The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination.     

Influence of DNA adsorption and DNA/cationic surfactant coadsorption on the interaction forces between hydrophobic surfaces

The forces between hydrophobic surfaces with physisorbed DNA are markedly and irreversibly altered by exposure to DNA/cetyltrimethylammonium bromide (CTAB) mixtures. In this colloidal probe atomic force microscopy study of the interactions between a hydrophobic polystyrene particle and an octadecyltrimethylethoxysilane-modified mica surface in sodium bromide solutions, we measure distinct changes in colloidal forces depending on the existence and state of an adsorbed layer of DNA or CTAB-DNA complexes. For bare hydrophobic surfaces, a monotonically attractive approach curve and very large adhesion are observed. When DNA is adsorbed at low bulk concentrations, a long-range repulsive force dominates the approach, but on retraction some adhesion persists and DNA bridging is clearly observed. When the DNA solution is replaced with a CTAB-DNA mixture at relative low CTAB concentration, the length scale of the repulsive force decreases, the adhesion due to hydrophobic interactions greatly decreases, and bridging events disappear. Finally, when the surface is rinsed with NaBr solution, the length scale of the repulsive interaction increases modestly, and only a very tiny adhesion remains. These pronounced changes in the force behavior are consistent with CTAB-induced DNA compaction accompanied by increased DNA adsorption, both of which are partially irreversible.     

The interaction between DNA and cationic lipid films at the air-water interface

The interaction between DNA and positively charged dioctadecyldimethylammonium bromide (DODAB) and DODAB/disteroylphosphatidylcholine (DSPC) monolayers at the air-aqueous interface was studied by a combination of the surface film balance and Brewster angle microscopy. In presence of DNA, the Pi-A isotherm of the cationic monolayer shifts to larger mean molecular areas due to the electrostatic interaction with DNA while the typical liquid expanded-liquid condensed phase transition for DODAB monolayers disappear and the monolayer remains to be in the liquid expanded phase. Furthermore, the morphology of the film dramatically changes, where the large dendritic-like condensed aggregates observed for DODAB monolayers vanish. The charge density of the monolayer was varied by using mixed monolayers with the zwitterionic DSPC and no large effect was observed on the interaction with DNA. By modeling the electrostatic interactions with the linearized Poisson-Boltzmann equation using the finite-element method and taking into account the assumption in the dielectric constants of the system, it was possible to corroborate the expansion of the cationic monolayer upon interaction with DNA as well as the fact that DNA does not seem to penetrate into the monolayer.     

Preparation,characterization of cationic terbium luminescent copolymer and its interaction with DNA

A complex of Tb³⁺, acrylic acid (AA), and 1, 10-phenanthroline (Phen) was synthesized. The structure and fluorescence of Tb(AA)₃Phen·H₂O was characterized with elemental analysis, FT-IR, and fluorescence spectroscopy. A novel copolymer containing rare earth complex, poly (METAC-co-NIPAm-co-Tb(AA)₃Phen·H₂O) (PMNTb), was prepared by free radical copolymerization in methanol with azodiisobutyronitrile (AIBN) as initiator. ¹H NMR, fluorescence spectroscopy, UV-vis spectroscopy, and TEM were used to characterize this copolymer. The interaction of PMNTb with DNA was studied by fluorescence spectroscopy, UV-vis spectroscopy, and agarose gel electrophoresis. The results of fluorescence, UV-vis absorption, and agarose electrophoresis indicated that the PMNTb could interact with DNA in an electrostatic bonding mode and the bonding constant was 3.4 × 10⁵ L mol⁻¹. The TEM observation showed that the PMNTb could form micelles in water solution; the efficient complexation of PMNTb with DNA occurred. The results of in vitro cytotoxicity indicated that PMNTb had good biocompability. These results laid the foundation for the further study in potential gene detective reagent and gene delivery carrier.     

