A columnar phase of dendritic lipid-based cationic liposome-DNA complexes for gene delivery: hexagonally ordered cylindrical micelles embedded in a DNA honeycomb lattice

Gene therapy holds great promise as a future approach to fighting disease and is explored in worldwide clinical trials. Cationic liposome (CL)-DNA complexes are a prevalent nonviral delivery vector, but their efficiency requires improvement and the understanding of their mechanism of action is incomplete. As part of our effort to investigate the structure-transfection efficiency relationships of self-assembled CL-DNA vectors, we have synthesized a new, highly charged (16+) multivalent cationic lipid, MVLBG2, with a dendritic headgroup. Our synthetic scheme allows facile variation of the headgroup charge and the spacer connecting hydrophobic and headgroup moieties as well as gram-scale synthesis. Complexes of DNA with mixtures of MVLBG2 and neutral 1,2-dioleoyl-sn-glycerophosphatidylcholine (DOPC) exhibit the well-known lamellar phase at 90 mol % DOPC. Starting at 20 mol % dendritic lipid, however, two novel nonlamellar phases are observed by synchrotron X-ray diffraction. The structure of one of these phases, present in a narrow range of composition around 25 mol % MVLBG2, has been solved. In this novel dual lattice structure, termed H(I)C, hexagonally arranged tubular lipid micelles are surrounded by DNA rods forming a three-dimensionally continuous substructure with honeycomb symmetry. Complexes in the H(I)C phase efficiently transfect mouse and human cells in culture. Their transfection efficiency, as well as that of the lamellar complexes containing only 10 mol% dendritic lipid, reaches and surpasses that of commercially available, optimized DOTAP-based complexes. In particular, complexes containing MVLBG2 are significantly more transfectant over the entire composition range in mouse embryonic fibroblasts, a cell line empirically known to be hard to transfect.     

Structural evolution of environmentally responsive cationic liposome-DNA complexes with a reducible lipid linker

Environmentally responsive materials (i.e., materials that respond to changes in their environment with a change in their properties or structure) are attracting increasing amounts of interest. We recently designed and synthesized a series of cleavable multivalent lipids (CMVLn, with n = 2-5 being the number of positive headgroup charges at full protonation) with a disulfide bond in the linker between their cationic headgroup and hydrophobic tails. The self-assembled complexes of the CMVLs and DNA are a prototypical environmentally responsive material, undergoing extensive structural rearrangement when exposed to reducing agents. We investigated the structural evolution of CMVL-DNA complexes at varied complex composition, temperature, and incubation time using small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS). A related lipid with a stable linker, TMVL4, was used as a control. In a nonreducing environment, CMVL-DNA complexes form the lamellar (L(α)(C)) phase, with DNA rods sandwiched between lipid bilayers. However, new self-assembled phases form when the disulfide linker is cleaved by dithiothreitol or the biologically relevant reducing agent glutathione. The released DNA and cleaved CMVL headgroups form a loosely organized phase, giving rise to a characteristic broad SAXS correlation profile. CMVLs with high headgroup charge also form condensed DNA bundles. Intriguingly, the cleaved hydrophobic tails of the CMVLs reassemble into tilted chain-ordered L(β') phases upon incubation at physiological temperature (37 °C), as indicated by characteristic WAXS peaks. X-ray scattering further reveals that two of the three phases (L(βF), L(βL), and L(βI)) constituting the L(β') phase coexist in these samples. The described system may have applications in lipid-based nanotechnologies.     

Electrostatics of DNA complexes with cationic lipid membranes

We present the exact solutions of the linear Poisson-Boltzmann equation for several problems relevant to electrostatics of DNA complexes with cationic lipids. We calculate the electrostatic potential and electrostatic energy for lamellar and inverted hexagonal phases, concentrating on the effects of dielectric boundaries. We compare our results for the complex energy with the known results of numerical solution of the nonlinear Poisson-Boltzmann equation. Using the solution for the lamellar phase, we calculate the compressibility modulus and compare our findings with the experimental data available. Also, we treat charge-charge interactions across, along, and between two low-dielectric membranes. We obtain an estimate for the strength of electrostatic interactions of one-dimensional DNA smectic layers across the lipid membrane. We discuss in the end some aspects of two-dimensional DNA condensation and DNA-DNA attraction in the DNA-lipid lamellar phase in the presence of di- and trivalent cations. We analyze the equilibrium DNA-DNA separations in lamellar complexes using the recently developed theory of electrostatic interactions of DNA helical charge motifs.     

