Isolation, Synthesis, and Characterization of CD47- and Integrin α4/β1-Modified Macrophage Membrane-Coated...

Atherosclerosis is a chronic inflammatory disease and a major contributor to cardiovascular morbidity and mortality. Targeted delivery of anti-inflammatory agents to atherosclerotic sites remains a significant challenge. Herein, we present a detailed protocol for the preparation and characterization of CD47- and integrin α4/β1-co-modified macrophage membrane-coated colchicine-loaded poly(lactic-co-glycolic acid) nanoparticles (MMM/COL NPs). The protocol includes macrophage modification via plasmid transfection and endothelin-1 stimulation, membrane isolation, NP fabrication, and membrane coating. The resulting MMM/COL NPs are evaluated using physicochemical characterization, protein expression analysis, and in vitro and in vivo assays. Functional validation includes assessment of cellular association with activated endothelial cells, macrophage uptake, anti-inflammatory effects in foam cells, and biodistribution in a murine atherosclerosis model. Additionally, key experimental parameters and quality control steps are highlighted to ensure reproducibility and consistency across laboratories. This protocol provides a robust and scalable approach for generating biomimetic NPs for targeted drug delivery applications in atherosclerosis research.