Effect And Mechanism Researches Of PLGA Nanoparticles On The Atherosclerotic Plaque Development Of ApoE^-/- Mouse

In recent years,the prevalence and mortality of cardiovascular diseases?CVD?in China have continued to increase,and atherosclerosis is the most important cause.Atherosclerosis is a systemic disease associated with lipid metabolism disorders.Its pathological features are that lipids in the blood enter the arterial wall and deposit in the intima to form atheromatous plaques,resulting in thickening and hardening of the arteries.Currently,combined treatment,medication and surgery are the common methods for treating CVD.The development of nanotechnology,it has brought new ideas to the prevention and treatment of atherosclerosis.Its unique physicochemical properties have shown great potential in nano-drug carriers and developers.For example,designing nanoparticles that target inflammatory cells such as macrophages to prevent and treat atherosclerosis;using nanoparticles and nanomaterials to deliver therapeutic molecules,stem cells,etc.to the ischemic myocardium site;nanoparticles developed by bacteria accurate imaging observation of the transplanted cells in vivo.In the future nanotechnology will play a more important role in the prevention and treatment of atherosclerosis.With the wide application of nanotechnology,people began to pay more attention to the safety of nanomaterials.At present,nanometer toxicology research has been formed,and the distribution,metabolism,accumulation and damage to organs caused by nanomaterials entering the human body have been deeply explored.Studies have shown that silica nanoparticles promote atherosclerosis,induce endothelial dysfunction and mononuclear cell infiltration.At the same time,the nanoparticles entering the body will cause an inflammatory reaction at the early stage of ingestion,and tend to accumulate in the vascular lesion site.So far,there were a few studies on the effects of polymer nanomaterials on atherosclerosis.Therefore,FDA-approved PLGA with good biocompatibility was used to study the process of atherosclerotic lesions in apoE^-/-mice after PLGA nanoparticles intravenous injection.The results of this study provide theoretical support for the safety of polymer nanomaterials in the application of atherosclerosis.PLGA nanoparticles were first prepared,their particle size was characterized,and their blood compatibility was evaluated.Then,the apoE^-/-mouse tail vein was continuously injected with PLGA nanoparticles,and the aortic sinus was taken for sectioning to observe the formation of plaque.The situation and the expression of related inflammatory factors TNF-?,IL-6,IL-10;further exploration of the uptake of PLGA nanoparticles and foam cell transformation by Raw264.7,the relevant mechanism of the influence of PLGA nanoparticles on the occurrence and development of atherosclerotic plaque were preliminarily explored.The main research contents and experimental results are as follows:?1?Preparation,characterization and blood compatibility evaluation of PLGA nanoparticles.PLGA nanoparticles were prepared by dialysis method and incubated with mouse serum to obtain PLGA@PC for DLS detection with particle sizes of 92.69±3.1 nm and 123.8±5.3 nm,respectively;zeta potentials were-31.6±2.8 mV and-12±3.5 mV;TEM detection showed that the prepared PLGA nanoparticles were uniform spheroids with a particle size of 95±1.9 nm and PLGA@PC was 110±3.7 nm,which was consistent with the DLS results.The hemolysis rate of PLGA nanoparticles was less than 5%,which was meet a national criterion.There was no significant difference in APTT,PT,TT and Fbg after PLGA nanoparticle incubation with platelet-poor plasma for 30 minutes.After incubation with platelet-rich plasma for 30 minutes,ELISA test the concentration of GMP-140 was measured without significant difference between all groups.Therefore,the prepared PLGA nanoparticles have no significant effect on coagulation