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 limited by a narrow therapeutic window and systemic side effects, necessitating targeted delivery systems to enhance accumulation at the plaque site while minimizing toxicity⁸.

Cell membrane–coated nanoparticles (NPs) have emerged as a powerful platform for targeted drug delivery, leveraging the natural biological functions of source cells^9,10,11. Among cell types involved in atherosclerosis, macrophages play a pivotal role^12,13,14. Integrin α4/β1, expressed on macrophages, mediates recruitment to atherosclerotic plaques by binding to vascular cell adhesion molecule-1 (VCAM-1) expressed on activated endothelial cells (ECs). CD47, a “don’t eat me” signal, enables evasion of phagocytosis by the mononuclear phagocyte system^15,16. By engineering macrophage membranes to overexpress these proteins compared to native macrophage membranes, one can create NPs with enhanced targeting via integrin α4/β1–VCAM-1 interaction and reduced phagocytosis via CD47-mediated signaling⁶. The dual modification strategy improves upon native membrane-coated NPs and ligand-based targeting systems by simultaneously enhancing targeting to activated ECs and reducing phagocytic clearance. Unlike native macrophage membranes which express only baseline levels of these proteins, our engineered membrane provides enhanced functionality without introducing synthetic targeting ligands that may trigger immunogenicity.

This protocol details the preparation and characterization of colchicine-loaded poly(lactic-co-glycolic acid) NPs (COL NPs) coated with CD47- and integrin α4/β1–co-modified macrophage membranes, generating modified macrophage membrane–coated NPs (MMM/COL NPs). We describe methods for the genetic and pharmacological modification of RAW 264.7 macrophages, isolation of modified membranes, preparation and coating of PLGA NPs, and comprehensive in vitro and in vivo functional assays. This method provides a reproducible framework for preparing and evaluating biomimetic NPs for targeted drug delivery applications in atherosclerosis research.

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 supplies.
2. Culture RAW 264.7 cells in a humidified incubator at 37°C with 5% CO₂ and maintain the cells until they reach approximately 4–5 × 10⁵ cells per well in a 6-well plate.
3. Perform CD47 modification
   1. Prepare plasmid DNA solution by mixing 2.5 µg of plasmid DNA with 5 µL of P3000 reagent in 125 µL Opti-MEM serum-free medium.
   2. Prepare transfection reagent solution by diluting 3.75 µL of transfection reagent in 125 µL of serum-free medium.
   3. Use these volumes for a 6-well plate format. Scale all components proportionally for other formats while maintaining a DNA:P3000 ratio of 1:2 (µg:µL) and a DNA:transfection reagent ratio of 1:1.5 (µg:µL).
      NOTE: For example, use 10 µg of DNA, 20 µL of P3000, and 15 µL of transfection reagent for 10 cm dishes.
   4. Combine the DNA solution with the transfection reagent solution and incubate for 15 min at 25°C.
   5. Add the transfection complexes to the cells in antibiotic-free DMEM supplemented with 10% FBS at approximately 2–2.5 × 10⁶ cells per 10 cm dish.
   6. Replace the medium with fresh complete medium containing antibiotics 6 h post-transfection.
4. Perform integrin α4/β1 modification
   1. Stimulate transfected or non-transfected cells with 100 nM ET-1 in serum-free medium for 24 h to upregulate integrin α4/β1 expression.
      NOTE: Verify modification efficiency by quantitative polymerase chain reaction (qPCR) and Western blot analysis for CD47, integrin α4, and integrin β1.

