Doctoral Dissertations

Author

Xiaohuai Zhou

Date of Award

5-1992

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Leaf Huang

Committee Members

David Brian, John Koontz, Daniel Roberts

Abstract

Studies described in this dissertation include the following four parts: 1) the development of a freeze-thaw method for an improved DNA encapsulation in pH-sensitive liposomes, 2) the development of lipopoly-L-lysine (LPLL) for an efficient DNA transfection, 3) the development of LPLL/dioleoylphosphatidylethanolamine (DOPE) liposomes for an efficient DNA transfection, and 4) an investigation of the mechanism by which LPLL/DOPE liposomes mediate DNA delivery. To overcome the problem of inefficient DNA encapsulation in anionic pH-sensitive liposomes, a freeze-thaw method for DNA entrapment was developed. Three cycles of freeze-thawing a mixture of DNA and empty sonicated liposomes resulted in an efficient DNA entrapment. Smaller plasmids were entrapped more efficiently than the larger ones. Unlike the reverse-phase evaporation method, this protocol did not cause significant damage to the plasmids. DNA-containing liposomes prepared by this method mediated transfection in cultured L929 cells. The transfection activity depended on the pH-sensitivity of liposomes and could be modulated by protein kinase C activators incorporated in the liposome membrane. These findings may help to improve our liposome system for further studies on gene therapy. In an effort to construct a novel targetable and efficient DNA carrier other than the anionic pH-sensitive immunoliposomes, a cationic lipid was synthesized by conjugating poly-L-lysine with a lipid. The resulting conjugate was termed LPLL. The synthesis of LPLL represents the first step in the construction of such a DNA carrier. LPLL if tested effective for DNA delivery would be incorporated with a targeting ligand to promote target-specific delivery. LPLL contained an average of two lipids per poly-L-lysine molecule (MW -3,000). It indeed mediated efficient DNA transfection. Under the optimized conditions, LPLL had a 3-fold higher activity than Lipofectin which is a widely used commercial preparation of cationic liposomes. Moreover, compared to Lipofectin the action of LPLL was more resistant to the inhibitory effect of serum. However, scraping of the treated cells was necessary for the transfection activity of LPLL. The mechanical treatment required for the transfection activity of LPLL could be avoided by mixing LPLL with a helper lipid, DOPE. The activity of LPLL/DOPE liposomes was systematically optimized with respect to the lipophilicity of LPLL, LPLL/DOPE ratio, DNA/liposome ratio, and the concentration of DNA/liposome complexes. Under the same conditions, liposomes containing lipopoly-D-lysine had a similar activity to those containing LPLL, indicating that the degradation of polylysine chain which should take place in lysosomes is not required for the release of DNA into the cytoplasm. The mechanism by which LPLL/DOPE liposomes mediated transfection was studied in more detail. It was found that cellular factors were involved in the formation of DNA/liposome complexes. The complexes entered the cytoplasm by either penetrating through the plasma membrane or destabilizing endosomes after being endocytosed. Our results posed questions on a previous hypothesis which suggested that cationic liposomes mediated DNA translocation via a fusion mechanism. These studies shall pave the way to the ultimate goal of targeted delivery of DNA for gene therapy.

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