The Effect Of OPN Targeted Nanoparticles Loaded With PPAR? Agonists On The Progression Of Atherosclerosis

Background and Objective: Cardiovascular disease is the leading cause of death worldwide. Atherosclerosis is the major pathology progress for the acute cardiovascular events. So the early diagnosis and effective treatment for the atherosclerotic plaque is critical to prevent from the malignant events of cardiovascular disease. Studies have shown that both of early migration and late apoptosis of vascular smooth muscle cells play important roles in the development of atherosclerotic plaques. The osteopontin (OPN) on smooth muscle cells has been reported as a molecular target for the diagnosis and treatment of plaques. The PPAR?agonists can effectively inhibit the migration and apoptosis of vascular smooth muscle cells in plaques. In recent years, nanodrugs have became the rsearch focus because they have many advantages such as the targeting, changing the hydrophobicity of the drug itself, reducing side effects diagnosis and treatment integration. It also provides a favorable tool for molecular targeted therapy of atherosclerosis. This study was to prepare the micelles targeted OPN and loaded PPAR? agonist GW501516 to verify the therapeutic effect on atherosclerotic plaques by in vitro cell experiments and in vivo experiments. For the treatment of coronary heart disease to provide new ideas.Methods: 1.The expression of OPN was confirmed by immunofluorescence and Western blot on MOVAS that was incubated with Oxidized low density lipoprotein(ox-LDL) for 24 hours. 2. Establishment of the atherosclerotic ApoE-/- mice model by left carotid artery compression and high fat diet for 4 months. And C57 mice were fed with normal diet as control group. After 4 months, the mice were randomly subjected to Hematoxylin - red staining (HE) and oil red staining to confirm the establishment of atherosclerosis model successfully. Immunohistochemical staining was applied to confirm the expression of OPN in animal models of atherosclerotic plaque. 3.Micells NPs-GW501516 containing GW501516 were prepared by the coprecipitation method.Non-targeted micelles conjugated antibodies to prepare OPN-NPs-GW501516.Characterization of nanoparticles was measured by TEM, DLS and ultraviolet spectrophotometer. The drug encapsulation percentage and loading efficiency of the targeted micelles were determined by high performance liquid chromatography.Immunofluorescence and flow cytometry were used to measure the targeting of nanoparticles to cells. Transwell experiments, cell scratches experiments, AO / EB staining, and flow cytomety verified the targeted micelles play a treatment effect on smooth muscle cells in vitro. The targeted effect of nanoparticles in animals was measured at different timepoints by using small animal fluorescence imaging after tail vein injection of anti-OPN-NPs-GW501516 and NPs-GW501516. The effect of anti-OPN-NPs-GW501516 was evaluated by mice were sacrificed and the carotid arteries were stained with oil stains after two weeks of treatment.Results: 1. Western blot, flow cytomety and immunofluorescence showed that the expression of OPN was significantly increased in the mouse smooth muscle cell line(MOVAS) incubated with oxidized low density lipoprotein (ox-LDL) (60 ?g / ml).2.. HE staining of the carotid artery in the AS model mice showed that the atherosclerosis was established successfully. Immunohistochemistry revealed that theexpression of OPN in the model group mice was significantly higher than that in the control group mice. The position of the OPN in the plaque is co-localized with a-SMA. 3. TEM showed that the size of Anti-OPN-NPs-GW501516 is uniform arround100nm. The hydrated particle size is about 140 nm by DLS and the zeta potential changes from 0 to negative after conjugated antibody. The UV absorption spectra of Anti-OPN-NPs-GW501516 proved that the antibody was successfully connected. The targeted micelles have a high entrapment efficiency and drug loading by the UV spectrophotometer measurements. Immunofluorescence and flow cytometry showed that the micelles have a targeted effect on SMC after incubation with ox-LDL. Some experiments such as transwell, cell scratches, AO / EB staining,flow cytometry and Western blot showed that the inhibitory effect of targeted micelles on the migration and apoptosis of smooth muscle cells in vitro was significantly better than the non - targeted micelles and simple GW501516. 4. In vivo small animal fluorescence imaging results showed that the targeted effect of Anti-OPN-NPs-GW501516 in mice was significantly better than NPs-GW501516 and the signal was the strongest after 6h injection. And Anti-OPN-NPs-GW501516 significantly inhibited the progression of atherosclerotic plaques in ApoE-/- mice.Conclusions: 1.The OPN in smooth muscle cells of atherosclerotic plaques can be used as a molecular target for the diagnosis of atherosclerosis. 2.Anti-OPN-NPs-GW501516 has the good targeting and therapeutic effect on atherosclerotic.