| Cholestatic liver disease（CLD）is a liver and biliary system disease caused by various factors that lead to obstruction of bile generation and flow,resulting in accumulation of toxic components in bile that disrupt liver function,cause metabolic disorders,and organic damage.CLD includes four important clinical stages:cholestasis,hepatitis,liver fibrosis,and cirrhosis.Currently,the treatment of CLD is focused on addressing the underlying causes and symptoms.Addressing the underlying causes is the most effective treatment,but only a small percentage of patients are eligible for this approach.Symptomatic treatment mainly involves using drugs to improve clinical symptoms and reduce liver inflammation.Ursodeoxycholic acid（UDCA）is the first-line drug for treating CLD in current clinical practice,but many patients have poor responses to it and it is not suitable for cholestatic liver disease which caused by genetic or immune factors.Although multiple new treatment targets have been identified and some drugs targeting these new targets have been approved for market,their therapeutic effects are limited,and some drugs have significant adverse reactions.Therefore,safe and effective drugs for cholestatic liver disease still need to be developed.Nucleic acid drugs have received great attention in recent years due to their broad targets,long-lasting effects,and rapid production capabilities.Among them,antisense oligonucleotide drugs show great potential.Micro RNA33（mi R33）is an intronic mi RNA encoded by the sterol regulatory element-binding protein 2 gene（Srebf2）.In recent years,it has been discovered that two important proteins,ABCB11 and ATP8B1,which are involved in the occurrence and development of cholestatic liver disease,are functional targets of mi R33.Based on this,this project aims to research idea of treating cholestatic liver disease by delivering antisense oligonucleotides targeting mi R33 to the liver site.A safe and effective nucleic acid drug delivery system has always been the key to the effectiveness of nucleic acid drugs.Polyethyleneimine（PEI）is a classical non-viral vector,but due to its positively charged characteristics,it has a relatively high level of cell toxicity.On the other hand,the use of focus microenvironment responsive drug delivery system is an effective strategy to improve the efficacy of different types of drugs and reduce their side effects,many studies have shown that phenylboronic acid pinacol ester has good reactive oxygen species（ROS）responsiveness.Local inflammation and oxidative stress-mediated excess ROS production are typical features of cholestatic liver disease.Therefore,this project aims to reduce the toxicity and to make it responsive to reactive oxygen species by using4-hydroxyphenylboronic acid pinacol ester（PBAP）to modify PEI and obtained the reactive oxygen species responsive material PBP.While simultaneously using a nanoprecipitation/self-assembly method to load Anti-mi R33 onto PBP,with the expectation of obtaining a nanomedicine with good physicochemical properties.The system will be characterized for particle size,Zeta potential,morphology,stability,and ROS responsiveness.Its biological activity will be evaluated in different cells and animal models in vitro and in vivo,and its safety in vivo will be evaluated.Methods1.Synthesis and characterization of the ROS-responsive material PBPMultiple PBAP molecules were grafted onto branched polyethyleneimine 600（PEI 600）to obtain the ROS responsive material PBP.The product structure was identified and the grafting ratio was calculated using 1H-NMR and FT-IR.2.Construction and physicochemical characterization of the reactive oxygen species-responsive antisense oligonucleotide nanomedicine APBP NP based on PBPVarious PBP-based reactive oxygen species-responsive nanomedicine（PBP NP）were prepared by fixing the amount of DSPE-PEG 2000 and phospholipids and varying the proportion of PBP.An appropriate proportion was selected by measuring the particle size,Polydispersity Index（PDI）value,and Zeta potential of different nanomedicines.The APBP nanomedicine was obtained by loading Anti-mi R33 into the selected PBP NP,and the particle size and Zeta potential of APBP NP were determined using a Malvern particle size analyzer.The morphology of PBP NP and APBP NP was observed using transmission electron microscopy,and the fluorescence intensity was measured using a fluorescence spectrophotometer to calculate the encapsulation efficiency of the nanomedicine.3.Stability evaluation of APBP NPNanomedicines with the same concentration were