Lp-PLA₂-Responsive Exosome Coating For Preventing Vascular In-Stent Restenosis Via Pro-Effercytosis

Efferocytosis is severely impaired inside atherosclerotic plaques,which causes that apoptotic or necrotic cells are unable to be cleared in time and inflammation resolution couldn’t be resolved.Then,a second necrosis occurs with atherosclerosis deteriorated.Through enhancing efferocytosis in plaques,such as CD47 antibody injection or mesenchymal stem cells injection,the atherosclerotic lesion was relieved.Considering the close relationship between in-stent neointima and pre-existing plaque features,mal-efferocytosis may contribute to in-stent restenosis,and pro-efferocytic vascular stent is promising to reduce the risk of in-stent restenosis occurrence.Therefore,this work focuses on the pro-efferocytic ability of mesenchymal stem cells-sourced exosomes,and the molecular mechanism was further studied.Based on the pathological characteristics of plaques while stenting,both in vitro and in vivo platforms were constructed for exosome and coating’s efficacy evaluation.An exsome-loaded pro-efferocytic coating that was bio-responsive was further designed and manufactured on vascular stent surface.By analyzing the content inside exosomes and comparing the differences of proteomics and miRNA profiles of exosome-treated macrophage,the pro-efferocytic ability of mesenchymal stem cells-sourced exosomes was assured,and it can modulate efferocytosis via multiple signaling ways to regulate efferocytic receptor interaction and actin rearrangement,including SLC2a1,STAT3/RAC1,miRNA mediated-CD300a pathway.In the meantime,via activating PPARγ/LXR/CD36 pathway,the foam cell differentiation was inhibited and the cytoplasmic overload caused by oxidized low-density lipoprotein was also relieved.Ox-LDL-based cell models were constructed and applied to studying the influence of mesenchymal stem cells-sourced exosome on plaque cells including smooth muscle cells and endothelial cells.Results showed that exosome was able to significantly downregulate the intracellular ROS levels of both cell types,and exosome promoted the transformation into contractile phenotype of smooth muscle cells,but the effects on nitric oxide generation ability and inflammatory regulation of endothelial cells were weak.Based on those cell models,three spheroids mimicking plaque components were fabricated for efferocytosis research,and exosome treatment enhanced the clearance of apoptotic cells and decreased the inflammatory level.On the basis of Lp-PLA₂activities positively related to plaque stages,exosome carrier was designed Lp-PLA₂-responsive.Phospholipid,as Lp-PLA₂ substrate,was adopted to manufacture liposome.The physical and anti-oxidative stability of liposome can be regulated by changingαtocopherol and cholesterol concentrations,wherein liposome with cholesterol equal/larger than 20%possessed better stability.Via manufacturing liposome into multiple ventricular vesicle,the hydrophilic space inside liposome was increased,which significantly improved the exosome loading ratio without impacting Lp-PLA₂ responsive behavior.Furthermore,an exosome-loaded bioresponsive coating was designed.Through amine-surface,coating was successfully grafted on material surface,on which phosphorus and sulfur elements were detected,and the water contact angle was decreased to 10°.Exosome was distributed evenly on the surface.The multiple ventricular vesicles endowed the coating the responsive ability to Lp-PLA₂,and on the coating,more than half vesicles were released in the presence of Lp-PLA₂ concentration at advanced plaque level.This coating contained natural amhipathic phospholipid and chondroitin sulfate molecule which possesses sulfo group and glycan chain,and it possess good biocompatibility including hemocompatibiity and cell biocompatibility.Through modifying the surface of vascular stent with exosome-loaded liposome vesicles,a vascular stent was obtained that was able to response to Lp-PLA₂to release exosome.In vivo safety evaluation demonstrated a weaker tissue stimulus than control,and the study on atherosclerotic rat model confirmed that the exosome-loaded vascular stent possessed pro-efferocytic effect and the neointima was significantly thinned.