ROS-responsive Biomimetic Nanoparticles For Potential Anti-atherosclerosis

Atherosclerosis（AS）,as a typical chronic inflammatory disease,is affecting the health of all the human beings.People with hypertension,diabetes,obesity,smoking history and genetic susceptibility genes are more likely to suffer from AS.At present,oral lipid-lowering drugs and interventional surgery,such as stent implantation,are the most used clinical practices to prevent AS.However,these treatments showed limited therapeutic outcome because the orally administered drugs have the shortages of low solubility,nonspecific distribution,and serious side effects,meanwhile,restenosis and acute thrombosis might happen after interventional surgery.In recent years,nanodrugs have attracted extensive attention due to their great advantages such as the improved drug solubility,the enhanced bioavailability and the reduced side effects.The development of nanodrugs provide new opportunities for the treatment of AS.However,the application of the synthetic drug delivery system in AS therapy is extremely limited,the main challenges remain in the short circulation time,lack of specific targeting ability and the poor drug release controllability.To solve the problems,we fabricated the macrophage membrane（MM）coated reactive oxygen species（ROS）-responsive drug delivery system based on the pathological condition of AS.The ROS-rich microenvironment of AS lesions could act as an intelligent"drug release switch".The boronic ester modified dextran（PCD）was designed as a carrier to load an anti-AS drug,rapamycin（RAP）,to construct the ROS-responsive nanodrugs（RNPs）,and RNPs were further coated with MM.The resulting ROS-responsive biomimetic nanoparticles（MM/RNPs）could reduce the clearance of the immune system and increasing its utilization in response to the"don’t eat me"signal sent by the functional molecule CD47 on MM.Furthermore,macrophages can be recruited to AS lesion site by inflammatory endothelial cells.Therefore,MM/RNPs could selectively accumulate in AS lesion site and release the drugs in response to ROS stimulation,reaching the enhanced therapeutic efficacy of AS.The main contents and results of this research are as follows.（1）The preparation and characterization of the ROS-responsive biomimetic NPs.The activated 4-phenylboronic acid pinacol ester（PBAP）was conjugated with dextran to synthesize the nano drug carrier,and its structure was confirmed by proton nuclear magnetic resonance（¹H-NMR）.The ROS responsiveness of the synthesized nano drug carrier was investigated in vitro.The drug-loaded NPs core was prepared using a nanoprecipitation method.MM was extracted by a mechanical breaking-gradient centrifugation method.And MM was coated on the surface of RNPs by a co-extruded method.Dynamic light scattering（DLS）measurement showed that the hydrodynamic diameter of MM/RNPs is about 156 nm and its surface is negatively charged.Transmission electron microscopy（TEM）results confirmed that MM/RNPs exhibited spherical structure,and the obvious core-shell structure was observed.SDS-PAGE and western blotting（WB）results showed that the coated NPs successfully retained the protein of MM.Moreover,in vitro drug release experiments showed that H₂O₂ could accelerate the drug release rate of the nano carrier,which achieved the effect of stimulus-response drug release.（2）In vitro efficacy of the ROS responsive biomimetic NPs.Cell proliferation experiments showed that MM/RNPs could effectively inhibit the proliferation of macrophages and smooth muscle cells in a dose-dependent manner.Confocal laser scanning microscopy（CLSM）and flow cytometry（FCM）confirmed that the coated NPs could escape the phagocytosis of macrophages.Furthermore,normal and induced human umbilical vein endothelial cells（HUVECs）were incubated with fluorescent labeled NPs to evaluate the targeting ability of MM-coated NPs to inflammatory ECs.The results showed that the MM-coated NPs could actively target inflammatory ECs.（3）The biocompatibility evaluation of the ROS responsive biomimetic NPs.The biocompatibility of MM/RNPs was detected by hemolysis test,cytotoxicity test（MTS）,platelet activation test and zebrafish embryo toxicity test.The MM-coated nanoparticles were found to be nontoxic in both cytotoxicity assay and in vivo toxicity evaluation.Furthermore,the hemolysis test indicated that both RNPs and MM/RNPs are anhemolytic,and platelet activation test confirmed that RNPs and MM/RNPs did’t affect the activation of platelet.Consequently,these results demonstrated that MM/RNPs have good biocompatibility.