EGFP-EGF1-conjugated poly(lactic-co-glycolic acid) nanoparticles as a carrier for the delivery of CCR2– shRNA to atherosclerotic macrophage in vitro

Atherosclerosis is a progressive disease characterized by lipid and fibrous accumulation in large and medium arteries and is a major cause of cardiovascular morbidity and mortality. Traditional risk factors and lifestyle modifications and lipid-lowering therapies reduce events, yet substantial residual risk persists, likely due to inflammation. Monocytes/macrophages are key effectors driving recruitment to activated endothelium, transmigration, proliferation, and propagation of inflammation within plaques. CCR2 is critical for monocyte recruitment; CCR2-deficient monocytes fail to respond to MCP-1 and adhere poorly to endothelium. Blocking CCR2 to inhibit monocyte recruitment is a promising strategy to ameliorate atherosclerosis and stabilize plaques. Prior work by the authors developed EGFP-EGF1-conjugated PLGA nanoparticles (ENPs) that target tissue factor (TF) and can mediate targeted delivery and gene silencing. Given elevated TF on atherosclerotic macrophages, the authors hypothesized that ENPs could adhere to atherosclerotic macrophages via TF binding and serve as carriers to deliver CCR2-shRNA, thereby manipulating monocyte/macrophage involvement in disease. They established an oxLDL-induced cellular model of atherosclerotic macrophages, assessed ENP uptake, constructed CCR2-shRNA, loaded it into ENPs, and evaluated CCR2 gene silencing.

CCR2's role in monocyte recruitment from bone marrow to inflamed tissues is well established; CCR2 knockout models show impaired monocyte migration and reduced atherosclerosis, underscoring CCR2 as a therapeutic target. Prior nanotherapies delivering siRNA or antagonists to CCR2 in monocytes/macrophages aimed to modulate recruitment, migration, proliferation, and differentiation, but were hindered by off-target effects and suppression of host defense, highlighting the need for specific monocyte/macrophage-targeting carriers. The authors previously created EGFP-EGF1-PLGA nanoparticles that specifically bind TF and demonstrated targeted delivery to injured brain endothelium, vessel smooth muscle cells, and plaques. Given high TF expression in circulating monocytes and plaque macrophages, ENPs may be suited for targeting in atherosclerosis.

- Nanoparticle preparation and characterization: PLGA nanoparticles (NPs) were prepared via a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method. EGFP-EGF1 fusion protein was conjugated to form ENPs. Two payloads were used: coumarin-6 (for uptake studies) and a CCR2-specific shRNA (for gene silencing). CCR2-shRNA loaded PLGA NPs, coumarin-6 loaded ENPs, and CCR2-shRNA loaded ENPs were prepared. Dynamic light scattering (DLS) measured mean particle size; zeta potential was measured in double-distilled water. Drug loading capacity (DLC) and drug encapsulation efficiency (DEE) for shRNA were determined: DLC = weight of shRNA in nanoparticles / total nanoparticle weight; DEE (%) = (actual shRNA loading / theoretical shRNA weight) × 100%. - Cells and reagents: RAW 264.7 cells cultured in RPMI 1640 (high glucose) with 10% FBS and antibiotics at 37 °C, 5% CO2. OxLDL used to induce an atherosclerotic phenotype. Primers for CCR2-shRNA construction and qPCR provided. - Cellular model establishment and time-course: RAW 264.7 cells (1×10^5/well, 6-well) were serum-starved and stimulated with oxLDL (50 µg/ml) to induce foam cell-like features. TF and CCR2 mRNA were measured by qPCR at 0, 0.5, 1, 2, 4, 8, 10, 12, and 24 h. GAPDH served as internal control. Primers for TF, CCR2, and GAPDH were specified. - Cellular uptake study: After 1 h oxLDL stimulation (50 µg/ml), cells were incubated with coumarin-6 loaded ENPs or NPs for 30 min at 37 °C. Cells were washed, fixed (4% paraformaldehyde), and analyzed by fluorescence microscopy and flow cytometry (FL1 channel). Untreated cells with empty nanoparticles served as controls. - In vitro transfection: OxLDL-stimulated RAW 264.7 cells (1×10^5/well, 70–80% confluence) were incubated for 6 h in serum-free medium with various formulations: blank NPs, ENPs, CCR2-shRNA-loaded NPs, CCR2-shRNA-loaded ENPs, or LTX+CCR2-shRNA; PBS served as negative control. CCR2 and TF expression were assessed by qPCR (24 h) and western blot (48 h). - Cytotoxicity: Cells (5×10^4/well, 24-well) were exposed for 24 h to PBS, CCR2-shRNA, EGF1, LTX, PLGA-NP, or ENP (n=4 wells/group). CCK-8 assay measured viability at 450 nm; viability normalized to blank cells. - Statistics: Data presented as mean ± SD. Student’s t-test for two-group comparisons; one-way ANOVA for multiple groups; significance threshold p ≤ 0.05.

