Design and characterization of novel anionic lipoplexes for the delivery of DNA-based therapeutics
Date of Completion
Health Sciences, Pharmacology|Health Sciences, Pharmacy
A novel anionic liposomal delivery system was designed based on DNA conformational changes observed upon divalent cation-mediated interactions of oligonucleotides with anionic liposomes using circular dichroism (CD). Sequence-specific solution conformations of oligonucleotides were identified by their characteristic CD spectral signatures. Oligonucleotide conformation underwent characteristic changes upon interaction with divalent cations that could be identified by characteristic changes in their CD spectrum. Conformational changes were specific to the oligonucleotide sequence and divalent cation, and resulted due to the stabilizing or destabilizing effects of divalent cations on oligonucleotide structure in solution. For example, divalent cations such as Ca2+ and Ba2+ stimulated the formation of G-DNA hyperstructures in oligonucleotides containing sequential guanines, whereas others such as Mn2+ led to complete obliteration of the CD signal. ^ CD also revealed that oligonucleotides underwent sequence and divalent cation specific changes upon divalent cation-mediated complexation with anionic liposomes. These changes were dissimilar to those induced by divalent cations alone. For example, calcium stimulated G-DNA formation in an oligonucleotide with sequential guanines, however in the presence of anionic liposomes, calcium interactions with this oligonucleotide led to the formation of a novel conformation. The liposome-complexed conformation of this oligonucleotide had some spectral characteristics of previously reported condensed C-DNA polymorphs, however other bands were also identified, thereby indicating that the overall conformation was unique. ^ Divalent cation-mediated complexation and condensation of oligonucleotides to anionic liposomes constituted the basis of the design of the liposomal DNA delivery vector. The delivery system consisted of transfection competent complexes (also known as lipoplexes) composed of DNA molecules, calcium, and mixtures of anionic and zwitterionic lipids. Transfection efficiency of the anionic lipoplex was evaluated using plasmid containing a transgene for the green fluorescent protein in a Chinese hamster ovary cell line. The anionic lipoplexes exhibited transfection efficiencies of ∼50% and were relatively nontoxic compared to the commercially available cationic liposomal formulation, Lipofectamine. Biophysical characterization of the lipoplexes demonstrated that formation of the lipoplex led to the conversion of the native B-DNA conformation of the plasmid DNA into the highly condensed Z-DNA form. Formation of the condensed DNA polymorph may explain the transfection capability of the system. ^
Patil, Siddhesh Dinanath, "Design and characterization of novel anionic lipoplexes for the delivery of DNA-based therapeutics" (2002). Doctoral Dissertations. AAI3076717.