co-incubated with H2O and PBS,and their stability was evaluated by measuring their particle size and Zeta-potential on the 0th,1st,2nd,4th,and 7th days.4.H2O2responsiveness evaluation of APBP NPThe nanomedicines were co-incubated with 0.1 m M,0.25 m M,0.5 m M,1 m M,10 m M H2O2,and PBS.The transmittance was measured using a UV spectrophotometer,and the hydrolysis curve was plotted.The changes in APBP NP and 1 m M H2O2co-incubated for 1hour were observed and evaluated to determine the nanomedicine’s responsiveness to H2O2.5.In vitro biological activity evaluation of APBP NPNanomedicines containing different concentrations of Cy3-Anti-mi R33 were co-incubated with Hep G2 cells and RAW264.7 cells for different periods.Flow cytometry and laser confocal microscopy were used to evaluate the cells’uptake of APBP NP.The effect of APBP NP on regulating bile flow in a cholestatic liver disease cell model was evaluated.6.Efficacy evaluation of APBP NP in a cholestatic liver disease mouse modelThe mouse cholestatic liver disease models were induced by feeding3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine（DDC）and bile duct ligation（BDL）.The efficacy of different doses of nanomedicine for cholestatic liver disease mice was evaluated by measuring changes in alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,alkaline phosphatase（ALP）,total bilirubin（T-Bil）,direct bilirubin（D-Bil）,and total bile acid（TBA）,and observing liver damage,fibrosis,and inflammatory infiltration in mouse liver slices.7.Preliminary mechanism study of APBP NP therapy for cholestatic liver diseaseUsing Anti-mi R33 and APBP NP with the same dosage of Anti-mi R33 to evaluate their ability to reduce ALP and TBA in a mouse model of cholestatic liver disease induced by DDC.The study aims to investigate the mechanism of APBP NP therapy for cholestatic liver disease.8.Safety evaluation of APBP NPSafety assessment of APBP NP is conducted by evaluating the changes in mouse body weight,organ index,blood routine,and kidney function after a single injection of high-dose nanoparticles.Results1.The ROS responsive material PBP was successfully synthesized,and the primary amine of PEI 600 was modified with PBAP.2.An reactive oxygen species responsive oligonucleotide nanomedicine,APBP NP,was successfully constructed.It has a regular shape,a uniform distribution,and can encapsulate Anti-mi R33 with an encapsulation efficiency more than 80%.3.Stability tests showed that APBP NP had good stability in both H2O and PBS,with no significant changes in particle size and Zeta potential within 7 days.4.The reactive oxygen species responsive experiment of APBP NP showed that the nanoparticles were sensitive to H2O2and could respond quickly at lower concentrations.5.Uptake experiments with different cells showed that various cells can take up APBP NP in a time-and dose-dependent manner.6.In vitro studies on the effect of APBP NP on bile flow showed that APBP NP can promote the expression of ABCB11 and ATP8B1 genes.7.In the treatment experiment of APBP NP on cholestatic liver disease mouse models induced by DDC and BDL,both models showed that APBP NP has good functions in reducing liver biochemical indicators,alleviating liver damage,and inhibiting fibrosis and inflammatory cell infiltration.8.In the preliminary mechanism study of APBP NP for treating cholestatic liver disease,Anti-mi R33 only showed a lowering effect on ALP but had no effect on TBA levels,APBP NP showed better results in reducing ALP and significantly reducing TBA levels.9.Safety tests of APBP NP in mice showed that the nanoparticles have good safety even at higher dosesConclusions1.Based on the ROS responsive material PBP,a nanomedicine that can load Anti-mi R33 was constructed.The physicochemical characterization showed that it has suitable particle size,Zeta potential,and a regular shape with a uniform distribution,and has good stability and reactive oxygen species responsiveness.2.APBP NP can be taken up by different cells related to the occurrence and development of cholestatic liver disease in a time-and dose-dependent manner,and cell experiments showed that it mainly promotes bile flow by increasing the expression of ABCB11 and ATP8B1.3.APBP NP has a good function in reducing liver biochemical indicators,improving liver tissue inflammation,and inhibiting liver fibrosis in different cholestatic liver disease models induced by different methods,and has great advantages in lowering bile acid levels.4.The preliminary in vivo safety tests showed that APBP NP has good biocompatibility and safety. |
