Delivery of macromolecules by cationic liposomes

Delivery of macromolecules such as DNA and protein to mammalian cells is a critical step for gene expression studies and for the new field of gene therapy. Cationic liposomes are useful and simple vehicles for carrying macromolecules into cells efficiently and reproducibly. New Formulations of cationic liposomes containing cationic cholesterol derivatives and the phospholipid dioleoyl phosphatidylethanolamine(DOPE) have been designed and tested for delivery of DNA to cells inculture in an effort to improve the efficiency and eliminate the toxic effect of cationic liposome formulations. Derivatives with a tertiary amine headgroup had efficient transfection activity, negligible cytotoxic activity, and no protein kinase C inhibition activity. In contrast, derivatives with a quaternary amine head group had no transfection activity and showed higher toxicity in addition to protein kinase C-inhibition activity. The Membrane stabilizing phospholipid, dioleoyl phosphatidylcholine (DOPC),did not replace the efficient role of DOPE (a membrane destabilizer) of transfection. The contribution of DOPE to superior transfection activity was studied in more detail which led to the development of a new DNAdelivery assay that demonstrated the essential and quantitative role ofDOPE in DNA delivery. DOPE was shown to be a major contributor to delivery of DNA into cells. Complete replacement of DOPE with DOPCabolished transfection while partial replacement had partial elimination of transfection activity. The lysosomotropic agent chloroquine also inhibited transfection suggesting that endocytosis is the major route of DNA delivery by cationic liposomes.

Cationic liposomes were also tested in the delivery of a model protein to mammalian cells. The protein delivered was the human immunodeficiency virus-1 (HIV-1) trans-activator protein (tat), a transcription factor that induces the expression of genes under the control of HIV-1 promoter element. To improve the delivery of tat, known to undergo cell surface binding, endocytosis, and inefficient entry into the cytosolic compartment., different cationic liposome formulations were successfully tested allowing up to 150-fold enhancement in tat delivery. Tat delivery was facilitated by the inclusion of DOPE, but not DOPC, in the liposome formulations. Similar to DNA delivery, tat protein delivery was dependent on high DOPE content in the liposome formulation. Co- internalization of tat and cationic liposomes was required for maximal delivery activity implicating a common compartment from which tat was released into the cytosol. This was proposed to be the endosome compartment based on the known membrane disrupting function of DOPE (especially under low, endocytic pH) and the natural endocytic fate of tat. To test the possibility of DNA and protein co-delivery, tat protein was complexed with DNA, coding for a reporter gene under the control of HIV-1 promoter, and cationic liposomes. Transfection of cells with the ternary complex showed detectable reporter gene expression with tat protein concentrations as low as 10 ng/ml. Gene induction was shown to be specific to tat-responsive promoter. The co-delivery system was applicable to different cell types using different cationic liposome formulations. This co-delivery strategy may be useful where transient and maximum gene expression may be necessary in certain applications such as cancer gene therapy.