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Experimental Therapeutics

Evaluation of the Antifungal and Wound-Healing Properties of a Novel Peptide-Based Bioadhesive Hydrogel Formulation

Ahmed S. Sultan, Taissa Vila, Eman Hefni, Amy J. Karlsson, Mary Ann Jabra-Rizk
Ahmed S. Sultan
aDepartment of Oncology and Diagnostic Sciences, Dental School, University of Maryland, Baltimore, Maryland, USA
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Taissa Vila
aDepartment of Oncology and Diagnostic Sciences, Dental School, University of Maryland, Baltimore, Maryland, USA
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Eman Hefni
aDepartment of Oncology and Diagnostic Sciences, Dental School, University of Maryland, Baltimore, Maryland, USA
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Amy J. Karlsson
bDepartment of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland, USA
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Mary Ann Jabra-Rizk
aDepartment of Oncology and Diagnostic Sciences, Dental School, University of Maryland, Baltimore, Maryland, USA
cDepartment of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore, Maryland, USA
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DOI: 10.1128/AAC.00888-19
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ABSTRACT

Oral candidiasis (OC) caused by the fungal pathogen Candida albicans is the most common opportunistic infection in immunocompromised populations. The dramatic increase in resistance to common antifungal agents has emphasized the importance of identifying alternative therapeutic options. Antimicrobial peptides have emerged as promising drug candidates due to their antimicrobial properties; specifically, histatin-5 (Hst-5), a peptide naturally produced and secreted by human salivary glands, has demonstrated potent activity against C. albicans. However, as we previously demonstrated vulnerability for Hst-5 to proteolysis by C. albicans proteolytic enzymes at specific amino acid residues, a new variant (K11R-K17R) was designed with amino acid substitutions at the identified cleavage sites. The new resistant peptide demonstrated no cytotoxicity to erythrocytes or human oral keratinocytes. To evaluate the potential of the new peptide for clinical application, we utilized our FDA-approved polymer-based bioadhesive hydrogel as a delivery system and developed a therapeutic formulation specifically designed for oral topical application. The new formulation was demonstrated to be effective against C. albicans strains resistant to the traditional antifungals, and the in vitro therapeutic efficacy was found to be comparable to that of the common topical antifungal agents in clinical use. Importantly, in addition to its antifungal properties, our findings also demonstrated that the new peptide variant induces cell proliferation and rapid cell migration of human oral keratinocytes, indicative of wound healing properties. The findings from this study support the progression of the novel formulation as a therapeutic agent against oral candidiasis, as well as a therapeutic modality for promoting wound healing.

INTRODUCTION

Candida albicans is a ubiquitous fungal species that commonly colonizes various mucosal sites, including oral surfaces (1–3). However, disruptions in the host immune status or local microenvironment perturbations can induce the commensal transition of C. albicans into a pathogen causing a wide range of infections, both mucosal and systemic, particularly in immunocompromised individuals (4, 5). Oral candidiasis (OC) is the most common manifestation of candidiasis and can occur in immunocompetent patients as a consequence of antibiotic therapy, steroid therapy, metabolic and hormonal disturbances, and denture wearing, among several other predisposing factors (6). With the continued increase in the incidence of C. albicans infections and resistance to the relatively limited number of available antifungal drugs, the prospect of preventing the C. albicans transition from commensal to pathogenic, thus precluding candidiasis, is becoming increasingly attractive.

Natural antimicrobial peptides (AMPs) have attracted significant attention as candidates for drug development due to their broad-spectrum antimicrobial and anti-inflammatory properties, lack of toxicity, and lack of development of drug resistance (7, 8). Histatin-5 (Hst-5), specifically, has demonstrated potent activity against C. albicans and other fungal species (9, 10). Hst-5, a member of the histatin family, is a 24-amino acid histidine-rich polypeptide secreted by human salivary glands and is present in saliva (11, 12). Hst-5 does not induce microbial resistance and is nontoxic to humans, which makes it an ideal candidate as a therapeutic agent against OC (13).

