Preparation Of Vitamin A Modified Liposome Targeting Hepatic Stellate Cell And Its Effects On Initiation And Progression Of Hepatocarcinoma

Background and AimsHepatocarcinoma is one of the malignant tumors with high morbidity and high mortality,and the most common type of hepatocarcinoma is hepatocellular carcinoma（HCC）.China is the"most severe area"affected by hepatocarcinoma.Hepatocarcinoma has become a major hidden danger threatening people’s life and health,and the therapy of hepatocarcinoma is faced with many problems including recurrence after surgery and chemotherapy resistance.Finding effective methods to intervene or treat HCC has been the focus of current research.The formation of primary liver cancer usually goes through the"liver cancer trilogy"of chronic inflammation,liver fibrosis,liver cirrhosis and hepatocarcinoma.Liver fibrosis is mainly caused by the abnormal accumulation of extracellular matrix（ECM）during the repairing of chronic inflammation-induced liver damages.At present,there is still no specific anti-fibrosis treatments.In the absence of effective treatments or in the long-term damage conditions,liver fibrosis may progress to cirrhosis and eventually lead to liver failure or cancer.Hepatic stellate cells（HSCs）are known as the major cell for excessive production and abnormal accumulation of ECM in the process of liver fibrosis.In the quiescent state,HSCs take up and store vitamin A（VA）from the blood.When the liver is damaged or stimulated by inflammatory factors,HSCs are activated and transform into myofibroblasts（MFB）,producing ECM components such as collagen and promoting liver fibrosis.HSC is also an important component of the tumor microenvironment（TME）,and more than 80%of liver tumors occur in a chronic inflammatory environment accompanied with liver fibrosis or cirrhosis.The change of TME is one of the important signs of the initiation of hepatocarcinoma.The microenvironment generated by HSC is conducive to tumor growth and angiogenesis,which is closely related with the initiation and progression of hepatocarcinoma.HSC has been reported as a potential target for the intervention of liver fibrosis.Therefore,we envisage that inhibiting the activation of HSCs or killing them may affect the process of liver fibrosis,regulate the TME,and ultimately inhibit the initiation and progression of HCC.It has been reported that since HSCs tend to take up and store VA from the blood,researchers at home and abroad prepared VA-modified nano-carriers to target HSCs during liver fibrosis,but there are few studies about the relevant treatment for primary liver cancer.Doxorubicin（DOX）widely kills tumor cells by inhibiting the synthesis of DNA and RNA,and its red fluorescence can be used for tracing or location in vivo and in vitro.It had been a traditional chemotherapy drug and is usually used in the clinical chemoembolization for liver cancer,but it has strong cardiotoxicity.Nanoliposomes are widely applied in many researches because of their high clinical acceptance,high biocompatibility.good pharmacokinetic parameters and easy surface modifications,which can improve the in vivo distribution of chemotherapy drugs,reduce systemic side effects,and enhance the drug delivery efficiency.In summary,in this study,liposomes were selected as the nanocarrier of DOX and VA was modified on the surface of liposomes to target HSCs and deliver DOX,and its effects on the uptake by HSC was observed.Subsequently,the effects of the prepared liposomes on the initiation and progression of liver fibrosis and HCC were observed in rat models,and its cellular and molecular effects were also studied to provide experimental phenomena for the further mechanism study and new strategies for the intervention or treatments of hepatocarcinoma.Methods and ResultsChapter 1 Preparation and characterization of vitamin A modified doxorubicin liposomes Doxorubicin liposomes（Dox-Lipo）,vitamin A modified doxorubicin liposomes（VA-Dox-Lipo）and blank liposomes were prepared by thin film hydration and ammonium sulfate gradient methods,and then characterized by particle sizes,Zeta potential,stability in vitro and TEM images.Liquid chromatography was used to detect the DOX concentration and encapsulation efficiency of liposomes,and immunofluorescence staining was used to evaluate the uptake of liposomes by