2. Isolation of modified macrophage membrane (MMM)

1. Harvest the modified RAW 264.7 cells using a cell scraper.
2. Centrifuge the cells at 900 × g for 5 min at 4°C.
3. Wash the cell pellet three times with cold 1× PBS.
4. Resuspend the cell pellet in 2 mL of pre-cooled hypotonic lysis buffer (10 mM of Tris-HCl, pH 8.0, 1 mM of KCl, 1.5 mM MgCl₂, and 1 mM of phenylmethylsulfonyl fluoride) and incubate the suspension on ice for 15 min.
5. Freeze the suspension in liquid nitrogen for 2 min, and then thaw it in a 37°C water bath for 5 min. Repeat the freeze–thaw cycle five times to fully lyse the cells.
   CAUTION: Liquid nitrogen can cause severe cryogenic burns. Handle using appropriate insulated gloves and face protection.
6. Centrifuge the lysate at 850 × g for 10 min at 4°C to remove nuclei and unbroken cells.
7. Centrifuge the collected supernatant at 15,000 × g for 30 min at 4°C. To obtain higher membrane purity, transfer the supernatant obtained after 15,000 × g centrifugation to ultracentrifuge tubes and centrifuge at 100,000 × g for 1 h at 4°C using an ultracentrifuge.
   CAUTION: Ensure ultracentrifuge tubes are properly balanced and rated for high-speed centrifugation to prevent rotor imbalance or failure.
   NOTE: Ultracentrifugation yields purified plasma membranes with reduced intracellular organelle contamination.
8. Collect the pellet containing MMM fragments and resuspend it in 300 µL of 1× PBS or lysis buffer to obtain a protein concentration of approximately 1–2 mg/mL.
9. Determine the membrane protein concentration using a BCA protein quantification assay kit.
   NOTE: Isolated membranes can be stored at −80°C for later use.

3. Preparation of colchicine-loaded PLGA NPs (COL NPs)

1. Dissolve 10 mg of PLGA (50:50, MW 90,000) and 1 mg of colchicine in 1 mL of dimethyl sulfoxide (DMSO).
   CAUTION: DMSO can penetrate skin and enhance absorption of other substances. Wear appropriate personal protective equipment (nitrile gloves, lab coat, and safety goggles) when handling. Collect DMSO-containing solutions as organic solvent waste and dispose through licensed hazardous waste disposal services. Colchicine is toxic and potentially teratogenic. Collect all colchicine-contaminated materials (solutions, gloves, and tubes) as hazardous pharmaceutical waste and dispose through licensed hazardous waste disposal services according to institutional and local regulations.
2. Stir the solution at 500 rpm using a magnetic stirrer with a 10 mm Teflon-coated stir bar at 25°C.
3. Add 4 mL of ultrapure water dropwise at approximately 1 mL/min using a syringe pump fitted with a 5 mL syringe and 26G needle at 25°C.
4. Continue stirring the mixture for 3 h to allow NP formation.
5. Transfer the NP suspension to a dialysis bag (MWCO 3,500 Da).
6. Dialyze against 2 L of distilled water at 4°C under gentle stirring at 200 rpm.
7. Continue dialysis for 24 h and replace the dialysis buffer completely at 6, 12, and 24 h to remove free colchicine and DMSO.
   CAUTION: Collect all organic solvents and reagents, including those from dialysis, as organic chemical waste. Do not dispose of it down the drain. Follow institutional hazardous waste disposal protocols.
8. Collect the dialyzed COL NP suspension and store at 4°C.
   ​NOTE: COL NPs can be stored at 4°C for short-term use (up to 3 days). For fluorescent labeling, incorporate 0.1% (w/w) of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) dye into the PLGA solution during Step 3.1.
9. Verify the removal of DMSO and free colchicine
   1. Collect the final dialysate (the buffer after the last change at 24 h) and analyze it using ultraviolet–visible (UV–vis) spectrophotometry.
   2. Scan the dialysate from 200 to 400 nm and compare it with a control sample (fresh dialysis buffer).
   3. Confirm that no absorbance peak is present at approximately 210 nm (characteristic of DMSO) or at 345 nm (characteristic of colchicine), indicating successful removal of residual solvent and free drug.
   4. Alternatively, determine the residual DMSO concentration using a DMSO assay kit according to the manufacturer's instructions. Ensure that the residual DMSO is below 0.1% (v/v) to avoid cytotoxicity in downstream assays.

