We propose a mechanism for oligonucleotide (ODN) release culture (1-4). The cationic liposomes form a polyelectrolyte complex with the ODN, protect them from nuclease degradation, enhance their cellular uptake, and improve ODN potency (1-4). Since Bennett's (2) earlier observation that cationic lipids alter intracellular localization of ODN, little has been published on the mechanism. This process is, nevertheless, critical for the delivery of ODN and polynucleotides (DNA) since it seems evident that these macromolecules have to dissociate from complexes to function. Moreover, the understanding of the process of intracellular release should have implications for improvements of ODN and polynucleotide cationic carriers and for in vivo therapeutic trials. In this study, we use fluorescent confocal microscopy to show that fluorescein-labeled ODN (F-ODN) separate from the rhodamine-labeled lipid [1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl); N-Rh-PE] and enters the nucleus leaving the fluorescent lipid in punctate cytoplasmic structures. We then show that anionic lipids cause a rapid release of ODN from the complex. Based on these results we propose a mechanism to account for ODN delivery by cationic lipid complexes.

We have designed a cationic amphipathic peptide, KALA (WEAKLAKALAKALAKHLAKALAKALKACEA), that binds to DNA, destabilizes membranes, and mediates DNA transfection. KALA undergoes a pH-dependent random coil to amphipathic alpha-helical conformational change as the pH is increased from 5.0 to 7.5. One face displays hydrophobic leucine residues, and the opposite face displays hydrophilic lysine residues. KALA-mediated release of entrapped aqueous contents from neutral and negatively charged liposomes increases with increasing helical content. KALA binds to oligonucleotides or plasmid DNA and retards their migration in gel electrophoresis. It displaces 50% of ethidium bromide from DNA at a charge ratio (+/-) of 0.9/1. In cultured cells, KALA assists oligonucleotide nuclear delivery when complexes are prepared at a 10/1 (+/-) charge ratio. KALA/DNA (10/1)(+/-) complexes mediate transfection of a variety of cell lines. The KALA sequence provides a starting point for a family of peptides that incorporate other functions to improve DNA delivery systems.

Nucleic acids carry the building plans of living systems. As such, they can be exploited to make cells produce a desired protein, or to shut down the expression of endogenous genes or even to repair defective genes. Hence, nucleic acids are unique substances for research and therapy. To exploit their potential, they need to be delivered into cells which can be a challenging task in many respects. During the last decade, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as magnetofection. In this review we summarize the progress and achievements in this field of research. We discuss magnetic formulations of vectors for nucleic acid delivery and their characterization, mechanisms of magnetofection, and the application of magnetofection in viral and nonviral nucleic acid delivery in cell culture and in animal models. We summarize results that have been obtained with using magnetofection in basic research and in preclinical animal models. Finally, we describe some of our recent work and end with some conclusions and perspectives.

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There are many very effective methods to introduce transcriptionally active DNA into viable cells but approaches to deliver functional proteins are limited. We have developed a lipid-mediated delivery system that can deliver functional proteins or other bioactive molecules into living cells. This delivery system is composed of a new trifluoroacetylated lipopolyamine (TFA-DODAPL) and dioleoyl phosphatidylethanolamine (DOPE). This cationic formulation successfully delivered antibodies, dextran sulfates, phycobiliproteins, albumin, and enzymes (␤-galactosidase and proteases) into the cytoplasm of numerous adherent and suspension cells. Two systems were used to demonstrate that the proteins were delivered in a functionally active form. First, intracellular ␤-galactosidase activity was clearly demonstrated within X-gal-stained cells after TFA-DODAPL:DOPE-mediated delivery of the enzyme. Second, the delivery system mediated delivery of several caspases (caspase 3, caspase 8, and granzyme B) into cultured cell lines and primary cells triggering apoptosis. Mechanistic studies showed that up to 100% of the protein mixed with the lipid formulation was captured into a lipid-protein complex, and up to 50% of the input protein associated with cells. This lipid-mediated transport system makes protein delivery into cultured cells as convenient, effective, and reliable as DNA transfection.