Dynamic Monocyte Differentiation Based Nanodrug For Inhibiting Progression Of Atherosclerotic Plaque

The continuous deposition of lipids and the aggravation of inflammation in the atherosclerotic artery can accelerate the progression of atherosclerotic plaque,which can lead to fatal cardiovascular and cerebrovascular events.The early intervention of plaque progression is clinically dependent on long-term drug administiation by regulating dy slipidemia and inhibiting macrophage-dense inflammation within the plaques.However,suboptimal intraplaque accumulation,along with severe off-target effects,remain formidable obstacles for the efficient pharmacotherapy of atherosclerosis.Exploiting the chemotaxis of monocytes into plaques and the dynamic differentiation of monocytes towards foam cells,we conceived a dynamic monocyte differentiation based intelligent nanodrug(nano-hitchhiker).Following intravenous injection,nano-hitchhikers were specifically engulfed by inflammatory monocytes in an efferocytosis-mimetic manner and selectively co-migrated with the monocytes towards the atherosclerotic plaques.Nanohitchhikers were readily activated to release the initially arrested curcumin in response to the differentiation of monocytes into foamy macrophages within the atherosclerotic plaques,thereby achieving precise treatment of atherosclerotic plaques.The contents of the thesis including the following aspects:1.Dynamic expression of legumain along with the differentiation of monocytesWe performed western blotting analysis and Immunofluorescence staining to analyze the dynamic expression of legumain in foam cells derived from RAW264.7 cells,THP-1 cells and bone-marrow derived monocytes(BMDMs).The protein levels of legumain in plaques were evaluated by immunohistochemical analysis.These results suggest that legumain is abundantly expressed after the differentiation of monocytes into foam cells.Besides,there is a positive correlation between legumain level and the accumulation of foam cells inside plaques.2.Preparation and characterization of nano-hitchhikersNano-hitchhikers were formulated following a two-step procedure,in which the CurAANC segment and phosphatidylserine(PtdSer)were first decorated onto HSA via covalent linkage and hydrophobic interaction.Next,nano-hitchhikers were fabricated by crosslinking the two types of decorated HSA.Characterization of nano-hitchhikers was conducted by high resolution mass spectrometry,nuclear magnetic resonance hydrogen spectrum,transmission electron microscope and dynamic light scattering.The legumain-sensitive liberation of curcumin was monitored by high-performance liquid chromatography.3.Nano-hitchhikers were selectively engulfed by circulating monocytes and co-migrated towards plaques with the monocyte shuttlesFlow cytometric analysis and confocal microscopy were performed to analyze the engulfinent of nano-hitchhikers by foam cells derived from RAW264.7 cells,THP-1 cells and BMDMs.The whole blood was harvested for flow cytometric analysis to evaluate the engulfinent of nano-hitchhikers by circulating monocytes.Nano-hitchhiker distribution in vivo was detected using a multimodal living animal imaging system.The results prove that nanohitchhikers can be selectively recognized and internalized by circulating inflammatory monocytes.Besides,the nano-hitchhikers can co-migrate towards plaques with the monocyte shuttles.4.Therapeutic efficacy and safety evaluation of nano-hitchhikersFirst therapeutic efficacy of nano-hitchhikers on atherosclerosis was assessed by ultrasound imaging and nuclear magnetic resonance imaging.Then,the lipid deposition and inflammatory factors inside plaques were analyzed by Oil-Red staining and immunofluorescence staining.Last,side effects of nano-hitchhikers were evaluated by avoirdupois monitoring,drug metabolism analysis,hematoxylin eosin staining,blood biochemistry analysis.These results illustrate that nano-hitchhiker can obviously decrease the distribution size and lipid deposition of plaques with no significant side effect.