4. Fabrication of membrane-coated NPs (MMM/COL NPs)

1. Sonicate the isolated MMM suspension (from step 2.7) for 20 min using a water bath sonicator at 40 kHz and 110 W. Maintain the sample volume at 1 mL in a 1.5 mL tube and temperature below 25°C using ice bath as needed.
2. Extrude the sonicated membrane suspension through a polycarbonate membrane filter with a pore size of 400 nm using a mini extruder to form membrane vesicles.
3. Mix the COL NPs (from step 3.5) with the MMM vesicles at a membrane protein-to-PLGA mass ratio of 1:1.
   1. Use a membrane suspension containing 10 mg of protein for 10 mg of PLGA as determined by protein quantification assay.
   2. Gently pipette mix the COL NPs with the MMM vesicles up and down 15 times using a 1 mL pipette.
4. Co-extrude the mixture through a polycarbonate membrane filter with a pore size of 200 nm and repeat the extrusion for 15 passes.
5. Collect the resulting suspension containing MMM/COL NPs and store it at 4°C for immediate use.

5. Characterization of NPs

1. Measure the hydrodynamic diameter, polydispersity index (PDI), and zeta potential using dynamic light scattering (DLS) under the following conditions: temperature, 25°C; backscatter angle, 173°; equilibration time, 2 min; and three replicate measurements per sample.
2. Dilute the samples 20-fold in deionized water (final concentration, approximately 0.1 mg/mL).
   NOTE: Dilution minimizes multiple scattering effects.
3. Visualize the core–shell structure of MMM/COL NPs using transmission electron microscopy (TEM).
   1. Place 5 µL of NP suspension (approximately 0.1 mg/mL) onto a glow-discharged, carbon-coated copper grid (300 mesh).
   2. Allow the sample to adsorb for 2 min at 25°C.
   3. Remove excess liquid by gently touching the edge of the grid with filter paper.
   4. Apply 5 µL of 1% uranyl acetate solution onto the grid and incubate for 1 min at 25°C.
   5. Remove excess stain with filter paper.
   6. Allow the grid to air-dry completely for at least 10 min before imaging.
      ​CAUTION: Uranyl acetate is toxic and radioactive. Handle with appropriate personal protective equipment (gloves, lab coat, and eye protection), and dispose of waste according to institutional hazardous-material guidelines.
4. Label the membrane with 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) dye during step 2 or by labeling intact cells prior to membrane isolation, and label the PLGA core with DiI.
5. Analyze the co-localization of DiO (green) and DiI (red) signals using confocal laser scanning microscopy (CLSM).
   1. Set excitation/emission parameters as follows: DiO (green), excitation 488 nm, emission 550 nm; DiI (red), excitation 561 nm, emission 620 nm.
   2. Use a 60× oil-immersion objective lens (numerical aperture, 1.4).
   3. Set the pinhole to 1 Airy unit.
   4. Adjust the detector gain to avoid pixel saturation.
   5. Acquire images at resolution of 1024 × 1024 pixels with 2× line averaging.
   6. Maintain the laser power below 5% to minimize photobleaching.
6. Analyze the presence of CD47, integrin α4, and integrin β1 on the MMM/COL NP surface using Western blot analysis.
7. Determine drug loading and release
   1. Lyse a 100 µL volume of NPs in DMSO and measure colchicine absorbance at 345 nm to determine drug loading and encapsulation efficiency.
   2. Place 1 mL of MMM/COL NP suspension (containing approximately 100 µg of colchicine) into a dialysis bag (MWCO, 3,500 Da).
   3. Seal the bag and immerse it in 50 mL of PBS (pH 7.4) containing 20% FBS in a 100 mL glass beaker.
   4. Maintain the release medium at 37°C under constant stirring at 100 rpm using a magnetic stirrer with a 20 mm Teflon-coated stir bar.
   5. Maintain sink conditions throughout the experiment.
      NOTE: Colchicine solubility in PBS with 20% FBS is >1 mg/mL; the maximum colchicine concentration in the release medium is <10% of saturation.
   6. At predetermined time points (0, 1, 2, 4, 8, 12, 24, 36, 48, and 62 h), withdraw 500 µL of release medium.
   7. Replace the withdrawn volume with an equal volume of fresh pre-warmed PBS (37°C) containing 20% FBS.
   8. Measure the colchicine concentration in the withdrawn samples using UV–Vis spectrophotometry at 345 nm.
   9. Perform all measurements in triplicate.