As an opportunistic pathogen, C. albicans is well adapted to its human host and possesses an array of virulence factors that contribute to its pathogenic potential. Most notable among the virulence factors are the secreted aspartyl proteinases (Saps), potent proteolytic enzymes that are capable of degrading several host proteins and are responsible for tissue invasion (14, 15). Our previous studies characterized a potential host immune evasion strategy by C. albicans via degradation of Hst-5 by the Sap enzymes, thereby, for the first time, identifying Hst-5 as a host substrate for the C. albicans Saps. Further, using high-pressure liquid chromatography (HPLC) and mass spectrometry analysis of the fragmented peptide, we identified the lysine residues at positions 11 and 17 on the 24-amino acid Hst-5 peptide to be the specific cleavage sites for the Sap enzymes (16). Based on these novel findings, and using Hst-5 as a blueprint, our efforts were directed toward engineering peptide derivatives of Hst-5 through amino acid substitutions to generate a stable variant peptide with retained antifungal activity that is resistant to proteolytic degradation (17). By targeting both vulnerable lysine residues, we were able to generate a double amino acid (K11R-K17R) mutant peptide that was immune to degradation.

To begin to explore the potential of developing Hst-5 and derivatives for therapeutic purposes, a feasible delivery system suitable for topical mucosal application is needed. As a proof-of-principle, we recently developed a polymer-based Hst-5 hydrogel formulation with bioadhesive properties specifically designed for oral application. The delivery system was fully evaluated for peptide release rate, stability, and potency and was shown to be efficacious in vivo in a mouse model of OC (18). In this study, we aimed to comparatively and comprehensively evaluate the double mutant peptide in vitro. Taken together, the findings indicated that the new peptide formulation possesses high killing potency against C. albicans, including strains resistant to common antifungals, and under certain conditions, activity was superior to that of the native Hst-5 peptide. Further, the peptide was established to lack toxicity to human cells and, importantly, induced rapid cell migration of oral keratinocytes, indicating additive wound-healing properties.

The development of novel alternative nontraditional therapeutic strategies for the prevention of OC is a very attractive concept. Specifically, the availability of an effective and safe oral topical antifungal with wound-healing properties that can be feasibly applied will be highly desirable to a multitude of cohorts of individuals susceptible to OC.

RESULTS

K11R-K17R and Hst-5 kill C. albicans in a cell density-dependent manner, with K11R-K17R exhibiting superior anticandidal potency.Based on CFU counts (Fig. 1A) and percent killing (Fig. 1B), both peptides were found to exert fungal killing ability inversely proportional to C. albicans cell density, with no (Hst-5) or minimal (K11R-K17R) killing noted when the absolute number of C. albicans cells incubated was 5 × 106 cells. Although no statistically significant differences (P > 0.05) between K11R-K17R and Hst-5 were observed at low cell densities, K11R-K17R consistently exhibited enhanced anticandidal potency against 5 × 105 C. albicans cells, with 61.3% mean killing potency (P < 0.001), compared to 33.8% for Hst-5 (P > 0.05). Almost complete killing of 5 × 102 C. albicans cells was observed with K11R-K17R (95.6%) and Hst-5 (97.8%) (P < 0.001). The vehicle polymer (HPMC) used in peptide formulation did not have any effect on C. albicans compared to phosphate-buffered saline (PBS).

FIG 1
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FIG 1

C. albicans cell density-dependent and exposure time-dependent susceptibility to formulations of K11R-K17R and Hst-5. (A, B) Based on CFU counts and percent killing, C. albicans cell density killing assays demonstrated significant killing ability for both peptides at 2 h that was inversely proportional to C. albicans cell density, with K11R-K17R exhibiting more potent killing against 5 × 105 cells. No killing and minimal killing were seen for Hst-5 and K11R-K17R, respectively, against 5 × 106 C. albicans cells. (C) Time-course killing assays using 5 × 102 C. albicans cells following 15-, 30-, and 120-min exposure demonstrated significant C. albicans killing by both peptide formulations in a manner proportional to the time of exposure, with maximum killing at 120 min and with K11R-K17R exhibiting significantly enhanced anticandidal potency at 15 and 30 min. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; ns, not significant.

K11R-K17R and Hst-5 kill C. albicans in a time-dependent manner with enhanced anticandidal potency for K11R-K17R.Based on CFU counts, the percent killing for both peptides was found to be proportional to the time of exposure of C. albicans cells to the peptides. Statistically significant differences between K11R-K17R and Hst-5 were observed, with K11R-K17R consistently exhibiting enhanced anticandidal potency at the earlier time points (15 and 30 min) (Fig. 1C); at 15 min, the mean killing potency was 26.9% and 88.5% for Hst-5 (P > 0.05) and K11R-K17R (P < 0.0001), respectively. At 30 min, the mean killing potency was 40.0% and 89.4% for Hst-5 (P < 0.001) and K11R-K17R (P < 0.0001), respectively. No differences were observed at 120 min (mean percent killing efficacy of K11R-K17R, 100%; Hst-5, 96.5%).

