Preparation And Characterization Of Drug-loaded Nanosystems Targeting Mitochondria Of Hepatic Stellate Cells

Hepatic fibrosis is a common pathological stage of various chronic liver diseases,which is mainly characterized by excessive accumulation of extracellular matrix proteins(ECM)in the liver to form scar tissue.Hepatic stellate cells(HSCs)play a key role in the pathological development of hepatic fibrosis and,upon activation,become a major source of ECM proteins.It has been shown that the metabolism and activity of mitochondria are enhanced in activated hepatic stellate cells(aHSCs),so nano-drug delivery systems targeting aHSCs and their mitochondria,intervening in the metabolism of aHSCs and their mitochondria,reversing aHSCs to the resting state or inducing apoptosis could be important methods for reversing liver fibrosis.In this thesis,a dual-target head micelle that can target both aHSC and aHSC mitochondria was prepared for the first time,and drug loading by this micelle can improve drug efficacy and reduce tissue toxicity,which is important for the study of targeted drug delivery system for liver fibrosis.The main research of this paper is as follows:1.Preparation and characterization of block polymers coupled with targeting ligands(VA-PEG-PCL,TPP-PEG-PCL):vitamin A acid as a targeting ligand for aHSC and triphenylphosphine(TPP)as a targeting ligand for mitochondria were coupled to polyethylene glycol-polycaprolactone(PEG-PCL)block copolymers,respectively.The chemical structures and properties of the polymers were characterized,and the results showed that the structures of the polymers were consistent with expectations.2.Preparation and characterization of VA-PEG-PCL/TPP-PEG-PCL micelles:The dual target-head micelles were prepared by thin film hydration method,and the particle size,morphology and critical micelle concentration of the micelles were investigated and their stability was verified.The results showed that the polymer micelles were homogeneous in particle size and morphology with good stability.We examined the biocompatibility of the blank micelles by CCK-8 method and hemolysis assay,and the results showed that the drug-loaded micelles had low cytotoxicity,the hemolysis rate was less than 5%,safe and non-toxic,and good biocompatibility;then we used camptothecin(CPT)as a mock drug to examine the drug loading,encapsulation rate,and drug release in PBS of the micelles;finally,we observed the cellular uptake of nile red-loaded micelles by fluorescence microscopy.The results showed that the drug loading performance of the dual-targeted micelles was good,with slow drug release in PBS and good targeting to aHSC.3.Preparation and characterization of folic acid-polyethylene glycol-polycaprolactone(FA-PEG-PCL)polymer and micelles:Firstly,FA was coupled on PEG-PCL polymer to verify its chemical structure,then FA-PEG-PCL polymer micelles were prepared and their particle size distribution was examined,and finally,the cellular targeting of polymer micelles was examined by fluorescence microscopy observation and flow cytometry.The results showed that the polymer micelles had good targeting to cells with high folic acid expression.The above study showed that VA-PEG-PCL/TPP-PEG-PCL dual target head micelles for mitochondrial targeting of aHSC and FA-PEG-PCL micelles for targeting of aHSC were successfully prepared in this thesis,and their chemical structures were well-defined,with good micelle properties and specific targeting effects on aHSC.