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 meth acrylate were synthesised and used for prep aration of polyelectrolyte complexes with calf thymus DNA intended for targ eted delivery of genes in vivo. In this study the effects of the speed of c omponent mixing. total concentration of polymers, ionic strength of solvent s, copolymer structure and content of HPR;IA in the copolymers on parameter s of the polyelectrolyte complexes was investigated. Static and dynamic lig ht scattering methods were used as a main tool for characterising these com plexes. The presence of HPMA units in the polycation had no significant eff ect on its ability to form complexes with DNA, but did affect molecular par ameters and aggregation (precipitation) of the complexes. The size of the c omplexes increases whereas their molecular weight decreases with increasing content of HPMA units. The density of the complexes decreases with increas ing HPMA content independently of the copolymer structure. In order to prep are stable DNA complexes containing single DNA molecule, the following rule s should be observed: (1) copolymers should have a content of HPMA units hi gher than 40%; (2) the DNA concentrations in solutions should be kept below 4.10(-5) g/ml and (3) both components should be mixed together in deionise d water. The stability of the complexes against precipitation in 0.15 M NaC l and the resistance of the complexed DNA to the action of nucleases was al so studied. Whereas DNA complexes of all copolymers showed very good nuclea se stability, the presence of a sufficiently high content of HPMA is necess ary for their good stability in 0.15 M NaCl. The investigation of the stabi lity and the interaction of DNA complexes in aqueous solutions of serum alb umin and dilute human blood serum revealed adsorption of biomacromolecules on DNA complexes accompanied by significant changes in the zeta-potential w hich finally resulted in formation of a "protein layer" and in undesirable precipitation of DNA complexes. In in vitro transfection experiments, the t ransfection efficiency of DNA complexes with copolymers was always higher t han that of the cationic homopolymer slightly increasing with increasing co ntent 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 observe d that DNA complexes produced greater gene expression than a direct microin jection of free DNA. The block copolymer complexes were also found to be mo re efficient than the corresponding simple polycation complexes. In the nuc lear microinjection, precisely the opposite behaviour was observed. (C) 100 0 Elsevier Science B.V. All rights reserved.