Poster O12 - Entrapment of pyruvate dehydrogenase in alginate hydrogel sheets towards a biofilm-eliminating wound dressing

Introduction: Biofilm infections present a significant challenge in wound management due to their remarkable tolerance to antimicrobial agents. Biofilms, which are present in 90% of chronic wounds, affect 2% of the U.S. population during their lifetimes and can be up to 1000 times less susceptible to antibiotics. This tolerance can significantly delay wound healing by prolonging inflammation. Currently, most therapeutic approaches using nanoparticles target either gram-positive or gram-negative bacteria, yet clinically, biofilms often contain both strains. Our previous research highlighted the crucial role of the metabolite pyruvate in both the formation and maintenance of biofilms in the wound environment [1]. Our novel work shows that enzymatic depletion of pyruvate disperses both gram-positive and gram-negative biofilms, making them as susceptible to antibiotics as planktonic cells [2]. In our studies, both in vitro and in vivo, we utilized pyruvate dehydrogenase (PDH) enzyme to induce this depletion. However, PDH is unstable at room temperature, posing challenges for commercial use. We have previously demonstrated that encapsulating PDH in polylactic-co-glycolic acid (PLGA) can stabilize its activity. Building on this, our current study aims to develop a wound dressing by encapsulating PDH within alginate hydrogels, a material that has long been used in FDA-approved wound dressing applications.

Materials and

Methods: We have synthesized a novel hydrogel-based wound dressing with varying concentrations of sodium alginate (Na-Alg) (2, 2.5, and 3% (w/v)), using calcium chloride (CaCl2) (0.2, 0.4, 0.6, 0.8, and 1 M) to encapsulate PDH. We investigated the effects of varying Na-Alg and CaCl2 concentrations on the hydrogel's mechanical properties through compression tests. The viscosity and gelation properties of the hydrogels were analyzed using a rheometer. Scanning electron microscopy (SEM) was employed to examine the pore sizes and structural integrity of the hydrogels. Another critical aspect of the hydrogels is their capacity to absorb wound exudate and pyruvate, essential for wound healing and adsorption of pyruvate to interact with PDH. Thus, the swelling behavior of the hydrogels in wound simulated fluid (WSF) was also studied. Additionally, we assessed the water retention capacity of the hydrogels. PDH (0, 2084, 2778, or 3473 mU) was encapsulated into hydrogels consisting of 2% (w/v) Na-Alg and 0.3 M CaCl2 (Alg2-Ca3). The presence and enzymatic activity of PDH within the hydrogel were quantified using a bicinchoninic acid (BCA) protein assay and ß-NADH absorption measurements, respectively.

Results, Conclusions, and Discussions: Results and

Discussion:
Increasing the concentration of Na-Alg consistently enhanced the compressive strength of the hydrogels, indicative of denser cross-linking within the material structure, which was also observed in SEM images (fig 1-a,b). In vivo studies reported the compressive modulus of 8.3 KPa for dermal cells, which is close to our hydrogels [3]. SEM images showed that hydrogels’ pore size are large enough to hold ~45 nm PDH enzyme complex, while the tortuosity of pores likely blocked PDH escape. This interconnected network of pores was designed to allow pyruvate and cofactors to be absorbed with the wound exudate. Despite increasing CaCl2 concentration, significant changes in compressive modulus were not observed, showing that we have passed the saturation point in cross-linking. Water retention and swelling behavior were also thoroughly examined, revealing that hydrogels with lower alginate concentrations absorbed more liquid initially but also exhibited higher rates of liquid loss over time, likely due to the larger pore sizes (fig 1-c). Conversely, higher concentrations of Na-Alg not only improved water retention capabilities but also maintained hydration levels within 84-94%, ideal for effective wound dressings. Viscosity analyses supported these results, showing a direct relationship between alginate concentration and viscosity, with the highest values observed at 3% Na-Alg concentration. These properties influence the gelation kinetics, critical for the hydrogel's performance. Rapid gelation observed upon CaCl2 addition confirmed the effective establishment of a three-dimensional polymeric network, essential for structural integrity and function in situ. By increasing PDH encapsulated from 2084 to 3473 mU, hydrogel enzymatic activity increased from 0.7 to 2.02 mU/ml. The BCA assay quantified enzyme encapsulated in the synthesized beads, showing a concentration-dependent trend and the average entrapment efficiency was 8.88% (fig 1-d), similar to the efficiency of PLGA nanoparticles (9%) [4].

Conclusion:
These findings suggest that the encapsulation of PDH in sodium alginate can potentially serve as a novel wound dressing that prevents and eliminates biofilm infections by pyruvate depletion. Future studies will focus on increasing the concentration of encapsulated PDH, optimizing PDH loading and efficiency, conducting biofilm studies in vitro and in vivo and evaluating cytotoxicity in human cells studies.

Acknowledgements (Optional): Acknowledgement:

Funding: NIH 1R56AI127815-01A1, University of Vermont, Ballenger Venture Funds, NSF CAREER AWARD
Special thanks to our collaborators: Dr. Karin Sauer (Binghamton University), Dr. Steve Davis (University of Miami), Dr. Rachael Floreani (UVM), and our supporting undergraduates Hannah Sherriff and Liam Orr.

References:
1. Sauer, K. and A.L. Doiron, Compositions and methods for disrupting biofilm formation and maintenance. 2023, Google Patents.
2. Goodwine, J., et al., Pyruvate-depleting conditions induce biofilm dispersion and enhance the efficacy of antibiotics in killing biofilms in vitr o and in vivo. Scientific reports, 2019. 9(1): p. 3763.
3. Zahouani, H., et al., Characterization of the mechanical properties of a dermal equivalent compared with human skin in vivo by indentation and static friction tests. Skin research and technology, 2009. 15(1): p. 68-76.
4. Han, C., et al., Enzyme-encapsulating polymeric nanoparticles: A potential adjunctive therapy in Pseudomonas aeruginosa biofilm-associated infection treatment. Colloids and Surfaces B: Biointerfaces, 2019. 184: p. 110512.