Photodynamic Eradication of Trichophyton Rubrum and Candida Albicans

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Article Photodynamic Eradication of Trichophyton rubrum and Candida albicans

Anton Valkov, Michael Zinigrad and Marina Nisnevitch *

Department of Chemical Engineering, Ariel University, Kiryat-ha-Mada, Ariel 4070000, Israel; [email protected] (A.V.); [email protected] (M.Z.) * Correspondence: [email protected]; Tel.: +972-3914-3042

Abstract: Conventional methods of onychomycosis treatment are ineffective in some cases because the cure of onychomycosis very often depends on the patient’s individual response to the treatment; therefore, there is a crucial need to research and develop new methods of onychomycosis therapy. One of the most innovative treatments is photodynamic therapy (PDT) using photosensitizers (PSs). However, effective treatment depends on the correct choice of photosensitizer and substances that improve the characteristics of the final formulation. The aim of our work was to find an effective formulation for the treatment of onychomycosis. To achieve this goal, we tested the effect of three types of PSs, rose Bengal (RB), malachite green oxalate (MGO), and methylene blue (MB), on Candida albicans. The most effective PS was RB, and so the study was continued with Trichophyton rubrum. Additional comparative studies were carried out on substances included in the formulation (urea and thiourea), focusing on their antifungal activity, which can improve penetration through the nail plate. The composition of the formulation that achieved 100% eradication of Trichophyton rubrum under our conditions consisted of 150 µM RB, 5% urea, and 0.5% thiourea in glycerol/water (70/30%, − w/w) solution. A white luminescent lamp was used as a light source (1.9 ± 0.1 mW cm 2). Stability of the formulation was checked. The selected formulation shows potential for future simplification Citation: Valkov, A.; Zinigrad, M.; and acceleration of PDT treatment of onychomycosis. Nisnevitch, M. Photodynamic Eradication of Trichophyton rubrum Keywords: photodynamic treatment; rose Bengal; malachite green; methylene blue; Trichophyton and Candida albicans. Pathogens 2021, rubrum; Candida albicans 10, 263. https://doi.org/10.3390/ pathogens10030263

Academic Editor: Ying-Lien Chen 1. Introduction Onychomycosis, fungal nail infection, is an acute and persistent problem in pub- Received: 29 January 2021 lic health. Epidemiological studies around the world have found a high incidence of Accepted: 21 February 2021 Published: 25 February 2021 onychomycosis in at least 10–30% of the general population [1,2], in more than 20% of those over 60 years old, in 50% of those over 70 years old, and in 30% of patients with

Publisher’s Note: MDPI stays neutral diabetes [3,4]. with regard to jurisdictional claims in Onychomycosis is often considered only an aesthetic problem. However, patients hav- published maps and institutional affil- ing onychomycosis along with a background of immunodeficiency or endocrine diseases iations. may develop widespread mycosis of the skin. Sometimes onychomycosis is accompanied by development of severe complications such as diabetic foot, extremity erysipelas, or lymphostasis. For this reason, onychomycosis treatment is strictly necessary and should be carried out promptly [5–7]. Clinical studies have shown that the effectiveness of systemic antimycotics after the end of treatment is 40–80%, and after 5 years only 14–50% [8]. It Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. was proven that the effectiveness of onychomycosis therapy can be increased by apply- This article is an open access article ing complex methods of treatment that combine antifungal drugs with agents affecting distributed under the terms and pathogenesis [9,10]. conditions of the Creative Commons Advanced methods of onychomycosis therapy include local or oral therapy and Attribution (CC BY) license (https:// surgical intervention [1,11]. However, even when using these complex methods, it is creativecommons.org/licenses/by/ difficult to achieve positive results, since drugs applied topically usually cannot penetrate 4.0/). into the nail plate to the site of infection, but instead are concentrated on its external

Pathogens 2021, 10, 263. https://doi.org/10.3390/pathogens10030263 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 263 2 of 13

surface [12]. Moreover, systemic use of antifungal drugs such as ketoconazole, terbinafine, and griseofulvin sometimes needs up to 12 months of treatment [13], and long-term application of these drugs carries a toxicological effect that can harm human health [14,15]. In addition, fungi may develop resistance to antifungal drugs under a long period of exposure. Surgical treatment is very unpopular, since it causes discomfort and pain in patients and is relatively ineffective since it leads to deformation of the treated nail plates and scar formation [16,17]. In recent years, alternate methods of onychomycosis treatment, such as photodynamic therapy (PDT), have been actively developed and tested [18]. PDT is a special field of light therapy. Compounds called photosensitizers (PSs) are applied to an infected site and undergo activation by visible light [19,20]. Illumination leads to production of active radicals, such as reactive oxygen species (ROS), which cause irreversible damage to microorganisms. The ability of PSs to eradicate microorganisms has been described by many authors [21–28]. PSs are effective against bacterial, viral, fungal, and protozoal infections [29]. To enhance the efficiency of PDT, it is necessary to select the PS formulation with optimal consistency in providing high-speed drug penetration into a nail plate. However, although necessary, this parameter is not a sufficient condition for successful local treatment of fungal infections. To assess the effectiveness of onychomycosis treatment with antifungal drugs in various forms (e.g., transfersomes, nanoemulsions, gels, creams, and ointments) and their combinations, with or without the addition of surfactants [30,31], the use of keratin biomembranes was proposed as a good substitute for hard-to-obtain human nails [32–34]. The keratin biomembranes have physical properties similar to nail plates and can be obtained with good reproducibility. This study aimed to find a promising formulation and conditions for effective PDT against fungal infection caused by Candida albicans and Trichophyton rubrum. Three water- soluble PSs were selected for antifungal studies, rose Bengal (RB), malachite green oxalate (MGO), and methylene blue (MB), all of which exhibit high antimicrobial activity [35,36]. These PSs were tested against fungi at different concentrations and in various formulations, with and without keratolytic agents. An appropriate formulation for photodynamic treatment of onychomycosis should include a photosensitizer and keratolytic agents to increase the permeability of nail plates to active agents. The formulation should also have a viscosity high enough to keep the PS on the nail surface and prevent its leakage to the nail sides. We also examined the stability of the proposed formulations.

2. Results and Discussion This work focused on a study of antifungal properties of photosensitizers, alone and in combination with factors affecting the permeation of drug molecules into a nail plate. Two fungi known to cause onychomycosis were examined: Candida albicans and Trichophyton rubrum. The former strain was tested in planktonic cultures and the latter on solid media including fabricated keratin membranes that model nail plates.

