Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic growth factor that is a primary stimulant of the development and maintenance of a vascular network in the vascularization of solid tumors. It has been reported that a blockade of VEGF-mediated angiogenesis is a powerful method for tumor regression. RNA interference represents a naturally occurring biological strategy for inhibition of gene expression. In mammalian systems, however, the in vivo application of small interfering RNA (siRNA) is severely limited by the instability and poor bioavailability of unmodified siRNA molecules. In this study, we tested the hypothesis that a hydrophobically modified protein transduction domain, cholesteryl oligo-d-arginine (Chol-R9), may stabilize and enhance tumor regression efficacy of the VEGF-targeting siRNA. The noncovalent complexation of a synthetic siRNA with Chol-R9 efficiently delivered siRNA into cells in vitro. Moreover, in a mouse model bearing a subcutaneous tumor, the local administration of complexed VEGF-targeting siRNA, but not of scrambled siRNA, led to the regression of the tumor. Hence, we propose a novel and simple system for the local in vivo application of siRNA through Chol-R9 for cancer therapy.
Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.
The development of biodegradable cationic polymers for use in somatic gene therapy is desirable because degradable polymers have the potential to overcome cellular toxicities that are related to the high charge densities of the polycationic delivery system. Therefore, to produce a biocompatible gene delivery vehicle, we have designed a novel biodegradable, high molecular weight multiblock copolymer (MBC) of the type (AB) n which consists of repeating units of low molecular weight poly(ethylene glycol) (PEG) conjugated to low molecular weight cationic poly(L-lysine) (PLL). PEG was used not only to impart steric stabilization properties onto the polymer/pDNA complexes but also to introduce biodegradable ester bond linkages into the backbone of the MBCs. Also, to improve the endosome-disrupting capabilities of the polymer, N,N-dimethylhistidine (His) was coupled at various mole ratios (5 mol % His, 9 mol % His, 16 mol % His, 22 mol % His) to the -amines of PLL to produce PEG-PLL-grafted-His (PEG-PLL-g-His) MBCs. Polymer screening revealed that MBCs with 16% His grafted (PEG-PLL-g-16% His) (31 kDa) produced the highest transfection efficiency with minimal cytotoxicity in murine smooth muscle cells (A7r5). The MBCs condensed plasmid DNA (pDNA) into nanostructures with an average particle size between 150 and 200 nm with no aggregation and surface charge of ∼4-45 mV. These MBCs also protected pDNA from endonuclease digestion for at least 2 h. The polymers showed exponential decay with a halflife (t 1/2) of ∼5 h in PBS, pH 7.4 at 37 °C. However, complexes incubated in PBS buffer showed complete stability up to 6 days despite the short polymer t1/2. The pK of the conjugated imidazoles was found to be 4.75 which would facilitate buffering at low pH environments of the late endosome/lysosome. Finally, the ability of the imidazoles to protonate and destabilize membrane vesicles was investigated by the use of bafilomycin A 1 which showed that the MBCs produced about five times higher transfection efficiency in vitro in A7r5 cells compared to the treated cells. This supports the function of histidine as an endosomal disrupting moiety. Therefore, these results suggest that biodegradable multiblock copolymers are promising candidates for long-term gene delivery.
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