6. In vitro functional assays

1. Seed human umbilical vein endothelial cells (HUVECs). Stimulate the cells with 10 ng/mL tumor necrosis factor alpha (TNF-α) for 24 h to upregulate VCAM-1.
2. Cellular association of MMM/DiI NPs with HUVECs
   1. Incubate the cells with DiI-labeled MMM/COL NPs (MMM/DiI NPs) at a concentration of 50 µg/mL (based on PLGA core mass) for 4 h at 37°C in a 5% CO₂ atmosphere.
   2. Wash the cells three times with pre-chilled PBS. Centrifuge the cells at 300 × g for 5 min at 4 °C after each wash.
   3. Resuspend the washed cells in PBS containing 0.5% bovine serum albumin (BSA). Adjust the final cell concentration to 1 × 10⁶ cells/mL.
   4. Set the instrument parameters for flow cytometry or CLSM. Detect DiI fluorescence using excitation at 549 nm and emission at 565 nm (PE channel).
   5. Acquire data from the samples. Acquire at least 10,000 events per sample for flow cytometry or capture representative fields for CLSM.
   6. Select five representative fields per sample from distinct regions, excluding edges, using a 60× oil immersion lens. Ensure that cells are intact and non-overlapping.
   7. Blind the samples prior to image acquisition. Select fields by randomly moving the stage rather than manually choosing specific areas.
   8. Apply identical threshold and analysis parameters to all samples under blinded conditions.
   9. Exclude cell debris using FSC-A/SSC-A gating. Select singlet cells using FSC-A/FSC-H gating.
   10. Select live cells using viability dye staining. Add 7-aminoactinomycin D (7-AAD) at 1 µg/mL, incubate for 5 min at 4°C in the dark, and use for live cell gating.
   11. Analyze fluorescence intensity to quantify cellular association. Use mean fluorescence intensity as the quantification metric. Perform data analysis using the FlowJo (v10.8.1) flow cytometry analysis software.
3. Cellular uptake of MMM/DiI NPs by RAW 264.7 macrophages
   1. Incubate DiI-labeled MMM/COL NPs (MMM/DiI NPs) with unmodified RAW 264.7 macrophages at a concentration of 50 µg/mL (based on PLGA core mass) for 4 h at 37°C in a 5% CO₂ atmosphere.
   2. Prepare the cells for analysis as described in Steps 6.2.2–6.2.3.
   3. Set instrument parameters for DiI detection using excitation at 549 nm and emission at 565 nm (PE channel).
   4. Acquire data from the samples. Acquire at least 10,000 events per sample.
   5. Exclude cell debris using FSC-A/SSC-A gating. Select singlet cells using FSC-A/FSC-H gating.
   6. Select live cells using viability dye staining. Add 7-AAD at 1 µg/mL, incubate for 5 min at 4 °C in the dark, and use for live cell gating.
   7. Analyze fluorescence intensity to quantify cellular uptake. Use mean fluorescence intensity as the quantification metric. Perform data analysis using flow cytometry analysis software.
4. Foam cell assay
   1. Differentiate RAW 264.7 cells into foam cells by incubating with oxidized low-density lipoprotein (oxLDL) at 50 µg/mL for 48 h. Confirm foam cell formation by Oil Red O staining, indicated by intracellular lipid accumulation.
   2. Treat foam cells with free colchicine, COL NPs, or MMM/COL NPs at an equivalent colchicine concentration of 1 µM. Incubate the cells for 24 h at 37°C in a humidified atmosphere containing 5% CO₂.
      NOTE: Select this concentration based on preliminary dose–response experiments evaluating cell viability and anti-inflammatory efficacy.
   3. Measure inflammatory cytokine levels using an enzyme-linked immunosorbent assay (ELISA).
      1. Collect cell culture supernatants immediately after treatment (24 h post-treatment).
      2. Centrifuge the plates at 300 × g for 5 min at 4°C to remove cell debris.
      3. Transfer the clarified supernatants to clean tubes and store at −80°C until analysis.
      4. Measure TNF-α and interleukin-6 levels using ELISA kits according to the manufacturer’s instructions.
      5. Perform all measurements in triplicate wells and repeat the experiment three independent times.
      6. Report cytokine concentrations as absolute values (pg/mL) without additional normalization, as cell seeding density and treatment conditions are consistent across all groups.

