O18 - Ex Vivo Model for the Evaluation of Secreted Aspartyl Protease-Responsive Hydrogels for Targeted Antifungal Delivery in Candida albicans-Infected Wounds

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Co-Author(s)

Camila C. Carvalho, BS

PhD Candidate
Brown University
Providence, Rhode Island, United States

Presenting Author(s)

Serly Chohmalian

Undergraduate Student
Brown University, United States

Co-Author(s)

KC

Kayla Campbell

Postdoc
Brown University, United States

Primary Investigator(s)

Anita Shukla, PhD

Elaine I. Savage Professor of Engineering
Brown University
Providence, Rhode Island, United States

Introduction: : Candida albicans is a leading cause of chronic wound infections [1]. While C. albicans is typically a commensal microbe, it can transition to a pathogenic state in response to microenvironmental disruptions, resulting in mucosal and systemic infections [2]. These infections are particularly challenging due to the ability of C. albicans to form resilient biofilms and resist conventional antifungal therapies [3]. Treatment is further hindered by the limited repertoire of antifungal drugs and rising antifungal resistance, emphasizing the need for new antifungal treatment approaches [4]. Secreted aspartyl proteases (Saps) are a key virulence factor produced by C. albicans; this enzyme family degrades host proteins and facilitates host tissue invasion [5,6]. We have developed a Saps-responsive hydrogel that enables C. albicans-triggered drug delivery by selective degradation-driven release in the presence of these enzymes. This hydrogel provides localized and controlled drug delivery at the site of infection. Compared to most antimicrobial hydrogels that respond to nonspecific stimuli, these hydrogels respond to pathogen-specific cues, potentially enabling more effective infection clearance while minimizing toxicity and resistance development [7,8]. We have previously demonstrated degradation of Saps-responsive hydrogels in suspension cultures containing C. albicans Saps, and here we developed an ex vivo porcine burn wound model to evaluate the performance of these hydrogels loaded with the liposomal antifungal, amphotericin B (AmBisome). We hypothesized that Saps-responsive hydrogels would undergo selective degradation in response to C. albicans Saps and achieve on-demand antifungal drug release within the ex vivo Candida albicans-infected wound model, leading to eradication of the infection.

Materials and

Methods: : Poly(ethylene glycol) (PEG)-based responsive and non-responsive hydrogels were synthesized via a thiol-maleimide reaction. Non-responsive hydrogels were fabricated using 4-arm PEG and PEG-dithiol, while responsive hydrogels were synthesized using 4-arm PEG and FK-10 (CFRKSKEKIGKC) peptide. The FK-10 peptide, derived from antimicrobial peptide LL-37, was selected for its susceptibility to cleavage by C. albicans Saps and was modified with terminal cysteine residues to enable crosslinking. Liposomal amphotericin B (AmBisome) was encapsulated within the hydrogel matrix to provide antifungal activity. The minimum inhibitory concentration (MIC50) of AmBisome against C. albicans was determined to be 6.25 µg/mL. For hydrogel loading, the highest possible drug concentration that did not interfere with gelation was used, resulting in an AmBisome concentration of 0.8 mg/mL within the hydrogel. The hydrogels were subsequently characterized through swelling studies, mesh size calculations, and compression testing.
To simulate Candida albicans-infected wounds and evaluate hydrogel performance, an ex vivo model was established using Yorkshire porcine skin. Burn wounds, 8 mm in diameter, were created on 2 × 2 cm skin sections following removal of the subcutaneous fat layer. Sterilization was performed using ethanol and ultraviolet (UV) irradiation. To initiate the infection, the sterilized skin was inoculated with a C. albicans culture (OD530 = 0.22–0.23). Responsive and non-responsive hydrogels were then applied to the infected samples, which were incubated at 37 °C until degradation of the responsive hydrogel was observed. Post-incubation, skin sections were homogenized and plated on CHROMagar Candida Plus to quantify colony forming units (CFUs) and assess fungal burden.

Results, Conclusions, and Discussions:: Successful synthesis of FK-10 was confirmed via liquid chromatography-mass spectrometry (Figure 1A). Reaction conditions for both responsive (R) and non-responsive (NR) hydrogels were optimized to ensure efficient crosslinking and comparable mechanical properties (Figure 1B). Both loaded and blank FK-10 R hydrogels exhibited higher swelling ratios than NR hydrogels, likely due to the presence of hydrophilic amino acids (e.g., serine, lysine, arginine) in the FK-10 crosslinker, which promote larger mesh structures and increased water uptake. Mesh size calculations confirmed the greater permeability of FK-10 hydrogels, with values for both R and NR formulations aligning with literature-reported ranges for PEG-based hydrogels (i.e., 5-50 nm) [9]. Compression testing showed that NR hydrogels had higher engineering stiffness than FK-10 hydrogels, which is expected given their denser crosslinking and lower water content.
To evaluate hydrogel degradation, an ex vivo porcine burn wound Candida albicans infection model was used. Following inoculation with actively growing C. albicans (OD530 = 0.22–0.23), blank FK-10 hydrogels fully degraded after 48 hours at 37 °C (Figure 1C), consistent with Saps-mediated cleavage, while blank NR hydrogels remained intact. However, no significant reduction in CFU counts was observed in either case, as the hydrogels lacked antifungal cargo. AmBisome-loaded FK-10 hydrogels also fully degraded after 48 hours (Figure 1C), releasing drug and achieving a 7-log reduction in fungal burden relative to the infected control, while loaded NR hydrogels did not degrade and showed no antifungal activity (Figure 1D). This work demonstrates the potential of pathogen-responsive hydrogels as an effective, localized therapeutic strategy for advanced wound care applications.

Acknowledgements and/or References (Optional): : [1] WHO fungal priority pathogens list to guide research, development and public health action. https://www.who.int/publications/i/item/9789240060241.
[2] Calderone, R. A. & Fonzi, W. A. Virulence factors of Candida albicans. Trends Microbiol 9, 327–335 (2001).
[3] Mayer, F. L., Wilson, D. & Hube, B. Candida albicans pathogenicity mechanisms. Virulence 4, 119–128 (2013).
[4] Lockhart, S. R., Chowdhary, A. & Gold, J. A. W. The rapid emergence of antifungal-resistant human-pathogenic fungi. Nat Rev Microbiol 21, 818–832 (2023).
[5] Gow, N. A. R., Van De Veerdonk, F. L., Brown, A. J. P. & Netea, M. G. Candida albicans morphogenesis and host defence: discriminating invasion from colonization. Nat Rev Microbiol 10, 112–122 (2012).
[6] Staib, P., Kretschmar, M., Nichterlein, T., Hof, H. & Morschhäuser, J. Differential activation of a Candida albicans virulence gene family during infection. Proceedings of the National Academy of Sciences 97, 6102–6107 (2000).
[7] Masteikova, R., Chalupova, Z. & Sklubalova, Z. Stimuli-sensitive hydrogels in controlled and sustained drug delivery. Medicina (B Aires) 39, 19–24 (2003).
[8] Bustamante-Torres, M. et al. Hydrogels classification according to the physical or chemical interactions and as stimuli-sensitive materials. Gels 7, 182 (2021).
[9] Richbourg, N. et al. Precise control of synthetic hydrogel network structure via linear, independent synthesis-swelling relationships. Science Advances 7, eabe3245 (2021).