A biomimetic membrane platform for predictive screening and rational engineering of endosomally escaped lipid...

The efficient delivery of CpG oligodeoxynucleotides (CpG ODN) adjuvants is constrained by the limited endosomal escape capability of lipid nanoparticles (LNPs). To evaluate this critical parameter, we constructed an in vitro escape evaluation platform that integrates serum protein corona simulation, a biomimetic phospholipid bilayer barrier, and pH regulation. This platform enables quantitative analysis of the transmembrane delivery mechanism of LNP-CpG ODN while overcoming limitations of cellular models for studying carrier-biointerface interactions. Through optimization of the biomimetic membrane formulation, an optimal balance between high vesicle stability (PDI < 0.18) and membrane fusion activity was achieved. Furthermore, a safer and more efficient preparation workflow was established by replacing chloroform with dichloromethane and eliminating the vacuum-drying step. Importantly, the results reported here identify the pH 6.0 microenvironment, which simulates late endosomal conditions, as the critical functional window for escape, with its efficiency showing a significant positive correlation with TLR9 pathway activation intensity (r = 0.8978, p < 0.0001). Overall, this work establishes a dual-parameter correlation framework linking escape efficiency to immune activity, providing a new paradigm for the rational design and optimization of LNP delivery systems.