Electrical driving of the surface memorization in dimerized nematics with short range smectic order

Electrical control of the surface memorization of an oriented smectic C texture in the temperature range of dimerized nematics with short range smectic C order (4-n-alkyloxybenzoic acids) is presented. It is demonstrated that by suitable choice of the electric field parameters and surface conditions it is possible to separate the thermal and electrical components of the total erasure activation energy. The significant role of the double charge electric layer in the mechanism of the surface memorization is verified. By the electrical control of the memorization, we have confirmed the electrical part of the modified Rapini-Papoular anchoring energy in the effective surface energy.     

Adsorption and desorption behaviors of cationic liposome-DNA complexes upon lipofection in inside and outside biomembrane models using a dynamic quasi-elastic laser scattering method.

The dynamic behaviors of cationic liposome-DNA complexes in inside and outside biomembrane models upon lipofection were investigated using the time-resolved quasi-elastic laser scattering (QELS) method. Inside and outside biomembrane models with similar phospholipid compositions to those in living cells were formed at a tetradecane/phosphate buffered saline (TD/PBS) interface. Cationic liposome-DNA complexes were injected into the buffer subphase, and their adsorption/desorption behaviors at the biomembrane models were monitored through changes in the interfacial tension. We found that the adsorption rate of the complexes increased 2.6 times more in the outside model than in the inside one. The adsorption rate of DNA alone did not show a remarkable difference from one side to the other; however, the adsorption rate of the cationic liposome alone showed a similar tendency to that of the liposome-DNA complex. These results indicated that the difference in lipid composition induced a different dynamic behavior of exogenous biomolecules and that the cationic liposomes played an important role in the faster incorporation of DNA into cells upon lipofection.     

Lamellar to inverted hexagonal mesophase transition in DNA complexes with calamitic, discotic, and cubic shaped cationic lipids

In this study, we report on the lipid tail molecular shape/size effect on the mesophase self-assembly behaviors of various cationic lipids complexed with double-stranded DNA. The molecular shape of the cationic lipids was tailored from rodlike (a cyanobiphenyl imidazolium salt) to discotic (a triphenylene imidazolium salt), and finally to cubic [a polyhedral oligomeric silsesquioxane (POSS) imidazolium salt]. An increase in the cross-sectional area of the hydrophobic tails with respect to the hydrophilic imidazolium head induced a negative spontaneous curvature of the cationic lipids. As a result, a morphological change from lamello-columnar (L(C)(alpha)) phase for the DNA-cyanobiphenyl imidazolium salt (DNA-rod) and DNA-triphenylene imidazolium salt (DNA-disk) complexes to an inverted hexagonal columnar (H(C)(II)) phase for the DNA-POSS imidazolium salt (DNA-cube) complex was observed. The DNA-rod complex had a typical smectic A (SmA) L(C)(alpha) morphology, whereas the DNA-disk complex had a double lamello-columnar liquid crystalline phase. However, when the lipid tail changed to POSS, an H(C)(II) morphology was achieved. These morphological changes were successfully characterized by X-ray diffraction and transmission electron microscopy. We expect that these liquid crystalline and crystalline DNA hybrid materials may become potential functional materials for various applications such as organic microelectronics and gene transfection.     