the rat HSCs in vivo and in vitro.The HSC toxicity of liposomes was studied by CCK-8 assay.The results showed that VA-Dox-Lipo could effectively enhance the uptake by HSCs both in vitro and in vivo.At a certain concentration,VA-Dox-Lipo was more effective in reducing cell viability of HSCs and inhibiting HSCs proliferation than free DOX and Dox-Lipo.The results of this chapter show that the prepared liposomes are in line with the project and can be used for subsequent experimental researches.Chapter 2 Study on the effect of vitamin A modified doxorubicin liposomes on hepatocarcinogenesisIn this chapter,we established a diethylnitrosamine（DEN）-induced rat model for liver fibrosis and primary HCC initiation,and administered Free DOX,DOX-Lipo and VA-Dox-Lipo for the rats via the tail vein at the stage of HCC initiation(6-9^th week since DEN induction began).After the rats were sacrificed at the 15^th week of HCC induction by DEN,the effects of the treatment groups on rats were observed by the detections of Sirius red staining,immunohistochemistry staining,H&E staining,q RT-PCR,Western Blotting,CCK-8 assay.We investigated the collagen staining images,activated HSCs localization,m RNA expression levels of fibrosis-related molecules,tumor numbers and maximum volume,tumor markers,serum tests of liver function,liver pathological structure,etc.The effects of various interventions on the activation and proliferation of hepatic progenitor cells（HPCs）in vitro and in vivo and their effects on rat liver stemness in vivo were also observed.The results indicated that VA-Dox-Lipo could reduce the degree of liver fibrosis in rats possibly by depleting HSCs or inhibiting their activation,improve the microenvironment before hepatocarcinogenesis,and then inhibit the proliferation of HPCs and the initiation of HCC.Chapter 3 Study on the effect of vitamin A modified doxorubicin liposome on the progression of hepatocellular carcinomaIn Chapter 3,we also used the DEN-induced HCC rat models.The rats were administered with Free DOX,DOX-Lipo and VA-Dox-Lipo at the stage of HCC progression which is between the 11-14^th week of DEN-induced HCC.The development of liver fibrosis and HCC in each group was studied by a series of experiments such as tissue section staining,q RT-PCR,and Western Blotting.We found that VA-Dox-Lipo could still reduce the amounts of activated HSCs and collagen deposition in the liver.Compared with free DOX,both Dox-Lipo and VA-Dox-Lipo could effectively reduce the m RNA levels of liver fibrosis-related molecules,improve liver function,and reduce cardiac toxicity of DOX.VA-Dox-Lipo significantly reduced the number and maximum volume of tumors,inhibited the proliferation and development of liver cancer cells in vivo,and reduced the stemness of liver cancer cells.The in vitro results also indicated that VA-Dox-Lipo was more effective in inhibiting the proliferation of HCC cells stimulated by the HSC’s culture supernatants.These results suggested that VA-Dox-Lipo may affect fibrosis-related TME by down-regulating the activation of HSCs and finally inhibit the proliferation and progression of HCC.ConclusionAfter the comprehensive analysis of the results in these chapters above,the conclusionscan be drawn and listed as follows:1.VA-Dox-Lipo with the particle size of about 200 nm were successfully prepared to enhance the uptake ability of HSCs in vitro and in vivo.VA-Dox-Lipo had the morphological characteristics of liposomes,with good particle size uniformity and stability,high DOX concentration and encapsulation efficiency,and possessed strong cytotoxicity of HSCs.2.The intervention of VA-Dox-Lipo at the stage of initiation and progression of HCC reduced the number and maximum volume of tumors and number of positive liver cancer markers,improved the liver function and pathological structure in DEN-induced HCC rats,and thereby inhibited the initiation and progression of HCC.At the same time,the number of activated HSC was effectively reduced,the degree of liver fibrosis induced by DEN was relieved,and the precancerous microenvironment and TME were regulated.3.The intervention of VA-Dox-Lipo in the stage of initiation and progression of HCC limited the hepatocarcinogenesis and HCC development by inhibiting the activation,proliferation and stemness of HPCs and tumor cells,respectively.