Cytoplasmic delivery of macromolecules VIA pH-sensitive liposomes

Author

Chen-Yen Wang

Date of Award

3-1987

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major Professor

Leaf Huang

Abstract

In this dissertation, it has been demonstrated that liposomes composed of dioleoylphosphatidylethanolamine, cholesterol and oleic acid (4:4:2) can effectively deliver the entrapped calcein to the cytoplasm of carrot protoplasts. These experiments were performed by using a fluorescent dye, calcein, as a marker of cytoplasmic delivery. The optimal ratio for uptake of pH-sensitive liposomes by protoplasts was 0.45μmole phospholipid per 6 X 10⁵ protoplasts. Addition of 11% PEG 6000 was essential for the optimal uptake of liposomes by plant protoplasts. The superiority of the new liposome composition over the conventional compositions was correlated with its high sensitivity to acidic environment.

These pH-sensitive liposomes were also used to deliver plasmid DNA to target mammalian cells in tissue culture or in a mouse model. In these studies, a cAMP regulatory sequence on the upstream of the rat PEPCK gene was used to control the expression of an exogenous gene in the plasmid. For tissue culture cells, HSV- thymidine kinase gene was delivered to mouse Ltk^- cells so that growth of the cells in the HAT medium was completely cAMP dependent. Maximal short-term expression of the viral thymidine kinase gene was obtained from the liposomes prepared with a pH-sensitive lipid composition, i.e. DOPE:chol:OA (4:4:2), and coated with monoclonal antibody specific to the target cells. The transformation efficiency mediated by the pH-sensitive immunoliposomes was significantly higher than that of the conventional calcium phosphate precipitation method. The long-term transformation efficiency via these liposomes was about 9%.

RDM-4 lymphoma cells grown in the peritoneal cavity of the nude mouse were used as a target for the immunoliposomes to deliver the bacterial chloramphenicol acetyltransferase (CAT) gene. Intraperitoneally injected liposomes, although not exclusively accumulated in the target cells, mediated a significant expression of the CAT activity in the target cells. Organs enriched with reticuloendothelial cells such as spleen did not show gene expression, despite significant uptake of the liposomes. Again, the pH-sensitive immunoliposomes mediated transformation gave higher CAT activity than the pH-insensitive ones did. The presence of the targeting antibody was also necessary for maximal gene delivery. The implications of this result in DNA mediated cancer chemotherapy and genetic therapy are discussed.

Recommended Citation

Wang, Chen-Yen, "Cytoplasmic delivery of macromolecules VIA pH-sensitive liposomes. " PhD diss., University of Tennessee, 1987.
https://trace.tennessee.edu/utk_graddiss/12181