Mesomorphic complexes of DNA with the mixtures of a cationic surfactant and a neutral lipid

Polyanionic DNA binds to cationic lipids to form electrostatic complexes exhibiting rich self-assembled structures. These types of complexes have been considered as a nonviral carrier in gene therapy and as a template for nanostructure construction. For the latter application where biocompatibility is not the key issue, replacement of cationic lipid by cationic surfactant is advantageous due to the wide availability of surfactant. Here we report the self-assembly behavior of the complexes of DNA with a cationic surfactant, dodecyltrimethylammonium bromide (DTAB), mixed with a neutral lipid, dioleoylphosphatidylethanolamine (DOPE), in fully hydrated state as a function of DTAB-to-DNA base pair molar ratio (x), DOPE-to-DTAB molar ratio (m) and temperature. The binary complexes of DNA with DTAB microphase separated to form hydrophilic and hydrophobic domains without long-range order. Incorporating DOPE into the complexes effectively strengthened the hydrophobic interaction and hence promoted the formations of long-range ordered mesophases, including a condensed multilamellar phase (L(alpha)(c)) at small to intermediate m (m approximately 6). The lyotropic mesophase transition with respect to the change of m was properly predicted by a formula for calculating the packing parameter of amphiphile mixture. In addition to the lyotropic transition, an unusual thermotropic order-order transition (OOT) between L(alpha)(c) and H(II)(c) phases was revealed for the isoelectric complex with m = 3. This OOT was thermally reversible and was postulated to be driven by the reduction of the effective headgroup area due to the release of trapped water molecules.     

Conjugation of testosterone modifies the interaction of mono-functional cationic platinum(II) complexes with DNA, causing significant alterations to the DNA helix

Previously a range of androgen conjugates with non-conventional platinum(II) complexes have been synthesised with the aim of enhancing cellular delivery, and which have shown increased cytotoxic activity compared with non-steroidal compounds (M. J. Hannon et al., Dalton Trans., 2010, DOI: 10.1039/c0dt00838a). To further study this, the complexes have been assessed for their ability to bind to and alter the structure of DNA. All platinum(II) complexes studied herein bind to model nucleo-bases and DNA, but to our surprise, testosterone-based complexes caused the DNA helix to undergo significant unwinding and bending, whereas non-steroidal control complexes caused minimal structural alterations. These effects are similar to those cisplatin induces on DNA structure despite the fact that these compounds produce a monofunctional lesion. This ability attributed to interactions between the DNA helix and bulky steroidal skeleton of testosterone, coupled with the enhanced cellular delivery induced by the steroid make the steroid approach an exciting way to explore non-conventional platinum drug delivery.     

Controlled complexation of plasmid DNA with cationic polymers: effect of surfactant on the complexation and stability of the complexes

The aggregation of the cationic polymer-plasmid DNA complexes of two commonly used polymers, polyethyleneimine (PEI) and poly-l-lysine (PLL) were systematically compared. The complexation was studied in 5% glucose solution at 25 degrees C using dynamic light scattering and isothermal titration calorimetry. The aggregation of the complexes was controlled by addition of the surfactant polyoxyethylene stearate (POES). The stability of the complexes was evaluated using dextran sulphate (DS) as relaxing agent. The relaxation of the complexes in the presence of DS was studied using agarose gel electrophoresis. This study elucidates the role of surfactant in controlling the size of the PEI/pDNA complex and reveals the differences of the two polymers as complexing agents.     

The effects of oligonucleotide overhangs on the surface hybridization in DNA films: an impedance study

While oligonucleotide hybridization and effects of nucleobase mismatches have been the intense focus of a number of electrochemical studies, the effects of the target strand length on the electrochemical response of oligonucleotide films have not been addressed yet. In this report, we have studied the electrochemical impedance of the oligonucleotide films having overhangs on either the target or the surface bound capture strand. Each system gives different impedance responses, which were interpreted with the help of modified Randles' equivalent. Results indicate that comparable sizes of target and capture strands ensure the higher hybridization efficiency and film order. The presence of nucleobase overhangs at the bottom of the film causes lower changes in charge transfer resistance (ΔR(CT)) after hybridization due to lower hybridization efficiency and presumably non-uniformity in the film. Nucleobase overhangs at the top of the film result in higher ΔR(CT) due to higher film order and accumulation of negative charges but appear not to cause any steric congestion.     