Efficacy of K11R-K17R against C. albicans azole-resistant strains.Similar to the efficacy against the standard susceptible strain, K11R-K17R was able to kill all C. albicans azole-resistant strains (Table 1) tested, with a range of mean killing potency of 71.5% to 92.2% (P < 0.0001) (Fig. 2A). Similar results were observed for Hst-5 (not shown).

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TABLE 1

The C. albicans antifungal-resistant strains used in this study

FIG 2
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FIG 2

Efficacy of the K11R-K17R formulation against antifungal-resistant C. albicans strains and comparative evaluation of efficacy to that of topical antifungal agents in clinical use. (A) Susceptibility assays were performed using characterized antifungal-resistant C. albicans strains and the susceptible wild-type strain. Based on CFU counts following 120-min incubation, the K11R-K17R formulation exhibited significant killing against all the resistant strains, comparable to that seen with the susceptible wild-type strain. (B) Clotrimazole cream and miconazole nitrate gel were included in killing assays for comparison with peptide formulations. Based on CFU counts and percent killing, the efficacies of both peptide formulations following 120 min were comparable to that seen with the traditional antifungal agents. Vehicle control gel demonstrated 0% killing. ****, P < 0.0001; ns, not significant; WT, wild type.

Efficacy of K11R-K17R and hst-5 compared to traditional commercially available topical antifungals.The percent killing based on recovered CFU indicated comparable killing efficacy for both peptides against C. albicans to that of traditional topical antifungals in clinical use (Fig. 2B). The mean killing potency for Hst-5 and K11R-K17R was 96.5 and 100%, respectively (P < 0.0001), which was comparable to 89.3% and 100% mean killing potency for 1% clotrimazole cream and 2% miconazole nitrate gel, respectively (P < 0.0001). However, the antifungal agents resulted in 100% killing of 5 × 105 C. albicans cells (data not shown), whereas the peptides exerted reduced killing at this cell density (34% and 61% for Hst-5 and K11R-K17R, respectively).

K11R-K17R and Hst-5 do not induce any hemolytic activity on human red blood cells.To demonstrate the lack of adverse effects for the peptides on host cells, hemolysis assays were performed. Following exposure of red blood cells to peptides, based on hemoglobin release measured by absorbance at 540 nm, no hemolytic effect was observed for either peptide at concentrations as high as 10 mg/ml (Fig. 3A). These experiments were performed using peptide solutions in PBS.

FIG 3
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FIG 3

Cytotoxicity assays to evaluate the effect of the K11R-K17R and Hst-5 peptides on mammalian cells. (A) Red blood cells were exposed to increasing concentrations of the peptides, and hemolysis was assessed. In addition, the antifungal amphotericin B was included for comparison, and PBS was used as a negative control and Triton-X as a positive control for lysis. Based on the measurement of hemoglobin released upon hemolysis at A540nm, neither of the peptides caused cell lysis at all the concentrations tested. In contrast, exposure to amphotericin B resulted in approximately 89% lysis under the same conditions. (B) Metabolic cytotoxicity assays were performed following treatment of NOK cells with the peptides over a period of 24 to 48 h. Based on spectrophotometric readings at A490nm and compared to PBS (starvation medium), the increase in cell metabolic activity induced by the peptides was seen at both time points; however, the increase was not statistically significant. (C) The proliferation of NOK cells was also evaluated at 24 h and 48 h of exposure to the peptides. Compared to the PBS control, although cell counts at 24 h did not increase with peptides, a pronounced increase in cell counts was seen at 48 h. No statistically significant differences were noted when K11R-K17R and Hst-5 were compared.

K11R-K17R and Hst-5 are nontoxic to oral mammalian cells.Results from cytotoxicity assays following treatment of normal oral keratinocyte (NOK) cells with K11R-K17R and Hst-5 indicated that both peptides are nontoxic to human cells. Interestingly, a slight increase in metabolic activity was observed for the cells treated with the peptides (Fig. 3B). These experiments were performed using peptide solutions in PBS.

