Alginate Hydrogels loaded with pyruvate dehydrogenase enzyme: a novel anti-biofilm wound dressing

Introduction: : Bacterial biofilm infections present a significant challenge in wound management due to their remarkable tolerance to antimicrobial agents. They are present in 90% of chronic wounds, affect approximately 2% of the U.S. population during their lifetimes, and can be up to 1000 times more tolerant to antibiotics than planktonic bacteria. As a result, it is crucial for wound dressings used in medical settings to discourage biofilm growth in addition to exhibiting physio-mechanical properties appropriate for a wound dressing. Alginate is a commonly used material in FDA-approved wound dressings and is known for its excellent biocompatibility, non-immunogenicity, high absorbent capacity, and ability to maintain a moist physiological environment. Despite its widespread clinical use, alginate promotes the development of biofilms [1]. Current strategies to overcome this limitation in alginate hydrogels include incorporating antibacterial nanomaterials such as silver or graphene oxide, adding nitric oxide donors or traditional antibacterial agents like honey, and loading bacteriophages into the hydrogel [2-9]. Most of these studies target either gram-positive or gram-negative bacteria; however, in clinical settings, biofilms often contain both types. Our previous research highlighted the critical role of the metabolite pyruvate in the formation and maintenance of both gram-positive and gram-negative biofilms in wound environments [10, 11]. Building on these findings, the current study aims to develop a wound dressing by encapsulating pyruvate dehydrogenase (PDH) within alginate hydrogels to overcome the current limitations of using alginate wound dressing on biofilm-prone wounds.

Materials and

Methods: : We synthesized a novel hydrogel-based wound dressing using varying concentrations of sodium alginate (Na-Alg) (2%, 2.5%, and 3% w/v) and calcium chloride (CaCl₂) (0.2, 0.4, 0.6, 0.8, and 1 M) to encapsulate PDH. The effects of different Na-Alg and CaCl₂ concentrations on the hydrogel's mechanical properties were evaluated through unconfined compression testing. Alginate hydrogels were tested using a DHR-2 rheometer to assess elastic modulus, calculated from the slope of the linear region (2–20% strain) of stress–strain curves. Viscosity and gelation behavior were analyzed using a rheometer, and pore size was examined via scanning electron microscopy (SEM). The capacity of the hydrogel to absorb simulated wound exudate and retain water was also assessed. Ten different units of PDH ranging from 0.05 to 1.1 units were loaded into hydrogels composed of 2.5% (w/v) Na-Alg and 0.4 M CaCl₂, and its enzymatic activity was measured. Storage stability was investigated using the β-NADH absorption assay, and enzyme release was studied using the bicinchoninic acid (BCA) protein and β-NADH absorption assays. Finally, the enzyme-loaded hydrogels were tested against biofilms by growing Pseudomonas aeruginosa PAO1 in LB broth and using it to form biofilms on an MBEC Assay® plate. After overnight incubation at 37°C and 110 rpm, biofilm-coated lids were transferred to wells containing 10-fold diluted LB with or without cofactors (2  mM β-NAD⁺, 2  mM CoA, 20  µM TPP, 50  µM MgCl₂) and incubated for 20 hours. Biofilms were then stained with 1% crystal violet, and biomass was quantified using UV-Vis spectrophotometry.

