Liposomes and immunoliposomes for delivery of macromolecules to human leukaemic cells

Abstract

Liposomes are simple phospholipid membrane vesicles which have been investigated as artificial membrane systems or as vehicles for the delivery of drugs or biologically active agents into cells or tissues.

Preparation of liposomes was by a combined sonication detergent dialysis method, avoiding the use of harsh organic solvents. This method resulted in large. unilamella vesicles with a high internal volume; making them ideal for entrapment of water soluble fluorescent marker dyes or oligonucleotides. These liposomes were relatively non-toxic to leukaemic cells over prolonged incubation periods.

By varying the lipid composition (DOPE or DOPC in combination with cholesterol and oleic acid), two types of liposomes were obtained: pH-sensitive and pH-insensitive. Both types of liposomes were able to fuse with cell membranes and be endocytosed. However, only pH-sensitive liposomes were more efficient in delivering their contents to the cytoplasm of leukaemic cells than pH-insensitive ones. The optimal conditions of incubation were determined for delivery into leukaemic cell lines. The liposomes were able to deliver more encapsulated calcein (a model drug system) to cells than free calcein alone. Of the eight different cell lines tested there was no difference in the percentage of cells which could uptake encapsulated fluorescein; however, the two larger myeloid cell lines appeared to be able to uptake more calcein per cell.

Liposomes have been found to fuse with any cell type. Thus as a drug delivery system they still have the disadvantage of non-specific delivery of entrapped contents. While immunoliposomes have shown some success in studies with various cell types in vitro, there is little information using this approach with human leukaemic cells. To improve the specificity of delivery, immunoliposomes were produced by using streptavidin to link biotinylated antibodies to the surface of liposomes containing biotin-phosphatidylethanolamine. Immunoliposomes could then be used to target against a specific cell Via an antigen-antibody reaction. The immunoliposomes produced in this study were shown to be specific, and combined with the pH-sensitive phospholipid composition proved to be an efficient method of cytoplasmic delivery of the fluorescent dye, calcein.

Exciting new areas of research include gene therapy and antisense therapy. To study the possibility of using liposomes to deliver macromolecules such as DNA to cells, the calcein was replaced with an oligo in liposomes and immunoliposomes. Similar results were obtained; the immunoliposomes could deliver more oligo to specifically targeted cells than ordinary liposomes or free oligo.

To date liposomes or immunoliposomes have been tested predominantly on plant and animal cells in vitro, with the occassional in vivo study, and few in vitro studies on human cells. This study has focused on using liposomes and immunoliposomes to deliver drugs or antisense oligos to specifically targeted human leukaemic cells. The application of this work has significant implications in human leukaemia therapy. By enclosing cytotoxic drugs within liposomes and immunoliposomes toxic side effects to normal cells and tissues can be reduced. The effective dose may also be reduced by delivering drugs via this system.