ENTRAPMENT OF NUCLEIC-ACIDS IN LIPOSOMES

The entrapment efficiency of three main methods used in the literature for the encapsulation of nucleic acids in liposomes were studied usin g 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. I n particular the reverse phase method, the dehydration/rehydration met hod, and the freeze/thawing method were compared to each other under s tandardised conditions, i.e. using in every case the same concentratio n of guest molecules (DNA, tRNA and ATP as low molecular weight analog ue) and equally extruded liposomes. The percentage of entrapment stric tly referred to the material localized inside the liposomes, i.e. part icular care was devoted to ruling out the contribution of the nucleic acid material bound to the outer surface of the liposomes: this was el iminated by extensive enzymatic digestion prior to column chromatograp hy. Depending on the conditions used, the percentage of the entrapped material varied between 10 and 54% of the initial amount. Further, the encapsulation efficiency was markedly affected by the salt concentrat ion, by the size of liposomes, but to a lower degree by the molecular weight of the guest molecules. In general, we observed that the freeze /thawing encapsulation procedure was the most efficient one. In a seco nd part of the work the freeze/thawing method was applied to encapsula te DNA (369 bp and 3368 bp, respectively) using liposomes obtained fro m POPC mixed with 1-10% charged cosurfactant, i.e. phosphatidylserine (PS) or didodecyldimethylammonium bromide (DDAB), respectively. Wherea s PS had no significant effect, the entrapment efficiency went lip to 60% in POPC/DDAB (97.5:2.5) liposomes. The large entrapment efficiency of DNA permits spectroscopic investigations of the DNA encapsulated i n the water pool of the liposomes. UV absorption and circular dichrois m spectra were practically the same as in water, indicating no appreci able perturbation of the electronic transitions or of the conformation of the entrapped biopolymer. This was in contrast to the DNA bound ex ternally to the POPC/DDAB liposomes which showed significant spectral changes with respect to DNA dissolved in water. (C) 1997 Elsevier Scie nce B.V.