Study On The Targetable And Intravenous-injectable Vectors For Efficient Gene Delivery

Gene therapy is becoming a promising approach for the treatment of disease with the more understanding of the genetic basis of disease. The success of gene therapy rests on development of a vector that can selectively and efficiently deliver a gene to the target cells with minimal toxicity. The vectors used to date can be classified into two groups: viral and non-viral vectors. Although viral vectors are relatively more efficient in gene transfection than non-viral methods, their toxicity and immunogenicity are the major concerns. The limitations of viral vectors make synthetic vectors an attractive alternative. Advantages of nonviral vectors include their nonimmunogenicity, low acute toxicity, simplicity, and feasibility to be produced on a large scale. Up to now, complexes formed between cationic liposomes and plasmid DNA (pDNA) have been the predominant non-viral vectors utilized in research. However, their utility in gene therapy is hampered by their poor colloidal stability, low tissue specificity and incompatibility with the abundance of negatively charged macromolccules present in the physiological environment.In an effort to seek an iv-injectable, targetable vector for gene transfer, we developed a novel non-viral gene delivery system, LPD (liposome-po'ycation-DNA complexes). Structurally, this formulation was virus-like panicles. The highly condensed core composed of protamine and DNA was surrounded by a lipid coat.Plasmid DNA was isolated and purified from DH5-αE. coli using theQiagen Giga Endo-free piasmid purification kit. DNA concentration and purity were quantified by UV absorbance at 260 nm and 280 nm on a GBCUV cintra lOe Spectrophotometer. The structural integrity and topology of purified DNA was analyzed by agarose gel electrophoresis.A simple and sensitive assay method for quantitative determination of DNA in LPD was developed by Fluorospectrophotometer with Hoechst 33/:58 as the fluorochrome. LPD were prepared by first mixing the plasmid DNA and protamine together. The resulted polyplexes were incubated for 10 min at room temperature, following the addition of preformed cationic or anionic liposomes. Univariate approach and central composite design (CCD) were employed to optimize the cationic and the anionic LPD formulations, respectively.LPD were prepared into lyophilized injection to improve their physical and biological stability. The lyophilization of LPD did not result in obvious change in morphology, particle size, zeta potential, pH and transfection efficiency in vitro. The average entrapping efficiency of lyophilized LPD was 93.61% with good redispersibility.The plasmid pORF-lacZ was employed as the reporter gene in the assay of transfection efficiency. The in vitro transfection efficiency of LPD in human lung cancer cells A431 was 36.25 ± 5.02 mU/mg protein, which was much higher than that of commercially available cationic liposomes Lipofectamine. The in vitro transfection efficiencies of LPD in Hepatoma ceils HepC.1 and SMMC-7721 were 18.71 + 1.36 and 28.84 + 3.62 mU/mg protein, respectively. The in vivo transfection experiments in mice demonstrated that LPD could be expressed in lung, spleen, kidney and liver after intravenous administration. Among the organs, lung had relatively hiuher expression, especially in the bronchial epithelium.The antitumor activity of lyophilized LPD (pORF-mlL 12) was investigated using C57 mice bearing lewis lung carcinoma. It was found that LPD (pORF-mIL12) could significantly inhibit tumor growth, compared with LPD (pORF-mcs) and the negative control. The survival of tumor-bearing mice could be further prolonged when LPD (pORF-mIL12)and cisplatin were delivered together.To develop a hepatoeyte-specific gene carrier, we synthesized a series of neutral cholesterylated thiogalactosi les with different spacer length la~d as the targetable ligand of liposomal carriers for liver parenchymal cells. The sugar moieties and cholesterol anchors were coupled by ether-linker containing ethylene glycol units. It was found that the length of the spacer between the anchor and galactose residues was important for the recognition of asialoglycoprotein receptor. LPDlc, which have the best targeting capability, are potentially useful gene carriers to liver parenchymal cells.Our research demonstrated that LPD was a stable, /v-injectable and easy to be prepared vector for gene delivery. This system may be a promi ing candidate to be further investigated in vivo for systemic gene delivery.