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

Macrophage membrane-coated nanoparticles (MM-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 M-like macrophages. This optimized workflow combines hypotonic lysis, Dounce homogenization, and differential centrifugation, yielding MM fractions with consistent protein, lipid, and DNA profiles. Building on this process, a detailed and reproducible method for preparing membrane-derived nanovesicles (MM-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 MM coating efficiency, different model nanocores (TPGS micelles, VD micelles, and PLGA NPs) were employed. Among these, semi-rigid hydrophobic PLGA NPs produced the most uniform and stable coatings. Furthermore, PLGA/MM-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.