Mechanism of destabilization of pH-sensitive liposomes and implications for cellular delivery

Author

David Collins

Date of Award

5-1989

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major Professor

Leaf Huang

Committee Members

John W. Koontz, W. D. Wicks, Solon Georghiou

Abstract

A model system for examining the pH-sensitive immunoliposome-cell interaction was developed. Diphtheria toxin fragment A (OTA) was encapsulated in liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and oleic acid (OA) and targeted to L929 cells. DTA, which cannot normally cross lipid membranes, was efficiently delivered by DOPE:OA immunoliposomes. The delivery process involved specific cell-surface binding and endocytosis of the immunoliposomes. Subsequent to endocytosis, acid-induced fusion between the immunoliposomes and the cellular endosomes occurred. The mechanism of fusion and destabilization was studied in detail for DOPE:OA liposomes and DOPE:palmitoyIhomocysteine (PHC) liposomes. Both systems could be destabilized by acidic pH and/or high temperature. For both systems, destabilization required close apposition of the liposome membranes, although leakage from non-aggregated DOPE:OA liposomes was detected. Following bilayer apposition, fusion occurred, leading to the formation of larger lamellar structures. Subsequent to fusion, the DOPE:OA system was shown to collapse into hexagonal phase, while DOPE:PHC did not form hexagonal phase under similar conditions. The intermediates in the bilayer-hexagonal phase transition in DOPE:OA could be trapped and visualized by addition of calcium to the system. The double-chain amphiphiles, dipalmitoylsuccinylglycerol (DPSG) and dioleoylsuccinylglycerol (DOSG) were prepared and combined with DOPE to construct pHsensitive liposomes. DOPE:DPSG and DOPE:DOSG were less acid-sensitive and more plasma stable than either DOPE:OA or DOPE:PHC liposomes. Both DOPE:DPSG and DOPE:DOSG immunoliposomes were able to fuse with the endosomes of L929 cells and efficiently deliver DTA. The possible biological implications of the differing acid-sensitivities and stabilities of the three classes of immunoliposomes was examined by monitoring the delivery kinetics of the immunoliposomes. DOPE:PHC immunoliposomes delivered early in the endocytic pathway, while DOPE:DPSG immunoliposomes delivered later in the pathway. The delivery kinetics of DOPE:OA liposomes were intermediate between those of the other two compositions and were complicated by cell-induced alterations of the immunoliposome composition and acid-sensitivity. These results suggest that pH-sensitive liposomes may be useful tools for studying the endocytic pathway.

Recommended Citation

Collins, David, "Mechanism of destabilization of pH-sensitive liposomes and implications for cellular delivery. " PhD diss., University of Tennessee, 1989.
https://trace.tennessee.edu/utk_graddiss/11633