2.1. Photodynamic Treatment of Planktonic Culture of Candida albicans First, the chosen PSs (RB, MGO, and MB) were tested on planktonic cultures of Candida albicans using a high concentration (500 µM) of each PS under prolonged illumination. For this purpose, the solutions were distributed into Petri dishes containing Candida albicans suspension and illuminated for 4 h by a white luminescent light. The experiment showed that only MGO and RB caused total eradication of Candida albicans cells, while MB was inactive (Figure1). In our previous studies, MB showed high photodynamic activity against Gram-positive and Gram-negative bacteria [22,35,37–39], however, in the case of fungi, this PS was unable to inhibit cell growth. Pathogens 2021, 10, x FOR PEER REVIEW 3 of 13

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against Gram-positive and Gram-negative bacteria [22,35,37–39], however, in the case of Pathogens 2021, 10, 263 againstfungi, Gram-positive this PS was unable and Gram-negative to inhibit cell growth. bacteria [22,35,37–39], however, in the case 3of of 13 fungi, this PS was unable to inhibit cell growth.

Figure 1. The effect of malachite green oxalate (MGO), rose Bengal (RB) and methylene blue (MB) Figure 1. The effect of malachite green oxalate (MGO), rose Bengal (RB) and methylene blue (MB) at at 500 μM concentration on planktonic cultures of Candida albicans under white-light illumination Figure500 1.µ MThe concentration effect of malachite on planktonic green oxalate cultures (MGO), of Candida rose Bengal albicans (RB)under and white-light methylene illuminationblue (MB) at at 500at 1.9 μM ± concentration0.1 mW/cm2 fluence on planktonic for 4 h. cultures of Candida albicans under white-light illumination 1.9 ± 0.1 mW/cm2 ﬂuence for 4 h. at 1.9 ± 0.1 mW/cm2 fluence for 4 h. Since only two PSs showed antifungal activity, the study was continued using MGO Since only two PSs showed antifungal activity, the study was continued using MGO andandSince RB RB onlyonly. only. two At At thePSs the next nextshowed stage, stage, antifungal their their photod photodynamic activity,ynamic the studyactivity activity was was was continued tested tested at at using different different MGO con- con- andcentrationscentrations RB only. At and and the during during next stage, various various their time time photod periods. ynamic The The activity effect effect of ofwas MGO MGO tested on on atCandida different albicans con- isis centrationsshownshown in and Figure during2 2a.a. Complete Completevarious time inactivation inactivation periods. of ofTheCandida Candida effect albicans albicansof MGOwas was on achieved achievedCandida using albicans using MGO MGO is at shownat250 250 µin Mμ FigureM concentration concentration 2a. Complete after after 4inactivation 4 h h of of illumination, illumination, of Candida compared compared albicans to towas 5050 µachievedμMM MGO,MGO, using which MGO caused at 250onlyonly μ Mpartial partial concentration cell cell destruction. destruction. after 4 hOver Over of illumination, shorter shorter time time compared periods, periods, even evento 50 250 250μM μµ MGO,MM MGO MGO which decreased decreased caused the onlycell partial concentration cell destruction. only by Over 0.5–2 shorter log10 values. time periods, The activity even of 250 RB μ againstM MGO Candida decreased albicans the is cell concentration only by 0.5–2 log10 values. The activity of RB against Candida albicans is cellillustratedillustrated concentration in in Figure Figure only 22b.byb. 0.5–2 The cells log10 were values. completelycompletely The activity eradicatederadicated of RB against atat concentrationsconcentrations Candida albicans ofof bothboth is 5050 illustratedandand 250 inμµM Figure RB RB after 2b. 30The min cells ofof were illumination,illumination, completely i.e., i.e., eradicated RB RB showed showed at a a signiﬁcantlyconcentrations significantly higher higher of both ability ability 50 in andinfungal 250 fungal μM cell RBcell eradication after eradication 30 min than thanof didillumination, did MGO. MGO. i.e., RB showed a significantly higher ability in fungal cell eradication than did MGO.

(a) (b) (a) (b) µ FigureFigure 2. 2. EffectEffect of of 50 50 and and 250 250 μMM of of (a (a) )MGO MGO and and (b (b) )RB RB on on planktonic planktonic culture culture of of CandidaCandida albicans albicans 2 ± 2 Figureunderunder 2. Effectwhite-light white-light of 50 andillumination illumination 250 μM of at at ( thea the) MGO fluence ﬂuence and of ( b1.9 ) RB ± 0.1 0.1on mW/cm planktonic mW/cm. . culture of Candida albicans under white-light illumination at the fluence of 1.9 ± 0.1 mW/cm2. Next, it was proven that the cells were eradicated by RB due to illumination. For this purpose, the photodynamic activity of RB against Candida albicans was compared to the same process in darkness. The results in Figure3 show that under illumination, 50 µM RB totally eradicated Candida albicans within 15 min, whereas in the dark, even after 30 min Pathogens 2021, 10, x FOR PEER REVIEW 4 of 13

Next, it was proven that the cells were eradicated by RB due to illumination. For this purpose, the photodynamic activity of RB against Candida albicans was compared to the Pathogens 2021, 10, 263 same process in darkness. The results in Figure 3 show that under illumination, 50 μM4 ofRB 13 totally eradicated Candida albicans within 15 min, whereas in the dark, even after 30 min the fungi cell concentration did not differ from that in the control series. Previously, the photodynamic effect of RB on planktonic cultures of Candida albicans was demonstrated the fungi cell concentration did not differ from that in the control series. Previously, the byphotodynamic Costa et al. [40] effect and of Freire RB on et planktonic al. [41]. Costa cultures et al. ofachievedCandida a albicans1.58 logwas10 reduction demonstrated in cell concentration under treatment with 5 μM RB, using a blue LED light at 455 ± 20 nm at the by Costa et al. [40] and Freire et al. [41]. Costa et al. achieved a 1.58 log10 reduction in cell 2 fluenceconcentration rate of under526 mW/cm treatment and with illumination 5 µM RB, for using 180 a s, blue which LED supplied light at 455an energy± 20 nm dose at theof 2 95fluence J/cm .rate Freire of 526et al. mW/cm used 12.52 and μM illumination RB under a green for 180 LED s, which light suppliedat 532 ± 10 an nm energy at a fluence dose of 2 2 rate95 J/cm of 2372. Freire mW/cm et al. and used the 12.5 sameµM RBillumination under a green time LED (energy light atdose 532 42.6± 10 J/cm nm at), athereby fluence achievingrate of 237 complete mW/cm cell2 and eradication. the same In illumination our study, timecomplete (energy cell doseeradication 42.6 J/cm was2 ),obtained thereby 2 atachieving a higher completeRB concentration cell eradication. (50 μM) Inbut our at study,much completelower light cell fluence eradication (2.0 mW/cm was obtained). In our at experiments,a higher RB concentrationusing 50 μM RB (50 illuminatedµM) but at muchby a white lower wi lightde-spectrum fluence (2.0 lamp mW/cm supplying2). In ouran energyexperiments, dose of using only 1.7 50 µJ/cmM RB2 enabled illuminated the complete by a white inhibition wide-spectrum of Candida lamp albicans supplying growth. an energy dose of only 1.7 J/cm2 enabled the complete inhibition of Candida albicans growth.

