Effects of BSA@LIR-PMF Nanoparticles on Cell Proliferation, Migration, Phagocytosis, and ROS Levels
The result of CCK8 analysis showed that BSA and BSA@PMF nanoparticles were non-toxic to cells in terms of cytotoxicity (Fig. 2A). The cell proliferation result analysis indicated that cells in the model group (ox-LDL (10 ug/ml) and high glucose condition (30 mmol/L) for 24 hours) showed abnormally increased proliferation compared to the control group (Fig. 2B). However, treatment with BSA@LIR, LIR, BSA@LIR, and BSA@LIR-PMF inhibited the abnormal proliferation of cells (Fig. 2B). Among these treatments, BSA@LIR-PMF showed the most significant effect (Fig. 2B). The cell migration result analysis showed that cell migration increased significantly in the model group compared to the control group. (Fig. 2C). However, treatment with LIR, BSA@LIR, and BSA@LIR-PMF effectively alleviated the level of ox-LDL-induced cell migration (Fig. 2C). On the other hand, BSA@PMF showed no obvious effect (Fig. 2C). The results indicated that LIR, BSA@LIR, and BSA@LIR-PMF can all inhibit the abnormal migration induced by ox-LDL, and BSA@LIR-PMF has the most significant effect (Fig. 2C). According to the cell phagocytosis analysis result, cells started to take up BSA@LIR-PMF nanoparticles after 4 hours, and the uptake amount increased with time (Fig. 2D E). Intracellular ROS detection results indicated that compared with the control group, the ROS levels of cells in the model group increased, but LIR, BSA@PMF, and BSA@LIR-PMF treatments significantly reduced the ROS levels of cells, with BSA@LIR-PMF having the most significant effect (Fig. 2F).
Effects of nanoparticles on lactate and ATP levels, oxidative phosphorylation, and glycolysis in vitro
The lactate level of cells in the model group increased and the ATP level decreased when compared to the Control group (Figs. 3A and 3B). However, the treatment with BSA@PMF, LIR, and BSA@LIR-PMF significantly reduced the lactate level of cells and increased the ATP level (Figs. 3A and 3B). Amongst these treatments, BSA@LIR-PMF had the most significant effect (Figs. 3A and 3B). Additionally, cellular energy metabolism levels were measured using the Seahorse XF24 Extracellular Flux Analyzer. Glycolysis was measured by analysis of extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation was measured by live cell real-time oxygen consumption rate (OCR). Compared with the control group, the model group had a decrease in OCR and an increase in ECAR. However, the treatment with BSA@PMF, LIR, and BSA@LIR-PMF restored oxidative phosphorylation and glycolysis (Figs. 3C and 3D).
Effect of nanoparticles on CHOP/EIF2a/GRP78 protein expression in vitro
The qPCR result analysis showed that the model group had increased gene expression of CHOP, GRP78, and eIF2a compared to the control group (Fig. 4A). However, treatment with BSA@PMF, LIR, and BSA@LIR-PMF significantly reduced the gene expression of CHOP, GRP78, and eIF2a (Fig. 4A). Notably, BSA@LIR-PMF treatment had the most significant effect (Fig. 4A). In the Western Blot result, the expression levels of endoplasmic reticulum stress-related proteins CHOP, eIF2a, and GRP78 were low in the normal group (Fig. 4B). However, after constructing the in-vitro model, the above proteins showed high expression (Fig. 4B). BSA-PMF did not show significant changes after the intervention, while the LIR intervention showed some improvement (Fig. 4B). After BSA@LIR-PMF intervention, the expression levels of the three proteins were the lowest, indicating that BSA@LIR-PMF can effectively inhibit endoplasmic reticulum stress (Fig. 4B).
BSA@LIR-PMF nanoparticles alleviate diabetic atherosclerosis-induced deleterious changes in vivo
Based on the results obtained from animal imaging, it was observed that there was no significant aggregation of nanoparticles that lacked platelet coating (BSA@LIR) in the aorta after 4 hours (Figure S1). However, after undergoing platelet coating, the nanoparticles (BSA@LIR-PMF) showed an improved targeting ability, as evidenced by their significant accumulation in the aorta after 4 hours (Figure S1). After 12 hours, the accumulation of BSA@LIR was slightly increased in the lesion area and more in the liver since the nanoparticles are primarily metabolized in the liver (Figure S1). On the other hand, BSA@LIR-PMF displayed a higher targeting effect as it significantly accumulated at the lesion site after 12 hours, which implies that it has a higher targeting ability compared to BSA@LIR (Figure S1).
