Cationic lipids are widely used for gene delivery, and inclusion of dioleoylphosphatidylethanolamine (DOPE) as a helper lipid in cationic lipid-DNA formulations often promotes transfection efficacy. To investigate the significance of DOPE's preference to adopt a hexagonal phase in the mechanism of transfection, the properties and transfection efficiencies of SAINT-2/DOPE lipoplexes were compared to those of lipoplexes containing lamellar-phase-forming dipalmitoylphosphatidylethanolamine (DPPE). After interaction with anionic vesicles, to simulate lipoplex-endosomal membrane interaction, SAINT-2/DOPE lipoplexes show a perfect hexagonal phase, whereas SAINT-2/DPPE lipoplexes form a mixed lamellar-hexagonal phase. The transition to the hexagonal phase is crucial for dissociation of DNA or oligonucleotides (ODN) from the lipoplexes. However, while the efficiencies of nucleic acid release from either complex were similar, SAINT-2/DOPE lipoplexes displayed a two- to threefold higher transfection efficiency or nuclear ODN delivery. Interestingly, rupture of endosomes following a cellular incubation with ODN-containing SAINT-2/DPPE complexes dramatically improved nuclear ODN delivery to a level that was similar to that observed for SAINT-2/DOPE complexes. Our data demonstrate that although hexagonal phase formation in lipoplexes is a prerequisite for nucleic acid release from the complex, it appears highly critical for accomplishing efficient translocation of nucleic acids across the endosomal membrane into the cytosol for transport to the nucleus.

Pyridinium amphiphiles, abbreviated as SAINT, are highly efficient vectors for delivery of DNA into cells. Within a group of structurally related compounds that differ in transfection capacity, we have investigated the role of the shape and structure of the pyridinium molecule on the stability of bilayers formed from a given SAINT and dioleoylphosphatidylethanolamine (DOPE) and on the polymorphism of SAINT/DOPE-DNA complexes. Using electron microscopy and small angle x-ray scattering, a relationship was established between the structure, stability, and morphology of the lipoplexes and their transfection efficiency. The structure with the lowest ratio of the cross-sectional area occupied by polar over hydrophobic domains (SAINT-2) formed the most unstable bilayers when mixed with DOPE and tended to convert into the hexagonal structure. In SAINT-2-containing lipoplexes, a hexagonal topology was apparent, provided that DOPE was present and complex assembly occurred in 150 mM NaCl. If not, a lamellar phase was obtained, as for lipoplexes prepared from geometrically more balanced SAINT structures. The hexagonal topology strongly promotes transfection efficiency, whereas a strongly reduced activity is seen for complexes displaying the lamellar topology. We conclude that in the DOPE-containing complexes the molecular shape and the nonbilayer preferences of the cationic lipid control the topology of the lipoplex and thereby the transfection efficiency.

Cationic liposomes are applied to transfer oligonucleotides (ODNs) into cells to regulate gene expression for gene therapeutic or cell biological purposes. In vivo, poly(ethylene glycol) (PEG)-lipid derivatives are employed to stabilize and prolong the circulation lifetime of nucleic acid-containing particles, and to improve targeting strategies. In this study, we have studied the effects of PEG-lipid analogues, i.e. PEG coupled to either phosphatidylethanolamine (PE) or ceramide, on cationic-lipid-DNA complex ('lipoplex') assembly and the mechanism of cationic-lipid-mediated delivery of ODNs in vitro. Inclusion of 10 mol% PEG-PE in ODN lipoplexes inhibited their internalization in Chinese hamster ovary cells by more than 70%. The intracellular fraction remained entrapped in the endosomal/lysosomal pathway, and no release of ODNs was apparent. Similar observations were made for complexes prepared from liposomes that contained PEG-ceramides. Interestingly, delivery resumed when lipoplexes had been externally coated with PEG-ceramides. In this case, the kinetics of delivery were dependent on the length of the ceramide acyl chain, consistent with a requirement for the PEG-lipid to dissociate from the complex. Moreover, although the chemical nature of the PEG-ceramides distinctly affected the net internalization of the complexes, impediment of delivery was largely related to an inhibitory effect of the PEG-lipid on the release of ODNs from the endosomal compartment. Cryo-electron microscopy and small-angle X-ray scattering revealed that the PEG-lipids stabilize the lamellar phase of the lipoplexes, while their acyl-chain-length-dependent transfer from the complex enables adaptation of the hexagonal phase. Within the endosomal compartment, this transition appears to be instrumental in causing the dissociation and cytosolic release of the ODNs for their nuclear homing.

Tridirectional conductivity in a polymeric material has been achieved. Nanoscale “wires” formed via hierarchical self‐organization of a basic block copolymer and a monomeric acid/alkylphenol complex establish a lamellar‐within‐lamellar superstructure (see Figure), resulting in a soft material with tunable conductivity and anisotropy.

