatous Amoebic Encephalitis (GAE). At the early stage of infection, AK presents symptoms such as eye redness, epithelial defects, photophobia and intense pain, but if no early and accurate diagnosis and effective treatment is performed, it could end in blindness or even in eye removal [2][3][4][5]. Additionally, severity of the infection depends on the Acanthamoeba strain, the cornea integrity and the host immune response [6].
Until now, there is no effective therapy for AK, but the recommended treatment regimen for AK includes a biguanide (0.02% polyhexamethylene biguanide (PHMB) or 0.02% chlorhexidine digluconate) accompanied with diamidine (0.1% propamidine isethionate, also known as Brolene, or 0.1% hexamidine, also known as Desomedine) [7,8]. Lately, voriconazole has been demonstrated to be effective against clinical strains of Acanthamoeba [9,10] and has also been successfully used in a clinical case of AK in Spain [11]. One of the main problems on AK recovery is the ability of Acanthamoeba to form an extremely resistant cyst phase. Therefore, the need for efficient therapies not only against the trophozoite form but also to this resistant stage is evident.
AK mostly affects contact lenses (CL) wearers and the reported cases are mostly related to the use of soft CL and their incorrect use and maintenance [12,13]. Moreover, the use of CL is also associated to other eye diseases, such as dry eye disease (DED) or Blepharitis. DED has been demonstrated as a predisposing factor to develop AK [14].
Systane ® Ultra (Systane ® ; Alcon, Fort Worth, TX, USA; Table 1) is one of the most widely used ocular lubricants in the USA [15]. In a recent study, the amoebicidal activity of this product was reported as well as its potential use for the prevention or even treatment of Acanthamoeba keratitis, or to contribute for an optimized solution [16]. Naviblef ® Daily Care (Novax ® Pharma, Monaco; Table 1) is a commercialized cleaning foam which contains tea tree oil (TTO) (0.02%) and chamomile oil (CO). This foam effectively removes excessive oil, pollen and other debris from the eyelids which increase the risk of irritation, blepharitis due to the presence of Demodex or bacteria as well as dry eye symptoms. Benzalkonium chloride (BAK) or its derivatives such as POLYQUAD ® is one of the most frequently used preservatives in ophthalmic solutions. The cytotoxic effects of BAK has been shown to induce inflammation on the ocular surface cells in numerous in vitro and in vivo models [17]. Systane ® Ultra, which contains POLYQUAD ® , has shown induction of inflammation markers. On the other hand, Naviblef ® Daily Care does not present any described toxic preservatives on its composition. In the present study, the in vitro anti-Acanthamoeba effect of different combinations of Systane ® Ultra and Naviblef ® Daily Care (Table 2) were tested as well as their cytotoxic effect against murine macrophages.

In Vitro Effect of Systane ® Ultra and Naviblef ® Daily Care Mixtures
Once the individual in vitro activity of each of the ophthalmic solutions was performed, the different combinations obtained with the Design-Expert ® software were tested against Acanthamoeba spp. (Figure 1), A. castellanii Neff cysts and murine macrophages ( Figure 2). All mixtures showed activity against Acanthamoeba spp., being the highest IC 50 values the ones for A. polyphaga and the lowest ones for A. castellanii Neff (Figure 1). In order to compare the in vitro activity against A. castellanii Neff trophozoites of each ophthalmic solution and their combinations, the percentage of Systane ® Ultra and Naviblef ® Daily Care was calculated for each mixture for all IC 50 values (Table 2). Moreover, we noticed a decrease in the quantity of each eye drop solution needed to inhibit 50% of amoebae growth when combinations were used. All IC 50 values against A. castellanii Neff cysts were higher than the IC 50 against trophozoites except in #2 and #11 (Figure 2), where the Systane ® Ultra and Naviblef ® Daily Care proportions were 0.43:0.43 (%:%) and 1.52:0.31 (%:%) respectively (Table 2). Both mixtures, #2 and #11 presented the lowest CC 50 (%) against murine macrophages, 37.39 ± 1.95% and 30.05 ± 3.58% respectively ( Figure 2).

