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

Atherosclerosis, a leading cause of cardiovascular disease, is characterized by lipid and fibrous element accumulation in arteries. Risk factors include elevated LDL, homocysteine, reduced HDL, metabolic syndromes, inflammation, hypertension, smoking, and unhealthy diets. While lifestyle changes and hyperlipidemia treatments are advocated, atherosclerosis remains a major cause of death globally. Inflammation plays a crucial role, with monocytes/macrophages being central effectors of innate immunity. Atherosclerosis begins with inflammatory monocyte recruitment to activated endothelial cells. These monocytes migrate through the endothelium, proliferate within the intima, and release inflammatory factors, further fueling the disease process. Chemokine (C-C motif) receptor 2 (CCR2) is essential for monocyte recruitment and migration. Blocking CCR2 is a promising therapeutic strategy. Nanotechnology offers potential for targeted siRNA or antagonist delivery to monocytes/macrophages, but challenges remain due to off-target effects and immune suppression. The authors previously developed EGFP-EGF1 nanoparticles (ENPs) with specific affinity to tissue factor (TF) protein. This study hypothesized that ENPs could effectively adhere to atherosclerotic macrophages via TF, serving as pharmaceutical carriers to target monocytes/macrophages in atherosclerosis.

The literature firmly establishes CCR2's role in monocyte recruitment and migration from bone marrow to inflammatory sites. CCR2 knockout mice show impaired monocyte migration and adhesion to endothelium, highlighting CCR2's importance in the monocyte-endothelium interaction. Blocking CCR2 to inhibit monocyte recruitment to atherosclerotic lesions is a promising approach to ameliorate atherosclerosis progression and plaque stabilization. Various nano-therapies have been devised for CCR2 targeting, but off-target effects and host defense suppression limit their success. Specific targeting nanocarriers are needed to improve efficacy and reduce side effects. Prior work demonstrated that ENPs mediate targeted TF-siRNA delivery to brain microvascular endothelial cells and exhibited specific binding to atherosclerotic vessel smooth muscle cells and plaques in animal models, suggesting their potential for targeting atherosclerotic macrophages.

The study used the *E. coli* strain BL21 (DE3) and plasmid pET-28a-EGFPEGF1. PLGA (50:50), M-PEG (MW 3000 Da), and Mal-PEG (MW 3400 Da) were purchased. Raw264.7 cells were cultured in RPMI 1640 with 10% FBS. CCR2-shRNA plasmids were constructed. Coumarin-6-loaded ENPs and CCR2-shRNA-loaded ENPs were synthesized using a double-emulsion method. Nanoparticle size and zeta potential were measured using DLS. Drug loading capacity (DLC) and drug encapsulation efficiency (DEE) were calculated. Atherosclerotic macrophage cellular models were established using oxLDL-stimulated Raw264.7 cells. TF and CCR2 mRNA levels were measured using real-time PCR at various time points. Cellular uptake of Coumarin-6-loaded ENPs and NPs was examined using fluorescence microscopy and flow cytometry. Raw264.7 cells were transfected with CCR2-shRNA-loaded nanoparticles, and CCR2 and TF expressions were determined by real-time PCR and Western blot. Cytotoxicity was assessed using the CCK-8 assay. Statistical analysis used Student's t-test and one-way ANOVA.

Coumarin-6 and CCR2-shRNA loaded NPs/ENPs had mean sizes around 100 nm and zeta potentials of around -10 mV. DEE for ENPs and NPs were similar (78.35% ± 0.34% and 79.37% ± 0.13%, respectively). OxLDL stimulation increased TF and CCR2 mRNA expression in Raw264.7 cells, peaking after 1 hour. Fluorescence microscopy and flow cytometry showed significantly greater ENP uptake by the cellular model compared to NPs. CCR2-shRNA-loaded ENPs and LTX effectively reduced CCR2 and TF mRNA levels, with ENPs being more efficient than NPs. CCR2-shRNA-loaded ENPs significantly inhibited CCR2 and TF protein expression, though less potently than LTX. The CCK-8 assay showed that CCR2-shRNA-loaded ENPs exhibited a favorable cytotoxic profile, with minimal effect on cell activity.

The findings demonstrate the successful development of ENPs as an effective carrier for targeted CCR2-shRNA delivery to atherosclerotic macrophages. The superior uptake of ENPs compared to NPs highlights the targeting efficiency conferred by the EGFP-EGF1 fusion protein's affinity for TF. The effective silencing of CCR2 expression by CCR2-shRNA-loaded ENPs, coupled with the favorable cytotoxicity profile, suggests a promising therapeutic approach for atherosclerosis. The observed coupling between TF and CCR2 expression warrants further investigation. This targeted approach addresses the limitations of non-specific anti-inflammatory therapies by directly targeting the inflammatory monocytes/macrophages involved in atherosclerosis progression.

This study successfully demonstrated the efficacy of EGFP-EGF1-conjugated PLGA nanoparticles as a targeted delivery system for CCR2-shRNA in atherosclerotic macrophages. The results highlight the potential of ENPs for reducing macrophage recruitment and inflammation in atherosclerosis. Future studies should focus on in vivo efficacy and safety testing, exploring the potential of this approach as a novel therapeutic strategy for atherosclerosis.

The study was conducted in vitro using a cellular model. Further in vivo studies are needed to confirm the findings and assess the therapeutic potential of ENPs in a more complex biological context. The mechanisms underlying the observed coupling between TF and CCR2 expression require further investigation. The long-term effects of ENP administration also need further evaluation.