Isolation of cell-free DNA from plasma by chromatography on short monolithic columns and quantification of non-apoptotic fragments by real-time polymerase chain reaction

Human plasma is an important medical substance and a raw material for production of various therapeutics. During blood sampling, storage and processing, genomic DNA is released into plasma from nucleated blood cells that are damaged in the course of the procedure. In order to determine the concentration of contaminating DNA in plasma, we developed a method for DNA isolation by using anion-exchange chromatography on a BIA Separations CIM (convective interaction media) diethylaminoethyl column. DNA was quantified by SYBR Green based real-time polymerase chain reaction. The concentration of cell-free, non-apoptotic DNA in plasma ranged between 0.06 and 22.5 ng/ml. As substantial volumes of plasma or whole blood are administered directly into the vascular system, a recipient is exposed to high amounts of cell-free DNA, several orders of magnitude higher than the amount found in other biologicals.     

Complexation of DNA with cationic gemini surfactant in aqueous solution

Interactions between DNA and the cationic gemini surfactant trimethylene-1,3-bis(dodecyldimethylammonium bromide) (12-3-12) in aqueous solution have been investigated by UV-vis transmittance, zeta potential, and fluorescence emission spectrum. Complexes of DNA and gemini surfactant are observed in which the negative charges of DNA are neutralized by cationic surfactants effectively. The DNA-induced micelle-like structure of the surfactant due to the electrostatic and hydrophobic interactions is determined by the fluorescence spectrum of pyrene. It is found that the critical aggregation concentration (CAC) for DNA/12-3-12 complexes depends little on the addition of sodium bromide (NaBr) because of the counterbalance salt effect. However, at high surfactant concentration, NaBr facilitates the formation of larger DNA/surfactant aggregates. Displacement of ethidium bromide (EB) by surfactant evidently illustrates the strong cooperative binding between surfactant and DNA. In contrast to that in the absence of surfactant, the added NaBr at high surfactant concentration influences not only the binding of surfactant with DNA, but also the stability of DNA/EB complex.     

Alginate polyelectrolyte complexes with cationic hydroxyethyl cellulose derivatives: Role of the block structure

It was shown that the block structure of alginate significantly affects the mechanism of formation and properties of its water-soluble polyelectrolyte complexes with cationic derivatives of hydroxyethyl cellulose. Alginate that contains residues of only mannuronic acid remains in solution in the form of coils, and the incorporation of guluronic acid residues gives rise to formation of a network structure. A difference in the structure of polyelectrolyte complexes is explained by different rigidities of blocks formed by residues of mannuronic and guluronic acids having different conformations of the pyranose ring.     

Robust packing in cyclopalladated primary amines: isomorphous crystal structures of four complexes with varying substitution patterns

The crystal structures of (SP‐4‐4)‐[rac‐2‐(1‐aminoethyl)phenyl‐κ²C¹,N]chlorido(pyridine‐κN)palladium(II), [Pd(C₈H₁₀N)Cl(C₅H₅N)], (I), (SP‐4‐4)‐[rac‐2‐(1‐aminoethyl)phenyl‐κ²C¹,N]bromido(pyridine‐κN)palladium(II), [PdBr(C₈H₁₀N)(C₅H₅N)], (II), (SP‐4‐4)‐[rac‐2‐(1‐aminoethyl)‐5‐bromophenyl‐κ²C¹,N]bromido(4‐methylpyridine‐κN)palladium(II), [PdBr(C₈H₉BrN)(C₆H₇N)], (III), and (SP‐4‐4)‐[rac‐2‐(1‐aminoethyl)‐5‐bromophenyl‐κ²C¹,N]iodido(4‐methylpyridine‐κN)palladium(II), [Pd(C₈H₉BrN)I(C₆H₇N)], (IV), are reported. The latter is the first iodide complex in this class of compounds. All four complexes crystallize in the same space group, viz.I4₁/a, with very similar lattice parameters a and more flexible lattice parameters c. Their packing corresponds to that of their enantiomerically pure congeners, which crystallize in the t2 subgroup I4₁.     

