This protocol enables the preparation and characterization of CD47- and integrin α4/β1–co-modified macrophage membrane–coated poly(lactic-co-glycolic acid) nanoparticles for targeted drug delivery applications.
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Method Article
This protocol enables the preparation and characterization of CD47- and integrin α4/β1–co-modified macrophage membrane–coated poly(lactic-co-glycolic acid) nanoparticles for targeted drug delivery applications.
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.
Atherosclerosis is a chronic inflammatory disease that leads to fatal cardiovascular events and stroke through arterial lumen stenosis and unstable plaque rupture, accounting for a significant global health burden1,2,3. Downregulation of inflammatory responses at the atherosclerotic site is a major therapeutic target4,5. Colchicine, a potent anti-inflammatory drug, has shown promise in reducing adverse cardiovascular events6,7. However, its clinical utility is....
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All animal procedures described in this protocol were approved by the Institutional Animal Care and Use Committee of Nanjing Drum Tower Hospital and followed the guidelines for the care and use of laboratory animals established by the National Institutes of Health.
1. Modification of RAW 264.7 macrophages
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DLS measurements were performed using a Zetasizer Nano ZS system at 25°C with a 173° backscatter angle. Samples were diluted 20-fold in deionized water (final concentration approximately 0.1 mg/mL) and equilibrated for 2 min before measurement. Each sample was measured in triplicate, and results are presented as mean ± SD. DLS and TEM reveal an increase in NP size following membrane coating and confirm the formation of a core–shell structure (Figure 1). COL NPs exhibit a hydrodynamic diamete.......
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This protocol provides a comprehensive method for constructing and applying CD47- and integrin α4/β1–co-modified macrophage membrane-coated NPs for targeted atherosclerosis therapy. The key advantages of this system include active targeting via the integrin α4/β1–VCAM-1 interaction and reduced macrophage uptake associated with CD47-mediated signaling, as supported by in vitro uptake assays (Figure 3) and in vivo biodistribution results (
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The authors declare no competing financial interests or other conflicts of interest.
The authors thank Dr. Jinxuan Zhao and Dr. Dr. Jiaqi Yu for their scientific advice, and the staff of PROMAB for their technical support.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 7-Aminoactinomycin D (7-AAD) | Thermo Fisher Scientific (USA) | A1310 | Viability dye for flow cytometry live/dead discrimination |
| Alexa Fluor 488-conjugated goat anti-rabbit IgG | Thermo Fisher Scientific (USA) | A-11008 | Secondary antibody for CD68 immunofluorescence staining |
| Anti-mouse CD47 antibody | Abcam (USA) | ab214453 | Used for Western blot analysis to verify CD47 expression |
| Anti-mouse integrin α4 antibody (B-2) | Santa Cruz Biotechnology (USA) | sc-376334 | Used for Western blot analysis to verify integrin α4 expression |
| Anti-mouse integrin β1 antibody (A-4) | Santa Cruz Biotechnology (USA) | sc-374429 | Used for Western blot analysis to verify integrin β1 expression |
| ApoE-/- mice | The Jackson Laboratory (USA) | N/A | Used for in vivo atherosclerosis model |
| BCA assay kit | Thermo Fisher Scientific (USA) | 23225 | Used to determine protein concentration |
| Black silicone plate | N/A | N/A | Used to mount aorta for ex vivo imaging |
| CD47 plasmid (pcDNA3.1(+)) | Generay Biotech (China) | G0177361-1 | Contains mouse Cd47 gene for overexpression |
| Colchicine | Sigma-Aldrich (USA) | C9754 | Therapeutic payload in nanoparticles |
| Confocal laser scanning microscope | Leica Microsystems | TCS SP8 | Used for fluorescence imaging and co-localization analysis |
| Citrate buffer (pH 6.0) | Thermo Fisher Scientific (USA) | N/A | Used for antigen retrieval in immunofluorescence staining |
