The Pharmaceutical Research On Antibody-conjugated Long-circulating Cationic Liposomes Mediating DNA And SiRNA Transfection

The women health in our country is threatened severely by the increasing incidence of breast cancer in recent years. Breast cancer has been one of the primary malignant tumor against women's health in our country.At present, surgical treatment,radiation therapy and chemotherapy are commonly the traditional treatment.Although these methods will enable patients to receive higher opportunity of survival or even cure, The questions of recurrence and distant metastasis to the scholars continue a major problem. With the rapid development of molecular biology and immunology techniques and the deeping knew to pathogenesis of breast cancer.Gene therapy treatment of tumor biology has gradually become an important part in the treatment of breast cancer,what is more,it showed a good value in application, and achieved certain results, so, gene therapy treatment is becoming a promising treatment option.Recently,the methods of gene delivery are gradually developing and have made great progresses.So the gene therapy of cancer is a hot spot of the biology and clinic medical currently.The gene delivery vectors and vectors associated immune response,cell toxicity and safety are the bottleneck of research and clinic application of gene therapy.The two main systems for DNA or siRNA delivery are viral and non-viral vectors is less than that by the viral vectors,non-viral systems are preferred in terms of safety,stability,the relative ease of large-scale production and characterization,and the lack of immunogenicity.The cationic liposomes (CL) is one of the most extensively researched non-virus vectors at present. Cationic liposomes have many advantages such as low toxicity/immunogenicity and potential for oncogenicity, size independent delivery of nucleic acids (from oligonucleotides to artificial chromosomes), ease of preparation, and quality control. But the primary disadvantages are the poor efficiency of transfection and failure of delivering therapeutic nucleic acids to their desired site of action in cells. Moreover, slow vector accumulation and consequently low vector concentration at target tissues have been identified as simple but strong barriers to effective gene delivery. So it is important to improve the target delivering of CL.It has been demonstrated that HER2 receptor was overexpressed in 25% to 30% patients with breast cancer.Amplification of HER2/neu gene has been shown to correlate with poor clinical prognosis for certain cancer patients.Overexpression of HER2 in breast cancer may imply that it resists to some conventional therapies such as chemotherapy or endocrinetherapy.The HER2/neu proto-oncogene encodes a 185 kDa protein HER2(p185),which belongs to epidermal growth factor receptor family.At present the main anti-HER2 therapy methods include:①Anti-HER2 monoclonal antibody,such as Tuastuzumab(Herceptin), which can inhibit HER2 receptor activation through competitive binding the membraneous HER2 receptors;②protein tyrosine kinase(PTK) inhibitor,for example,Gifitinib(Iressa) inhibite epidermal growth factor receptor(EGFR) selectively;③Antisense gene techniques,such as antisense oligonucleotides and ribozyme e.g.Nevertheless,on account of the severe side effects of anti-HER2 monoclonal antibody and PTK inhibitor and the week genetic expression suppression efficiency of antisense gene techniques,they could not achieve satisfactory clinical result.Clearly,the development of more effective treatment is needed to improve the outcome for this devastating cancer.In the present formulation, the rhuMAbHER2 Fab' to the HER2 antigen on breast cancer cells is used to target the pegylated immunoliposome carrying the plasmid DNA or siRNA to tissues in vivo that high express HER2.We have completed the pharmaceutical research on antibody-conjugated long-circulating cationic liposomes mediating DNA and siRNA transfection.Our study aims to examine the effect of the molar ratio of DC-Chol and DOPE, PEGylation and serum on the plasmid DNA (pDNA) and siRNA (small interfering RNA) transfection of DC-Chol/DOPE liposomes prepared by the dry-film method. The results showed that the most efficient DC-Chol/DOPE liposomes for pDNA or siRNA delivery were at a 1:2 or 1:1 molar ratio of DC-Chol to DOPE respectively, and increasing the weight ratio of DC-Chol to siRNA resulted in increased siRNA transfection efficiency. However, the pDNA transfection efficiency decreased accompanied with the increased weight ratio of DC-chol to pDNA. The effect of PEGylation on transfection efficiency of DC-Chol/DOPE liposomes was also investigated. Consistent with previous results, PEGylation decreased siRNA and pDNA transfection efficiency. In PEGylated DC-Chol/DOPE liposomes, increased weight ratio of DC-chol to pDNA did not lead to higher pDNA transfection efficiency, whereas increased weight ratio resulted in increased siRNA transfection efficiency. Furthermore, the serum did not inhibit the pDNA and siRNA transfection efficiency of DC-chol/DOPE liposomes, which did not induce remarkable cytotoxicity against SK-BR3 cells. In conclusion, our results elucidated the influence factors of DC-chol/DOPE liposomes transfection efficiency and would reveal that in DC-chol/DOPE liposomes, siRNA and DNA transfection mechanisms were quietly different.At last we successfully prepared the HER2-targeted cationic liposome(Liposomes conjugated with Anti-HER2 Fab',HER2-Flip). This study will use the rhuMAbHER2 Fab' to the HER2 antigen as a targeting ligand to conjugated cationic liposome surface which has 4% PEG——the manner of Antibody targeting.It was confirmed that the HER2-Flip have a high specific expression in SK-BR3 of breast cancer cell and endocytosis, Real-time PCR experiments proved that it can silence the expression of specific gene VEGF specific gene.