In Vitro Effect of Systane ® Ultra and Naviblef ® Daily Care Mixtures
Once the individual in vitro activity of each of the ophthalmic solutions was performed, the different combinations obtained with the Design-Expert ® software were tested against Acanthamoeba spp. (Figure 1), A. castellanii Neff cysts and murine macrophages ( Figure 2). All mixtures showed activity against Acanthamoeba spp., being the highest IC50 values the ones for A. polyphaga and the lowest ones for A. castellanii Neff (Figure 1). In order to compare the in vitro activity against A. castellanii Neff trophozoites of each ophthalmic solution and their combinations, the percentage of Systane ® Ultra and Naviblef ® Daily Care was calculated for each mixture for all IC50 values (Table 2). Moreover, we noticed a decrease in the quantity of each eye drop solution needed to inhibit 50% of amoebae growth when combinations were used. All IC50 values against A. castellanii Neff cysts were higher than the IC50 against trophozoites except in #2 and #11 (Figure 2), where the Systane ® Ultra and Naviblef ® Daily Care proportions were 0.43:0.43 (%:%) and 1.52:0.31 (%:%) respectively ( Table  2). Both mixtures, #2 and #11 presented the lowest CC50 (%) against murine macrophages, 37.39 ± 1.95% and 30.05 ± 3.58% respectively ( Figure 2). In vitro anti-Acanthamoeba spp activity

Response Surface Analysis
The IC50 and CC50 variations using different proportions of compounds are also shown using mixture contour plots ( Figure 3). The maximum percentage for each factor (each compound) is represented in a corner of an equilateral triangle. The edges of the triangle represent the twocomponent blends. Each point within the triangle represents the three-component blends. The center of the triangle represents the mixture in equal proportion. Mixtures of Systane ® Ultra and Naviblef ® Daily Care presented lower IC50 values against trophozoites than both products separately, but this effect is even higher against cysts.

Discussion
Systane ® Ultra combines polyethylene glycol 400 (0.4%) with propylene glycol (0.3%) lubricants in an aqueous formulation with an HP-Guar demulcent designed to afford a prolonged lubrication to the ocular surface [15]. Both of these compounds have been reported to be effective against

Response Surface Analysis
The IC 50 and CC 50 variations using different proportions of compounds are also shown using mixture contour plots ( Figure 3). The maximum percentage for each factor (each compound) is represented in a corner of an equilateral triangle. The edges of the triangle represent the two-component blends. Each point within the triangle represents the three-component blends. The center of the triangle represents the mixture in equal proportion. Mixtures of Systane ® Ultra and Naviblef ® Daily Care presented lower IC 50 values against trophozoites than both products separately, but this effect is even higher against cysts. . The discharge of these afferents may regulate basal tear secretion by sensing eye wetness [17].
Dysfunction of TRPM8-mediated sensing of evaporation-induced temperature and osmolarity change in the corneal surface has been suggested as a possible pathophysiological mechanism in DED [15]. TRPM8 knock-out mice showed a reduction in basal tear secretion [8]. Corcoran et al. [18] showed that perception of cold was modified in patients with DED compared to healthy subjects. The altered sensitivity was not seen in corneal mechanoreceptors. Recently, Alcalde et al. [19] found in studies on mice that aging impairs activity of high-threshold cold thermoreceptors, which makes the ocular surface more sensitive to stimuli and ocular irritation, and to tearing. These findings substantiate the hyperactivity of the cold thermoreceptor on the cornea as one of the risk factors that cause abnormal lacrimation and contribute to the high incidence of DED in aged people. It is not clear if the manifestations of changes seen in temperature sensitivity of DED patients are triggered by the disease process or just epiphenomenon. The consensus view at this time is that abnormal TRPM8 reactivity on the cornea triggers irritation.

TRP Channels Related to Ocular Pain in DED
According to a TFOS DEWS II report, the revised definition of DED included neurosensory abnormalities emphasizing the importance of neural regulation of tearing as well as pain sensing [1,15]. Researchers focus on corneal nociceptors (polymodal nociceptors) as initiators of DED pathophysiology [15,20]. The coding of sensory neural circuits has been intensively studied [21]. The distributions and morphological specialization related to the function of TRPV1, and TRPM8 ion

