Cerium Oxide-based Multi-functional Nanoprobes For Accurate Diagnosis And Treatment Of Atherosclerosis

Cardiovascular and cerebrovascular disease are major culprit threatening human health and become the primary cause of morbidity and mortality worldwide.Atherosclerosis is the disease process that underlies cardio-and cerebrovascular events.However,lacking of methods for effectively identify and intervene vulnerable plaque,major adverse cardiovascular event,such as heart attack and stroke,are still in a high level due to the sudden rupture of vulnerable plaque.Current technologies can't achieve high-resolution imaging of vulnerable plaque,which decides that they are not suitable for early detection of atherosclerosis.Traditional approaches?such as surgical bypass?are costly and not altogether without risk.In addition,the anti-atherogenic drugs face serious limitations such as potential side effects,uncontrollable release manner and the lack of targeting ability,thus hindering its further use in atherosclerosis therapy.Therefore,development of in-situ,non-invasive,high sensitive imaging platform and accurate treatment of vulnerable plaques have become a focus in the research of atherosclerosis.Atherosclerosis vulnerable plaques is characterized by the accumulation of macrophages.Macrophages of plaques are activated by inflammation and produce a large amount of reactive oxygen species,especially hydrogen peroxide?H₂O₂?,which leads to the destruction of intracellular redox equilibrium and eventually causes plaque rupture.Thus,H₂O₂ can serve as an important molecular marker for identifying and detecting vulnerable plaques.The lack of plaque targeting ability and slow response to H₂O₂ of current techniques make it impossible to dynamically monitor H₂O₂ content in plaques.In addition,the vascular smooth muscle cells are important factors to maintain the stability of plaques.The stability of plaques will decline if the autophagy of cells is abnormal.The mammalian rapamycin?mTOR?pathway is one of the major pathways in regulating autophagy.Inhibiting mTOR function activates the autophagy process,reduce lipid droplets accumulation and then impedes foam cell formation.However,excessive or insufficient activation of autophagy will provoke cell death and plaque destabilization.In this way,imaging tools with highly specificity and sensitivity allowing direct visualization of H₂O₂ in situ is urgent to be developed,and it is also important to develop steady,biocompatible and controlled methods for regulating mTOR signaling pathways to maintain the stability of plaques and prevent plaque rupture.Nanotechnology has shown their powerful ability to achieve this goal.The advantages?such as high specific surface area and easy to modify?of nanomaterials make them have a supernormal adsorption capacity and chemical reactivity.As a result,nanoprobes/gene delivery carriers can be constructed because nanomaterials can adsorb protein or DNA through electrostatic adsorption or metal coordination methods.The nanoprobes/gene delivery carriers have become a powerful tool with the high sensitive detection of biological molecules and precise regulation of cell unction to improve the effect of treatment.Due to cerium oxide nanowires?CeO₂ NWs?have one-dimensional nanostructures with high aspect ratio,good biocompatibility and a high fluorescence quenching efficiency,non-toxic,easy to modify,it is an ideal nanoplatfroms for construction of nanoprobe/gene delivery vectors.Based on high permeability and difference in H₂O₂ contents of the atherosclerotic plaque,CeO₂ NWs-based nanoprobe or gene delivery vector can be developed for the detection of H₂O₂ and controlled-release of gene in the atherosclerotic plaque.They will achieve highly recognition of atherosclerotic plaque and halt its progression,which become the holes of achieving accurate of diagnosis and treatment of atherosclerosis.Herein,by virtue of the different adsorption affinities of CeO₂ NWs toward DNA and H₂O₂,we constructed a fast,highly sensitive and highly selective detection H₂O₂-response nanoprobes for evaluating risk stratification and a visually,controlled-release nanoscale gene carriers for accurate treatment of vulnerable plaques:1.To achieve high resolution imaging of H₂O₂ in activated macrophage of vulnerable plaque,we constructed a dual-targeted CeO₂ nanoprobe that is assembled by CeO₂ NWs and FAM dye-labeled DNA through the metal coordination.The nanoprobe functionalized with dual targeting ligands,folic acid and CD 36 antibody,to specifically target macrophage with high expression of folate receptor?FR-??and fatty acid transport protein?CD36?.The dual-targeted CeO₂ nanoprobe displayed non-fluorescence signal due to highly efficient quenching of the FAM on CeO₂ NWs surface.The dual-targeted CeO₂ nanoprobe was efficiently taken up by macrophage into the cytosol brings H₂O₂ and the nanoprobe into close contact.The fluorescence exhibited a significant recovery due to the higher coordination reactivity between H₂O₂ and Ce⁴⁺than that between DNA and Ce⁴⁺.The FA/CD36-CeO₂-DNA nanoprobe has shown rapid response,high sensitivity,and selectivity for H₂O₂.Using this novel nanoprobe,a better assessment of the inflammation level of macrophage and a more accurate evaluation of the extent of atherosclerotic lesions can be obtained.2.We engineered a cerium oxide nanowire?CeO₂ NW?-based RNA interference?RNAi?oligonucleotide delivery nanoplatform for the effective silencing of mTOR and treatment of atherosclerosis.This nanoplatform is composed of the following three key components:?i?a stabilin-2-specific peptide ligand?S2P?to improve plaque targeting and penetration;?ii?polyethylene glycosylation?PEGylation?to extend in vivo circulation time;and?iii?a high aspect ratio CeO₂ core to facilitate endosome escape and ensure“on-demand”release of the RNAi payloads through competitive coordination of cytosolic hydrogen peroxide?H₂O₂?.Systemic administration of the nanoplatforms efficiently targeted stabilin-2-expressing plaque and suppressed mTOR expression,which significantly rescued the impaired autophagy and inhibited the atherosclerotic lesion progression in apolipoprotein E-deficient?ApoE^-/^-?mice fed with a high-fat diet.These results demonstrated that this H₂O₂-responsive and plaque-penetrating nanoplatform can be a potent and safe tool for gene therapy of atherosclerosis.