Isolation, Synthesis, and Characterization of CD47- and Integrin α4/β1–Modified Macrophage Membrane–Coated Poly(lactic-co-glycolic acid) Nanoparticles

Summary

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.

Abstract

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.

Introduction

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 burden^1,2,3. Downregulation of inflammatory responses at the atherosclerotic site is a major therapeutic target^4,5. Colchicine, a potent anti-inflammatory drug, has shown promise in reducing adverse cardiovascular events^6,7. However, its clinical utility is....

Protocol

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

1. Prepare RAW 264.7 cells, complete Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin, CD47 overexpression plasmid, Lipofectamine 3000 transfection reagent, endothelin-1 (ET-1), phosphate-buffered saline (PBS), and sterile tissue culture sup....

Results

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.......

Discussion

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 (

Materials

List of materials used in this article

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