K11R-K17R and Hst-5 promote mammalian cell proliferation.Based on findings from the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) metabolic assay, peptides were tested for their ability to induce cell proliferation. Results from label-free direct cell counting, compared to PBS control at the 24-h time point, demonstrated that the peptides maintained cell counts. However, there was a significant increase in cell counts at 48 h, with approximately 16% and 36% increases from the 24-h time point for Hst-5 and K11R-K17R, respectively (Fig. 3C). These experiments were performed using peptide solutions in PBS.

K11R-K17R and Hst-5 promote wound healing by increasing cell migration.Since wound healing activity has been reported for some members of the histatin family of peptides, we designed migration assays using NOK cells to explore whether Hst-5 and K11R-K17R exhibit wound-healing properties at concentrations used in the gel formulation (2 mg/ml) and at concentrations physiologically present in saliva (0.05 mg/ml). Migration of cells treated with the peptides was monitored over a period of 24 h, and at each time point, migration was evaluated microscopically and quantitatively. Results demonstrated that Hst-5 and K11R-K17R at concentrations of 2 mg/ml comparably and significantly induced cell migration (90.2% to 94.5% compared to the PBS control) following 8 h of incubation (P < 0.0001) (Fig. 4A and B). Microscopic images indicated profound cell migration as evidenced by cell spreading, with keratinocytes extending their cytoplasmic processes to attach to adjacent cells via desmosomal linkage of the reciprocating processes, and thus, reepithelialization is demonstrated by the resultant gap closure (Fig. 4C). Further, keratinocytes also appeared to have enhanced metabolic activity as evidenced by the increased nuclear mitotic activity. Conversely, no extension of cytoplasmic processes and, thus, no cell migration were seen with the PBS control. Microscopically, peptide precipitation was seen at 2-mg/ml concentrations, manifested as uniform small dark precipitates within the gap; however, no effect on cell migration was seen when Hst-5 and K11R-K17R were used at physiologic concentrations of 0.05 mg/ml (Fig. 5). When monitored over a 24-h period, no significant increase in wound closure activity was observed for either peptide after 8 h (Fig. 5). These experiments were performed using peptide solutions in PBS.

FIG 4
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FIG 4

Microscopic and quantitative analysis of the wound-healing properties of the K11R-K17R and Hst-5 peptides. (A) Representative images and (B) quantitative analysis of the wound-healing process after an 8-h exposure of NOK cells to the peptides (2 mg/ml). Findings demonstrated significant activity in cell migration into the gap (wound) area (within the red lines) induced by the peptides, indicative of wound healing. ****, P < 0.0001. (C) Magnified image of NOK cells demonstrating their migration into the wound area (red arrows) after treatment with K11R-K17R for 8 h; the gray area within the gap represents deposits of peptides. PBS was used as a negative control for cell migration (0%). No significant differences were noted between the two peptides in their abilities to induce cell migration.

FIG 5
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FIG 5

Time course evaluation of the wound-healing process by the K11R-K17R and Hst-5 peptides. Representative images depicting the migration of NOK cells over time demonstrating significant wound healing at the 8-h time point. No significant increase in the level of cell migration was seen after 8 h. PBS was used as a negative control for cell migration.

DISCUSSION

The increase in the number of predisposed populations coupled with the emergence of resistance to the standard antifungal agents highlights the need for identifying alternative, natural nontoxic strategies to treat fungal infections that lack the side effects of existing therapies. Formulations of traditional antifungals, such as nystatin, clotrimazole, and miconazole, are commonly prescribed for topical treatment of OC with various success rates (19). However, although they have proven efficacy against Candida, miconazole can interact with anticoagulants and increase the risk of bleeding complications (19), and both nystatin and miconazole have been reported to cause gastrointestinal upset secondary to their use. Moreover, many of the topical formulations of commercially available antifungal agents such as clotrimazole are alcohol-based and contain unwanted preservatives. Many of these topical antifungals contain sucrose to make them more palatable for patients; however, the absence of sucrose is of particular importance in terms of oral health, given its close association with the development of dental caries (20). In contrast, our topical peptide formulation has the advantage in that it is sucrose, alcohol, and preservative free. It is important to note that, unlike both peptides, the antifungals tested were efficacious at high C. albicans cell densities. However, these densities are indicative of active infections which warrant antifungal treatment; in contrast, the potential of the formulation is as a product that can be feasibly and safely applied topically as a preventative prophylactic agent by keeping Candida at commensal levels in the oral cavity, which is reported to be in the range of 1 × 102 cells/ml (21).

