Block and graft copolymers and NanoGel copolymer networks for DNA delivery into cell.

Self-assembling complexes from nucleic acids and synthetic polymers are evaluated for plasmid and oligonucleotide (oligo) delivery. Polycations having linear, branched, dendritic. block- or graft copolymer architectures are used in these studies. All these molecules bind to nucleic acids due to formation of cooperative systems of salt bonds between the cationic groups of the polycation and phosphate groups of the DNA. To improve solubility of the DNA/polycation complexes, cationic block and graft copolymers containing segments from polycations and non-ionic soluble polymers, for example, poly(ethylene oxide) (PEO) were developed. Binding of these copolymers with short DNA chains, such as oligos, results in formation of species containing hydrophobic sites from neutralized DNA polycation complex and hydrophilic sites from PEO. These species spontaneously associate into polyion complex micelles with a hydrophobic core from neutralized polyions and a hydrophilic shell from PEO. Such complexes are very small (10-40 nm) and stable in solution despite complete neutralization of charge. They reveal significant activity with oligos in vitro and in vivo. Binding of cationic copolymers to plasmid DNA forms larger (70-200 nm) complexes. which are practically inactive in cell transfection studies. It is likely that PEO prevents binding of these complexes with the cell membranes ("stealth effect"). However attaching specific ligands to the PEO-corona can produce complexes, which are both stable in solution and bind to target cells. The most efficient complexes were obtained when PEO in the cationic copolymer was replaced with membrane-active PEO-b-poly(propylene oxide)-b-PEO molecules (Pluronic 123). Such complexes exhibited elevated levels of transgene expression in liver following systemic administration in mice. To increase stability of the complexes, NanoGel 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 mixing with NanoGel suspension, which results in the formation of small particles (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 intestinal cells (Caco-2) suggesting potential usefulness of these systems for oral administration of oligos. In conclusion the approaches using polycations for gene delivery for the design of gene transfer complexes that exhibit a very broad range of physicochemical and biological properties, which is essential for design of a new generation of more effective non-viral gene delivery systems.

Self-assembly of polyamine-poly(ethylene glycol) copolymers with phosphorothioate oligonucleotides.

The cationic copolymers for DNA delivery were synthesized by conjugating poly(ethylene glycol) (PEG) and polyamines: polyspermine (PSP) and polyethyleneimine (PEI). These molecules spontaneously form electrostatic complexes with a model 24-mer phoshorothioate oligonucleotide, T24 (PS-ODN). The copolymer complexes are water soluble. This is a marked contrast with the complexes formed by nonmodified PSP and PEI, which immediately precipitate out of solution. The potentiometric titration study suggests that the amino groups of the copolymers form a cooperative system of salt bonds with the thiophosphate groups of the PS-ODN. The PEG-PEI complexes are stable at physiological pH and ionic strengths. The PEG-PSP complexes are less stable in the presence of the low molecular mass electrolytes compared to the PEG-PEI complexes. The dynamic light scattering and transmission electron microscopy demonstrate that the complex particles are small-ca. 12 nm for PEG-PSP and ca. 32 nm for PEG-PEI. They can be lyophilized and redissolved or stored in solution for up to several months without changing size. The study suggests that as a result of formulation with the PEG-PEI the interactions of PS-ODNs with serum proteins (using the example of bovine serum albumin) are decreased and PS-ODN is protected against nuclease degradation. The simplicity of preparation and long shelf life make these systems attractive as potential pharmaceutical formulations for oligonucleotides.

Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents.

Cationic copolymers consisting of polycations linked to non-ionic polymers are evaluated as non-viral gene delivery systems. These copolymers are known to produce soluble complexes with DNA, but only a few studies have characterized the transfection activity of these complexes. This work reports the synthesis and characterization of a series of cationic copolymers obtained by grafting the polyethyleneimine (PEI) with non-ionic polyethers, poly (ethylene oxide) (PEO) or Pluronic 123 (P123). The PEO-PEI conjugates differ in the molecular mass of PEI (2 kDa and 25 kDa) and the degree of modification of PEI with PEO. All of these conjugates form complexes upon mixing with plasmids, which are stable in aqueous dispersion for several days. The sizes of the particles formed in these systems vary from 70 to 200 nm depending on the composition of the complex. However, transfection activity of these systems is much lower than that of PEI (25 kDa) or Superfect as assessed in in vitro transfection experiments utilizing a luciferase reporter expression in Cos-7 cells as a model system. In contrast, conjugate of P123 with PEI (2 kDa) mixed with free P123 (9:1(wt)) forms small and stable complexes with DNA (110 nm) that exhibit high transfection activity in vitro. Furthermore, gene expression is observed in spleen, heart, lungs and liver 24 h after i.v. injection of this complex in mice. Compared to 1,2-bis(oleoyloxy)-(trimethylammonio) propane:cholesterol (DOTAP:Chol) and PEI (25 kDa) transfection systems, the P123-PEI system reveals a more uniform distribution of gene expression between these organs, allowing a significant improvement of gene expression in liver.