Figure 3. Treatment of Candida albicans planktonic culture by 50 µM RB under white-light illumination Figureat the ﬂuence3. Treatment of 1.9 of± Candida0.1 mW/cm albicans−2 (L) planktonic and in the culture dark (D). by 50 μM RB under white-light illumination at the fluence of 1.9 ± 0.1 mW/cm−2 (L) and in the dark (D). 2.2. Photodynamic Treatment of Trichophyton rubrum 2.2. PhotodynamicIn this part ofTreatment the study, of Trichophyton we tested two rubrum prospective photosensitizers, MGO and RB, againstIn this the part fungus of theTrichophyton study, we rubrumtested tw. Ino addition,prospective we photosensitizers, examined the antifungal MGO and effect RB, againstof two the common fungus keratolytic Trichophyton agents, rubrum urea. In and addition, thiourea we [examined42]. Solutions the antifungal of the agents effect were of twoprepared common in akeratolytic water base agents, or a glycerol/water urea and thiour (70/30%,ea [42]. Solutionsw/w) mixture. of the First,agents we were studied pre- paredthe photodynamic in a water base effects or a of glycerol/water RB and MGO in(70/30%, aqueous w/w solutions) mixture. at various First, we concentrations studied the photodynamic(Figures4 and5 effects, respectively). of RB and Figure MGO4 ina,b aqueou shows the solutions effect of at RB various on the concentrations cells of Trichophy- (Fig- ureston rubrum4 and 5,under respectively). illumination Figure and 4a,b in theshow dark, the respectively.effect of RB on The the layout cells of of Trichophyton wells in the rubrumexperiment under is illumination shown in Figure and4 inc. the Complete dark, respectively. inhibition of TheTrichophyton layout of rubrumwells ingrowth the exper- was imentachieved is shown in the presencein Figure of 4c. 50 CompleteµM RB under inhibition illumination of Trichophyton (Figure4a). rubrum RB had growth no effect was on achievedTrichophyton in the rubrum presencein the of dark.50 μM RB under illumination (Figure 4a). RB had no effect on TrichophytonIt is worth rubrum mentioning in the dark. that the cells of Candida albicans were also inhibited by RB at this concentration (Figure2b). In the work of Houang et al. [ 43], it was shown that RB at the concentration of 140 µM can kill virtually all (99.99 ± 0.01%) spores of Trichophyton rubrum under irradiation by a 532 nm laser at 40 mW for 10 min (101 J/cm2 ﬂuence). In clinical studies, RB was applied at the concentration of 140 µM under laser irradiation at 2.55 W/cm2 against toenail onychomycosis in vivo and after 5 treatments for 3 months complete clinical and mycological clearance was achieved [44]. A similar experiment was performed using MGO, and the results showed that this PS also totally inhibited the growth of Trichophyton rubrum at 50 µM, but unlike RB, there was no difference between the results obtained under illumination and in the dark (Figure5a,b, respectively). Figure5c presents the layout of wells in this experiment. Since the effect of

MGO on fungal cells could not be assigned to its photodynamic activity, the study was continued using only RB as a PS. Pathogens 2021, 10, x FOR PEER REVIEW 5 of 13

(a) (b) (c) Pathogens 2021, 10, x FOR PEER REVIEW 5 of 13 Pathogens 2021, 10, 263 5 of 13 Figure 4. Effect of aqueous solutions of RB at various concentrations on growth of Trichophyton rubrum on Yeast Mold (YM) agar: (a) under illumination of 1.9 ± 0.1 mW/cm2 for 30 min and (b) in the dark. The layout of wells in the experiment (c): control wells A1 and B1—untreated culture of Trichophyton rubrum; control C1—agar medium without fungal culture; the remaining wells—agar medium with applied aqueous solutions of RB at concentrations specified in the scheme.

It is worth mentioning that the cells of Candida albicans were also inhibited by RB at this concentration (Figure 2b). In the work of Houang et al. [43], it was shown that RB at the concentration of 140 μM can kill virtually all (99.99 ± 0.01%) spores of Trichophyton rubrum under irradiation by a 532 nm laser at 40 mW for 10 min (101 J/cm2 fluence). In clinical studies, RB was applied at the concentration of 140 μM under laser irradiation at 2.55 W/cm2 against toenail onychomycosis in vivo and after 5 treatments for 3 months complete clinical and mycological clearance was achieved [44]. (a) A similar experiment(b) was performed using MGO, and(c) the results showed that this PS also totally inhibited the growth of Trichophyton rubrum at 50 μM, but unlike RB, there Figure 4.4. EffectEffect ofof aqueousaqueous solutions solutionswas of ofno RB RB difference at at various various concentrationsbetween concentrations the results on on growth growth obtain of Trichophyton ofed Trichophyton under illumination rubrum rubrumon Yeast on and Yeast Mold in the Mold (YM) dark (Figure 2 agar:(YM) (agar:a) under (a) under illumination illumination of 1.9 5a,b,of± 1.90.1 respectively).± mW/cm 0.1 mW/cmfor2 30for Figure min 30 min and 5c and ( bpresents) in(b) the in the dark. the dark. layo The The layoutut oflayout wells of wells of inwells inthis the in experiment. the experiment experiment (cSince): the ef- control(c): control wells wells A1 andA1 and B1—untreated B1—untreatedfect culture of culture MGO of Trichophyton ofon Trichophyton fungal rubrumcells rubrum could; control; controlnot C1—agar be C1—agarassigned medium medium to its without photodynamic without fungal fungal culture; activity, culture; the the study the remaining wells—agar medium with applied aqueous solutions of RB at concentrations specified in the scheme. remaining wells—agar medium withwas applied continued aqueous using solutions only RB of RBas a at PS. concentrations speciﬁed in the scheme. It is worth mentioning that the cells of Candida albicans were also inhibited by RB at this concentration (Figure 2b). In the work of Houang et al. [43], it was shown that RB at the concentration of 140 μM can kill virtually all (99.99 ± 0.01%) spores of Trichophyton rubrum under irradiation by a 532 nm laser at 40 mW for 10 min (101 J/cm2 fluence). In clinical studies, RB was applied at the concentration of 140 μM under laser irradiation at 2.55 W/cm2 against toenail onychomycosis in vivo and after 5 treatments for 3 months complete clinical and mycological clearance was achieved [44]. A similar experiment was performed using MGO, and the results showed that this PS also totally inhibited the growth of Trichophyton rubrum at 50 μM, but unlike RB, there (a) was no difference between (theb) results obtained under illumination(c) and in the dark (Figure

