Altered organ accumulation of oligonucleotides using polyethyleneimine grafted with poly(ethylene oxide) or pluronic as carriers.

Passive targeting provides a simple strategy based on natural properties of the carriers to deliver DNA molecules to desired compartments. Polyethylenimine (PEI) is a potent non-viral system that has been known to deliver efficiently both plasmids and oligonucleotides (ODNs) in vitro. However, in vivo systemic administration of DNA/PEI complexes has encountered significant difficulties because these complexes are toxic and have low biodistribution in target tissues. This study evaluates PEI grafted with poly(ethylene oxide) (PEO(8K)-g-PEI(2K)) and PEI grafted with non-ionic amphiphilic block copolymer, Pluronic P85 (P85-g-PEI(2K)) as carriers for systemic delivery of ODNs. Following i.v. injection an antisense ODN formulated with PEO(8K)-g-PEI(2K) accumulated mainly in kidneys, while the same ODN formulated with P85-g-PEI(2K) was found almost exclusively in the liver. Furthermore, in the case of the animals injected with the P85-g-PEI(2K)-based complexes most of the ODN was found in hepatocytes, while only a minor portion of ODN was found in the lymphocyte/monocyte populations. The results of this study suggest that formulating ODN with PEO(8K)-g-PEI(2K) and P85-g-PEI(2K) carriers allows targeting of the ODN to the liver or kidneys, respectively. The variation in the tissue distribution of ODN observed with the two carriers is probably due to the different hydrophilic-lipophilic balance of the polyether chains grafted to PEI in these molecules. Therefore, polyether-grafted PEI carriers provide a simple way to enhance ODN accumulation in a desired compartment without the need of a specific targeting moiety.

Inducing neutrophil recruitment in the liver of ICAM-1-deficient mice using polyethyleneimine grafted with Pluronic P123 as an organ-specific carrier for transgenic ICAM-1.

Coordinated expression of cell adhesion molecules and chemokines on the surface of vascular endothelium is responsible for the homing of immune effector cells to targeted sites. One way to attract non-activated immune cells to targeted organs is to use transgenically expressed adhesion molecules responsible for leukocyte recruitment. We have previously shown that polyethyleneimine (PEI) grafted with non-ionic amphiphilic Pluronic P123 block copolymer (P123PEI) modifies biodistribution of plasmid DNA toward the liver. In the present study, a P123PEI-formulated plasmid carrying the gene encoding for the murine ICAM-1 molecule was injected i.v. into transgenic ICAM-1-deficient mice. The RT-PCR analysis of ICAM-1 mRNA expression showed that P123PEI induced a dose-dependent expression of ICAM-1 in the liver. Furthermore, this expression of ICAM-1 induced neutrophil invasion in the liver, while no such invasion was observed in mice injected with formulated control plasmid or naked DNA. These results suggest that P123PEI allows functional transgene expression in the liver following i.v. injection and that ICAM-1 could be used to enhance immune response locally by attracting immune effector cells.

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.