Engineering Stability: Cholesterol-Modulated Liposome Response to Physical and Chemical Stressors for Enhanced Antimicrobial Activity

<b>Background/Objectives</b>: Liposomes are promising carriers for enhancing antibiotic delivery, but their stability under various stress conditions is crucial for clinical applicability. <b>Methods</b>: This study aimed to evaluate the physicochemical and antimicrobial stability of streptomycin-loaded liposomes subjected to mechanical (bath ultrasonication, probe ultrasonication, ultra-turrax homogenization), thermal (freezing, heating), and chemical (H₂O₂, Triton X-100, sodium dodecyl sulfate-SDS) stressors. <b>Results</b>: Isolated mechanical stresses (probe ultrasonication, ultrasonic bath, and ultra-turrax) did not significantly affect hydrodynamic diameter (DH), polydispersity index (PdI), or Zeta potential (ZP) (<i>p</i> > 0.05). In contrast, combined ultrasound-freezing stress induced marked destabilization, with DH increasing from ~110 nm to 362 nm (Lc0uf) and from 109 nm to 334 nm (Lc1uf), accompanied by PdI increases from ~0.13 to 0.37-0.41 and a ZP shift in Lc1uf from -43.1 mV to -60.1 mV. Thermal exposure at 75 °C and freezing at -30 °C caused no significant changes in DH or PdI, whereas freezing at -80 °C led to severe destabilization, with over fourfold increases in DH and fivefold increases in PdI; the cholesterol-free formulation (Lc0t-80) reached ~664 nm and a PdI of 0.609. Chemical oxidation with 1% H₂O₂ did not affect DH, PdI, or ZP, while surfactants (1% Triton X-100 and 1% SDS) significantly altered PdI and ZP in a cholesterol-dependent manner. MIC assays showed that formulations stressed by freezing at -80 °C or by combined ultrasound-freezing retained activity against <i>Escherichia coli</i> (MIC = 50 µg/mL), whereas SDS abolished activity. For <i>Staphylococcus aureus</i>, all stressed formulations remained active, and SDS reduced the MIC from 12.5 to 5.625 µg/mL. <b>Conclusions</b>: These findings demonstrate the robustness and stress-specific stabilization of these liposomal formulations, confirming that the antibiotic's activity is preserved, which highlights their potential for therapeutic use.