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

Semipermeable Polymer Vesicle (PICsome) Self-Assembled in Aqueous Medium from a Pair of Oppositely Charged Block Copolymers: Physiologically Stable Micro-/Nanocontainers of Water-Soluble Macromolecules

A new entity of polymer vesicle with a polyion complex (PIC) membrane, a PICsome, was prepared by simple mixing of a pair of oppositely charged block copolymers, composed of biocompatible PEG and poly(amino acid)s, in an aqueous medium. Flow particle image analysis revealed the formation of spherical particles with a size range up to 10 μm. Observation by dark-field and confocal laser scanning microscopes clearly confirmed that the PICsome has a hollow structure with an inner-water phase, in which FITC-dextran emitting green fluorescence was successfully encapsulated simply by the simultaneous mixing with the block copolymers. Confocal laser scanning microscopic observation and spectral analysis revealed the smooth penetration of a low molecular weight fluorescent dye (TRITC; MW = 443.5) emitting red fluorescence into the FITC-dextran encapsulated PICsome to give the PICsome image with a merged color of yellows, indicating the semipermeable nature of the PICsome membrane. The PICsomes showed appreciable physiological stability even in the presence of serum proteins, suggesting their feasibility in biomedical fields such as carriers of therapeutic compounds and compartments for diagnostic enzymes

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-<i>n</i>-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

Bundled Assembly of Helical Nanostructures in Polymeric Micelles Loaded with Platinum Drugs Enhancing Therapeutic Efficiency against Pancreatic Tumor

Supramolecular assemblies of amphiphilic block copolymers having polypeptide segments offer significant advantages for tailoring spatial arrangement based on secondary structures in their optically active backbones. Here, we demonstrated the critical effect of α-helix bundles in cisplatin-conjugated poly(l- (or d-)glutamate) [P(l(or d)Glu)-CDDP] segment on the packaging of poly(ethylene glycol) (PEG)-P(l(or d)Glu)-CDDP block copolymers in the core of polymeric micelles (CDDP/m) and enhanced micelle tolerability to harsh <i>in vivo</i> conditions for accomplishing appreciable antitumor efficacy against intractable pancreatic tumor by systemic injection. CDDP/m prepared from optically inactive PEG-poly(d,l-glutamate) (P(d,lGlu)), gradually disintegrated in the bloodstream, resulting in increased accumulation in liver and spleen and reduced antitumor efficacy. Alternatively, CDDP/m from optically active PEG-P(l(or d)Glu) maintained micelle structure during circulation, and eventually attained selective tumor accumulation while reducing nonspecific distribution to liver and spleen. Circular dichroism and small-angle X-ray scattering measurements indicated regular bundled assembly of α-helices in the core of CDDP/m from PEG-P(l(or d)Glu), which is suggested to stabilize the micelle structure against dilution in physiological condition. CDDP/m suffered corrosion by chlorides in medium, yet the optically active micelles with α-helix bundles kept the micelle structure for prolonged time, with slowly releasing unimers and dimers from the surface of the bundled core in an erosion-like process, as verified by ultracentrifugation analysis. This is in sharp contrast with the abrupt disintegration of CDDP/m from PEG-P(d,lGlu) without secondary structures. The tailored assembly in the core of the polymeric micelles through regular arrangement of constituting segments is key to overcome their undesirable disintegration in bloodstream, thereby achieving efficient delivery of loaded drugs into target tissues

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-<i>b</i>-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

Morphology Control in Water of Polyion Complex Nanoarchitectures of Double-Hydrophilic Charged Block Copolymers through Composition Tuning and Thermal Treatment

