CELLULAR UPTAKE AND LOCALIZATION OF LIPOSOMAL-METHYLPHOSPHONATE OLIGODEOXYNUCLEOTIDES

Nuclease digestion and intracellular delivery are major factors limiti ng the potential use of oligodeoxynucleotides as antisense molecules. Structural analogues of phosphodiester oligodeoxynucleotides, such as phosphorothioates and methylphosphonates, are resistant to nuclease de gradation and can still bind to their mRNA targets. However, their lim ited ability to escape from the endosomal/lysosomal compartments and r each the intracellular sites of action have dampened their potential c linical application. To circumvent this problem we have incorporated m ethylphosphonate oligodeoxynucleotides into liposomes. We found that t he level of uptake of liposome-incorporated methylphosphonate oligodeo xynucleotides is time and concentration dependent. Maximal uptake occu rred at 8 h when 4-8 mu M liposome-incorporated methylphosphonate olig odeoxynucleotides was added. Approximately 50% of liposome-incorporate d methylphosphonate oligodeoxynucleotides were retained in cells after 24 h of incubation. Using fluorescent microscopy, intracellular fluor escence could be seen within 2.5 h of incubation. Diffused fluorescenc e was found throughout the cytoplasm, suggesting that the liposome-inc orporated methylphosphonate oligodeoxynucleotides were not confined wi thin the endosomal/lysosomal structures. We conclude that liposomes ca n effectively deliver methylphosphonate oligodeoxynucleotides to the c ytoplasm, which is the major intracellular site of action for translat ional arrest.