Results, Conclusions, and Discussions:: Results and

Discussion:
Fig1-a shows that the hydrogels demonstrated a tunable compressive modulus ranging from 20 to 80 kPa, which falls within the range of commercial wound dressings [12, 13]. In water retention and swelling studies, hydrogels with lower alginate concentrations absorbed more liquid initially but exhibited higher rates of liquid loss over time, likely due to larger pore sizes, which was shown in SEM images. In contrast, higher Na-Alg concentrations not only improved water retention but also maintained hydration levels within 84–94%, which is ideal for wound dressings. Rheometry showed a direct relationship between alginate concentration and viscosity. After 24 hours of storage at 4 °C, there was no significant reduction in hydrogel activity. After one week of storage, the hydrogels retained approximately 60% of their initial activity. As shown in Fig1-c, after 12 hours of incubation in buffer, only approximately 20–30% of the enzyme was released, indicating that the majority of the enzyme remained entrapped within the matrix. In biofilm study, hydrogels containing varying concentrations of PDH (0.35 to 0.85 U) were exposed to biofilms and their antibiofilm activity was compared with a commercially available alginate dressing (Dimora). This concentration range was selected based on prior enzyme activity studies (fig1-b), which showed that peak activity was achieved when 0.85U of PDH was added to 65 l of alginate. Hydrogel containing 0.85 U of PDH showed a significant reduction in biofilm biomass, with 41.44% compared to media alone and 81.94% compared to a commercial alginate dressing. The free PDH sample led to an even greater reduction in biomass. However, we believe that the encapsulated PDH system has the potential to perform comparably, if not better, under optimized conditions. A limitation of this study was the small hydrogel volume (65  µl) used for biofilm testing, constrained by the 96-well plate format and PEG-coated lids. Larger volumes could encapsulate more enzyme and retain higher activity.

Conclusion:
The encapsulation of PDH in sodium alginate can prevent and eliminate biofilm infections by pyruvate depletion, a novel and impactful addition to clinical wound dressing technologies.

Acknowledgements and/or References (Optional)::

Funding: NSF CAREER AWARD 2341706
Special thanks to our collaborators: Dr. Karin Sauer (Binghamton University) and Dr. Rachael Floreani (UVM), and our supporting undergraduates Hannah Sherriff and Liam Orr.
Reference:
1. Phillips, P.L., et al., Antimicrobial dressing efficacy against mature Pseudomonas aeruginosa biofilm on porcine skin explants. International wound journal, 2015. 12(4): p. 469-483.
2. Xie, F., et al., Alginate foam gel modified by graphene oxide for wound dressing. International Journal of Biological Macromolecules, 2022. 223: p. 391-403.
3. Porter, G.C., et al., AgNP/Alginate Nanocomposite hydrogel for antimicrobial and antibiofilm applications. Carbohydrate polymers, 2021. 251: p. 117017.
4. Singh, R. and D. Singh, Radiation synthesis of PVP/alginate hydrogel containing nanosilver as wound dressing. Journal of Materials Science: Materials in Medicine, 2012. 23(11): p. 2649-2658.
5. Urzedo, A.L., et al., Cytotoxicity and antibacterial activity of alginate hydrogel containing nitric oxide donor and silver nanoparticles for topical applications. ACS Biomaterials Science & Engineering, 2020. 6(4): p. 2117-2134.
6. Hasan, N., et al., Diethylenetriamine/NONOate-doped alginate hydrogel with sustained nitric oxide release and minimal toxicity to accelerate healing of MRSA-infected wounds. Carbohydrate polymers, 2021. 270: p. 118387.
7. Nazeri, S., et al., Evaluation of effectiveness of honey-based alginate hydrogel on wound healing in rat model. Journal of Applied Biotechnology Reports, 2015. 2(3): p. 293-297.
8. Ali, H.R., C. Valdivia, and D. Negus, Bacteriophage-embedded and coated alginate layers inhibit biofilm formation by clinical strains of Klebsiella pneumoniae. Journal of Applied Microbiology, 2025. 136(5): p. lxaf099.
9. Shiue, S.-J., F.-S. Syu, and H.-Y. Lin, Two types of bacteriophage-modified alginate hydrogels as antibacterial coatings for implants. Journal of the Taiwan Institute of Chemical Engineers, 2022. 134: p. 104353.
10. Sauer, K. and A.L. Doiron, Compositions and methods for disrupting biofilm formation and maintenance. 2023, Google Patents.
11. 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.
12. Malektaj, H., A.D. Drozdov, and J. deClaville Christiansen, Mechanical properties of alginate hydrogels cross-linked with multivalent cations. Polymers, 2023. 15(14): p. 3012.
13. Boddupalli, A. and K.M. Bratlie, Second harmonic generation microscopy of collagen organization in tunable, environmentally responsive alginate hydrogels. Biomaterials science, 2019. 7(3): p. 1188-1199.