Fluidity as a key determinant of stability in PEGylated lipid nanoparticles loaded with a TLR7 agonist

Abstract

TLR7 agonists are low molecular weight immunomodulators that can rapidly diffuse from the site of administration, often leading to undesired systemic inflammatory effects. To mitigate toxicity and broaden therapeutic applicability, imiquimod (IMQ), a widely used TLR7 agonist, was encapsulated in lipid-based nanocarriers (LNCs). A fractional factorial design (24−1) was employed to examine the influence of formulation variables—liquid lipid (LL), solid lipid (SL), lipid molar ratio, and surfactant—on particle size, encapsulation efficiency, colloidal stability, and cytotoxicity in bone marrow-derived macrophages (BMDMs) and erythrocytes. Screening indicated that LL type and SL/LL ratio were the most critical factors affecting both physicochemical and biological properties. Based on these insights, a refined study focused on C10–C18 triglycerides as SL and PEG40-stearate as surfactant, while varying LL (oleic acid or isostearic acid) at two SL/LL molar ratios (2.5:1 and 1:2.5). Structural and biophysical analyses (DSC, SAXS, DPH and Laurdan fluorescence) showed that lipid shell fluidity dictated PEG conformation at the particle surface: rigid shells promoted a hydrated brush-like PEG layer, whereas fluid shells yielded a collapsed, less stable arrangement. The optimal formulation (triglycerides: oleic acid, 2.5:1) generated nanoparticles of 45 nm with efficient IMQ encapsulation and low cytotoxicity. This system effectively reprogrammed BMDMs toward a pro-inflammatory (M1) phenotype, confirmed by gene and cytokine expression. Altogether, these results highlight the importance of formulation design and nanostructural characterization in developing nanocarrier systems that enable safer and more versatile delivery of potent immunotherapeutics such as IMQ