The Research Of Nano-liposome Targeting At Hepatocyte Mediated By HBVpreS1Peptide (2-21Aa)

Background:The liver is the vital organ concerning to energy conversion, digestion, excretion,immune and detoxification process, which is involved in a variety of chemical synthesisand metabolism in the human body. The liver diseases are common clinicalfrequently-occurring diseases and some of them such as viral hepatitis, non-alcoholic fattyhepatitis, liver fibrosis, liver cirrhosis and hepatocellular carcinoma (HCC) greatly affecthuman health. China is one of the hardest-hit areas where viral hepatitis accounted for asignificant proportion. There are about120million HBV carriers and10million cases ofhepatitis C infection. Studies have found that nearly80%of primary liver cancer are causedby viral hepatitis. The current clinical treatment of liver disease includes biological agents,chemical drugs surgery, minimally invasive treatment and liver transplantation. Concerningdrug as an important treatment strategy which aims to deliver drugs to the liver, remove thelesion site of pathogenic viruses and repair the damaged liver cells and liver tissue.However, most of the clinical drugs for treatment of liver diseases are limited to applicationdue to less distribution, poor pharmacokinetic properties and side effects. Therefore, toexplore the effective methods and strategies of liver disease treatment is a major problemwhich needs to be settled.The hepatic targeted drug delivery systems (HTDDS) are new strategy for drug controlledrelease. It is able to deliver drugs to the diseased tissue and cells effectively. It can weaken theside effects and evidently minimize administration frequency, markely reduce dose,which playsa positive role in liver disease treatment.HTDDS can be divided into active type and passivetype. The passive type generally uses polymeric material of good biocompatibility, low toxicityto load drug, and fulfill the function of targeted delivery by using enhanced permeation andretention effect in Systemic metabolic and devour uptake role of liver reticuloendothelial system.The active type modifies the carrier and transfers drugs to specific tissues and cells. It forms a common mode which function itself byspecific binding.Content:With the deepening understanding of the liver tissue structure, people constantly findnew targets for the liver cells, which provides more sites for targeted therapy of liver disease.Using mass spectrometry with double affinity purification technology, scientists in Chinareveals that Sodium Taurocholate Cotransporting Polypeptide (NTCP) is a functional receptorfor HBV and HDV and the targeted probe is polypeptide derived from HBV PreS1antigen.HBV belongs to the family Hepadnaviridae, Its envelope protein preS1is essential tocombine with the liver cell, and the specific binding site is part of the peptide ofHBVpreS1/2-48. The reports show that the special peptide HBVpreS1modified withacetylation and nutmeg acylation can successfully protect the primary human hepatocytes,primary tupaia hepatocytes and the induced HepRG cells from being infected with HBV.The9-21amino acids of HBVpreS1is a highly conserved sequence with combination ofblocking ability. And the nutmeg acylating modified peptide HBVpreS1/2-48myris the mosteffective blocking inhibiting polypeptide. HBVpreS1/2-21myris proved to be the shortestpeptide which can effectively block the infection and the sequence ismyr-GTNLSVPNPLGFFPDHQLDP-NH2, which prompted us HBVpreS1is the specificligand of NTCP and we can design a new type of liver targeting drug delivery systemthrough the specificity binding with receptor and ligand.Method：The overall design of this project is that we take HBVpreS1/2-21myras targetingmedium, long circulating liposomes as drug carrier, Fluorescein Sodium as fluorescencepattern evaluation drug, and product a new drug delivery system HBVpreS1/2-21myr-FS-LCLby the ethanol injection method.First of all, we synthesized peptide HBVpreS1by using microwave peptide synthesizerand characterize the peptide product by HPLC, MS and ITMS. And the peptide wasmodified by acetylation and myristoylation, gaining the productmyr-GTNLSVPNPLGFFPDHQLDP-NH2. Then we marked it with FITC for targetingvalidation and coupled with Mal-PEG2000-DSPE to get HBVpreS1/2-21myr-PEG2000-DSPEas a targeted medium..Then, taken HBVpreS1/2-21myr-PEG2000-DSPE as one of components, the liposomes were made by the ethanol injection method. The liposome encapsulating fluorescein sodium(FS), HBVpreS1/2-21myr-FS-LCL, was produced and then its physical and chemicalcharacteristics were detected using nanoparticle size analyzer and transmission electronmicroscope (TEM). We used the high performance liquid chromatography (HPLC) toanalyze the encapsulation efficiencies, and took further research about stability of liposomeswhich were dealed with High pressure homogenizer and kept in different temperature.Finally, we built cells, HepG2-NTCP, LO2-NTCP, HEK293-NTCP, and gained theprimary tupaia hepatocytes (PTH) or primary rat hepatocytes (PRH) from the animals.These cells were used in analyzing the target ability of the HBVpreS1/2-21myr-FITC andHBVpreS1/2-21myr-FS-LCL by RT-PCR, flow cytometry and confocal laser scanningmicrosope, which evaluated the target ability from the level of protein and mRNA.Result:(1) The product of polypeptide synthesis had a high purity, simple technology, highefficiency, and was easy to operate.The molecular weight and sequence of polypeptidesynthesis was in accord with the expected result.(2) We successfully modified the peptide by FITC and DSPE-PEG2000-Mal.(3) The peptide HBVpreS1/2-21myrcould specifically target the hepatocyte by thereceptor NTCP.(4) We found that the average particle sizes of the liposomes were (94.09±9.2) nm anddispersed evenly in the quasi-circular shape, consistent with what shown under the TEM,and the encapsulation efficiencies of the liposomes were (89.32±1.02)%.(5) The cell models of HepG2-NTCP, LO2-NTCP, HEK293-NTCP, PTH and PRHwere suit to evaluate the targeting ability of the HBVpreS1/2-21myrandHBVpreS1/2-21myr-FS-LCL.(6)HBVpreS/2-21myr-FS-LCLcouldspecificallytargetthehepatocytebythereceptorNTCP.Conclusion:The HBVpreS1/2-21myr-FS-LCL was a nano-liposome with high encapsulationefficiency, and could specifically target the hepatocyte by the receptor NTCP, and it mightbe a novel drug delivery system for the therapy of liver diseases.