5a,b, respectively). Figure 5c presents the layout of wells in this experiment. Since the ef- FigureFigure 5. Effect 5. Effect of aqueous of aqueous solutions solutions of MGO of MGO at various at various concentrations concentrations on growthon growth of Trichophyton of Trichophyton rubrum rubrumon on YM YM agar: agar: (a) fect of MGO on2 fungal cells could not be assigned to its photodynamic activity, the study (a) underunder illumination illumination of 1.9 of± 1.90.1 ± mW/cm0.1 mW/cmfor2 for 30 min30 min and and (b) in(b the) in dark.the dark. The layoutThe layout of wells of wells in the in experiment the experiment (c): control (c): control wells A1wells and A1 B1—untreated and B1—untreatedwas culture continued culture of Trichophyton of usingTrichophyton rubrum only RB; rubrum control as a; C1—agarPS.control C1—agar medium medium without without fungal culture; fungal theculture; remaining the remain- wells—agaring wells—agar medium with medium applied with aqueous applied solutions aqueous ofsolutions MGO at of concentrations MGO at concentrations speciﬁed in specified the scheme. in the scheme.

Two agentsTwo agents known known to enhance to enhance the permeability the permeabilit of nails,y of nails, urea andurea thioureaand thiourea [42,45 [42,45],], Pathogens 2021, 10, x FOR PEER REVIEW were tested for antifungal activity as well. The experiments were performed6 of as 13 in the case were tested for antifungal activity as well. The experiments were performed as in the case of theof PSs. the FiguresPSs. Figures6 and 76 showand 7 theshow antifungal the antifungal effects effects of thiourea of thiourea and urea, and respectively,urea, respectively, whenwhen applied applied in aqueous in aqueous solutions solutions to agar to mediumagar medium with pre-seededwith pre-seeded fungi. fungi.

(a) (b) (c)

Figure 5. Effect of aqueous solutions of MGO at various concentrations on growth of Trichophyton rubrum on YM agar: (a) under illumination of 1.9 ± 0.1 mW/cm2 for 30 min and (b) in the dark. The layout of wells in the experiment (c): control wells A1 and B1—untreated culture of Trichophyton rubrum; control C1—agar medium without fungal culture; the remaining wells—agar medium with applied aqueous solutions of MGO at concentrations specified in the scheme.

Two agents(a known) to enhance the permeabilit(by) of nails, urea and thiourea [42,45], were tested for antifungal activity as well. The experiments were performed as in the case Figure 6. Effect of thiourea at various concentrations on growth of Trichophyton rubrum on YM agar Figure 6. Effectof the of PSs. thiourea Figures at various 6 and concentrations 7 show the antifungal on growth effects of Trichophyton of thiourea rubrum and on urea, YM respectively, agar mediummedium (a). A( alayout). A layout of wells of wells (b) containing (b) containing aqueous aqueous solutions solutions of thiourea of thiourea applied applied to agar to agar medium at when applied in aqueous solutions to agar medium with pre-seeded fungi. medium atconcentrations concentrations speciﬁed specified in in the the scheme scheme (%, (%,w/v w/v). Control). Control wells wells contained contained no no thiourea. thiourea. The experiment The experimentwas performed was performed under under illumination illumination of 1.9 of± 0.11.9 mW± 0.1 cmmW−2 cmfor−230 for min. 30 min.

(a) (b)

Figure 7. Effect of urea at various concentrations on growth of Trichophyton rubrum on YM agar medium (a). A layout of wells (b) containing aqueous solutions of thiourea applied to agar medium at concentrations specified in the scheme (%, w/v). Control wells contained no urea. The experiment was performed under illumination of 1.9 ± 0.1 mW/cm2 for 30 min.

The results showed that thiourea exhibited strong antifungal effects at concentrations above 3% (Figure 6), while urea totally inhibited the growth of Trichophyton rubrum at concentrations of above 25% (Figure 7). Previous in vivo studies showed contradictory data on the antifungal effect of urea. Bunyaratavej at al. [46] reported a partial (32%) cure of onychomycosis when using a cream containing 40% urea. However, Escalante et al. [47] found that application of the same 40% urea cream led to an onychomycosis mycological cure in 8.3% of cases. At the next stage of our study, we tested the antifungal activity of RB, a mixture of urea and thiourea, and a mixture of RB, urea and thiourea in a glycerol/water solution. Figure 8 shows the effect of RB in a glycerol/water mixture on Trichophyton rubrum. Fungal growth was totally inhibited at RB concentrations above 200 μM. Although it is not seen distinctly in the photograph in Figure 8a, at concentrations of 50 and 100 μM there was a proliferation of fungal cells.

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(a) (b)

Figure 6. Effect of thiourea at various concentrations on growth of Trichophyton rubrum on YM Pathogens 2021, 10agar, 263 medium (a). A layout of wells (b) containing aqueous solutions of thiourea applied to agar 6 of 13 medium at concentrations specified in the scheme (%, w/v). Control wells contained no thiourea. The experiment was performed under illumination of 1.9 ± 0.1 mW cm−2 for 30 min.

(a) (b)

Figure 7. Effect of ureaFigure at various 7. Effect concentrations of urea at various on growth concentrations of Trichophyton on growth rubrum of onTrichophyton YM agar rubrum on YM agar medium (a). A layoutmedium of wells (a ).(b A) containing layout of wells aqueous (b) containing solutions of aqueous thiourea solutions applied of to thiourea agar me- applied to agar medium dium at concentrationsat concentrations specified in the speciﬁed scheme in (%, the w/v scheme). Control (%, w/v wells). Control contained wells no contained urea. The noex- urea. The experiment periment was performedwas performed under illumination under illumination of 1.9 ± 0.1 of mW/cm 1.9 ± 0.12 for mW/cm 30 min.2 for 30 min.