Of significance, we investigated the susceptibility of various C. albicans antifungal-resistant strains (Table 1) to the proposed resistant peptide variant K11R-K17R. We found the peptide to be potent against strains with various mechanisms of resistance, namely, overexpression of the ERG11 gene, the ABC transporter genes CDR1 and CDR2, and the facilitator gene MDR1 (Fig. 2A) (22). Resistance to AMPs in general is not common; however, Hst-5 was shown to be transported out of C. albicans cells by the Flu1 efflux pump. A recent study investigated whether C. albicans can develop resistance to Hst-5 by screening a library of C. albicans strains and found that fluconazole-resistant strains with increased upregulation of the multidrug efflux pump MDR1 and FLU1 genes were found to be simultaneously resistant to Hst-5 (23). In our study, a resistant isolate with upregulation in the MDR1 gene was found to be equally susceptible to Hst-5, but we did not test a strain with upregulation in the FLU1 gene.

Using recombinant Hst-5 and several peptide variants, a previous study evaluating the structure-function relationship of the peptides with regard to their candidacidal activity indicated that Lys-13 and Arg-22 in the sequence of Hst-5 were important for candidacidal activity (24, 25). However, as our prior work identified for the first time susceptibility of Hst-5 to lysis by C. albicans-secreted aspartic proteases (Saps) at the Lys-11 and Lys-17 residues, we substituted these 2 lysines while retaining the Lys-13 and Arg-22 residues. Other synthetic analogues of Hst-5 were previously developed, such as dhvar1, dhvar2, and dhvar4; however, these derivatives were found to be hemolytic to red blood cells (26). In contrast, our peptide proved to be nonhemolytic (Fig. 3A) and nontoxic to human oral keratinocytes (Fig. 3B).

In addition to antimicrobial activity, wound-healing properties have also been described for some AMPs. It is commonly accepted that wounds in the oral mucosa heal faster and more efficiently than those on the skin and that saliva accelerates the reepithelialization process via increased keratinocyte migration and proliferation (27). These findings are supported by evidence from salivary dysfunction (reduced salivary levels) and xerostomia models demonstrating impaired oral wound healing under these conditions (28). Studies by Oudhoff et al. demonstrated that human saliva promotes the migration of cultured human oral keratinocytes in vitro and identified histatins as the main salivary factors promoting the migration (29, 30). Not all histatins promote cell migration, however, and so far, histatin‐1, histatin‐2, and histatin‐3, but not Hst-5, have been shown to promote the migration of oral keratinocytes in vitro, although the receptors involved in histatin‐dependent migration of epithelial cells remains unknown (31). An interesting finding from our experiments indicated a significant increase in oral keratinocyte cell proliferation induced by the peptides at their concentration in the formulation, raising the question of whether the peptides possess wound-healing properties (Fig. 3C).

The phenomena of cell proliferation and migration are the basis of reepithelialization and restoring the functional integrity of the epithelial barriers (31). Therefore, we designed cell migration-based wound-healing experiments using different peptide concentrations, with concentrations of what is reported in saliva (0.05 mg/ml) and 2 mg/ml, which is the concentration in our formulation. Our results were in line with previous studies for Hst-5, where at near physiologic concentrations (50 μg/ml or 0.05 mg/ml), Hst-5 did not impact cell migration. However, as our aim was to explore the various aspects of the therapeutic potential of our formulation in addition to antifungal capability, we performed wound-healing studies using the same peptide concentrations found in the gel (2 mg/ml). The findings from these experiments indicated that Hst-5 and K11R-K17R at 2 mg/ml induced rapid cell migration and significant wound-healing activity as early as 8 h following exposure of the cells to the peptides (Fig. 4). Although the exact mechanism behind the ability of the peptides to rapidly induce cell migration is yet to be fully investigated, it has been hypothesized that cell migration secondary to Hst-1 and Hst-2 occurs by activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway through involvement of a G-protein-coupled receptor (29, 30). This mechanism may also apply to Hst-5 and K11R-K17R; however, this warrants further investigation. Nevertheless, the demonstration of the potency of the K11R-K17R peptide to enhance oral keratinocyte proliferation and migration at the concentration used in our formulation is an added advantage. This is particularly relevant for individuals who suffer from salivary dysfunction and therefore lack the benefit of saliva’s natural wound-healing activity, such as Sjogren’s syndrome patients, who are markedly predisposed to OC (32).

