Significant difference between sirolimus and paclitaxel nanoparticles in anti-proliferation effect in normoxia and hypoxia: The basis of better selection of atherosclerosis treatment

Abstract

Compared with paclitaxel, sirolimus has been more used in the treatment of vascular restenosis gradually as an anti-proliferative drug, but few basic studies have elucidated its mechanism. The anti-proliferative effects of sirolimus or paclitaxel have been demonstrated by numerous studies under normoxia, but few studies have been achieved focusing hypoxia. In this study, porcine carotid artery injury model and classical cobalt chloride hypoxia cell model were established. Sirolimus nanoparticles (SRM-NPs), paclitaxel nanoparticles (PTX-NPs) and blank nanoparticles (Blank-NPs) were prepared respectively. The effect of RPM-NPs on the degree of stenosis, proliferative index and the expression of PCNA after 28 days of porcine carotid artery injury model was evaluated. Compared with saline group and SRM groups, SRM-NPs group suppressed vascular stenosis, proliferative index and the expression of PCNA (P < 0.01 and P < 0.05). Endothelial cell (EC) and smooth muscle cell (SMC) were pre-treated with cobaltous chloride, followed by SRM-NPs, PTX-NPs, Blank-NPs or PBS control treating, the effects on cell proliferation, HIF-1 expression and glycolysis were detected. SRM-NPs could inhibit EC and SMC proliferation under hypoxia, while PTX-NPs couldn't (P < 0.001). Significant differences between sirolimus and paclitaxel NPs in anti-proliferation effect under normoxia and hypoxia may due to the different inhibitory effects on HIF-1α expression and glycolysis. In conclusion, these results suggest that sirolimus can inhibit the proliferation of hypoxic cells more effectively than paclitaxel. These observations may provide a basis for understanding clinical vascular stenosis therapeutic differences between rapamycin and paclitaxel.

Keywords: Atherosclerosis; Glycolysis; HIF-1α; Hypoxia; Paclitaxel; Sirolimus.

Graphical abstract

Fig. 1

Characterization of Blank-NPs, SRM-NPs and PTX-NPs in vitro. (A) The particle size of them was (204.7 ± 1.1) nm, (214.3 ± 0.6) nm and (216.9 ± 0.6) nm, the PDI value was (0.099 ± 0.008), (0.098 ± 0.022) and (0.129 ± 0.046), and the surface potential was - (12.05 0.35) mV, - (12.10 ± 0.14) mV and - (14.35 ± 0.21) mV. (B) Stability test showed that the Blank-NPs, SRM-NPs and PTX-NPs had good stability within 21 d. (C) In vitro release results showed that the system could achieve relatively similar release of SRM-NPs and PTX-NPs. (D) Typical images of three kinds of nanoparticles under TEM, NPs were spherical.

Fig. 2

Cytocompatibility of Blank-NPs and uptake of Coumarin-6 NPs by cells. Cytocompatibility testing and cell uptake of Blank-NPs were detected. Within 24 h–72 h, Blank-NPs had almost no inhibitory effect on the growth of EC and SMC in normoxia (A, a) and hypoxia (B, a), and the cell survival rate was basically above 75%. Coumarin-6 NPs can be successfully swallowed into cells by EC and SMC under normoxia (A, b) and hypoxia (B, b), and lysosomal escape has been achieved. (A,b & B,b)The white bars represent 20 μm, blue stands for DAPI and green represents coumarin 6. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

The result of histopathological evaluation. (A), the condition of intima hyperplasia; (B), degree of stenosis; (C), proliferative index; (D), the expression of PCNA.

Fig. 4

Comparison of cell proliferation inhibition under normoxia and hypoxia. At 24 h, 48 h and 72 h, the inhibitory effects of SRM-NPs and PTX-NPs on EC and SMC proliferation were compared under normoxia (A) or hypoxia (B) within 24 h–72 h. The results suggested that paclitaxel seemed only inhibited the proliferation of normoxic cells. On the contrary, sirolimus-eluting stents could inhibit the proliferation of both hypoxic and normoxic cells.

Fig. 5

Effect on HIF-1α expression of SRM-NPs and PTX-NPs under normoxia. The expression of HIF-1α was detected by cellular immunofluorescence assay and Western blot. The inhibition of HIF-1α expression by Blank-NPs, SRMS -NPs and PTX-NPs at low-dose (100 μg/mL) and high-dose (1000 μg/mL) was detected by laser confocal microscopy. (A), at various concentrations (50, 100, 500, 1000, 2000 μg/mL) was detected by Western blot (B) and quantified by Image J software(C). (A)The white bars represent 20 μm, blue stands for DAPI and green represents HIF-1α. Our results suggested that PTX-NPs couldn't inhibit the expression of HIF-1α, but RPM-NPs can achieve this. Cellular immunofluorescence assay also supported similar conclusion. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Effects on cell glycolysis of Blank-NPs, SRMS -NPs and PTX-NPs under hypoxia. The effects of Blank-NPs, SRMS -NPs and PTX-NPs on EC, SMC glycolysis at low-dose (100 μg/mL) and high-dose (1000 μg/mL) were detected by real-time hippocampal energy metabolism analyzer X24. Among them, the left column is real-time measurement curve of cell glycolysis metabolism, and the right column is quantitative analysis result. Wave software was used for quantitative analysis. The results showed that glycolysis ability of SRM-NPs group was lower than that of both Blank-NPs and PTX-NPs groups at high doses. In addition, there was no significant difference in glycolysis among PTX-NPs, Blank-NPs and Control groups.

Fig. 7

Schematic diagram of mechanism. Sirolimus and paclitaxel were delivered by 3s-PLGA nano-delivery system and lysosomal escape was achieved. After that, sirolimus successfully inhibited the expression of HIF-1α and glycolysis, cell proliferation was inhibited, while paclitaxel could not achieve a significant inhibitory effect. (Step1 stands for cellular uptake, Step 2 stands for lysosome escape, Step 3 stands for disassembling, Red Cross stands for inhibition and red arrow stands for Inhibited pathway in EC and SMC). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)