Panning Peptides Targeting To Hepatocellular Carcinoma And The Targeting Study Of X1/MePEG-PLA-CS Nanoparticles

Hepatocellular carcinoma(HCC) is one of the most common and malignant tumors, and it has the second mortality rate among malignant tumors in China. Thus, and treatment of HCC is extremely critical. The treatment of HCC include liver resection, orthotopic liver transplantation (OLT), transcatheter arterial chemoembolization (TACE), percutaneous ethanol injection (PEI), radiofrequency ablation (RFA), microwave ablation (MCT), frozen surgery, radiotherapy, anti-hormone therapy, biological therapy and traditional Chinese medicine treatment etc. At present, surgical resection of primary liver cancer is still the preferred method. However, after radical resection, high recurrence rate after 5 years and high metastasis ratio are still problems in clinical therapy for HCC.In recent years, gene therapy has become popular in biological science and clinical research as the gene transfer techniques matures day by day. Nanoparticle is developing as a new non-viral gene delivery vector. Gene therapeutical molecules molecules, such as DNA, RNA, are are encapsulated into or loaded on the surface of nanoparticles. At the same time, specific ligands and monoclonal antibodies are linked to the surface of nanoparticles. The nanopartieles/DNA complexes can be taken into target cells by receptor-mediated endocytosis, so that the effective target gene therapy is achieved.Therefore, we screened the peptides that bind specifically to the hepatoma cells using phage display peptides library, and identified the specificity of the phages to hepatoma cells. We also constructed a gene delivery system with high efficiency of gene transfer, targeting ability, efficiency of protection to DNA from DNase degradation and good biocompatibility, especially high stability and the ability to prolong gene transfer. Our research established good scientific foundation for the application of the nanoparticle-mediated gene delivery and gene therapy.Chapter oneScreening for hepatocellular carcinoma cell specific binding peptidesObjective:To screen the peptides that bind specifically to the hepatoma cells using in vitro and in vivo phage display technology.Methods:(1) Three rounds of panning were conducted in vitro, which were targeted at HepG2 cell lines. Experimental Animal Models of hepatoma were established on nude mouse, and one round of panning was conducted in vivo.(2) After 4 rounds of panning,30 phage clones picked randomly were sequenced to identify the consensus sequence.Results:(1) After 4 rounds of panning in vitro, phages that bind to the HepG2 cells were enriched from 7.2×103pfu at the first round to 2.5×106pfu at the third round of panning (with an increase of more than 300-fold); the output/input ratio was also increased from 4.8×10-6 at the first round to 1.67x 10"3 at the third round of panning.(2) After panning from hepatoma cells in vitro and in vivo,30 phage clones were randomly picked and sequenced.4 sequences were obtained from these 30 clones. X1 was repeated 19 times, X2 was repeated 3 times, X3 was repeated 4 times, and X4 was repeated 1 time. Among them, X1 (QSFASLTDPRVL) was the most repeats. Low homology between the peptide sequences was displayed by the phages and no known proteins was identified by BLAST analysis.Conclusions:Peptides that can specifically bind to hepato-carcinoma cells both in vitro and in vivo were selected from 12-phage display library by in vitro and in vivo phage display technology. Chapter TwoIdentification of binding specificity of X1 phage and peptide SA1 to Hepatoma cellsObjective:To identify the affinity of X1 phage and peptide SA1 to hepatoma carcinoma cells in order to make nano-drug carrier with high target to hepatoma in the future.Methods:(1) The affinity of phages with hepatoma cells was examined by enzyme-linked immunosorbent assay (ELISA).(2) Immunocytochemical staining were performed to determine the specificity of the phages to hepatoma cells, and immunohistochemical staining was used to examine the binding specificity of the phages to hepatoma tissues.(3) Immunoflurescence microscopy was used to study the binding of synthesized peptides to hepatoma cells.(4) Identifying the targeting effectiveness of phages by injection in vivo and immunohistochemical staining.Results:(1) The phages selected by screening all have specific binding affinity to hepatoma cells, and the highest positive is the phage X1 (QSFASLTDPRVL) the most repeats. (2) Immunocytochemical staining and immunohistochemical staining suggested that X1 phage preferably binds to hepatoma cells rather than controls, and phage was also found to be able to bind to hepatoma tissue sections.(3) Using immunofluorescence microscopy, fluorescence labeled FITC-SA1 peptide was observed on the membrane and in the perinuclear cytoplasm of hepatoma cells.(4) The X1 phage shows higher enrichment in tissue of tumor than normal tissue, it could specifically bind to hepatoma tissues after blood circulation.Conclusions:The peptide X1 (QSFASLTDPRVL) has the best targeting ability among all candidates. It can bind to hepatocarcinoma cells and hepatocarcinoma tissues, but not delete normal liver cells. And this peptide also has the specific binding ability in vivo; it can accumulate to the tumor tissues rapidly through the blood vessel system.Chapter ThreeThe preparation and targeting study of X1/MePEG-PLA-CS nanoparticleObjective:To construct an idea gene delivery system with high efficacy of gene transfer, targeting ability, effect of protection to DNA from DNase degradation and good biocompatibility, especially high stability and the ability to prolong gene transfer.Methods:(1) Using copolymer methoxypolyethyleneglycol-PLA (MePEG-PLA), and chitosan (CS) to prepare MePEG-PLA-CS nanoparticle. Detect the particle diameter and efficiency of DNA and cytotoxicity.(2) Coupling the peptide X1 and MePEG-PLA, observing the effect of transfection efficiency with X1/MePEG-PLA-CS nanoparticle.Results:(1) The particle diameter of MePEG-PLA-CS is 103.2±9.6 nm, the surface bands positive charge, efficiency of DNA is 93.2±2.9%, MePEG-PLA-CS/DNA complexes could protect the DNA from degradation of DNaseⅠ.In our research,we found that MePEG-PLA-CS nanoparticles had no obvious cell toxicity to L-02 cells at proper concentration,but cell toxicity could be showed at high concentration.(2) The MePEG-PLA-CS nanoparticle that binds phage X1 has higher transfection efficiency compared with MePEG-PLA-CS nanoparticle.Conclusions:The MePEG-PLA-CS is a stable nanoparticle and had higher transfection efficiency while with much lowert oxicity in vivo. The MePEG-PLA-CS nanoparticle binding with phage X1 has higher transfection efficiency compared with MePEG-PLA-CS nanoparticle. Furtherly targeting to hepatoma carcinoma cell, established experimental fundament for Diagnosis and treatment of HCC.