Polyplex Micelles with Double-Protective Compartments of Hydrophilic Shell and Thermoswitchable Palisade of Poly(oxazoline)-Based Block Copolymers for Promoted Gene Transfection.

Improving the stability of polyplex micelles under physiological conditions is a critical issue for promoting gene transfection efficiencies. To this end, hydrophobic palisade was installed between the inner core of packaged plasmid DNA (pDNA) and the hydrophilic shell of polyplex micelles using a triblock copolymer consisting of hydrophilic poly(2-ethyl-2-oxazoline), thermoswitchable amphiphilic poly(2-n-propyl-2-oxazoline) (PnPrOx) and cationic poly(L-lysine). The two-step preparation procedure, mixing the triblock copolymer with pDNA below the lower critical solution temperature (LCST) of PnPrOx, followed by incubation above the LCST to form a hydrophobic palisade of the collapsed PnPrOx segment, induced the formation of spatially aligned hydrophilic-hydrophobic double-protected polyplex micelles. The prepared polyplex micelles exhibited significant tolerance against attacks from nuclease and polyanions compared to those without hydrophobic palisades, thereby promoting gene transfection. These results corroborated the utility of amphiphilic poly(oxazoline) as a molecular thermal switch to improve the stability of polyplex gene carriers relevant for physiological applications.

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.

Dual environment-responsive polyplex carriers for enhanced intracellular delivery of plasmid DNA.

In this study, we describe a multifunctional, nontoxic delivery vehicle with dual-environment sensitivity to deliver plasmid DNA (pDNA) into the cytoplasm of cells. This delivery vehicle was designed to be destabilized by reduction of disulfide cross-links in the intracellular environment and also to contain pH-sensitive membrane-destabilizing activity in acidic late endosomal/lysosomal compartments to allow escape of pDNA into the cell cytoplasm. Polyion complex formation was used to form ternary polyplexes using ionic polymers containing specific chemistries to achieve functional demands. First, template binary polyplexes were formed by association of cationic poly(l-lysine) containing thiol groups (PLys(PDP)) with pDNA and were subsequently cross-linked by disulfide formation for increased stability. Then, binary cross-linked polyplexes were coated with a pH-sensitive membrane-active polyanion, poly(ethylene glycol)-b-poly(aspartamide(DET-Aco)) (PEG-PAsp(DET-Aco)), to produce ternary cross-linked polyplexes. PEG-PAsp(DET-Aco) comprises two repeating units of aminoethylene in PAsp side chains and primary amines modified with anionic cis-aconitic groups. PEG-PAsp(DET-Aco) degrades at acidic pH to generate the parent PEG-PAsp(DET) polymer, which is active toward late endosomal/lysosomal membranes and thus can assist in the endosomal escape of pDNA following endocytosis. Binary/ternary cross-linked polyplexes remained stable toward counter polyanion exchange with dextran sulfate, but released pDNA following disulfide reduction. Ternary cross-linked polyplexes formed by addition of PEG-PAsp(DET-Aco) resulted in enhanced gene transfection efficiency in cultured cells (Huh-7 and HUVEC) without associated cytotoxicity. The enhanced gene transfection was found to be correlated with improved endosomal escape by observation of intracellular trafficking using confocal laser scanning microscopy. This multifunctional ternary cross-linked polyplex demonstrates the successful design of a gene delivery vehicle utilizing intracellular stimuli, and is a promising platform for further development toward practical use.

Antiangiogenic gene therapy of experimental pancreatic tumor by sFlt-1 plasmid DNA carried by RGD-modified crosslinked polyplex micelles.

