Pharmaceutical and biological properties of poly(amino acid)/DNA polyplexes.
Physicochemical properties of polyplexes formed between pRSVlacZ and poly(amino acid)s were investigated as a paradigm of more complex, synthetic virus-like, DNA delivery systems, that are of interest to many gene delivery laboratories. We observed the interaction between polymer and DNA using ethidium exclusion, and determined the size distributions and the zeta potentials of polyplexes. We correlated these properties with their fundamental interactions with cultured B16 murine melanoma cells, and the resulting efficiency of transfection. A variety of poly(amino acid)s each condensed DNA to produce particles with mean hydrodynamic diameters of approximately 100 nm (a typical span of a population was 80-120nm). Poly(amino acid) polyplexes were unstable in electrolyte solutions such as cell culture media. The apparent particle size increased in electrolyte, depending on the charge ratio, to diameters up to 700 nm. This was thought to be due to aggregation, since neutral particles were most sensitive. When the charge ratio (+/-) exceeded unity polyplexes had positive zeta potentials (which peaked at approximately +30 mV), bound non-specifically to cells, were internalised and in the presence of an endosomolytic agent were able to transfect cells. Though all cationic poly(amino acid)s investigated formed polyplexes with similar physical properties, their biological properties were significantly different. Polyplexes prepared with poly-L-ornithine were the most effective transfection agents, but poly(lys-co-ala, 1: 1) systems appeared to be inactive. This may reflect the differences in uncoupling of DNA and polymer, which is expected to be necessary for passage through the nuclear pore. Uncoupling of polycation and DNA was investigated by exposing the complexes to dextran sulphate. Release of DNA was detected by increased fluorescence at 600 nm in the presence of ethidium. Release of DNA was incomplete from polyplexes formed with high molecular weight polylysine. This may explain the lower levels of transfection observed with high molecular weight polylysine. The significance of these observations for design of advanced non-viral gene delivery systems is discussed.
Lucas
P
P
Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, UK.
Milroy
D A
DA
Thomas
B J
BJ
Moss
S H
SH
Pouton
C W
CW
D000596:Amino Acids; D000818:Animals; D002412:Cations; D004247:DNA / Q000008:administration & dosage*; D004996:Ethidium; D018014:Gene Transfer Techniques; D008545:Melanoma / Q000235:genetics; D051379:Mice; D011108:Polymers; D014162:Transfection; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D002412:Cations; D011108:Polymers; D004247:DNA; D004996:Ethidium