H15 - Designing a wound dressing material to deliver an anti-biofilm enzyme: Physicomechanical Properties of Oxidized Alginate-Chitosan Composite Sheets

Introduction: : Wound dressings are critical medical devices that promote healing by maintaining moisture in the wound environment and protecting against infections in wounds. Bacterial biofilm infections pose a major obstacle to wound care due to their exceptional tolerance to antimicrobial treatments. Found in 90% of chronic wounds, biofilms can be up to 1000 times more tolerant to antibiotics and delay healing. Previous work has found that the metabolite pyruvate to be essential in both the development and persistence of biofilms within wound environments [1]. To induce biofilm dispersion in wounds, pyruvate dehydrogenase (PDH) was entrapped in alginate hydrogels, a common wound dressing material [2]. However, due to the negative charge on alginate, this hydrogel was only able to encapsulate 10% of the introduced PDH. To increase loading, a hydrogel composed of carboxymethyl cellulose (CMC) crosslinked with oxidized sodium alginate (OAlg) is being designed to optimize the balance of charges in the material for high encapsulation of PDH for the purposes of treating biofilms. This study demonstrates that OAlg:CMC has similar mechanical properties to those observed in alginate hydrogels, and continuing work is evaluating the antibacterial properties of the hydrogels.

Materials and

Methods: : To make OAlg:CMC hydrogels, sodium alginate (1.8% w/v) was oxidized with sodium periodate (NaIO₄), based on the molar ratio of NaIO₄ to uronic acid units in alginate. The required NaIO₄ was added to the alginate solution to react for 24 hours in the dark at room temperature to generate 10% and 25% OAlg. The reaction was then quenched by adding a molar equivalent of ethylene glycol to NaIO4. Dialysis (6-8 kDa MWCO) was performed using deionized water with 12 water bath changes over four days. The solution was then frozen before being lyophilized to yield a dry OAlg powder. The degree of oxidation (OD) was measured via the absorbance of unused sodium periodate at 486 nm [3, 4] and 1H-NMR analysis done at the concentration of 10 mg/ml in D2O. We have synthesized a novel hydrogel-based wound dressing with varying concentrations of 10% and 25% OAlg (2, 2.5, and 3% (w/v) combined with CMC (1 and 1.5% (w/v)), using ionic crosslinker calcium chloride (CaCl2, 1 M). The effects of varying CMC and OAlg concentrations and degree of oxidation on the compressive modulus of hydrogels were investigated. The capacity of a hydrogel for absorption of wound exudate and pyruvate is essential for wound healing and adsorption of pyruvate to interact with PDH. Thus, the swelling behavior of the hydrogels in wound simulated fluid was also studied. Finally, the water retention capacity of the hydrogels was assessed.

Results, Conclusions, and Discussions:: Both quantitative and qualitative analyses confirmed the OD of OAlg. UV-Vis spectroscopy revealed that the theoretical ODs of 10% and 25% corresponded to measured values of 9.94% and 24.9%, respectively. In Figure 1A, 1H NMR spectroscopy detected signals at 5.47 ppm and 5.67 ppm in the spectra for 10% and 25% OAlg, respectively, attributed to hemiacetal structures formed by intramolecular reactions between aldehyde and hydroxyl groups, which were more pronounced in 25% OAlg. Hydrogels were generated using OAlg:CMC ratios of 1:3, 1:2, 1:1, 2:1, 3:1, and 4:1. Ratios of 3:1 and 4:1 demonstrated better structural integrity. 3:1 was selected for further optimization due to incorporating more CMC. Unconfined compression tests were conducted on hydrogels made with 10% and 25% OD OAlg (2, 2.5, and 3% (w/v)) and CMC (1 and 1.5% (w/v)) in order to determine compressive moduli. As shown in Figure 1B, hydrogels using 25% OD OAlg showed a significantly reduced compressive modulus, whereas 10% OD OAlg maintained a compressive modulus similar to 2.5% (w/v) alginate hydrogel controls. Consequently, 10% OD OAlg was chosen for subsequent swelling and weight loss studies. As shown in Figure 1C and D, swelling studies revealed that hydrogels composed of 10% OD 3% (w/v) OAlg crosslinked with 1% (w/v) CMC (ratio 3:1) exhibited swelling and water retention comparable to alginate controls. All other formulations performed 100% less in swelling which was statistically different from alginate controls (p < 0.0001). Thus, 10% OD 3% OAlg:1% CMC hydrogels were identified as having the best strength and swelling properties for future studies.These findings demonstrate that OAlg:CMC hydrogels, at a 3:1 ratio using 10% OD 3% (w/v) OAlg and 1% (w/v) CMC, exhibit mechanical properties in strength and swelling behavior to similar OAlg:CMC wound dressings [5]. This suggests that the inclusion of CMC does not significantly compromise structural integrity while potentially enhancing functional versatility by including an additional antimicrobial compound. Future studies will focus on further characterization of OAlg:CMC hydrogels incorporating PDH enzyme.

Acknowledgements and/or References (Optional)::

Funding: NSF CAREER AWARD 2341706
Special thanks to our collaborators: Dr. Karin Sauer (Binghamton University) and Dr. Rachel Floreani (University of Vermont).

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 vitro and in vivo. Scientific reports, 2019. 9(1): p. 3763.
3. Mousavi, A., et al., Biohybrid oxidized alginate/myocardial extracellular matrix injectable hydrogels with improved electromechanical properties for cardiac tissue engineering. International journal of biological macromolecules, 2021. 180: p. 692-708.
4. Gomez, C.G., et al., Oxidation of sodium alginate and characterization of the oxidized derivatives. Carbohydrate polymers, 2007. 67(3): p. 296-304.
5. Mohabatpour, F., et al., Self-Crosslinkable Oxidized Alginate-Carboxymethyl Chitosan Hydrogels as an Injectable Cell Carrier for In Vitro Dental Enamel Regeneration. Journal of Functional Biomaterials, 2022. 13(2): p. 71.