It is important to note that Hst-5 was also included in our studies to investigate whether in addition to resistance to proteolysis, K11R-K17R possesses other advantages over the native peptide. In terms of antifungal ability, K11R-K17R exhibited enhanced anticandidal potency against 5 × 105 C. albicans cells (P < 0.001) compared to Hst-5 (P > 0.05), likely due to its tolerance of the C. albicans Saps. Significantly, when we comparatively tested antifungal ability over time, K11R-K17R triggered cell death within 15 min of exposure, approximately 61.5% faster than Hst-5. Additionally, effects on keratinocyte cell proliferation and metabolic activity were also higher for K11R-K17R than for Hst-5, although no significant differences in effect on cell migration were seen. It is important to note that an inhibitor of cell proliferation was included in the cell migration assays in order to specifically explore the effect on cell migration.

Collectively, the findings from this study support the potential of the novel K11R-K17R formulation as a therapeutic agent targeting OC. Further, the peptide may have applicability as a component of artificial saliva for patients with salivary dysfunction, and the formulation may serve as an adjuvant agent to traditional antifungal agents for active infections. In conclusion, coupled with the wound-healing potential of the formulation and the proteolytic degradation advantage of the peptide, this nontoxic formulation has enhanced candidacidal activity and is a viable and promising therapeutic option. However, animal models that closely mimic the pathological conditions in human subjects are warranted to establish the full potential of the formulation as a therapeutic agent. These studies are under way in our laboratory.

MATERIALS AND METHODS

Reagents.Clotrimazole cream, USP 1% (Rite Aid, Camp Hill, PA), miconazole nitrate gel 2% (wt/wt) (Daktarin gel), MTS proliferation assay (Promega, Madison, WI) were used in the study.

Peptide hydrogel formulation.Hst-5 and the K11R-K17R peptide (Table 2) were synthesized by GenScript (Piscataway, NJ) with ≥95% purity. The double mutant K11R-K17R peptide was comparatively and fully evaluated for resistance against proteolysis by C. albicans and individual secreted proteases as described in the patent application passed to allowance (to be issued soon) (Jabra-Rizk, 2017 United States Patent Application Publication [US 2017/0291930 A1], “Histatin-5 Based Synthetic Peptides And Uses Thereof”) and by S. Ikonomova et al. (manuscript submitted). Lyophilized peptides were reconstituted in 1 mM PBS and incorporated in 4% (wt/wt) hydroxypropyl methylcellulose (HPMC) powder under vigorous stirring for 30 min to render the polymer bioadhesive hydrogel formulation. This HPMC concentration was chosen based on our previous demonstration that 4% generates optimum viscosity for oral application in mice (18). A vehicle gel with no peptide was similarly made using 4% (wt/wt) HPMC powder in 1 mM PBS and used as control. Details on optimization of the bioadhesive hydrogel in terms of the viscosity, shear stress, and peptide release have been previously published by our group (18). Hst-5 and K11R-K17R were used in the hydrogel formulations at the predetermined final concentration of 2 mg/ml. In experiments that precluded the use of the hydrogel formulation, peptides were suspended in 1 mM sterile PBS buffer solution.

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TABLE 2

Peptide sequences used in this study

Strains and growth conditions.The C. albicans wild-type reference strain SC5314 (33) was used in all experiments. The following C. albicans antifungal-resistant strains were used where indicated: TW00313, TW00315, TW00304, and TW00636 (22). These clinical isolates are characterized by their mechanisms of drug resistance as listed in Table 1. All isolates were maintained on yeast-peptone-dextrose (YPD) agar plates (Difco Laboratories). For experiments, cultures were grown in YPD broth overnight at 30°C with shaking. Cells were harvested, washed with 1 mM PBS, and then resuspended in PBS to the required final cell density.