The results showedThe that results thiourea showed exhibited that thiourea strong antifungal exhibited strongeffects antifungalat concentrations effects at concentrations above 3% (Figureabove 6), while 3% (Figureurea totally6), while inhibited urea totallythe growth inhibited of Trichophyton the growth rubrum of Trichophyton at rubrum at concentrations ofconcentrations above 25% (Figure of above 7). Prev 25%ious (Figure in vivo7). Previous studies showedin vivo contradictorystudies showed contradictory data on the antifungaldata oneffect the of antifungal urea. Bunyaratavej effect of urea. at al. Bunyaratavej [46] reported at a al. partial [46] reported (32%) cure a partial (32%) cure of onychomycosis when using a cream containing 40% urea. However, Escalante et al. [47] of onychomycosis when using a cream containing 40% urea. However, Escalante et al. [47] found that application of the same 40% urea cream led to an onychomycosis mycological found that application of the same 40% urea cream led to an onychomycosis mycological cure in 8.3% of cases. cure in 8.3% of cases. At the next stage of our study, we tested the antifungal activity of RB, a mixture of At the next stageurea andof our thiourea, study, we and tested a mixture the antifungal of RB, urea activity and thiourea of RB, a in mixture a glycerol/water of solution. urea and thiourea,Figure and 8a showsmixture the of effect RB, ofurea RB and in a thiourea glycerol/water in a glycerol/water mixture on Trichophyton solution. rubrum. Fungal Pathogens 2021, 10, x FOR FigurePEER REVIEW 8 shows thegrowth effect of was RB totally in a glycerol/water inhibited at RBmixture concentrations on Trichophyton above rubrum 200 µM.. Fungal Although 7it of is 13 not seen

growth was totallydistinctly inhibited in at the RB photograph concentrations in Figure above8a, 200 at concentrationsμM. Although ofit is 50 not and seen 100 µM there was a distinctly in the photographproliferation in of Figure fungal 8a, cells. at concentrations of 50 and 100 μM there was a proliferation of fungal cells.

(a) (b)

Figure 8. FigureEffect of 8. glycerol/waterEffect of glycerol/water (70/30%, w/w (70/30%,) solutionsw/w) solutionsof RB at various of RB at concentrations various concentrations on on growth 2 growth ofof TrichophytonTrichophyton rubrum rubrum onon YM YM agar, agar, (a) (undera) under illumination illumination of 1.9 of ± 1.9 0.1± mW/cm0.1 mW/cm2 for 30 min.for 30 min. A A layout layoutof wells of (b wells): control (b): wells control A1, wells B1, and A1, C1—untreated B1, and C1—untreated culture of cultureTrichophyton of Trichophyton rubrum; the rubrum ; the remainder—agarremainder—agar medium mediumwith applied with glycerol/water applied glycerol/water solutions solutions of RB at ofconcentrations RB at concentrations specified speciﬁed in in the scheme.the scheme.

It shouldIt be should mentioned be mentioned that this thatinhibitory this inhibitory concentration concentration of 200 μM of 200is muchµM ishigher much higher than thatthan of RB that in ofan RBaqueous in an aqueoussolution solution(50 μM). (50Weµ assumeM). We that assume such that a difference such a difference between between these resultsthese may results be due may to be the due different to the different amounts amounts of dissolved of dissolved oxygen in oxygen the solutions. in the solutions. Oxygen Oxygensolubility solubility decreases decreases linearly linearlywith an withincrease an increaseof glycerol of glycerolfraction fractionin glycerol/water in glycerol/water mixtures [48], so the oxygen concentration may be insufficient for providing effective photodynamic activity of PSs acting according to the Type II mechanism. This mechanism involves energy transfer from a PS in a triplet excited state (3PS*) to dissolved molecular oxygen in a triplet ground state (3O2), leading to a formation of oxygen in a singlet excited state (1O2), which causes irreversible damage to microorganisms [49,50]. After determining the concentrations of thiourea and urea in aqueous solution that inhibit growth of Trichophyton rubrum, the antifungal effects of their mixture in a glycerol/water solution was tested. Since urea and thiourea inhibited Trichophyton rubrum at concentrations of 25% and 3%, respectively, which is an approximate ratio of 10:1, this ratio was chosen as a baseline for all dilutions in this experiment. The results are presented in Figure 9. Interestingly, upon mixing urea and thiourea, the inhibiting of fungal growth occurred at much lower concentrations of these components than when applying each of them separately. The same inhibitory effects on Trichophyton rubrum obtained at the above-mentioned concentrations of urea and thiourea when applied separately (25% and 3%, respectively) was achieved by their mixture at concentrations of only 10% and 1%, respectively (Figure 9). The results obtained in the dark were identical to those obtained under illumination (data not shown). It appears that the mixture caused a synergistic antifungal effect despite the addition of glycerol to the solution.

(a) (b)

Figure 9. Effect of urea/thiourea in various concentrations on growth of Trichophyton rubrum on YM agar medium (a). Layout of wells (b) containing glycerol/water (70/30%, w/w) solutions of

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Pathogens 2021, 10, 263 7 of 13 (a) (b)

