Stimuli-responsive release of covalently attached Omega-3 fatty acids from electrospun chitosan membranes

Long-chain ω-3 polyunsaturated fatty acids (ω-3 PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), possess strong anti-inflammatory and antimicrobial properties, but their therapeutic potential is severely limited by high susceptibility to oxidative degradation. Covalent immobilization onto biopolymers using cleavable ester linkages offers a route to prevent oxidation while enabling stimuli-responsive payload delivery. In this work, a transesterification-based covalent conjugation approach was developed to graft DHA and EPA onto electrospun chitosan membranes (ESCM) to achieve controlled bioactive release tailored for wound healing. DHA and EPA were conjugated to ESCM via transesterification, and successful ester bond formation was verified using Fourier-transform infrared spectroscopy (FTIR). The degree of substitution and surface loading densities were quantified via High-Performance Liquid Chromatography (HPLC), X-ray Photoelectron Spectroscopy (XPS), and acid-base titration, while surface wettability was characterized by static water contact angle measurements. In vitro release kinetics were monitored over five days in neutral phosphate-buffered saline (PBS), acidic acetate-buffered saline (ABS), and PBS containing Pseudomonas lipase. Indirect cytocompatibility and cell migration were assessed using L929 mouse fibroblasts in transwell co-cultures evaluated with luminescence viability assays and live/dead fluorescence staining. Antimicrobial efficacy was determined against both planktonic and biofilm forms of Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa . FTIR spectra confirmed successful ester linkage of DHA and EPA onto the electrospun chitosan nanofiber surfaces. The resulting functionalized membranes displayed a hydrophobic water contact angle of 127±1°, and quantitative HPLC analysis confirmed high surface substitution with approximate loadings of ~25 µg/cm 2 DHA and ~13 µg/cm 2 EPA. Elution assays demonstrated responsive release profiles, showing no measurable lipid leaching in neutral PBS, but pronounced release under acidic conditions (ABS) and enzymatically active conditions (PBS with lipase). In cell culture assays, the conjugated membranes showed high biocompatibility by maintaining greater than 90% L929 fibroblast viability while supporting cellular migration. Furthermore, the membranes significantly suppressed planktonic growth and biofilm formation of MRSA, although no significant inhibitory activity was observed against P. aeruginosa . Covalently conjugating DHA and EPA to ESCM resolves the primary clinical limitation of PUFA oxidation while preserving their therapeutic efficacy. By leveraging localized acidic pH and bacterial lipase activity as trigger mechanisms, the scaffold facilitates on-demand, targeted release of active lipids within hostile wound microenvironments. Supported by high cytocompatibility with dermal fibroblasts and targeted bactericidal action against MRSA biofilms, this composite membrane represents a viable bioactive dressing for the management of infected and chronic wounds.