Two Kinds Of Curcumin-loaded Angelica Sinensis Polysaccharide Nanoparticles For The Prevention Of Acute Alcoholic Liver Injury

Currently,polysaccharide and its derivatives as drug carriers are widely studied.Angelica sinensis polysaccharide（ASP）,one of the major active ingredients in the traditional Chinese medicine Angelica sinensis,can be used as a drug carrier due to its excellent water solubility,good biodegradability,low cost and easy modification.Compared with the complexity through the introduction of targeting groups,polysaccharides with an intrinsic targeting capability are simpler and more suitable for the design of targeted drug carrier based on polysaccharide.In this study,the synthesis of two kinds of curcumin（Cur）loaded nanoparticles using ASP as an instinctive liver targeting drug delivery carrier were maninly investigated and their applications in acute alcoholic liver damage were studied.Firstly,an amphiphilic ASP derivative,composed of hydrophilic ASP and hydrophobic cholesterol,was synthesized using succinic acid as a linker.The synthesis of ASP derivatives（ASP-CHEMS）was confirmed by ¹H-NMR and FT-IR.The critical micelle concentration of ASP-CHEMS was determined to be 4.6×10^-3 mg/m L by pyrene fluorescence probe method,indicating ASP derivative had good self-aggregation behavior in aqueous solution.Cur was chosen as a model drug and Cur-loaded nanomicelles（Cur/ACNPs）were prepared by dialysis method.The encapsulation of Cur was proved by FT-IR、XRD and DSC method.The results showed that Cur was loaded in the hydrophobic core of ACNPs and exisited in amorphous state.The properties of micelles were investigated by dynamic light scattering and transmission electron microscopy.The results showed that ACNPs and Cur/ACNPs were spherical in shape,with the particle size range of 200～260nm and the Zeta potential around-20 m V.The ACNPs presented good stability within 7days.Photostability studies demonstrated that ACNPs could act as a barrier to protect Cur from irradiation.In vitro release results indicated that ACNPs had a potential application as a sustained release carrier for Cur.The cell uptake was evaluated on human liver cancer Hep G2 cells and human normal liver LO2 cells by confocal microscopy and flow cytometry.It was found that ACNPs could improve cellular uptake of drugs.At the same time,ACNPs had high affinity for asialoglycoprotein receptor（ASGPR）on the surface of hepatocytes and could be taken by cells through ASGPR-mediated endocytosis pathway.In vivo bioimaging verified the active liver targeting of ACNPs,and the tissue distribution of Cur/ACNPs results confirmed that ACNPs enhanced the accululation of Cur in the liver.The liver protective effect of Cur/ACNPs was further investigated on the acute alcoholic liver injury mouse model in vivo and the alcohol-induced LO2 cells injury model in vitro.It was found that Cur/ACNPs could inhibit the effect of alcohol on biochemical markers,and alleviate the alcohol-induced injury by activating Nrf2 signaling pathway and increasing the expression of downstream proteins GCLC,HO-1,NQO1.Furthermore,nanocomposites with Cur were prepared using ASP as a carrier based on the non-covalent interaction between polysaccharide and polyphenol.According to the properties of nanocomposites（Cur-ASP）prepared using different organic solvents or different addition sequence,adding actone solution of Cur into ASP aqueous solution was selected for the fabrication of Cur-ASP.The results of FT-IR proved Cur-ASP was successfully prepared,and Raman spectroscopy further proved that there were interactions between Cur and ASP.DSC results showed that Cur was successfully captured by ASP,and exsisted in the form of amorphous structure.Observed under transmission electron microscopy and scanning electron microscopy,the shape of ASP was irregular and aggregated,and Cur-ASP was spherical in shape,indicating that the nanocomposites were successfully prepared.The characterization of Cur-ASP was studied by dynamic light scattering,and the results indicated that the average particle size was around 250 nm with a Zeta potential about-20 m V.The in vitro release results indicated that Cur-ASP had a sustained release effect on Cur.The stabilities test of Cur-ASP under the conditions of storage at 4℃,UV light irradiation or 60℃proved that Cur-ASP had good storage stability,light stability and thermal stability.In vitro DPPH free radical scavenging experiment and ABTS free radical scavenging experiment showed that Cur-ASP maintained the free radical scavenging ability of Cur and ASP,and had antioxidant activity.Cell uptake experiments and in vivo bioimaging experiments domenstrated that Cur-ASP maintained the affinity of ASP to ASGPR and had active liver targeting.Safety tests indicated that ASP had good blood compatibility while could improve the blood compatibility of Cur,and the system safety of Cur-ASP was good.A mouse model of acute alcoholic liver injury was established to investigate the protective effect of ASP,Free Cur,Cur-ASP,and the mixture of Cur and ASP on the liver.By comparing the changes of mouse state,serum biochemical indexes,liver biochemical indexes and lipid metabolism,it was found that Cur-ASP could maintain the liver protective effect of ASP and Cur,and had better liver protective effect.In summary,in this study,two kinds of ASP-based nanoparticles with active liver targeting were prepared using water-soluble ASP as the basic material and Cur as the model drug,and their physicochemical properties,safety,target ability and pharmacological effects were further studied in vitro and in vivo.The experimental results showed that ASP with intrinsic liver targeting were expected to be a carrier material for liver disease targeted therapy.