Figure 8. Effect of glycerol/water (70/30%, w/w) solutions of RB at various concentrations on growth of Trichophytonmixtures [48 rubrum], so theon YM oxygen agar, ( concentrationa) under illumination may beof 1.9 insufﬁcient ± 0.1 mW/cm for2 for providing 30 min. effective A layout ofphotodynamic wells (b): control activity wells A1, of PSsB1, and acting C1—untreated according toculture the Type of Trichophyton II mechanism. rubrum This; themechanism remainder—agarinvolves medium energy with transfer applied from glycerol/water a PS in a tripletsolutions excited of RB stateat concentrations (3PS*) to dissolved specified molecular in the scheme. 3 oxygen in a triplet ground state ( O2), leading to a formation of oxygen in a singlet excited 1 state ( O2), which causes irreversible damage to microorganisms [49,50]. It should beAfter mentioned determining that this the concentrationsinhibitory concentration of thiourea of and 200 ureaμM is in much aqueous higher solution that than that ofinhibit RB in growthan aqueous of Trichophyton solution (50 rubrumμM). We, the assume antifungal that such effects a difference of their mixturebetween in a glyc- these resultserol/water may be solutiondue to the was different tested. Sinceamounts urea of and dissolved thiourea oxygen inhibited in theTrichophyton solutions. rubrum at Oxygen solubilityconcentrations decreases of 25% linearly and 3%,with respectively, an increase whichof glycerol is an fraction approximate in glycerol/water ratio of 10:1, this ratio mixtures [48],was so chosen the oxygen as a baseline concentration for all dilutions may be insufficient in this experiment. for providing The resultseffective are pho- presented in todynamicFigure activity9. Interestingly,of PSs acting upon according mixing to ureathe Type and thiourea,II mechanism. the inhibiting This mechanism of fungal growth involves energyoccurred transfer at much from lower a PS concentrationsin a triplet excited of these state components(3PS*) to dissolved than when molecular applying each oxygen inof a triplet them separately.ground state The (3O same2), leading inhibitory to a formation effects on ofTrichophyton oxygen in a singlet rubrum excitedobtained at the state (1O2),above-mentioned which causes irreversible concentrations damage of to urea microorganisms and thioureawhen [49,50]. applied separately (25% and After3%, determining respectively) the concentrations was achieved byof theirthiourea mixture and urea at concentrations in aqueous solution of only that 10% and 1%, respectively (Figure9). The results obtained in the dark were identical to those obtained inhibit growth of Trichophyton rubrum, the antifungal effects of their mixture in a glyc- under illumination (data not shown). It appears that the mixture caused a synergistic erol/water solution was tested. Since urea and thiourea inhibited Trichophyton rubrum at antifungal effect despite the addition of glycerol to the solution. concentrations of 25% and 3%, respectively, which is an approximate ratio of 10:1, this Finally, we tested a formulation containing all three successful compounds (RB, urea, ratio was chosenand thiourea) as a baseline in glycerol/water for all dilutions (70/30%, in this experiment.w/w) solutions The at results various are concentrations, presented with in Figure 9.Trichophyton Interestingly, rubrum, upon undermixing illumination urea and thiourea, and in the inhibiting dark. Figure of fungal 10 shows growth the results of occurred atthis much experiment. lower concentrations Under illumination, of these total components inhibition than of fungal when growth applying was each observed of in the them separately.formulation The containingsame inhibitory 150 µ Meffects RB, 5% on urea, Trichophyton and 0.5% rubrum thiourea obtained (Figure 10ata). theFigure 10b above-mentionedshows that concentrations without illumination of urea and the formulationthiourea when rendered applied no separately antifungal (25% effect. and The mixture 3%, respectively)of urea was and achieved thiourea atby thetheir concentrations mixture at concentrations applied in this of only experiment 10% and did 1%, not inhibit respectivelyTrichophyton (Figure 9). rubrum, The resultsand theobtained inhibiting in the concentration dark were identical of RB in thisto those formulation obtained was lower under illuminationthan when (data RB wasnot shown). applied It alone. appears Therefore, that the themixture presence caused of ureaa synergistic and thiourea an- even in tifungal effectsub-inhibiting despite the concentrations addition of glycerol contributed to theto solution. the overall antifungal effect.

(a) (b)

Figure 9. EffectFigure of 9.urea/thioureaEffect of urea/thiourea in various concentrations in various concentrations on growth of onTrichophyton growth of rubrumTrichophyton on rubrum on YM agar mediumYM agar (a). medium Layout of (a ).wells Layout (b) containing of wells (glycerol/waterb) containing (70/30%, glycerol/water w/w) solutions (70/30%, of w/w) solutions of urea/thiourea applied onto agar medium at concentrations speciﬁed in the scheme (%, w/v). Control wells contained no urea/thiourea. The experiment was performed under illumination of 1.9 ± 0.1 mW/cm2 for 30 min.

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urea/thiourea applied onto agar medium at concentrations specified in the scheme (%, w/v). Con- trol wells contained no urea/thiourea. The experiment was performed under illumination of 1.9 ± 0.1 mW/cm2 for 30 min.

Finally, we tested a formulation containing all three successful compounds (RB, urea, and thiourea) in glycerol/water (70/30%, w/w) solutions at various concentrations, with Trichophyton rubrum, under illumination and in the dark. Figure 10 shows the results of this experiment. Under illumination, total inhibition of fungal growth was observed in the formulation containing 150 μM RB, 5% urea, and 0.5% thiourea (Figure 10a). Figure 10b shows that without illumination the formulation rendered no antifungal effect. The mixture of urea and thiourea at the concentrations applied in this experiment did not inhibit Pathogens 2021, 10, 263 Trichophyton rubrum, and the inhibiting concentration of RB in this formulation was lower8 of 13 than when RB was applied alone. Therefore, the presence of urea and thiourea even in sub-inhibiting concentrations contributed to the overall antifungal effect.

(a) (b) (c)

Figure 10. Effect of RB,RB, urea,urea, andand thioureathiourea atat variousvarious concentrationsconcentrations ininglycerol/water glycerol/water (70/30%,(70/30%, w/w)) solutions on the growth of Trichophyton rubrumrubrum onon YMYM agar:agar: ((aa)) underunder illumination illumination at at 1.9 1.9± ± 0.10.1 mW/cmmW/cm22 forfor 30 30 min min and and ( (b)) in the dark. A layout of wells ( c): control control wells wells A1, A1, B1, B1, and and C1—untreated C1—untreated culture culture of of Trichophyton rubrum ; the remainder—agar medium with applied glycerol/water solutions solutions of of RB, RB, urea, urea, and thiourea at concentrations specified speciﬁed in the scheme.

