Development and Characterization of LL37 Antimicrobial-Peptide-Loaded Chitosan Nanoparticles: An Antimicrobial...

CSNPs synthesized via the ionic gelation method have emerged as a promising nanoplatform in diverse fields such as pharmaceuticals, nanotechnology, and polymer science due to their biocompatibility, ease of fabrication, and tunable properties. This study focuses on the development and characterization of LL37-loaded CSNPs, designed to enhance antibacterial efficacy while maintaining biocompatibility. This study pioneers a systematic loading optimization approach by evaluating the encapsulation efficiency (%EE) of antimicrobial peptide LL37 across multiple concentrations (7.5, 15, and 30 µg/mL), thereby identifying the formulation that maximizes peptide incorporation while preserving controlled release characteristics. The multi-concentration analysis establishes a new methodological benchmark for peptide delivery system development. To achieve this, CSNPs were optimized for size and stability by adjusting parameters such as the chitosan concentration, pH, and stabilizer. LL37, a potent antimicrobial peptide, was successfully encapsulated into CSNPs at concentrations of 7.5, 15, and 30 µg/mL, yielding formulations with favorable physicochemical properties. Dynamic light scattering (DLS) and Zeta sizer analyses revealed that blank CSNPs exhibited an average particle size of 180.40 ± 2.16 nm, a zeta potential (ZP) of +40.57 ± 1.82 mV, and a polydispersity index (PDI) of 0.289. In contrast, 15-LL37-CSNPs demonstrated an increased size of 210.9 ± 2.59 nm with an enhanced zeta potential of +51.21 ± 0.93 mV, indicating an improved stability and interaction potential. Field emission scanning electron microscopy (FE-SEM) analyses exhibited the round shaped morphology of nanoparticles. The release profile of LL37 exhibited a concentration-dependent rate and showed the best fit with the first-order kinetic model. Cytocompatibility assessments using the XTT assay confirmed that both blank and LL37-loaded CSNPs did not exhibit cytotoxicity on keratinocyte cells across a range of concentrations (150 µg/mL to 0.29 µg/mL). Notably, LL37-loaded CSNPs demonstrated significant antibacterial activity against E. coli and S. aureus, with the 15-LL37-CSNP formulation exhibiting superior efficacy. Overall, these findings highlight the potential of LL37-CSNPs as a versatile antibacterial delivery system with applications in drug delivery, wound healing, and tissue engineering.