Disulfide crosslinked polyplex micelles with RGD peptides were formed through ion complexation of thiolated c(RGDfK)-poly(ethylene glycol)-block-poly(L-lysine) (c(RGDfK)-PEG-P(Lys-SH)) and plasmid DNA encoding sFlt-1 and tested for their therapeutic effect in BxPC3 pancreatic adenocarcinoma tumor bearing mice. These micelles, systemically injected, demonstrated significant inhibition of tumor growth up to day 18, as a result of the antiangiogenic effect that was confirmed by vascular density measurements. Significant therapeutic activity of the 15% crosslinked micelle (c(RGDfK)-PEG-P(Lys-SH15)) was achieved by combined effect of increased tumor accumulation, interaction with endothelial cells and enhanced intracellular uptake through receptor-mediated endocytosis. These results suggest that RGD targeted crosslinked polyplex micelles can be effective plasmid DNA carriers for antiangiogenic gene therapy.

Versatile and selective synthesis of "click chemistry" compatible heterobifunctional poly(ethylene glycol)s possessing azide and alkyne functionalities.

Versatile route for "click chemistry" compatible heterobifunctional PEGs was established through preparation of alpha-tetrahydropyranyloxy-omega-hydroxyl poly(ethylene glycol) (THP-PEG-OH) via ring-opening polymerization of ethylene oxide using 2-(tetrahydro-2H-pyran-2-yloxy)ethanol as an initiator, followed by the functionalization of omega-OH group to either the azido or alkyne group. Quantitative azidation of THP-PEG-OH was confirmed from the analysis of molecular functionality of the derivatives. While the conversion efficiency of omega-alkynation was appropriately 70%, the unreacted THP-PEG-OH fraction was successfully removed by ion-exchange chromatography after the carboxylation of the hydroxyl group with succinic anhydride. Then, the protecting group of the alpha-end, THP, was removed in mild acidic media, followed by two- or three-step modification of the resulting alpha-hydroxyl group to primary amino or thiol groups. Consequently, "click chemistry" compatible heterobifunctional PEG derivatives (X-PEG-Y; X = NH(2) and SH, Y =Azide and Alkyne) were synthesized with high efficiency and controlled molecular weight.

pDNA/poly(L-lysine) Polyplexes Functionalized with a pH-Sensitive Charge-Conversional Poly(aspartamide) Derivative for Controlled Gene Delivery to Human Umbilical Vein Endothelial Cells.

An efficient endosome-escaping function was integrated into the polyplex of plasmid DNA (pDNA) with poly(L-lysine) (PLys) to improve its gene transfection efficiency through electrostatic coating with charge-conversional polymer (CCP). CCP showed charge-conversional function responding to endosomal pH, leading to the release of pDNA/PLys polyplex into the cytoplasm. The cells took up the intact CCP-integrated ternary polyplex, which exerted appreciably higher transfection efficiency with lower cytotoxicity than pDNA/PLys polyplex against human umbilical vein endothelial cells (HUVECs). This is consistent with the facilitated endosomal escape of the CCP-integrated ternary polyplex compared to the pDNA/PLys polyplex as directly observed with confocal laser-scanning microscopy.

A facile synthesis of azido-terminated heterobifunctional poly(ethylene glycol)s for "click" conjugation.

New azido-terminated heterobifunctional poly(ethylene glycol) (PEG) derivatives having primary amine and carboxyl end groups, (Azide-PEG-NH 2 and Azide-PEG-COOH, respectively) were synthesized with high efficiency. An alpha-allyl-omega-hydroxyl PEG was prepared as the first step to Azide-PEG-X (X = NH 2 and COOH) through the ring-opening polymerization of ethylene oxide (EO) with allyl alcohol as an initiator, followed by two-step modification of the hydroxyl end to an azido group. To introduce primary amino or carboxyl functional groups, amination and carboxylation reactions of the allyl terminal ends was then conducted by a radical addition of thiol compounds. Molecular functionalities of both ends of the PEG derivatives thus prepared were characterized by (1)H, (13)C NMR, and MALDI-TOF MS spectra, validating that the reaction proceeded quantitatively. The terminal azido functionality is available to conjugate various ligands with an alkyne group through the 1,3-dipolar cycloaddition reaction condition ("click chemistry").