7. In vivo therapeutic application in an atherosclerosis model

NOTE: All animal procedures must be approved by the relevant Institutional Animal Care and Use Committee.

1. Establish animal model
   1. Establish a vulnerable atherosclerotic plaque model in 8-week-old male apolipoprotein E knockout (ApoE⁻/⁻) mice (C57BL/6 background).
   2. Feed mice a high-fat diet containing 15% of fat and 0.25% of cholesterol ad libitum for 1 week prior to surgery and continue for 4 weeks post-surgery.
   3. Anesthetize mice with isoflurane (2%–3% for induction and 1.5%–2% for maintenance) and administer buprenorphine (0.1 mg/kg, subcutaneously) for analgesia during carotid artery constriction surgery.
   4. Constrict the left common carotid artery using a 0.3 mm diameter needle placed alongside the artery. Tie a 6-0 silk ligature around both the artery and needle, and then remove the needle to create partial constriction.
   5. Confirm vulnerable plaque formation at 4 weeks post-surgery by histological assessment.
   6. Perform Hematoxylin and Eosin (H&E), Oil Red O, and Masson’s trichrome staining to evaluate plaque morphology, lipid deposition, and collagen content.
   7. Define vulnerable plaques by plaque area > 40% of vessel cross-sectional area, fibrous cap thickness < 120 µm, lipid core area > 40% of total plaque area, and substantial CD68-positive macrophage infiltration.
2. Inject 100 µL of DiI-labeled MMM/COL NPs intravenously via the tail vein at a dose of 2 mg/kg (based on PLGA core mass).
   1. Image mice at 1 day post-injection using an in vivo imaging system to assess whole-body biodistribution.
      1. Anesthetize mice with isoflurane (3% for induction and 1.5%–2% for maintenance, with oxygen flow rates of 1 and 0.5 L/min, respectively). Monitor anesthesia depth by respiratory rate and toe-pinch reflex.
      2. Position mice supine on a 37°C heated stage to maintain body temperature during imaging.
      3. Acquire fluorescence images using DiI settings (excitation 549 nm and emission 565 nm) with a CY3 or TRITC filter set. Use auto-exposure mode (typically 50–200 ms) and epifluorescence acquisition.
      4. Capture corresponding brightfield images concurrently for anatomical reference at a resolution of 1024 × 1024 pixels.
   2. Harvest major organs and carotid arteries for ex vivo fluorescence imaging.
      1. Anesthetize mice with isoflurane (3% for induction and 2.5%–3% for maintenance).
      2. Perfuse mice with 20 mL of ice-cold PBS (pH 7.4) via left ventricular puncture at a flow rate of 5 mL/min to remove residual blood and unbound NPs.
      3. Harvest organs (heart, aorta, liver, spleen, kidney, and lung) immediately after perfusion at 24 h post-injection.
      4. Rinse harvested organs in ice-cold PBS to remove surface blood.
      5. Open the aorta longitudinally and pin it flat (endothelial side up) onto a black silicone plate using stainless steel pins.
      6. Keep tissues in ice-cold PBS and perform ex vivo fluorescence imaging within 30 min of harvest. Remove excess PBS from the tissue surface immediately before imaging.
3. Perform dosing and treatment
   1. Determine colchicine dose from preliminary dose-response study (0.01–0.45 mg/kg, n = 7/group). Select 0.05 mg/kg of colchicine as the therapeutic dose.
   2. Administer 100 µL via tail vein injection, corresponding to a NP dose of 2 mg/kg (PLGA core mass) and colchicine dose of 0.05 mg/kg. Inject once daily for 14 consecutive days.
   3. Randomize mice into treatment groups (PBS, free colchicine, and MMM/COL NPs) using a random number generator. Perform all quantifications by two observers blinded to treatment groups.
   4. Apply humane endpoints, including >20% body weight loss, impaired mobility, or signs of distress.
4. Perform endpoint analysis
