(358a) Fungi-Responsive Hydrogel Drug Delivery Systems

Introduction: Wounds are readily infected by fungi, which causes increased morbidity and mortality. Candida spp. are among the most common fungi responsible for wound infections [1]. Antifungal toxicity, poor water solubility, and recent increases in drug resistance, make these infections difficult to treat. The United States Centers for Disease Control and Prevention (CDC) has called the increased resistance to antimicrobials a global threat. One of the main factors that has contributed to an increase in drug resistant Candida is uncontrolled administration of antifungals. Recently, C. auris a multi-drug resistant species, has garnered attention as it has already spread to 30 countries with a mortality rate of 60% [2]. There is a need for smart, controlled antifungal delivery technologies that respond to their environment and release soluble drug locally only in the presence of virulent microbes. We have developed a responsive hydrogel system to combat topical wound fungal infections that degrades, and releases encapsulated antifungal therapeutics in response to secreted aspartic proteases (Saps). These Saps are produced and released by virulent Candida, aiding in tissue invasion. Antifungal loaded liposomes, which can allow for better drug dispersion throughout a wound and improved biofilm penetration [3], were incorporated into these hydrogels. The hydrogel was formulated by incorporating a peptide that is specifically cleaved by Saps into a poly(ethylene glycol) (PEG) backbone, trapping the loaded antifungal liposomes until the hydrogel is degraded in the presence of Saps. This technology thereby eliminates unnecessary exposure to antifungals, helping prevent drug resistance and off-site toxicity (Figure 1A).

Materials and Methods: The 8 amino acid peptide sequence LRF(p-NO₂)↓FLAPK (LFFK) [4] was synthesized using solid phase peptide synthesis with Fmoc chemistry and characterized using mass spectrometry. The peptide was conjugated to PEG acrylate functionalized with succinimidyl valerate under basic conditions. Conjugation was confirmed using size exclusion chromatography (SEC). The responsive hydrogel system was fabricated via free radical photopolymerization of acrylate-PEG-LFFK-PEG-acrylate (PK) using photoinitiator, eosin Y, and co-initiator, triethanolamine. The encapsulated therapeutic, liposomal amphotericin B (AmB) (i.e., AmBisome) or liposomal anidulafungin, was added prior to photopolymerization. Hydrogels were degraded at 37°C while shaking in sodium citrate buffer (SCB) (pH 4.4) with Saps extracted from C. albicans ATCC 10231, at concentrations (2 mg/mL) that mimic physiologically relevant Sap proteolytic activity as previously determined from clinical isolates [5]. For in vitro testing, 10⁶ CFU/mL of C. albicans 10231 were incubated with the hydrogels in yeast carbon base media supplemented with albumin at 37°C while shaking. Every 24 hours 1% of the culture medium was removed and Candia burden was quantified.

Results and Discussion: We successfully synthesized Candida responsive hydrogels. As seen in Figure 1B, the estimated hydrogel mesh size typically decreased with increasing polymer concentration. No significant differences were observed between blank and AmBisome loaded PK or non-responsive PEG hydrogels. After 2 hours of Sap exposure in solution, 10% (w/v) PK hydrogels loaded with AmBisome, released 72.9 ± 5.3% of the total loaded AmB; the release rate was comparable to the release of the PEG degradation products (~68% released at 2 hours). When compared to blank PK hydrogels, the PEG degradation product quantified after 2 hours was also similar at 64.5 ± 2.0%. When 10% (w/v) PK hydrogels loaded with AmBisome were cyclically exposed to Saps and buffer (Figure 1C) we observed significant differences in PEG and AmB release between the two conditions. In order to tune hydrogel degradation rate and subsequently drug release rates, PK concentration was varied. After incubation in Saps at 37°C, 5%, 10%, and 15% (w/v) PK hydrogels loaded with AmBisome degraded within 4, 15, and 38 hours, respectively (Figure 1D). PK hydrogels that remained in SCB at 37°C released less than 2% of the total loaded drug during this time, further confirming that drug release is controlled by hydrogel degradation.

The 10% (w/v) PK hydrogels loaded with AmBisome were examined for their antifungal efficacy against C. albicans in solution; after 48 hours, these hydrogels were able to fully eradicate the infection (0 colony forming units, CFU). Cultures with non-responsive 10% (w/v) PEG hydrogels loaded with AmBisome or anidulafungin liposomes retained a fungal burden of approximately 10⁵ CFU/mL over 5 days (Figure 1E). PK hydrogel-Candida specificity was also tested. The hydrogels degraded and released AmBisome in the supernatant culture of C. tropicalis and C. albicans ∆SAP2 (note, Sap2 is the most abundantly secreted Sap isoenzyme). Hydrogels did not release AmBisome in culture supernatants of C. glabrata, C. krusei (which also showed minimal proteolytic activity), and C. albicans ∆SAP1-3, suggesting that Saps 1 and/or 3 are required for hydrogel degradation. PK hydrogel stability was also assessed in murine wound fluid which contains other proteolytic enzymes such as matrix metalloproteinases which are active in the wound healing process. Hydrogels remained stable in murine wound fluid for up to 7 days with no discernable differences in hydrogel diameter when compared to PK hydrogels in 1× phosphate buffered saline. At day 7, 10¹² CFU/mL of C. albicans was introduced to the wound fluid and hydrogels degraded within 24 hours (Figure 1F).

Conclusions: We developed a target-triggered hydrogel system that responds to Saps secreted by pathogenic C. albicans allowing us to locally deliver controlled doses of antifungals in a manner that may delay drug resistance, reduce toxicity and improve therapeutic efficacy. Degradation and drug release rates can be readily modulated by changing PK concentration. The hydrogels effectively kill pathogenic Candida and remain stable in non-infected wound fluid. Future work includes in vivo studies of this hydrogel system in murine flesh wound models.

References: 1. Jarvis W.R. Clin Infect Dis, 1995. 20(6): p. 1526-1530. 2. CDC. General Information about Candida auris. 2018. 3. Forier K. J Control Release. 2014; 190, 607-623. 4. Fusek MP. Biochemistry. 1994; 33(32), 9791-9799. 5. Schreiber B. Diagn Microbiol Infect Dis. 1985; 3(1), 1-5.

Figure Caption:

Candida responsive hydrogels. (A) Responsive hydrogel structure and degradation scheme. (B) Hydrogel mesh size with and without AmBisome (****p < 0.0001, **p < 0.01, two-way ANOVA with Tukey’s post-hoc analysis, n = 3). (C) AmB and PEG release for 10% (w/v) PK hydrogels exposed to alternating Saps and buffer (****p < 0.0001, **p < 0.01, *p < 0.05, two-way ANOVA with Tukey’s post-hoc analysis, n = 3). (D) AmB release from 5, 10, and 15% (w/v) PK hydrogels in Saps over time. (E) C. albicans burden over time after introducing 10% (w/v) PK hydrogels with or without therapeutic at time = 0 hrs (96 hrs: A×B,C: n.s.; two-way ANOVA, Tukey’s post-hoc analysis, n=3). (F) Percent AmBisome remaining in 10% (w/v) PK hydrogels incubated in Candida spp. supernatant over time (12 hrs: A,D×C,E,F; B×A,C,D,E,F: p < 0.0001; 24 hrs: A,B,DxC,E,F: p < 0.0001; two-way ANOVA, Tukey’s post-hoc analysis, n = 3).