A Reproducible Workflow for Macrophage Membrane Isolation and Nanocore Selection Toward Bioinspired Nanoparticles Targeting Immunologically Cold Tumors

Macrophage membrane-coated nanoparticles (M₂M-NPs) offer a promising strategy for targeting immunologically "cold" tumors resistant to conventional therapies. However, workflows for membrane isolation and NP coating remain limited and often lack reproducibility. Herein, a multi-step process was established for isolating plasma membranes from human M₂-like macrophages. This optimized workflow combines hypotonic lysis, Dounce homogenization, and differential centrifugation, yielding M₂M fractions with consistent protein, lipid, and DNA profiles. Building on this process, a detailed and reproducible method for preparing membrane-derived nanovesicles (M₂M-NVs) was developed, and these nanovesicles were thoroughly characterized in terms of morphology, particle size, and stability under various short-term storage conditions. The incorporation of fluorescent lipids and cholesterol was essential for efficient extrusion and enhanced nanovesicle stability. To systematically evaluate the influence of nanocore dynamics and hydrophobicity on M₂M coating efficiency, different model nanocores (TPGS micelles, VD₃ micelles, and PLGA NPs) were employed. Among these, semi-rigid hydrophobic PLGA NPs produced the most uniform and stable coatings. Furthermore, PLGA/M₂M-NPs loaded with paclitaxel demonstrated high colloidal stability, excellent hemocompatibility, enhanced immune evasion, and selective cytotoxicity against triple-negative breast cancer, pancreatic, and glioblastoma cells compared with free paclitaxel and the clinical approved nanoformulation (Abraxane). Collectively, this reproducible workflow offers a reliable foundation for engineering macrophage membrane-based biomimetic NPs and advances their translational potential for treating immunologically "cold" tumors.