- Nanoparticle characteristics: Mean sizes approximately 100 nm with slightly negative zeta potentials (~ −10 mV). Specifically: Coumarin-6-ENP 105.06 ± 2.32 nm, −10.61 ± 1.01 mV; Coumarin-6-NP 98.86 ± 1.42 nm, −10.31 ± 2.18 mV; CCR2-shRNA-ENP 106.08 ± 1.33 nm, −10.35 ± 3.09 mV; CCR2-shRNA-NP 99.25 ± 2.52 nm, −10.53 ± 1.73 mV (n=3). DEE for CCR2-shRNA: ENP 78.35 ± 0.34%, NP 79.37 ± 0.13%; DLC: ENP 1.33 ± 0.02 µg/mg, NP 1.34 ± 0.03 µg/mg (n=3). - OxLDL induction: In RAW 264.7 cells stimulated with oxLDL (50 µg/ml), TF and CCR2 mRNA increased, peaking at ~1 h. After 2 h, CCR2 decreased back to baseline; TF decreased but remained relatively elevated. The 1 h time point was selected for subsequent experiments. - Uptake: Fluorescence microscopy showed stronger intracellular fluorescence with coumarin-6 ENPs vs NPs after 30 min. Flow cytometry confirmed significantly higher fluorescence intensity in ENP-treated cells than NP-treated cells (p<0.01), indicating enhanced uptake via TF targeting. - Gene silencing (qPCR): OxLDL increased CCR2 and TF mRNA, remaining elevated at 24 h. Treatment with CCR2-shRNA-ENPs significantly reduced CCR2 and TF mRNA compared to oxLDL alone and to CCR2-shRNA-NPs; CCR2-shRNA delivered by LTX also reduced both transcripts. Empty ENP reduced TF but not CCR2 mRNA. - Protein expression (western blot): OxLDL increased CCR2 and TF proteins. CCR2-shRNA delivered by LTX showed the strongest inhibition of CCR2 protein; CCR2-shRNA-ENPs and CCR2-shRNA-NPs also significantly reduced CCR2 protein, with ENPs more effective than NPs. TF protein levels were likewise reduced by CCR2-shRNA treatments. - Cytotoxicity: ENPs and CCR2-shRNA-loaded ENPs showed favorable cytotoxicity profiles comparable to controls (no significant toxicity). LTX and LTX+CCR2-shRNA reduced cell viability; CCR2-shRNA alone (0.1 µg) also decreased viability.

The study demonstrates that EGFP-EGF1-conjugated PLGA nanoparticles (ENPs) efficiently target oxLDL-activated macrophages via tissue factor binding, leading to enhanced cellular uptake relative to non-targeted PLGA NPs. Delivery of CCR2-shRNA with ENPs effectively suppressed CCR2 mRNA and protein, and also reduced TF expression in the oxLDL-induced macrophage model, suggesting potential coupling of CCR2 and TF pathways. These findings address the need for macrophage-targeted RNAi delivery to modulate monocyte/macrophage recruitment and inflammation in atherosclerosis. The ENP platform achieved effective gene silencing with minimal cytotoxicity, supporting its relevance as a targeted carrier to inflammatory monocytes/macrophages, which could contribute to reducing macrophage migration and plaque inflammation.

EGFP-EGF1-conjugated PLGA nanoparticles specifically target tissue factor-expressing, oxLDL-activated macrophages, exhibit superior uptake compared to non-targeted NPs, and efficiently deliver CCR2-shRNA to suppress CCR2 expression at mRNA and protein levels with low cytotoxicity. These results indicate that ENPs are a promising carrier for targeted delivery of CCR2-shRNA to inflammatory monocytes/macrophages for potential atherosclerosis therapy.