The design, synthesis, and characterization of a novel poly(ethylene glycol) gene delivery system

Advancement in delivery of genetic therapeutics for clinical applications requires the development of effective gene delivery vehicles with low toxicity. Viral based vehicles are the dominant approach for DNA delivery in clinical studies; however concerns regarding the safety of viral vehicles have resulted in increasing interest in non-viral vehicles. Polyethylenimine (PEI), a highly investigated non-viral vehicle, is effective at packaging DNA and can be modified with functional groups to mimic viral trafficking. However, clinical use of PEI vehicles has been limited by toxicity and non-specific protein adsorption which result in rapid clearance and low in vivo efficiency. Conjugating polyethylene glycol (PEG) to the surface of PEI/DNA particles is known to reduce toxicity and increase clearance time. But, surface conjugated PEG decreases transfection efficiency due to PEG shielding. This research describes the development of a novel PEG gene delivery vehicle consisting of a PEG backbone coupled with multiple functional peptides, which has low non-specific protein adsorption and toxicity. Flexible PEG arms, rather than a PEG shield, did not inhibit the activity of functional peptides coupled to the PEG backbone. Rational design was used to determine the most efficient combination of functional peptides on a four arm PEG-based vehicle. DNA binding peptides (DBP), cell targeting ligand (CTL), endosomal escape peptides (EEP), or nuclear localization signals (NLS), were coupled to PEG tetraacrylate (PEG-TA) using a Michael-type addition. Dynamic light scattering showed PEG/DNA particles ranging from 200-350 nm in diameter with near neutral zeta potentials (+/- 10mV). Coupling CTLs, EEPs, or NLS peptides to PEG-DBP vehicles increased transfection efficiency of PEG/DNA particles up to 85% as efficient as PEI. A combination of both PEG-DBP-EEP vehicles and PEG-DBP-NLS vehicles mixed with DNA was 3 times more effective than PEI vehicles. Analysis of PEG-based vehicle intracellular trafficking determined that nuclear localization is the rate-limiting step and should be the focus of future vehicle design. In conclusion, these studies described the development of multifunctional PEG-based vehicles which are more efficient and less toxic alternatives to PEI vehicles for non-viral gene delivery.