   1. Euthanize mice 24 h after the final injection. Harvest carotid arteries and aortas. Assess plaque burden by en face Oil Red O staining of aortas.
   2. Quantify plaque area
      1. Quantify plaque area as Oil Red O-positive area percentage of total aortic lumen area in cross-sections (five sections per mouse) using image analysis software.
      2. Open the image in the ImageJ image analysis software.
      3. Click Image → Type → 8-bit to convert the image to grayscale.
      4. Click Image → Adjust → Threshold. Select the Otsu method and apply the threshold to identify Oil Red O-positive regions.
      5. Apply the same threshold parameters to all sample images and record the threshold settings for verification.
      6. Click Analyze → Set Measurements and select Area.
      7. Click Analyze → Measure to quantify the selected regions.
      8. Export the quantified data.
   3. Evaluate plaque stability
      1. Evaluate plaque stability by histological staining of carotid cross-sections using H&E, Oil Red O, and Masson’s trichrome staining. Quantify collagen as the Masson’s trichrome-positive blue area percentage of plaque area.
      2. Prepare paraffin-embedded sections at a thickness of 5 µm and optimal cutting temperature–embedded frozen sections at a thickness of 8 µm.
      3. Perform H&E staining by deparaffinizing and rehydrating sections, followed by hematoxylin staining for 5 min and eosin staining for 2 min at room temperature (RT; 23°C ± 2°C).
      4. Perform Oil Red O staining on frozen sections by incubating with Oil Red O working solution for 15 min at RT. Differentiate sections in 60% isopropanol for 2 s and rinse with distilled water.
      5. Perform Masson’s trichrome staining according to the kit instructions by incubating sections with Weigert’s iron hematoxylin for 10 min, Ponceau-acid fuchsin for 5 min, phosphomolybdic acid for 5 min, and aniline blue for 5 min at RT.
      6. Open histological images in image analysis software.
      7. Apply color thresholding to identify collagen-positive regions. Perform automatic thresholding and apply the same parameters to all sample images.
      8. Record threshold settings for verification.
      9. Define total plaque area as the sum of all plaque areas within the vascular intima.
      10. Measure plaque area by manually tracing plaque boundaries or by using automatic threshold segmentation to identify all plaque regions.
      11. Measure collagen-positive area.
      12. Calculate collagen content as collagen-positive area divided by total plaque area × 100%.
   4. Assess macrophage content
      1. Perform CD68 immunofluorescence staining to assess macrophage content. Quantify macrophage content as the CD68-positive area percentage of plaque area.
      2. Prepare tissue sections (5 µm thickness for paraffin-embedded sections). Perform antigen retrieval using citrate buffer (pH 6.0) under high pressure for 2 min.
      3. Block sections with 5% BSA for 30 min at RT.
      4. Incubate sections with rabbit anti-mouse CD68 monoclonal primary antibody (clone D4B9C, dilution 1:200 in 1% BSA) overnight at 4°C.
      5. Incubate sections with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:500) for 1 h at RT.
      6. Counterstain nuclei with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min.
      7. Open fluorescence images in image analysis software.
      8. Identify CD68-positive regions using thresholding. Perform automatic thresholding and apply the same parameters to all sample images.
      9. Record threshold settings for verification.
      10. Measure CD68-positive area.
      11. Calculate macrophage content as CD68-positive area divided by total plaque area × 100%.

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