No abstract

Both efficiency and safety profiles are crucial for promotion of gene delivery systems towards practical applications. A promising template system was previously developed based on block catiomer of poly(ethylene glycol) (PEG)-b-poly{N'-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide}-cholesteryl [PEG-PAsp(DET)-cholesteryl] with strategies of ligand conjugation at the α-terminus for specific affinity to the targeted cells and cholesteryl conjugation at the ω-terminus for structural stabilization to obtain systemic retention. Aiming for advocating this formulation towards practical applications, in the current study, the binding profile of this polymer to plasmid DNA (pDNA) was carefully studied to address an issue of toxicity origin. Quantification of free polymer composition confirmed that the toxicity mainly results from unbound polymer and polyplex micelle itself has negligible toxicity. This evaluation allowed for identifying an optimal condition to prepare safe polyplex micelles for systemic application that possess maximal polymer-binding but exclude free polymers. The identified polyplex micelles then faced a drawback of limited transfection efficiency due to the absence of free polymer, which is an acknowledged tendency found in various synthetic gene carriers. Thus, series of functional components was strategically compiled to improve the transfection efficiency such as attachment of cyclic (Arg-Gly-Asp) (cRGD) peptide as a ligand onto the polyplex micelles to facilitate cellular uptake, use of endosome membrane disruptive catiomer of PAsp(DET) for facilitating endosome escape along with use of the conjugated cholesteryl group to amplify the effect of PAsp(DET) on membrane disruption, so as to obtain efficient transfection. The mechanistic investigation respecting the appreciated pH dependent protonation behavior of PAsp(DET) permitted to depict an intriguing scenario how the block catiomers manage to escape from the endosome entrapment in response to the pH gradient. Subsequent systemic application to the pancreatic tumor demonstrated a capability of vascular targeting mediated by the cRGD ligand, which was directly confirmed based on in situ confocal laser scanning microscopy observation. Encouraging this result, the vascular targeting to transfect a secretable anti-angiogenic gene was attempted to treat the intractable pancreatic tumor with anticipation that the strategy could circumvent the intrinsic physiological barriers derived from hypovascular and fibrotic characters. The obtained therapeutic efficiency demonstrates promising utilities of the proposed formulation as a safe systemic gene delivery carrier in practical use.

A critical role of polyethylene glycol (PEG) crowding in the packaging of plasmid DNA (pDNA) into polyplex micelles (PMs) was investigated using a series of PEG-b-poly(l-lysine) (PEG-PLys) block copolymers with varying molecular weights of both PEG and PLys segments. Rod-shaped PMs preferentially formed when the tethered PEG chains covering pDNA in a precondensed state were dense enough to overlap one another (reduced tethering density (RTD) > 1), whereas globular PMs were obtained when they were not overlapped (RTD < 1). These results submitted a scheme that steric repulsive effect of PEG regulated packaging pathways of pDNA either through folding into rod-shape or collapsing into globular depending on whether the PEG chains are overlapped or not. The rod-shaped PMs gave significantly higher gene expression efficacies in a cell-free system compared to the globular PMs, demonstrating the practical relevance of regulating packaging structure of pDNA for developing efficient gene delivery systems.

No abstract

Selectively spooling single plasmid DNA (pDNA), as a giant polyelectrolyte, into a nanosized toroidal structure or folding it into a rod-like structure has been accomplished by polyion complexation with block catiomers to form polymeric micelles in varying NaCl concentrations. The interactive potency between the pDNA and block catiomers was determined to play a critical role in defining the ultimate structure of the pDNA; the formation of toroidal or rod-like structures was achieved by complexation in 600 or 0 mM NaCl solutions, respectively. Compared with the rod-like structure, the toroidal structure possessed superior biological functions capable not only of elevating in vitro transcription but also of elevating in vivo gene transduction efficiency. This demonstrated the great utility of the toroidal pDNA packaging as a distinct structured gene carrier. Furthermore, the fact that the NaCl concentration at which the toroidal structure was specifically formed corresponds to seawater stimulates interest in this ordered nanostructure as a possible inherent structure for DNA.