VA-functionalized Polyaminaglycoside Targeting Hepatic Stellate Cells Delivery Of ShRNA-TGFβ₁ For Hepatic Fibrosis Therapy

Liver fibrosis is a scar repair response to liver injury and is an important factor in causing chronic liver disease.Early stage liver fibrosis is highly dynamic and reversible,but as it worsens,it can further progress to irreversible cirrhosis and even liver cancer.Therefore,taking appropriate anti-fibrotic measures is the key to treating chronic liver disease.Since hepatic stellate cells（HSCs）play a dominant role in the development and progression of liver fibrosis,slowing down or reversing the activation and proliferation of HSCs is a potential approach to alleviate or even reverse liver fibrosis.However,existing antifibrotic therapeutic agents lack the ability to be released rapidly and selectively within HSCs.In contrast,vitamin A（VA）can specifically bind to RBP receptors on the cell membranes of HSCs mediated by retinol binding protein（RBP）to achieve localization and targeting of HSCs.This provides a promising avenue for the development of drugs targeting HSCs.However,the current anti-fibrotic drugs designed using VA to target HSCs functionally,their biocompatibility and gene delivery capabilities are in urgent need of improvement.Therefore,in Chapter 2 work,a degradable branched cationic nucleic acid delivery vector（SS-HPVA）targeting HSCs was constructed based on a low toxicity and high transfection capacity branched polyaminoglycan（SS-HPT）and HSCs targeting molecule VA.Physicochemical characterization such as NMR,IR,elemental analysis,and biochemical characterization such as particle size potential,agarose gel electrophoresis,and atomic force microscopy（AFM）confirmed the successful preparation and sensitive reduction response of SS-HPVA;cytotoxicity and transfection experiments demonstrated the low toxicity and high transfection properties of SS-HPVA;furthermore,flow,laser confocal and targeted transfection experiments were used to illustrate the role of SS-HPVA and RBP in the development of SS-HPVA.The dose-effect relationship between VA and RBP in the targeting of HSCs by SS-HPVA was elaborated using flow,laser confocal and targeted transfection experiments.In conclusion,the screening resulted in a delivery vector with great potential for HSCs targeting gene therapy.In addition,transforming growth factor（TGFβ₁）,the most potent known pro-fibrotic cytokine,is involved in almost all key steps in the development of liver fibrosis.The current use of TGFβ₁ antibodies,soluble receptors or small molecule inhibitors to impair the production of TGFβ₁ can inhibit the progression of liver fibrosis to some extent,but there are also problems of low regulatory efficiency and side effects.In addition,systemic inhibition of TGFβ₁expression can also accelerate the proliferation of related cells and tumor response.Therefore,the search for reliable and effective targeted regulatory systems is a top priority in the development of TGFβ₁-blocking drugs.In Chapter 3 work,the SS-HPVA delivery gene silencing plasmid sh RNA-TGFβ₁with excellent performance in Chapter 2 was utilized to treat liver fibrosis.In vitro experiments,Q-PCR,WB and IFC confirmed the downregulation of TGFβ₁ and its downstream liver fibrosis-related factors by the SS-HPVA/sh RNA-TGFβ₁ system;while in vivo experiments,multispectral photoacoustic tomography and in vivo fluorescence imaging verified the SS-HPVA/psh RNA-TGFβ₁ gene silencing system has excellent liver targeting ability in animals,especially in liver fibrosis models.Moreover,the system was confirmed by non-invasive high-frequency ultrasound and histopathology from multiple angles to demonstrate that the gene therapy system can effectively reverse liver fibrosis with no toxic side effects on all organs and has good potential for in vivo application.In summary,using the principle of VA targeting on HSCs,we innovatively proposed an efficient liver fibrosis reversal system,which opens up new prospects for clinical liver fibrosis treatment research.