| Dialysis tubing (MWCO 3,500 Da) | Sigma-Aldrich (USA) | PURD35030 | Used for nanoparticle purification |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich (USA) | D2447 | Solvent for nanoparticle preparation |
| DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) | Beyotime (China) | C1036 | Fluorescent labeling of nanoparticle core |
| DiO (3,3′-dioctadecyloxacarbocyanine perchlorate) | Beyotime (China) | C1038 | Fluorescent labeling of macrophage membrane |
| DiR | Beyotime (China) | Y239280 | Used for in vivo fluorescence imaging |
| DAPI | Thermo Fisher Scientific (USA) | D3571 | Nuclear counterstain for fluorescence imaging |
| Dulbecco’s Modified Eagle Medium (DMEM) | Thermo Fisher Scientific (USA) | 11965092 | Cell culture medium |
| Dynamic light scattering analyzer (Zetasizer Nano ZS) | Malvern Panalytical (UK) | N/A | Used for dynamic light scattering (DLS) measurements of nanoparticle size, PDI, and zeta potential |
| ELISA kit | R&D Systems (USA) | Depends on target cytokine | Used to quantify inflammatory cytokines |
| Endothelin-1 (ET-1) | Sigma-Aldrich (USA) | E7764 | Used to stimulate integrin α4/β1 expression |
| Fetal bovine serum (FBS) | Gibco (Thermo Fisher Scientific, USA) | 10091148 | Supplement for cell culture medium |
| Flow cytometer | BD Biosciences (USA) | FACSCalibur | Used for fluorescence signal acquisition |
| Flow cytometry analysis software (FlowJo v10.8.1) | BD Biosciences (USA) | N/A | Used for flow cytometry data analysis |
| Human umbilical vein endothelial cells (HUVECs) | FuHeng Biotechnologies (China) | FH1122 | Used for in vitro targeting assays |
| Image analysis software (ImageJ) | National Institutes of Health (USA) | N/A | Used for quantitative image analysis |
| In vivo imaging system | PerkinElmer (USA) | IVIS Lumina III | Used for whole-body fluorescence imaging |
| Isopropanol (60%) | Sigma-Aldrich (USA) | 34863 | Used for differentiation during Oil Red O staining |
| Lipofectamine 3000 | Thermo Fisher Scientific (USA) | L3000001 | Transfection reagent |
| Magnetic stirrer and stir bar | N/A | N/A | Used for nanoparticle preparation |
| Masson’s Trichrome stain kit | Abcam (USA) | ab150686 | Used for collagen staining |
| Mini extruder | Avanti Polar Lipids (USA) | 610000 | Used for membrane extrusion |
| Murine RAW 264.7 cell line | FuHeng Biotechnologies (China) | FH0328 | Source of macrophages |
| Murine smooth muscle cell line | FuHeng Biotechnologies (China) | FH-Y063 | Used for safety evaluation |
| Oil Red O | Sigma-Aldrich (USA) | O0625 | Used for lipid staining |
| Opti-MEM | Thermo Fisher Scientific (USA) | 31985062 | Reduced-serum medium for transfection |
| Oxidized low-density lipoprotein (Ox-LDL) | Yiyuan Biotechnologies (China) | YB-002 | Used to induce foam cell formation |
| Penicillin–streptomycin | Thermo Fisher Scientific (USA) | 15140122 | Antibiotic supplement for cell culture |
| Phenylmethylsulfonyl fluoride (PMSF) | Sigma-Aldrich (USA) | 78830 | Protease inhibitor |
| Phosphate-buffered saline (PBS) | Thermo Fisher Scientific (USA) | 10010023 | Washing and dilution buffer |
| Poly(lactic-co-glycolic acid) (PLGA, 50:50; MW 90,000) | Dalian Meilun Biotechnology (China) | MB5649-1 | Polymer for nanoparticle fabrication |
| Polycarbonate membrane filters (200 nm, 400 nm) | Whatman (UK) | 800282 | Used for extrusion |
| Potassium chloride (KCl) | Sigma-Aldrich (USA) | P9333 | Component of lysis buffer |
| P3000 reagent | Thermo Fisher Scientific (USA) | L3000001 | Transfection enhancer |
| Rotary microtome | Leica Biosystems (Germany) | RM2235 | Used for paraffin sectioning |
| Syringe pump | Harvard Apparatus (USA) | 70-3007 | Used for controlled nanoparticle formation |
| Transmission electron microscope (TEM) | Thermo Fisher Scientific (USA) | Talos F200S | Used for nanoparticle imaging |
| Tris-HCl | Sigma-Aldrich (USA) | T2663 | Buffer component |
| Tumor necrosis factor alpha (TNF-α) | R&D Systems (USA) | 410-MT | Used to activate endothelial cells |
| Ultrapure water | Milli-Q (Merck, USA) | ZMQSV0T01 | Used for nanoparticle preparation and dialysis |