2.3. Stability Stability of of Formulations Formulations To examine the stability of th thee selected antifungal formulation over over time, time, eight eight solu- solu- tions,tions, containing each each component alone alone or or a mixture of all three components in aqueous and glycerol/water solutions solutions (Table (Table 11),), were preparedprepared atat thethe samesame concentrationsconcentrations ofof stockstock solutions used for the photodynamicphotodynamic experiments described above. The solutions were incubatedincubated under under shaking in the darkdark andand monitoredmonitored byby measuringmeasuring theirtheir UV/VisUV/Vis spectra over time. The The monitoring monitoring of of the solution solutionss was performed at wavelengthswavelengths of maximal λ absorption, λmax (Table1 1).). WithWith regard regard to to the the four four water-based water-based and and three three glycerol/water glycerol/water solutions (samples 1–4 and 6–8 in Table1, respectively), no changes in their spectra were solutions (samples 1–4 and 6–8 in Table 1, respectively), no changes in their spectra were observed during 32 days of monitoring; therefore, it was concluded that these samples observed during 32 days of monitoring; therefore, it was concluded that these samples were stable over time. However, the spectrum of the urea solution in the glycerol/water were stable over time. However, the spectrum of the urea solution in the glycerol/water mixture (sample 5 in Table1) changed after the 32-day incubation, displaying a new peak mixtureat 272 nm (sample (Figure 5 11in a),Table probably 1) changed due toafter decomposition the 32-day incubation, of the urea. displaying Since the a antifungal new peak atformulation 272 nm (Figure suggested 11a), asprobably optimal due contains to deco allmposition three compounds of the urea. (urea, Since thiourea, the antifungal and RB) formulationin the glycerol/water suggested mixture, as optimal we contains assume thatall three urea compounds may decompose (urea, in thiourea, this formulation and RB) inas the well. glycerol/water Nevertheless, mixture, its spectra we assume (sample that 8, Figureurea may 11b) decompose did not register in this anyformulation additional as well.peak, Nevertheless, or even a shoulder, its spectra in the (sample region 8, of Figure 250–300 11b) nm. did Therefore, not register we any concluded additional that peak, this orformulation even a shoulder, demonstrates in the region good overallof 250–300 stability. nm. Therefore, we concluded that this formulation demonstrates good overall stability. Table 1. Composition and wavelengths of maximal absorption (λmax) of samples in experiments for testing stability of formulations.

Solvent Sample Number Urea (g) Thiourea (g) Rose Bengal (g) λmax (nm) 1 0.1 190

1 2 0.1 196, 236 Water 3 0.1 212, 546 4 0.1 0.1 0.1 230, 546 5 0.1 202 Glycerol/Water 1 6 0.1 230 70/30%, w/w 7 0.1 214, 556 8 0.1 0.1 0.1 230, 556 1 Components were dissolved in 10 mL of the solvent.

We assume that in vivo experiments will show the suggested formulation to be ef- ﬁcient against onychomycosis. Moreover, using an ordinary white luminescent lamp as proposed here contrasts with most studies on photodynamic inhibition of fungi, where excitation of PSs is performed by lasers at speciﬁc wavelengths. This difference should simplify future applications of our suggested formulation for onychomycosis treatment in everyday environments. Pathogens 2021, 10, 263 9 of 13

To complete the determination of optimal conditions of the treatment, our group is Pathogens 2021, 10, x FOR PEER REVIEW 9 of 13 currently carrying out studies on PS permeation into keratin biomembranes that model nail plates. These studies are attempting to test the effect of the keratolytic agents (urea and thiourea) on delivery of PSs into the nail plate.

(a) (b)

Figure 11. UV-visibleFigure 11. absorption UV-visible spectra absorption of the spectra urea solution of the urea (a) andsolution the formulation (a) and the formulation containing RB,containing urea, and thiourea ◦ (b) in a glycerol/waterRB, urea, (70/30%,and thioureaw/w )(b solution) in a glycerol/water before (blue line) (70/30%, and afterw/w) 32 solution days of before incubation (blue (blackline) and dotted after line) 32 at 30 C in the dark. days of incubation (black dotted line) at 30 °C in the dark.

We assume3. Materials that in vivo and experiments Methods will show the suggested formulation to be effi- cient against3.1. onychomycosis. Fungal Strains Moreover, using an ordinary white luminescent lamp as proposed here contrastsTwo fungal with strainsmost studiesCandida on albicans photodynamic(ATCC 90028) inhibition and ofTrichophyton fungi, where rubrum (ATCC excitation of MYA-4438)PSs is performed were used by lasers in this at study. specific wavelengths. This difference should simplify future applications of our suggested formulation for onychomycosis treatment in everyday environments.3.2. Preparation and Growth of Fungi To complete Candidathe determination albicans was of seeded optimal onto conditions DifcoTM ofYeast the Moldtreatment, (YM) our agar group (BD, Franklin is Lakes, currently carryingNJ, USA) out andstudies incubated on PS forpermea 48 htion at 30 into◦C; keratin 2–3 colonies biomembranes of yeast were that seeded model into DifcoTM nail plates. TheseYM broth studies (BD) are and attempting incubated to undertest the shaking effect of at the 200 keratolytic rpm for 24 agents h at 30 (urea◦C. The obtained and thiourea)inoculum on delivery was of dilutedPSs into with the nail saline plate. (0.90% w/v of NaCl) to obtain a suspension having OD530 = 0.10–0.16. This OD was measured using a spectrophotometer (Genesys 10S UV- Table 1. CompositionVIS, Thermo and wavelengths Fisher, Waltham, of maximal MA, absorption USA), and (λ itsmax) value of samples corresponded in experiments to cell concentration for testing stabilityof 1–3 of× formulations.106 cells/ml according to the 0.5 McFarland standard method [51]. ◦ 2 Solvent SampleTrichophyton Number rubrum Ureawas (g) grown Thiourea for 14 (g) days Rose on YMBengal agar (g) at 30λmaxC. (nm) Samples of 0.5 cm of dermatophyte1 culture0.1 were cut off from the agar plates with a190 scalpel, suspended ◦ in 10 mL of2 YM broth and incubated0.1 under shaking at 200 rpm196, for 236 48 h at 30 C. The Water1 obtained suspensions3 were filtered through a sterile0.1 cotton gauze212, pad, 546 which retained hyphal fragments4 but permitted0.1 the passage0.1 of dermatophyte0.1 microconidia.230, 546 5 0.1 202 Glycerol/Water3.3.1 Photodynamic6 Treatment of Planktonic0.1 Cultures of Candida albicans 230 70/30%, w/w Stock7 solutions of RB, MGO, and MB hydrate (Sigma-Aldrich,0.1 214, St. Louis,556 MO, USA) in water were8 prepared at the0.1 concentration0.1 of 4.5 mM.0.1 Aliquots of the230, stock 556 solutions were 1 Componentsadded were dissolved to 10 mL in of 10Candida mL of the albicans solvent.suspension at the concentration of 2–8 × 106 cells/mL in a Petri dish, to achieve a final PS concentration of 500 µM. In some experiments, RB 3. Materials and Methods MGO solutions were added to 10 mL of Candida albicans suspension to obtain final 3.1. Fungal Strainsconcentrations of 50, 250, or 500 µM. The cell suspensions with MB were exposed to light Two fungalfor 4strains h, and Candida samples albicans withRB (ATCC and MGO90028)were and Trichophyton exposed for rubrum 0.5, 1, 2,(ATCC 3, and 4 h, using an 18 W white luminescent lamp with an emissions range of 400–700 nm, providing a MYA-4438) were used in this study. light intensity of 1.9 ± 0.1 mW/cm2 at ambient temperature. Illuminance was measured µ 3.2. Preparationby and a LX-102 Growth light of Fungi meter (Lutron, Taipei, Taiwan). After irradiation, 100 L aliquots of each sample in 3–4 decimal dilutions were spread over YM agar plates with a Drigalsky TM Candida spreader,albicans was incubated seeded onto at 37 Difco◦C overnight, Yeast Mold and (YM) then theagar colony-forming (BD, Franklin Lakes, units (CFU) were NJ, USA) and incubated for 48 h at 30 °C; 2–3 colonies of yeast were seeded into DifcoTM YM broth (BD) and incubated under shaking at 200 rpm for 24 h at 30°C. The obtained inoculum was diluted with saline (0.90% w/v of NaCl) to obtain a suspension having OD530 = 0.10–0.16. This OD was measured using a spectrophotometer (Genesys 10S UV-VIS,

