Poly(ethylene glycol) Crowding as Critical Factor To Determine pDNA Packaging Scheme into Polyplex Micelles for Enhanced Gene Expression.

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.

Rod-to-Globule Transition of pDNA/PEG-Poly(l-Lysine) Polyplex Micelles Induced by a Collapsed Balance Between DNA Rigidity and PEG Crowdedness.

The role of poly(ethylene-glycol) (PEG) in rod-shaped polyplex micelle structures, having a characteristic core of folded plasmid DNA (pDNA) and a shell of tethered PEG chains, is investigated using PEG-detachable polyplex micelles. Rod shapes undergo change to compacted globule shapes by removal of PEG from polyplex micelles prepared from block copolymer with acid-labile linkage between PEG and poly(l-lysine) (PLys) through exposure to acidic milieu. This structural change supports the previous investigation on the rod shapes that PEG shell prevents the DNA structure from being globule shaped as the most favored structure in minimizing surface area. Noteworthy, despite the PEG is continuously depleted, the structural change does not occur in gradual shortening manner but the rod shapes keep their length unchanged and abruptly transform into globule shapes. Analysis of PEG density reveals the transition occurred when tethered PEG of rod shapes has decreased to a critical crowdedness, i.e., discontacted with neighboring PEG, which eventually illuminates another contribution, rigidity of DNA packaged as bundle in the rod shapes, in addition to the steric repulsion of PEG, in sustaining rod shapes. This investigation affirms significant role of PEG and also DNA rigidity as bundle in the formation of rod-shaped structures enduring the quest of compaction of charge-neutralized DNA in the polyplex micelles.

Targeted decationized polyplexes for siRNA delivery.

The applicability of small interfering RNA (siRNA) in future therapies depends on the availability of safe and efficient carrier systems. Ideally, siRNA delivery requires a system that is stable in the circulation but upon specific uptake into target cells can rapidly release its cargo into the cytoplasm. Previously, we evaluated a novel generation of carrier systems ("decationized" polyplexes) for DNA delivery, and it was shown that folate targeted decationized polyplexes had an excellent safety profile and showed intracellular triggered release upon cell specific uptake. Targeted decationized polyplexes consist of a core of disulfide cross-linked poly(hydroxypropyl methacrylamide) (pHPMA) stably entrapping nucleic acids and a shell of poly(ethylene glycol) (PEG) decorated with folate molecules. In the present study, the applicability of folate targeted decationized polyplexes for siRNA delivery was investigated. This required optimization of the carrier system particularly regarding the cross-linking density of the core of the polyplexes. Stable and nanosized siRNA decationized polyplexes were successfully prepared by optimizing the cross-link density of their core. Upon incubation in human plasma, a significant portion of siRNA remained entrapped in the decationized polyplexes as determined by fluorescence correlation spectroscopy (FCS). When tested in a folate receptor overexpressing cell line stably expressing luciferase, Skov3-luc, sequence specific gene silencing was observed. As expected, neither interference on the intrinsic luciferase expression nor on the cell metabolic activity (determined by XTT) was induced by the free-polymer or the siRNA polyplexes. In conclusion, targeted decationized polyplexes are safe and stable carriers that interact with the targeted cells and rapidly disassemble upon cell entry making them promising siRNA delivery systems.

A tadpole-shaped gene carrier with distinct phase segregation in a ternary polymeric micelle.

A distinct tadpole-shaped nanostructure characterized by a spherical head and an extended shaft was identified in a single plasmid DNA (pDNA)-based polymeric micelle. The tadpole-shaped structure was constructed by adding anionic chondroitin sulfate (CS) to the rod-shaped polyplex micelle containing a single pDNA molecule packaged by the PEG-polycation block copolymer through their electrostatic self-assembly. The complex consequently developed a novel structure composed of segregated domains of the CS-rich inflated head and CS-poor folded DNA tail. Hence, this tadpole structure can be regarded as evidence that distinct phase segregation occurred in a single polymeric micelle containing pDNA.

Effect of shear stress on structure and function of polyplex micelles from poly(ethylene glycol)-poly(l-lysine) block copolymers as systemic gene delivery carrier.

Structural stability of polyplex micelles (PMs), prepared from plasmid DNA (pDNA) and poly(ethylene glycol)-b-poly(l-lysine) block catiomer (PEG-PLys), was evaluated in terms of their resistance against shear stress. When exposed to shear stress at magnitudes typically present in the blood stream, structural deterioration was observed in PMs owing to the partial removal of PEG-PLys strands. Eventually, impaired PEG coverage of the polyplex core led to accelerated degradation by nucleases, implying that structural deterioration by shear stress in blood stream may be a major cause of rapid clearance of PMs from blood circulation. To address this issue, introduction of disulfide crosslinking into the PM core was shown to be an efficient strategy, which successfully mitigated unfavorable effects of shear stress. Furthermore, improved in vivo blood retention profile and subsequently enhanced antitumor efficacy in systemic treatment of pancreatic adenocarcinoma were confirmed for the crosslinked PMs loaded with pDNA encoding an anti-angiogenic protein, suggesting that high stability under the shear stress during blood circulation may be a critical factor in systemically applicable gene delivery systems.

Polyplex micelle installing intracellular self-processing functionalities without free catiomers for safe and efficient systemic gene therapy through tumor vasculature targeting.

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.