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However, workflows for membrane isolation and NP coating remain limited and often lack reproducibility. Herein, a multi\u2010step process was established for isolating plasma membranes from human M\n                    <jats:sub>2<\/jats:sub>\n                    \u2010like macrophages. This optimized workflow combines hypotonic lysis, Dounce homogenization, and differential centrifugation, yielding M\n                    <jats:sub>2<\/jats:sub>\n                    M fractions with consistent protein, lipid, and DNA profiles. Building on this process, a detailed and reproducible method for preparing membrane\u2010derived nanovesicles (M\n                    <jats:sub>2<\/jats:sub>\n                    M\u2010NVs) was developed, and these nanovesicles were thoroughly characterized in terms of morphology, particle size, and stability under various short\u2010term 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 M\n                    <jats:sub>2<\/jats:sub>\n                    M coating efficiency, different model nanocores (TPGS micelles, VD\n                    <jats:sub>3<\/jats:sub>\n                    micelles, and PLGA NPs) were employed. Among these, semi\u2010rigid hydrophobic PLGA NPs produced the most uniform and stable coatings. Furthermore, PLGA\/M\n                    <jats:sub>2<\/jats:sub>\n                    M\u2010NPs loaded with paclitaxel demonstrated high colloidal stability, excellent hemocompatibility, enhanced immune evasion, and selective cytotoxicity against triple\u2010negative 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\u2013based biomimetic NPs and advances their translational potential for treating immunologically \u201ccold\u201d tumors.\n                  <\/jats:p>","DOI":"10.1002\/smtd.202501731","type":"journal-article","created":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T09:35:11Z","timestamp":1770888911000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A Reproducible Workflow for Macrophage Membrane Isolation and Nanocore Selection Toward Bioinspired Nanoparticles Targeting Immunologically Cold Tumors"],"prefix":"10.1002","volume":"10","author":[{"given":"Diana","family":"Peixoto","sequence":"first","affiliation":[{"name":"Departamento de Farmacolog\u00eda Farmacia y Tecnolog\u00eda Farmac\u00e9utica I+D Farma (GI\u20101645) Faculty of Pharmacy iMATUS and Health Research Institute of Santiago de Compostela (IDIS) University of Santiago de Compostela  Santiago Spain"},{"name":"Department of Pharmaceutical Technology Faculty of Pharmacy of the University of Coimbra University of Coimbra  Coimbra Portugal"},{"name":"REQUIMTE\/LAQV Group of Pharmaceutical Technology Faculty of Pharmacy of the University of Coimbra University of Coimbra  Coimbra Portugal"},{"name":"Comprehensive Health Research Centre NOVA Medical School Faculdade De Ci\u00eancias M\u00e9dicas NMS FCM Universidade Nova de Lisboa  Lisboa Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patricia","family":"Diaz\u2010Rodriguez","sequence":"additional","affiliation":[{"name":"Departamento de Farmacolog\u00eda Farmacia y Tecnolog\u00eda Farmac\u00e9utica I+D Farma (GI\u20101645) Faculty of Pharmacy iMATUS and Health Research Institute of Santiago de Compostela (IDIS) University of Santiago de Compostela  Santiago 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Pharmaceutical Technology Faculty of Pharmacy of the University of Coimbra University of Coimbra  Coimbra Portugal"},{"name":"REQUIMTE\/LAQV Group of Pharmaceutical Technology Faculty of Pharmacy of the University of Coimbra University of Coimbra  Coimbra Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8422-6792","authenticated-orcid":false,"given":"Jo\u00e3o","family":"Conde","sequence":"additional","affiliation":[{"name":"Comprehensive Health Research Centre NOVA Medical School Faculdade De Ci\u00eancias M\u00e9dicas NMS FCM Universidade Nova de Lisboa  Lisboa Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carmen","family":"Alvarez\u2010Lorenzo","sequence":"additional","affiliation":[{"name":"Departamento de Farmacolog\u00eda Farmacia y Tecnolog\u00eda Farmac\u00e9utica I+D Farma (GI\u20101645) Faculty of Pharmacy iMATUS and Health Research Institute of Santiago de Compostela (IDIS) University of 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