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counted. These experiments aimed to test the photodynamic character of the PS activity compared to activity without exposure to light. In the control experiments, samples of Candida albicans suspension without addition of PS were used.

3.4. Photodynamic Treatment of Trichophyton rubrum The schematic presentation of experiments in the photodynamic treatment of Tri- chophyton rubrum is shown in Scheme1. First, 2 mL portions of YM agar were dispensed into each well of a 12-well plate. After hardening of the agar medium, 100 µL aliquots of Trichophyton rubrum suspension were added on the surface. After that, 100 µL of PS solutions, in water or in a glycerol/water (70/30%, w/w) mixture, were applied to the surface after two-fold dilutions. In the case of water solutions, the ﬁnal concentration in those added onto the agar surface ranged between 800 to 3.12 µM for RB, 200 to 0.78 µM for MGO, 25% to 5% (w/v) for urea, and 7% to 0.06% (w/v) for thiourea. In the case of glycerol/water solutions, the ﬁnal concentration on the agar surface ranged between 800 Pathogens 2021, 10, x FOR PEER REVIEW 11 of 13 to 3.12 µM for RB, and 20/2% to 0.078/0.008% (w/v) for urea/thiourea. The concentration in the three-component formulation (RB/urea/thiourea) was between 150 µM/5%/0.5% and 0.6 µM/0.02%/0.002% (w/v), respectively.

Scheme 1. General presentation of the experiment on the photodynamic treatment of Trichophyton rubrum. Scheme 1. General presentation of the experiment on the photodynamic treatment of Trichophyton rubrum. After addition of Trichophyton rubrum suspensions and the solutions of components 3.5. Stability of Formulations speciﬁed above, the 12-well plate was illuminated for 30 min with a white luminescent lamp atTwo the lightsolvents ﬂuence were ofused 1.9 to± prepare0.1 mW/cm eight2 so. Alllutions; experiments four of them were were performed water-based at ambient (samples 1–4) and four were based on a glycerol/water (70/30%, w/w) mixture (samples 5– temperature. After illumination, the plates were incubated in the dark at 30 ◦C for 7 days 8). Table 1 presents the prepared solutions, containing 0.1 g of each compound (urea, thi- andourea, examined or RB, and visually the three for compounds fungal growth. together The) dissolved same experiments in 10 mL of each were solvent. carried All out in parallelthe solutions without were light incubated exposure, under and shaking all experiments at 200 rpm were for 32 duplicated. days at 30 °C in the dark. Aliquots of 100 μL from each solution were sampled at the beginning of the experiment, 3.5.and Stability after 4, of 7, Formulations and 32 days of incubation. Before examination, the samples were diluted withTwo water, solvents and their were spectra used were to prepare then regist eightered solutions;using a 1 cm four quartz of themcuvette were in the water-based range (samplesof 190–1100 1–4) nm and with four steps were of 2 based nm. Further on a glycerol/water monitoring of the (70/30%, solutionsw/w was) performed mixture (samples at 5–8).wavelengths Table1 presents of maximal the absorption prepared λ solutions,max (Table containing 1). 0.1 g of each compound (urea, thiourea, or RB, and the three compounds together) dissolved in 10 mL of each solvent. All the3.6. solutions Statistical were Methods incubated under shaking at 200 rpm for 32 days at 30 ◦C in the dark. AliquotsEach of experiment 100 µL from was each carried solution out in were duplicate sampled and then at the repeated beginning to confirm of the the experiment, re- andproducibility after 4, 7, andof results. 32 days The ofaverage incubation. value ± BeforeSD was examination,calculated. the samples were diluted with water, and their spectra were then registered using a 1 cm quartz cuvette in the range 4. Conclusions of 190–1100 nm with steps of 2 nm. Further monitoring of the solutions was performed at wavelengthsThree photosensitizers, of maximal absorption RB, MB, λandmax MGO, (Table were1). tested for antifungal activity, but only RB was active against Trichophyton rubrum and Candida albicans under illumination. To find a formulation suitable for onychomycosis treatment, urea and thiourea were added to RB as factors for potential enhancement of nail plate permeability. The most effective formulation for inhibiting fungal growth contained 150 μM RB, 5% urea, and 0.5% thiourea in a glycerol/water (70/30%, w/w) solution. The formulation was stable for at least one month of storage at 30 °C.

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3.6. Statistical Methods Each experiment was carried out in duplicate and then repeated to conﬁrm the reproducibility of results. The average value ± SD was calculated.

4. Conclusions Three photosensitizers, RB, MB, and MGO, were tested for antifungal activity, but only RB was active against Trichophyton rubrum and Candida albicans under illumination. To ﬁnd a formulation suitable for onychomycosis treatment, urea and thiourea were added to RB as factors for potential enhancement of nail plate permeability. The most effective formulation for inhibiting fungal growth contained 150 µM RB, 5% urea, and 0.5% thiourea in a glycerol/water (70/30%, w/w) solution. The formulation was stable for at least one month of storage at 30 ◦C.

Author Contributions: Conceptualization, M.N.; methodology, M.N. and A.V.; investigation, A.V.; resources, M.N. and M.Z.; data curation, A.V.; writing—original draft preparation, A.V.; writing— review and editing, M.N. and M.Z.; supervision, M.N. and M.Z.; project administration, M.N. and M.Z.; funding acquisition, M.N. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Research Authority of the Ariel University, Israel (Grant RA1700000353). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. Acknowledgments: We acknowledge the Research Authority of the Ariel University, Israel, for supporting this research. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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