Characteristics of proflavine solubilized in complexes of anionic polymers with a cationic surfactant

Absorption and emission spectra of 3,6-diaminoacridine (proflavine) are reported in mixed solutions of dodecyltrimethylammonium bromide (DTAB) with various polyelectrolytes including the sodium salts of poly(acrylic acid) (PAA). poly(methacrylic acid) (PMA), poly(styrenesulfonic acid) (PSS), poly(garacturonic acid) (pectate), and the alternating copolymers of maleic acid with ethylene (PMA-E) and styrene (PMA-S). The spectral change indicates the association of the dye (blue-shift) on these polyions except on PSS, the easy dissociation of the aggregated form into the monomeric form and the solubilization into the hydrophobic PMA-S/DTAB complex (red-shift), the little dissociation in the PAA/DTAB, PMA/DTAB and PMA-E/DTAB complexes, and the liberation of the bound dye in the case of pectate/DTAB complexes. In the PSS system, the strong interaction of the dye with the styrene groups induces the completely different spectral behavior. These results are discussed with the cooperative binding of the dye and the surfactant ion.     

Spectroscopic investigation of cationic comb-type copolymers/DNA interaction: interpolyelectrolyte complex enhancement synchronized with DNA hybridization

We have demonstrated that cationic comb-type copolymers consisting of a polycation backbone and abundant grafts of water-soluble polymers stabilize DNA hybrids. Furthermore, the copolymers were found to accelerate strand exchange reaction between a double-stranded DNA and its complementary single-stranded DNA. In this article, we investigated the effects of PLL-g-Dex on base pairs of a self-complementary DNA octamer, d(GGAATTCC). The soluble interpolyelectrolyte complex (IPEC) between the DNA and copolymer allowed us to characterize the complex by using spectroscopic methods under physiological ionic condition. Chemical shifts of nucleobase proton signals were not changed by PLL-g-Dex. Furthermore, the copolymer slightly changed the von't Hoff DeltaH accompanying the helix-coil transition of the octamer. These results indicated that the base pairs of the duplex DNA in the IPEC were not perturbed by the polycationic copolymer. It was obviously shown by temperature dependencies of proton and phosphorus NMR spectra that DNA/copolymer interaction was considerably enhanced in response to ds DNA formation. An increase in the density and total number of DNA negative charges upon hybrid formation likely caused the higher affinity of the copolymer with the ds form over that of the copolymer with the ss form. The IPEC formation of CCCs with DNA, however, seems highly sensitive to the coil-helix transition of the DNA.     

Atomistic simulation of discotic liquid crystals: transition from isotropic to columnar phase example

Molecular dynamics simulations at atomistic level have been performed on a metal-porphyrazine complex. Starting from an isotropic state, the system was cooled until transition from isotropic to columnar phase was observed; no nematic phase was encountered. Many tools were utilized to follow the system evolution: order parameter, g(r), g(||)(r(||)), g(c)(r(||)), g(perpendicular)(r(perpendicular)), g(2)(r), also density and energy changes. Very long runs were required to get reliable results, times greater than 40 ns of simulation. The structure of columnar phase was analyzed and the organization of molecules in the columns was investigated, along with the role of conformation of side chains. We found that in columnar phase the molecules are tilted versus the column axis and the conformation of side chains changes during the phase transition to allow this kind of organization; moreover the direction of columns axes is different from that of the director.     

The use of capillary electrophoresis with entangled polymer matrix to analyse plasmid DNA and a cationic lipid/cholesterol liposome: DNA complex

Summary A liposome based gene therapeutic product is being developed for the treatment of cystic fibrosis. The product comprises a complex of plasmid DNA and a cationic lipid/cholesterol liposome. Using CE with an entangled polymer matrix, routine separations of linear, supercoiled and open-circle conformers of DNA in plasmid DNA and the liposome complex have been performed. The CE method has been used to support novel quality control and process validation for the manufacture of plasmid DNA and to monitor the degradation of DNA in the liposome complex. Significant features of the method are simple sample preparation and the use of direct UV detection, avoiding the use of potentially mutagenic reagents.