To further explore the mechanism of diabetic atherosclerosis, we developed a mouse model (Fig. 5A) and performed a series of experiments.
the weight of the model group was lower than that of the control group (Figure S2A), but the blood glucose content of the model group was significantly higher than that of the control group(P < 0.01) (Figure S2B); After the mice were harvested, their aortas were stained with oil red (Fig. 5B-5C). The analysis results showed that the stained plaques in the aortas of the mice in the model group were deposited and distributed throughout the aorta, indicating that the diabetic atherosclerosis model was successfully constructed (Fig. 5B-5C). After nano-treatment, the results showed that there was no significant reduction in aortic artery plaques in the BSA-PMF group, but there was a partial reduction in LIR (Fig. 5B-5C). However, the plaques in the BSA@LIR and BSA@LIR-PMF groups were significantly reduced (Fig. 5B-5C). This indicates that nanoparticles have a certain therapeutic effect and can effectively alleviate aortic disease (Fig. 5B-5C). Among the groups, BSA@LIR-PMF had the best therapeutic effect due to its precise targeting (Fig. 5B-5C).
Oil red dyeing result analysis showed that compared to the control group, the model group had obvious lipid droplets (Fig. 5B-5C). However, treatments with BSA@PMF, LIR, BSA@LIR, and BSA@LIR-PMF significantly reduced lipid droplet deposition (Fig. 5B-5C). The most significant effect was observed with BSA@LIR-PMF treatment (Fig. 5C,5F). H&E staining showed that the model group had obvious calcification lesions, with a thickened aortic valve, disordered smooth muscle cell arrangement, and loss of original structure (Fig. 5D,5G). Treatments with BSA@PMF, LIR, and BSA@LIR resulted in gradually arranged cells and a few loose and deformed cells. After BSA@LIR-PMF treatment, the intima was smooth and the structure was intact (Fig. 5D, 5G). Masson staining revealed that in the model group, surrounding fibrous tissue was disordered and showed cell infiltration (Fig. 5E). After treatment with BSA@PMF, LIR, BSA@LIR, and BSA@LIR-PMF, fibrous tissue was arranged in order, with no infiltration of blood vessels and inflammatory cells (Fig. 5E,5H). (Fig. 5D ).
BSA@LIR-PMF nanoparticles regulate apoptosis and endothelial and vascular functions in diabetic atherosclerosis in vivo
Immunofluorescence of Caspase-3, α-SMA, and CD31 were analyzed and compared between the Control group and the model group. The expression of Caspase-3, α-SMA, and CD31 increased in the model group (Fig. 6A-6B). However, after treatment with BSA@PMF, LIR, BSA@LIR, and BSA@LIR-PMF, the expression of Caspase-3, α-SMA, and CD31 decreased (Fig. 6A-6B). The most significant effect was observed with BSA@LIR-PMF treatment (Figs. 6A and 6B). These results suggested that BSA@LIR-PMF nanoparticles regulate apoptosis and endothelial function in diabetic atherosclerosis in vivo.
BSA@LIR-PMF nanoparticles regulate metabolism- and reticulum endoplasmic-associated markers in diabetic atherosclerosis in vivo
In addition, serum was separated from arterial blood to measure the concentration of biochemical indicators such as TC, TG, HDL, and LDL (Fig. 7A). Compared to the Control group, the levels of TC, TG, and LDL were significantly increased in the model group (Fig. 7A). Nevertheless, no significant change in HDL level was recorded (Fig. 7A). On the other hand, BSA@PMF, LIR, BSA@LIR, and BSA@LIR-PMF groups showed remarkably decreased levels of TG, TC, and LDL (Fig. 7A). Among them, BSA@LIR-PMF had the most significant effect (Fig. 7A).
The qRT-PCR and Western Blot analysis indicated that the tested nanoparticles were effective in reducing endoplasmic reticulum stress(Fig. 7B and C). Specifically, BSA@LIR-PMF treatment had the most significant effect in reducing IRE1α, CHOP and Caspase-3 gene expression, and promoting PGC1α and CPT1 gene expression, as compared to the Control group (Fig. 7B and C). The study found that the group with diabetic atherosclerosis experienced high levels of endoplasmic reticulum stress, as indicated by the higher expression levels of IRE1α, CHOP and Casepase-3 proteins, and decreased expression of PGC1α and CPT1 genes compared to the normal group (Fig. 7B and C). BSA-PMF intervention did not show significant therapeutic effects, whereas LIR treatment effectively reduced the level of endoplasmic reticulum stress, leading to reduced expression levels of IRE1α, CHOP and Casepase-3, and an increase in the expression levels of PGC1α and CPT1. (Fig. 7B and C). The BSA@LIR-PMF nanoparticles were found to effectively inhibit endoplasmic reticulum stress and improve diabetic atherosclerosis.
The H&E results of the heart, liver, spleen, lung, and kidney in each group after different nano-treatments showed no obvious lesions and verified infiltration, indicating that the nanoparticles have good biological safety (Fig. 8).