In vitro evaluation of the anticandidal efficacy of the formulation.In order to demonstrate the killing potency of the formulation, C. albicans cell concentration-dependent and exposure time-dependent killing assays were performed. For these experiments, 5 × 102, 5 × 103, 5 × 104, 5 × 105, or 5 × 106 C. albicans total cells (volume, 5 μl) were added to 20 μl of Hst-5 (2 mg/ml) gel, K11R-K17R (2 mg/ml) gel, or vehicle gel in 1-ml Eppendorf tubes, and reaction mixtures were incubated at 37°C for 120 min. For time-dependent killing, experiments were performed as described using 5 × 102 C. albicans cells, and reaction mixtures were incubated for 15, 30, or 120 min. Following incubation, reactions were emulsified in PBS and serially diluted in the same buffer, and aliquots were plated on YPD agar and incubated for 24 h at 37°C for viable colony enumeration. Results were presented as percent killing based on CFU/ml values, with vehicle control gel representing 0% killing. In order to ascertain the lack of adverse effect for the gel polymer on C. albicans, experiments were performed using the liquid formulation of the peptide in 1 mM PBS, and no effect on C. albicans viability was noted for the vehicle polymer. In addition, to compare the efficacy of the peptides to those of topical antifungals in clinical use, in vitro killing assays were performed by incubating 5 × 102 C. albicans cells in either control gel, Hst-5 (2 mg/ml) gel, K11R-K17R (2 mg/ml) gel, 1% clotrimazole cream, or 2% miconazole nitrate gel.

Evaluation of peptide cytotoxicity.Cytotoxicity assays were performed in order to demonstrate the lack of adverse effects for the K11R-K17R to the host using the following two established cytotoxicity assays: (i) red blood cell hemolysis assay and (ii) MTS tetrazolium metabolic activity assay. For these experiments, peptides were suspended in 10 mM PBS and used in reactions at the final concentrations desired.

Red blood cell hemolytic assay.K11R-K17R and Hst-5 at concentrations ranging from 0.5 to 10 mg/ml were tested in a red blood cell hemolysis cytotoxicity assay as previously described (24). Briefly, human erythrocytes from healthy individuals (Interstate Blood Bank, Inc., Memphis, TN) were centrifuged for 5 min at 4°C (900 × g), and harvested erythrocytes were washed three times in 10 mM PBS to remove hemoglobin released from lysed erythrocytes. Following incubation for 20 min at 37°C, a 1% (vol/vol) erythrocyte/PBS suspension was prepared. The antifungal amphotericin B was also included, at concentrations ranging from 0.25 to 10 mg/ml. We added 75 μl of either K11R-K17R, Hst-5, or amphotericin B to 75 μl of 1% (vol/vol) erythrocyte/PBS suspension in a flat-bottom 96-well plate. We used 10 mM PBS and 20% Triton-X to indicate 0% and 100% hemolysis, respectively. Following incubation for 60 min at 37°C, the 96-well plate was centrifuged for 5 min at 1,800 rpm, 100 μl of the supernatant from each well was transferred to the wells of a new flat-bottom 96-well plate, and hemoglobin release (i.e., cell lysis) was evaluated by measuring the absorbance at 540 nm (A540nm; Epoch microplate spectrophotometer; BioTek Instruments, Inc., Winooski, VT).

Human oral keratinocyte toxicity assay.For these studies, human-derived spontaneously immortalized normal oral keratinocyte (NOK) cells (kindly provided by Silvio Gutkind via the laboratory of Abraham Schneider) were used as a normal oral keratinocyte cell line. The effect of the peptide on NOK cell metabolic activity was evaluated using the MTS metabolic assay. Cells were seeded in 96-well plates at a cell density of 5,000 cells/well in defined keratinocyte serum-free medium (D-SFM) and incubated overnight at 37°C with 5% CO2. Following incubation, cells were inoculated with either PBS or the peptides (K11R-K17R, 2 mg/ml; Hst-5, 2 mg/ml) and incubated for 24 or 48 h at 37°C in 5% CO2. Following incubation, 20 μl of MTS reagent was added to each well, and 96-well plates were foil-covered and incubated for 60 min at 37°C prior to measurement of absorbance at 490 nm (A490nm) using a plate reader (Epoch microplate spectrophotometer; BioTek Instruments, Inc., Winooski, VT). The data were normalized to the PBS control.

