Anti-Oxidative, Anti-Apoptotic, and M2 Polarized DSPC Liposome Nanoparticles for Selective Treatment of Atherosclerosis

Abstract

Purpose: Oxidative stress is one of the main pathogenic factors of atherosclerosis. However, no antioxidants have brought positive effects on the treatment of atherosclerosis. To selectively treat atherosclerosis, various means such as antioxidation, anti-apoptosis, and M2 polarization are used. The ultimate goal is that multiple regulatory pathways can help to treat atherosclerosis.

Patients and methods: In this study, Simvastatin (SIM) as a model drug, EGCG as an antioxidant agent, and distearyl phosphatidylcholine (DSPC) as major carriers were used to make liposome nanoparticles (SE-LNPs). The cytotoxicity, phagocytosis, antioxidant, and anti-apoptotic properties of nanoparticles were tested in vitro. ApoE-/- atherosclerotic mice were treated with nanoparticles. The changes of aortic Oil red staining, blood lipid, HE, and Masson sections of the aortic root were observed.

Results: SE-LNPs exhibited a sustained release profile, potentially enabling the accumulation of the majority amount of drugs at the atherosclerotic plaque. The phagocytosis effect was stronger in RAW. The anti-oxidative and anti-apoptotic effects of the formulation were verified in vitro. SE-LNPs promoted the polarization of M2 macrophages. The therapeutic effect of SE-LNPs was assessed in the ApoE-/- mice model of atherosclerosis. SE-LNPs reduced reactive oxygen species and lipids in vivo. The results of Oil red staining, blood lipid, HE, and Masson sections of the aortic root showed the recovery of the focus.

Conclusion: Studies have shown that SE-LNPs could resist oxidation, and apoptosis, promote M2 polarization, and reduce blood lipids and lesions, which is a reliable and selective treatment for atherosclerosis.

Keywords: inhibit inflammation; macrophage polarization; reduce apoptosis; scavenging ROS.

Graphical abstract

Figure 1

Composition and function diagram of liposome nanoparticles (SE-LNPs). Liposome nanoparticles SE-LNP were composed of the model drug (simvastatin), EGCG, DSPC, and DSPE-PEG, which were used to treat atherosclerosis through tail vein injection in mice. Liposome nanoparticles could reduce inflammatory factors, reduce ROS, inhibit apoptosis, promote proliferation, promote M2 induction, and improve the therapeutic effect of free drugs at the same time of lowering lipids. It was a new promising therapeutic strategy.

Figure 2

Basic properties of liposome nanoparticles. (A) The size distribution of LNPs and SE-LNPs. (B)The TEM image of SE-LNPs in PBS. (C) The TEM image of SE-LNPs in H₂O₂. (D) The release behavior of SE-LNPs in PBS or H₂O₂ (H₂O₂ was added after three days). (E) Cell survival rate under different groups (12.5, 25, 50, 100 μg/mL). (F) Phagocytosis of free Rho and Rho-LNP by EC and RAW cells. (G) Fluorescence percentage of quantitative phagocytosis. (H) Average fluorescence intensity of quantitative phagocytosis. *p < 0.01, **p < 0.05, ***p < 0.01.

Figure 3

ROS responsiveness of the preparation. (A) Removal percentage of H₂O₂ in vitro. (B) Intracellular ROS clearance in RAW. (C) Fluorescence percentage of quantitative ROS in RAW. (D) Average fluorescence intensity of quantitative ROS in RAW. *p < 0.01, **p < 0.05, ***p < 0.01.

Figure 4

Anti-apoptotic effect of liposome nanoparticles. (A) Representative fluorescent pictures by TUNEL staining of all groups in RAW. Scale bar=100 μm. (B) Fluorescence percentage of quantitative TUNEL positive cells in RAW. (C) Average fluorescence intensity of quantitative TUNEL positive cells in RAW. *p < 0.01, ***p < 0.01.

Figure 5

The effect of liposome nanoparticles on M2 polarization. (A) Morphological changes of cells after incubating by all groups. Scale bar=20 μm. (B) Representative fluorescent pictures by CD206 staining of all groups in RAW. Scale bar=40 μm. (C) Fluorescence percentage of quantitative CD206 positive cells in RAW. (D) Average fluorescence intensity of CD206 in RAW. **p < 0.05, ***p < 0.01.

Figure 6

Therapeutic effect of SE-LNPs in ApoE−/− mice. (A) Oil Red O-stained aortas from mice after treatment with different groups. (B) Quantitative analysis of the plaque area in the aortas. (C–F) The levels of blood lipids (TC, TG, LDL-C, HDL-C) in vivo. *p < 0.01, **p < 0.05, ***p < 0.01.

Figure 7

Histochemistry analyses of aortic root sections from ApoE−/−mice after different treatments. (A) Representative images of aortic root sections stained with HE. (B) Quantitative analysis of the necrotic core area relative to plaque area. (C) Representative images of aortic root sections stained with Masson. (D) Quantitative analysis of the plaque collagen area relative to the plaque area. *p < 0.01, **p < 0.05.