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

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.

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.

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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.