DNA delivery systems based on complexes of DNA with synthetic polycations and their copolymers.
Block and graft copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-(trimethylammonio)ethyl methacrylate were synthesised and used for preparation of polyelectrolyte complexes with calf thymus DNA intended for targeted delivery of genes in vivo. In this study the effects of the speed of component mixing, total concentration of polymers, ionic strength of solvents, copolymer structure and content of HPMA in the copolymers on parameters of the polyelectrolyte complexes was investigated. Static and dynamic light scattering methods were used as a main tool for characterising these complexes. The presence of HPMA units in the polycation had no significant effect on its ability to form complexes with DNA, but did affect molecular parameters and aggregation (precipitation) of the complexes. The size of the complexes increases whereas their molecular weight decreases with increasing content of HPMA units. The density of the complexes decreases with increasing HPMA content independently of the copolymer structure. In order to prepare stable DNA complexes containing single DNA molecule, the following rules should be observed: (1) copolymers should have a content of HPMA units higher than 40%; (2) the DNA concentrations in solutions should be kept below 4 x 10(-5) g/ml and (3) both components should be mixed together in deionized water. The stability of the complexes against precipitation in 0.15 M NaCl and the resistance of the complexed DNA to the action of nucleases was also studied. Whereas DNA complexes of all copolymers showed very good nuclease stability, the presence of a sufficiently high content of HPMA is necessary for their good stability in 0.15 M NaCl. The investigation of the stability and the interaction of DNA complexes in aqueous solutions of serum albumin and dilute human blood serum revealed adsorption of biomacromolecules on DNA complexes accompanied by significant changes in the zeta-potential which finally resulted in formation of a "protein layer" and in undesirable precipitation of DNA complexes. In in vitro transfection experiments, the transfection efficiency of DNA complexes with copolymers was always higher than that of the cationic homopolymer slightly increasing with increasing content of HPMA in the copolymers but being about 10-100-times lower than the complexes DNA-poly(L-lysine. In the cytoplasmic injections, it was observed that DNA complexes produced greater gene expression than a direct microinjection of free DNA. The block copolymer complexes were also found to be more efficient than the corresponding simple polycation complexes. In the nuclear microinjection, precisely the opposite behaviour was observed.
Oupický
D
D
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Square 2, 162 06, Prague, Czech Republic.
Konák
C
C
Ulbrich
K
K
Wolfert
M A
MA
Seymour
L W
LW
D000178:Acrylamides / Q000138:chemical synthesis / Q000737:chemistry; D000328:Adult; D000818:Animals; D002264:Carboxylic Acids / Q000737:chemistry; D002412:Cations; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry; D003851:Deoxyribonucleases / Q000378:metabolism; D018014:Gene Transfer Techniques / Q000295:instrumentation*; D006801:Humans; D008027:Light; D008297:Male; D008689:Methacrylates / Q000138:chemical synthesis / Q000737:chemistry; D009865:Oocytes / Q000378:metabolism; D011092:Polyethylene Glycols; D011108:Polymers / Q000138:chemical synthesis* / Q000737:chemistry; D011145:Polyvinyls / Q000138:chemical synthesis / Q000737:chemistry; D012542:Scattering, Radiation; D012995:Solubility; D014162:Transfection; D014981:Xenopus
C120889:2-(trimethylammonio)ethyl methacrylate; D000178:Acrylamides; D002264:Carboxylic Acids; D002412:Cations; D008689:Methacrylates; D011108:Polymers; D011145:Polyvinyls; C008996:poly(vinylamine); D011092:Polyethylene Glycols; D004247:DNA; D003851:Deoxyribonucleases; C032976:N-(2-hydroxypropyl)methacrylamide