α-Pinene and β-Sitosterol co-loaded into Alginate/Gelatin-functionalized niosome: Synergistic enhanced antibacterial and anti-biofilm activities

Background The rise of antibiotic resistance in infectious disease, particularly from carbapenem-resistant Klebsiella pneumoniae (CRKP) and methicillin-resistant Staphylococcus aureus (MRSA), poses a significant global health challenge. This study aimed to fabricate and evaluate a novel biocompatible hydrogel scaffold (Nio-PIN/STL@SC) incorporating niosomes loaded with α-pinene (PIN) and β-sitosterol (STL) to enhance antibacterial and anti-biofilm activities against MRSA and CRKP isolates. Methods Niosomes containing PIN and STL (Nio-PIN/STL) were prepared using the thin-layer hydration method and then incorporated into an alginate/gelatin hydrogel scaffold via a straightforward crosslinking reaction, forming Nio-PIN/STL@SC. The physicochemical properties of the scaffolds were characterized using DLS, SEM, TEM, and FTIR. In vitro assays assessed drug release, swelling, degradation, stability, cytotoxicity, antibacterial efficacy (MIC, disk diffusion, time-kill assay), anti-biofilm activity (CV assay, MBEC), and the expression of biofilm-related genes. Results The Nio-PIN/STL@SC formulation exhibited nanoscale particle size (263.5 ± 5.2 nm) with favorable encapsulation efficiency (EE) (78.2 ± 1.18% for PIN and 79.7 ± 1.35% for STL) and a sustained biphasic drug release profile. The incorporation of Nio-PIN/STL enhanced the mechanical properties and reduced the degradation rate of the hydrogel. Nio-PIN/STL@SC demonstrated significantly enhanced antibacterial and anti-biofilm activities against MRSA and CRKP isolates compared to free PIN, STL, and their combination, as evidenced by lower MIC and MBEC values and larger inhibition zones. Furthermore, Nio-PIN/STL@SC treatment led to a significant downregulation of biofilm-associated genes ( icaA , icaD in MRSA; mrkA , mrkD , fimA in CRKP) and showed reduced cytotoxicity against normal human cells sub-MIC concentrations. Conclusions The developed Nio-PIN/STL@SC hydrogel scaffold demonstrates a promising drug delivery system for the synergistic enhancement of antibacterial and anti-biofilm activities against multidrug-resistant pathogens like MRSA and CRKP with reduced cytotoxic effects, suggesting its potential for treating bacterial infections. Further in vivo studies are warranted to validate these findings.