Discussion
Systane ® Ultra combines polyethylene glycol 400 (0.4%) with propylene glycol (0.3%) lubricants in an aqueous formulation with an HP-Guar demulcent designed to afford a prolonged lubrication to the ocular surface [15]. Both of these compounds have been reported to be effective against different bacteria species [18,19] and, on the other hand, propylene glycol also presents a remarkable preservative effect [20,21]. A previous study from our laboratory has shown the anti-Acanthamoeba activity of Systane ® Ultra and its induction of programmed cell death (PCD) mechanism [16]. In addition, the presence of a POLYQUAD ® , which has been demonstrated as capable to activate and elevate the secretion of inflammation markers, is a disadvantage in the ocular use of Systane ® Ultra. However, Naviblef ® Daily Care does not present any described toxic preservatives on its composition but its individual IC 50 is significantly lower than the one of Systane ® Ultra in the three tested strains (Table 3). Different studies have reported the activity of some Naviblef ® Daily Care components, such as TTO and chamomile derivatives, against Acanthamoeba [22,23] or AK disorders [24]. The A. griffini strain (genotype T3) was isolated in October 2013 from a severe case of keratitis from both a contact lens and a corneal scrape (Association to prevent Blindness in Mexico, Luis Sanchez Bulnes Hospital, Mexico City, Mexico) [25]. Chlorhexidine is the recommended treatment regimen for AK and in the study of González-Robles and colleagues this strain presented an important resistance to chlorhexidine, while it was more sensitive to voriconazole. In the present study we have reported an IC 50 value of 7.99 ± 1.25% and 1.13 ± 0.16% of Systane ® Ultra and Naviblef ® Daily Care respectively for the same strain (Table 3).
Regarding the mixtures, the #4 mixture was the most active against trophozoites of the three Acanthamoeba tested strains (Figure 1). However, this #4 mixture was one of the most toxic for macrophages ( Figure 2). Concerning cytotoxicity, the mixture who has presented the lowest CC 50 value was #6, which has also presented moderate in vitro activity against Acanthamoeba spp. trophozoites and A. castellanii cysts. Regarding to experiment #4, 4.97% of Systane ® Ultra was needed to inhibit the growth of 50% of A. castellanii Neff trophozoites when it is used individually. However, only a 2.53% of Systane ® Ultra was required to produce the same effect when it was combined with Naviblef ® Daily Care (Table 2). Therefore, in the experiment #4, the Systane ® Ultra amount has been reduced to the half. In the same experiment, the percentage of Naviblef ® Daily Care in IC 50 against A. castellanii Neff trophozoites has been also reduce three times, from 0.68% alone to 0.21% in the mixture. On the other hand, in the less cytotoxic #6 mixture, the value of Naviblef ® Daily Care has been also reduced, from 0.68% to 0.04% (Table 2). Despite the #6 mixture was not the most active one, both IC 50 values against trophozoites and cysts are moderate (Table 2 and Figure 2A, respectively). Thus, it is important to highlight that the combination of Systane ® Ultra and Naviblef ® Daily Care reduce the amount of product needed to produce the same effect as their individual use. In contrast, both #2 and #11 mixtures have presented the highest cytotoxicity against the murine macrophages.

Chemicals
In this study the Systane ® Ultra ophthalmic solution and the Naviblef ® Daily Care cleaning foam were selected to evaluate their anti-Acanthamoeba in vitro activity (Table 3).

Macrophage Cell Line
In order to evaluate the cytotoxicity in mammal cells, in this work the macrophages J774A.1 (ATCC # TIB-67) strain was used. The in vitro effect of Systane ® Ultra and Naviblef ® Daily Care against the three tested Acanthamoeba strains (A. castellanii Neff, A. polyphaga (ATCC ® 30461™) and A. griffini), was evaluated using a colorimetric assay based on the alamarBlue TM Cell Viability Reagent (Invitrogen by Thermo Fisher Scientific), as it has previously described [7,16,26,27]. As first step, the Acanthamoeba culture was seeded in triplicate on a 96-well microtiter plates (50µL from a stock solution of 5 × 10 4 cells/mL). After the trophozoites were attached to the bottom of the well, 50 µL of serial dilutions of the products were added to the 96-wells plate. As a negative control we have used Acanthamoeba spp. trophozoites in PYG medium. Finally, the alamarBlue TM was placed onto each well at 10% as a final concentration. Then, plates were incubated during 96 h at 28 • C with a soft agitation and finally analyzed in the EnSpire ® Multimode Plate Reader (Perkin Elmer, Madrid, Spain) using emitted florescence at 570/585 nm. The inhibitory concentration 50 (IC 50 ) was calculated by no linear regression analysis with 95% confidence limits using Sigma Plot 12.0 statistical analysis software (Systat Software). All the experiments were carried out three times each in triplicate, calculating the mean values.