Polyion complexes (PICs) are attractive as eco-friendly materials, because they offer simple and fast preparation methods to exert various functionalities in aqueous medium. However, control of nanoarchitectures in PIC materials has not been fully realized, except for the case of micelles and unilamellar vesicles formed from block ionomers. Here, the procedure to control PIC nanoarchitectures with various morphologies was established for the first time by careful tuning in the composition of PICs made from PEG-based block-ionomers with a varying amount of homoionomers as additive to modulate the PEG weight fraction (<i>f</i>_PEG) in the obtained PICs. Accordingly, the variation in <i>f</i>_PEG from 12.1% to 6.5% induced vigorous transition in the microphase separated structures of PICs basically from micelle to lamella via cylindrical network. Notably, uniformed lamella with alternative layers of PEG and PIC domains was found at elevated temperature (70 °C), which, by lowering temperature, reversibly transformed to cylindrical PIC network apparently with connected aqueous channel in mesoscopic scale

Anti-tumor efficacy of the polyplex micelle-based DNA vaccine in mice harboring peritoneal and subcutaneous tumors.

<p>(A) Vaccination schedule of polyplex micelles encapsulating therapeutic genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101854#pone-0101854-t001" target="_blank">Table 1</a>) in the peritoneal dissemination model of CT26 tumors. (B) There were significant increases in the survival rates of SART3/CD40L+GM-CSF, GM-CSF, and SART3+GM-CSF groups (<i>P</i><0.0001, n = 18; <i>P</i><0.01, n = 10; <i>P</i><0.01, n = 7 vs. mock control, n = 19, respectively; left panel). Long-term surviving mice were only obtained by vaccination of polyplex micelles with SART3, CD40L, and GM-CSF genes. No significant improvement in survival was detected by transfection of SART3 or CD40L genes alone (n = 6 and 10; right panel). (C) Vaccination schedule of polyplex micelles with therapeutic genes in subcutaneous CT26 and LLC tumor models. (D) Polyplex micelles with SART3, CD40L, and GM-CSF genes (n = 7 and 9), but not other transgenes (n = 5−6), significantly decreased the weights of CT26 (left panel) and LLC subcutaneous tumors (right panel) compared with those in the mock control (n = 6 and 7). *<i>P</i><0.01.</p

Quantification of Poly(ethylene glycol) Crowding on Nanodiamonds toward Quantum Biosensor for Improved Prevention Effects on Protein Adsorption and Lung Accumulation

Nanometer-sized diamonds (NDs) containing nitrogen vacancy centers have garnered significant attention as potential quantum sensors for reading various types of physicochemical information in vitro and in vivo. However, NDs intrinsically aggregate when placed in biological environments, hampering their sensing capacities. To address this issue, the grafting of hydrophilic polymers onto the surface of NDs has been demonstrated considering their excellent ability to prevent protein adsorption. To this end, crowding of the grafted chains plays a crucial role because it is directly associated with the antiadsorption effect of proteins; however, its quantitative evaluation has not been reported previously. In this study, we graft poly­(ethylene glycol) (PEG) with various molecular weights onto NDs, determine their crowding using a gas adsorption technique, and disclose the cross-correlation between the pH in the grafting reaction, crowding density, molecular weight, and the prevention effect on protein adsorption. PEG-grafted NDs exhibit a pronounced effect on the prevention of lung accumulation after intravenous injection in mice. PEG crowding was compared to that calculated by using a diameter determined by dynamic light scattering (DLS) assuming a sphere

Protective effect of the polyplex micelle-based DNA vaccine on lung metastasis of LLC tumors.

<p>(A) Hematoxylin and eosin staining of lung tissues showed that lung metastatic nodules were highly developed by day 28 after subcutaneous injection of LLC cancer cells in the mock control (4/4 cases), but not present in the SART3/CD40L+GM-CSF vaccine group (0/4 cases). (B) Lung tissues were immunostained with anti-CD4 or CD8a antibodies. Increased infiltration of CD4⁺ and CD8a⁺ T cells into the lung beds was observed in the SART3/CD40L+GM-CSF group. *<i>P</i><0.05, n = 4.</p

Therapeutic genes encapsulated by polyplex micelles and the survival periods of mice with peritoneal dissemination of CT26 tumors.

<p>SART3: squamous cell carcinoma antigen recognized by T cells 3; GM-CSF: granulocyte macrophage colony-stimulating factor. *<i>P</i><0.05, **<i>P</i><0.01, and ^†<i>P</i><0.0001 vs. Mock control.</p