Evaluation of the effect of the peptides on the proliferation of oral keratinocyte cells.NOK cell proliferation experiments were performed using BioTek Cytation 5 label-free direct cell counting. This label-free method is advantageous over fluorescent-labeled methods in that there is no alteration of cell properties and there are no cytotoxic effects on the cells. NOK cells were seeded in 96-well plates at a cell density of 5,000 cells/well in D-SFM and incubated overnight at 37°C with 5% CO2. Following incubation, control (PBS starvation medium), K11R-K17R, or Hst-5 was added to the cells at 2 mg/ml final concentration, and cell proliferation was monitored and readings recorded at 24 and 48 h.

Evaluation of wound-healing activity.As some histatins were reported to exhibit wound-healing properties, cell migration assays were performed using the Ibidi culture insert system to assess peptide wound-healing capability (Fig. 6). For these experiments, NOK cells were seeded in 24-well plates in D-SFM with Ibidi culture inserts (Ibidi USA, Inc., Madison, WI), which were positioned in the center of each well. The advantage of this method over the standard scratch assay is that Ibidi culture inserts ensure uniform reproducible defined 500-μm cell-free gaps that allow no leakage during cultivation. An equal number of NOK cells (2 × 104 cells/ml) were added to the reservoirs within the insert and incubated at 37°C with 5% CO2. Following attachment, the inserts were gently removed to create a gap of ∼500 μm. In order to attribute any observed results specifically to migration and not to an increase in cell proliferation, cells were pretreated with mitomycin C (5 μg/ml for 2 h) and then incubated in D-SFM with a 1:1 equal proportion of either the PBS vehicle control, Hst-5, or K11R-K17R. Hst-5 and K11R-K17R were tested at 2 mg/ml and at the physiologic concentrations reported for Hst-5 in saliva of healthy individuals (0.05 mg/ml) (34). A baseline measurement of the area of the gap was taken at 0 h, and subsequent measurements were obtained at 8, 18, and 24 h using BioTek Cytation 5. GenV image software was used to determine the percent confluence within the gap area.

FIG 6
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FIG 6

Cell migration assay. The Ibidi culture insert system was used. Inserts were first positioned in the center of wells in a 24-well plate. NOK cells in D-SFM were seeded in each insert reservoir. Following attachment, inserts were removed to create uniform 500-μm cell-free gaps. The medium was removed, and cells were then incubated in D-SFM with a 1:1 equal proportion of either PBS (control) or the test peptides. Cell migration was monitored and imaged over time by light microscopy, and serial imaging was performed using BioTek Cytation 5.

Data analysis.All experiments were performed on at least 3 separate occasions and in triplicate where applicable, and quantitative data were expressed as mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). Student’s unpaired t test was used to compare differences between two samples. Ordinary one-way analysis of variance (ANOVA) was used for multiple comparisons. Post hoc analysis was performed with Bonferroni and Šídák tests when indicated. Significance was considered at P values of <0.05.

ACKNOWLEDGMENT

We thank Silvio Gutkind at the University of California, San Diego, and Abraham Schneider at the University of Maryland, Baltimore, for providing us with the NOK cells. We also thank Theodore White at the University of Missouri, Kansas City, for the C. albicans-resistant strains and Alexandra M. Rizk for assistance with the figures.

This work was supported by a National Institutes of Health grant under award number DE028693 (NIDCR) to M. A. Jabra-Rizk.

FOOTNOTES

    • Received 29 April 2019.
    • Returned for modification 2 July 2019.
    • Accepted 14 July 2019.
    • Accepted manuscript posted online 22 July 2019.
  • Copyright © 2019 American Society for Microbiology.

All Rights Reserved.

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Evaluation of the Antifungal and Wound-Healing Properties of a Novel Peptide-Based Bioadhesive Hydrogel Formulation
Ahmed S. Sultan, Taissa Vila, Eman Hefni, Amy J. Karlsson, Mary Ann Jabra-Rizk
Antimicrobial Agents and Chemotherapy Sep 2019, 63 (10) e00888-19; DOI: 10.1128/AAC.00888-19

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Evaluation of the Antifungal and Wound-Healing Properties of a Novel Peptide-Based Bioadhesive Hydrogel Formulation
Ahmed S. Sultan, Taissa Vila, Eman Hefni, Amy J. Karlsson, Mary Ann Jabra-Rizk
Antimicrobial Agents and Chemotherapy Sep 2019, 63 (10) e00888-19; DOI: 10.1128/AAC.00888-19
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KEYWORDS

Candida albicans
antimicrobial peptides
formulation
hydrogel
topical
wound healing

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