In Vitro Effect of Systane ® Ultra and Naviblef ® Daily Care Against the Cyst Stage of A. castellanii Neff Strain
A. castellanii Neff cysts were prepared as it has been described before [8,28]. Mature cysts were harvested and washed using PYG medium, obtaining a 5 × 10 4 cysts/mL solution. This cysts suspension was seeded in triplicate jointly with serial dilutions of each ophthalmic solution and the plates were incubated for 168 h at 28 • C. Then, the plate was centrifuge and the supernatant, which contained each ophthalmic solution dilution, was removed and PYG was added. Finally, alamarBlue TM reagent was added at 10% as a final concentration, and the plates were incubated and analyzed as it was described above.
After two weeks, an inverted microscope EVOS ® FL Cell Imaging System (ThermoFisher) was used to evaluate the excystment capacity after stopping the treatment. As a negative control we have used A. castellanii cysts incubated with PYG medium.

In Vitro Effect Against Acanthamoeba spp. Trophozoites of Systane ® Ultra and Naviblef ® Daily Care Mixtures
Once the IC 50 of each compound was calculated, the Design-Expert ® Software Version 10. Design-Expert ® software was used to provide experimental design, model building, calculation of equations, data analysis, and ideal process settings for top performance and discover optimal product formulations. In the present study, the software designed thirteen different mixtures of both compounds (Table 4), considering the IC 50 already obtained for each one. These mixtures were carried out in double distilled sterilized water. The in vitro activity of the obtained aqueous mixtures was carried out in the three Acanthamoeba strains as described in Section 4.3.1.

In Vitro Effect of Systane ® Ultra and Naviblef ® Daily Care Mixtures Against Acanthamoeba castellanii Neff Cysts Evaluated by the Alamarblue™ Colorimetric Assay
A. castellanii Neff cysts were prepared as it has been described before [8,27] and the protocol designed by Sifaoui and colleagues in 2018 [16] was followed. Briefly, mature cysts were harvested and washed using PYG medium, obtaining a 5 × 10 4 cysts/mL solution. Of this cyst's solution, 50 µL were seeded in triplicate into a 96-well microtiter plate wells, which contain 50 µL from serial dilutions of each combination. As a negative control we have used A. castellanii cysts incubated with PYG medium. Finally, alamarBlue TM Reagent was placed into each well at 10% and the plates were incubated for 144 h at 28 • C. The plates were measured and analyzed as it has been described in previous sections.
In Vitro Cytotoxic Effect in Murine Macrophages J774A.1 (ATCC # TIB-67) of Systane ® Ultra and Naviblef ® Daily Care Mixtures In order to evaluate the cytotoxicity of each new mixture of the commercialized compounds, the colorimetric assay based on the alamarBlue™ Reagent (Life Technologies, Madrid, Spain) was carried out. Fifty microliters from a stock solution of 10 5 macrophages/mL were added in 96-well microtiter plate wells, allowing them to adhere to the bottom. Thus, 50 µL of serial dilutions of the mixtures were added to the wells. At last, the alamarBlue Reagent™ was placed into each well at 10% and the plates were incubated for 24 h at 37 • C in presence of CO 2 at 5%. The plates were measured using the EnSpire ® Multimode Plate Reader as in the anti-Acanthamoeba assays. After all, the cytotoxic concentration 50% (CC 50 ) was obtained by no linear regression analysis with 95% confidence limits using Sigma Plot 12.0 statistical analysis software as it was explained above. All the experiments were carried out three times each in duplicate, calculating the mean values.

Conclusions
In conclusion, in a mixture of Systane ® Ultra and Naviblef ® Daily Care, the maximum concentration of Systane ® Ultra showed an increased value of the IC 50 against A. castellanii Neff trophozoites ( Figure 3A), and this effect is higher against A. castellanii Neff cysts ( Figure 3B). However, the diagram of the CC 50 shows a clear cytotoxicity effect of the Naviblef ® Daily Care ( Figure 3C). Naviblef ® Daily Care is a non-greasy and non-irritant formulation and it is recommended to be used as maintenance care, for patients that have followed the blepharitis treatment with Naviblef ® Intensive Care. Naviblef ® Daily Care continues providing protection against re-infestation from Demodex and maintains eyelid cleanliness. However, in the Naviblef ® Daily Care directions for use, they recommend rinsing the eyelashes and eyelids with warm water after each application. Tap water has been demonstrated as an important source of free-living amoeba (FLA), such as Acanthamoeba spp. [1,5,28]. Among the main symptoms of blepharitis, watery eyes, greasy eyelids and the appearance of skin flakes favor the establishment of pathogenic microorganisms as FLA. Both conditions could be an important risk factor to suffer AK. Despite the high toxicity effect of the present drug towards both Acanthamoeba and macrophages, due to its external application, it is recommended to use it as preventive tool, to eliminate possible eyelids microorganisms.