Nijland, H.; Polushkin, E.; Brinke, G. ten; Mäki-Ontto, R.; Ikkala, O.; M"ki-Ontto, R.; Maki-Ontto, RA facile concept to prepare nano-objects based on self-organizing polymeric supramolecules (also called supramolecular block copolymers) is demonstrated using one particular shape, i.e., nanorods or nanofibers. Cylindrical aggregates consisting of a polystyrene (PS) core and a thin corona of poly(4-vinylpyridine) (P4VP) chains are prepared from P4VP-(pentadecylphenol)-block-PS comb-coil supramolecules. Diblock copolymers P4VP-block-PS are stoichiometrically (with respect to the number of pyridine groups) hydrogen-bonded with pentadecylphenol (PDP) amphiphiles to yield P4VP(PDP)-block-PS comb-coil supramolecules. By the selection of appropriate relative block lengths for P4VP(PDP) and PS, a micro-phaseseparated cylindrical morphology of PS cylinders inside a P4VP(PDP) matrix is obtained. Subsequent removal of the amphiphiles, accounting for ca. 75% of the matrix material, results in cylinders with a core of PS and a corona of easily adjustable thickness of P4VP. The advantages of the present concept compared to the existing procedures are discussed.

Cationic lipids are widely used for gene transfection, but their mechanism of action is still poorly understood. To improve this knowledge, a structure-function study was carried out with two pyridinium-based lipid analogs with identical headgroups but differing in alkyl chain (un)saturation, i.e., SAINT-2 (diC18:1) and SAINT-5 (diC18:0). Although both amphiphiles display transfection activity per se, DOPE strongly promotes SAINT-2-mediated transfection, but not that of SAINT-5, despite the fact that DOPE effectively facilitates plasmid dissociation from either lipoplex. This difference appears to correlate with membrane stiffness, dictated by the cationic lipid packing in the donor liposomes, which governs the kinetics of lipid recruitment by the plasmid upon lipoplex assembly. Because of its interaction with the relatively rigid SAINT-5 membranes, the plasmid becomes inappropriately condensed, which results in formation of structurally deformed lipoplexes. This structural deformation does not affect its cellular uptake but, rather, hampers plasmid translocation across endosomal and/or nuclear membranes. This is inferred from the observation that both lipoplexes effectively translocate much smaller oligonucleotides into cells. In fact, SAINT-5/DOPE-mediated transfection is greatly improved when, before lipoplex assembly, the plasmid is stabilized by condensation with polylysine. The results emphasize a role of the structural shape of the plasmid in gaining cytosolic/nuclear access. Moreover, it has been proposed that such a translocation is promoted when the lipoplex adopts the hexagonal phase, and data are presented that demonstrate that the lamellar SAINT-5/DOPE lipoplex adopts such a phase after its interaction with acidic phospholipid-containing membranes.

Large-amplitude oscillatory shear alignment of a hierarchically ordered system of combshaped supramolecules and the preparation of core-corona nanorods thereof are reported. The supramolecules consist of nearly symmetric polystyrene-b-poly(4-vinylpyridine) diblock copolymers with pentadecylphenol side chains hydrogen bonded to the poly(4-vinylpyridine) blocks. The polystyrene blocks, accounting for approximately 20% (w/w) of the supramolecules, are segregated in hexagonally ordered cylinders inside a matrix formed by the comb-shaped poly(4-vinylpyridine)-based blocks. The matrix further self-organizes in a layered structure below the order-disorder transition temperature of 70 °C. Thus, a cylinder-within-lamellar morphology is formed. Imposing shear leads to considerable alignment of both structures. The cylinders align along the shear flow with the (10) plane parallel with respect to the shear plane. The matrix layers align perpendicular to the cylinders, transverse to the flow. After elimination of pentadecylphenol by dialysis, crew-cut rods of 20-25 nm in diameter and 5-10 µm in length are obtained.

The terrace formation behavior of chloroform vapor annealed thin films of asymmetric, low molecular weight comb-shaped supramolecules consisting of a short polystyrene (PS) block and a long supramolecular block of poly(4-vinylpyridine) (P4VP) hydrogen bonded with pentadecylphenol (PDP) on silicon oxide (SiO 2) was examined with atomic force microscopy. During annealing, PS microphase separated from the disordered P4VP(PDP) comb, resulting in the formation of terraces of parallelly oriented microdomains of PS in a matrix of P4VP. Upon evaporation of the solvent, the P4VP(PDP) combs dropped below their order-disorder transition, and formed alternating layers of P4VP and PDP, which for high P4VP(PDP) fractions were also oriented parallel to the substrate. This resulted in terraces of the short P4VP(PDP) length scale within terraces of the PS-P4VP long length scale. Washing away PDP from the thin films with ethanol provided an effective means of studying the morphology of the lowest terrace of the thin films and, for a particular system, also resulted in a uniform monolayer of cylinders with a PS core and a P4VP corona.