function,platelets activation,and have good blood compatibility.?2?The effect of PLGA nanoparticles on the formation of atherosclerotic plaque in apoE^-/-mice.There were two groups,high-fat diet and PLGA nanoparticles intravenous injection for 12 weeks,another one is 8-week high-fat diet then plus PLGA nanoparticles intravenous injection for 4 weeks.Using C57 mice and 5%glucose water as control.Aortic sinus was taken for biopsy oil red O staining.ApoE^-/--12w-Glu group?16.99±1.8%?and apoE^-/--12w-PLGA group?23.24±0.8%?plaque area exists significant difference.There is a significant difference in plaque area between apoE^-/--8w-HFD+4w-Glu group?16.95±1.1%?and apoE^-/--8w-HFD+4w-PLGA group?22.03±1.4%?,suggesting that the formation of atherosclerotic plaques in apoE^-/-mice is promoted by the combination of PLGA nanoparticles and a high-fat diet.?3?The effect of PLGA nanoparticles on the inflammatory response of apoE^-/-mice with atherosclerotic lesions.The apoE^-/-mouse 12w high-fat diet formed plaques and then injected with DiI-labeled PLGA nanoparticles.After 24 hours,the aortic sinus was taken for immunofluorescence staining of CD68 and CD11b.The results showed that PLGA nanoparticles and plaque inflammation sites were found.The co-localized red fluorescent signal indicates that the PLGA nanoparticles will be enriched in the plaque.At the same time,immunohistochemical staining of TNF-?,IL-6 and IL-10 was performed on the aortic sinus of the two experimental groups.The results showed no significant difference in the experimental groups that the levels of TNF-?and IL-6 were in the C57-12w-Glu and C57-12w-PLGA two groups,as well as apoE^-/--8w-HFD+4w-Glu and apoE^-/--8w-HFD+4w-PLGA;there were significant differences between the two groups of apoE^-/--12w-Glu and apoE^-/--12w-PLGA?P<0.05?.IL-10 was lower in C57-12w-Glu and C57-12w-PLGA groups;there was a significant difference in IL-10 content between C57-12w-PLGA and apoE^-/--12w-PLGA?P<0.05?.There was a significant difference in IL-10 content between C57-12w-PLGA and apoE^-/--8w-HFD+4w-PLGA?P<0.01?.Indicating continuous long-term injection of PLGA under a high-fat diet,nanoparticles can promote the inflammatory response of plaques in apoE^-/-mice.PLGA nanoparticles and high-fat diet have a long-term chronic synergistic effect on the development of atherosclerosis lesions.?4?Effects of PLGA nanoparticles and their protein corona on macrophage activity and foam cell formation.Raw264.7 cells were treated with different concentrations?0,50,100,200,400?g/ml?of PLGA nanoparticles and PLGA@PC.It was found that the activity of Raw264.7 cells decreased with the increasing of nanoparticles concentration.After treatment for 24 h and 48 h,400?g/ml of PLGA nanoparticles and PLGA@PC showed significant differences in the viability of Raw264.7 cells?P<0.01?;100?g/ml PLGA nanoparticles and PLGA@PC treated Raw264.7 cells after 0.5 h,2 h and 4 h.With the increase of time,the uptake of PLGA nanoparticles and PLGA@PC increased by Raw264.7,but at the same time point,Raw264.7 had lower intake of PLGA@PC than PLGA.Raw264.7 cells were treated with 50?g/ml ox-LDL,when the concentration of PLGA nanoparticles was not less than 400?g/ml,the CE/TC?%?value of Raw264.7 cells was significantly different?P<0.01?;the concentration of PLGA@PC was 200?g/ml and 400?g/ml,the CE/TC?%?values were extremely significant?P<0.01?.PLGA nanoparticles and PLGA@PC promote the conversion of Raw264.7 to foam cells,and the effect of PLGA@PC was stronger than that of PLGA nanoparticles.The results of this study indicate that the prepared PLGA nanoparticles have good blood compatibility and can promote the formation of atherosclerotic plaques in apoE^-/-mice under the combined action of high-fat diet.The mechanism may be related to PLGA nanoparticles increase the expression of TNF-?,IL-6 and other inflammation-related factors in plaques,and promote the transformation of macrophage cells into foam cell.