Block and graft copolymers and Nanogel (TM) copolymer networks for DNA delivery into cell

Self-assembling complexes from nucleic acids and synthetic polymers are eva luated for plasmid and oligonucleotide (oligo) delivery. Polycations having linear, branched, dendritic, block- or graft copolymer architectures are u sed in these studies. All these molecules bind to nucleic acids due to form ation of cooperative systems of salt bonds between the cationic groups of t he polycation and phosphate groups of the DNA. To improve solubility of the DNA/polycation complexes, cationic block and graft copolymers containing s egments from polycations and non-ionic soluble polymers, for example, poly( ethylene oxide) (PEO) were developed. Binding of these copolymers with shor t DNA chains, such as oligos. results in formation of species containing hy drophobic sites from neutralized DNA-polycation complex and hydrophilic sir es from PEG. These species spontaneously associate into polyion complex mic elles with a hydrophobic core from neutralized polyions and a hydrophilic s hell from PEG. Such complexes are very small (10-40 nm) and stable in solut ion despite complete neutralization of charge. They reveal significant acti vity with oligos in vitro and in vivo. Binding of cationic copolymers to pl asmid DNA forms larger (70-200 nm) complexes, which are practically inactiv e in cell transfection studies. It is likely that PEO prevents binding of t hese complexes with the cell membranes ("stealth effect"). However attachin g specific ligands to the PEO-corona can produce complexes, which are both stable in solution and bind to target cells. The most efficient complexes w ere obtained when PEO in the cationic copolymer was replaced with membrane- active PEO-b-poly( propylene oxide)-b-PEO molecules (Pluronic 123), Such co mplexes exhibited elevated levels of transgene expression in liver followin g systemic administration in mice, To increase stability of the complexes, NanoGel(TM) carriers were developed that represent small hydrogel particles synthesized by cross-linking of PEI with double end activated PEO using an emulsification/solvent evaporation technique. Oligos are immobilized by mi xing with NanoGel(TM) suspension. which results in the formation of small p articles (80 nm). Oligos incorporated in NanoGel are able to reach targets within the cell and suppress gene expression in a sequence-specific fashion . Further, loaded NanoGel particles cross-polarized monolayers of intestina l cells (Caco-2) suggesting potential usefulness of these systems for oral administration of oligos. In conclusion the approaches using polycations fo r gene delivery for the design of gene transfer complexes thar exhibit a ve ry broad range of physicochemical and biological properties, which is essen tial for design of a new generation of more effective non-viral gene delive ry systems.