A dialkynoyl analogue of DOPE improves gene transfer of lower-charged, cationic lipoplexes

Positively-charged gene delivery agents, such as cationic liposomes, typically prepared by mixing a cationic lipid and a neutral lipid in a 1 : 1 molar ratio, exhibit a fundamental flaw: on the one hand, the charge encourages cell uptake; on the other hand, the charge leads to aggregation in vivo with anionic serum components. We herein report a more phase-stable analogue of the zwitterionic and fusogenic lipid DOPE that allows for the reduction of the cationic lipid component of the liposome from 50 to 9 mol% with almost no apparent loss in transfection activity. This reduction in charge may induce important in vivo stability whilst still imparting high cell uptake and transgene expression.     

Meso-substituted cationic porphyrins of biological interest. Photophysical and physicochemical properties in solution and bound to liposomes

A series of cationic porphyrins with 1-4 positive charges are studied: mono(N-methyl-4-pyridyl)triphenylporphine chloride [Mono], cis(N-methyl-4-pyridyl)diphenylporphine chloride [Cis], tri(N-methyl-4-pyridyl)monophenylporphine chloride [Tri] and tetra(N-methyl-4-pyridyl)porphine chloride [Tetra]. Their photophysical properties are measured in small unilamellar vesicles and compared with those in homogeneous solution. Liposomes of L-alpha-dimyristoyl-phosphatidylcholine (100 nm diameter) and L-alpha-dipalmitoyl-phosphatidylcholine (50 nm diameter) in phosphate-buffered saline (pH = 7.4) or D2O 0.15 M NaCl were used. The effect of the medium microheterogeinity is discussed. The triplet quantum yields in liposomes for all the porphyrins are about 0.7, similar to the value obtained for Tetra in aqueous media. The singlet molecular oxygen quantum yields for the hydrophilic compounds Tri and Tetra are greater than those of the hydrophobic ones, Mono and Cis. Also, association constants (KL) of the dyes to liposomes and their localization within the membranes are determined from fluorescence and fluorescence polarization measurements, respectively. KL values are in the range of 10(4)-10(5) M-1 for all the compounds, indicating that hydrophobic and coulombic interactions between porphyrins and liposomes are responsible for the dye association. Fluorescence polarization experiments indicate that Mono and Cis can penetrate into the lipidic phase, and that Tri and Tetra are located near the polar heads of the lipidic molecules.     

Study on interaction between cationic polystyrene nanoparticles and DNA, and the detection of DNA by resonance light scattering technology

Monodisperse cationic polystyrene nanoparticles (PS-NPs) were prepared through microemulsion polymerization. Photon correlation spectrometry and transmission electron microscopy revealed that these cationic PS-NPs have a z-average particle size of 30 nm. Size analysis, circular dichroism spectroscopy and transmission electron microscopy indicate that the PS-NPs nanoparticles undergo electrostatic interaction with DNA to form a PS–DNA complex that obviously enhances the resonance light scattering (RLS) signal. A method is presented where the PS-NPs act as a probe for the detection of DNA by RLS. The method is convenient, sensitive, reproducible, and enables high-throughput. The PS-NPs are inexpensive and exhibit low toxicity.     

Immobilization of liposomes onto quartz crystal microbalance to detect interaction between liposomes and proteins

To study the interaction between liposomes and proteins, intact liposomes were immobilized on a metal planar support by chemical binding and/or bioaffinity using a quartz crystal microbalance (QCM). A large decrease in the resonance frequency of quartz crystal was observed when the QCM, modified by a self-assembled monolayer (SAM) of carboxythiol, was added to liposome solutions. The stable chemical immobilization of intact liposomes onto SAM was judged according to the degree with which adsorbed mass depended on the prepared size of liposomes, as well as on the activation time of SAMs when amino-coupling was introduced, where the liposome coverage of electrodes was 69+/-8% in optimal conditions. When avidin-biotin binding was used on amino-coupling liposome layers, liposome immobilization finally reached 168% coverage of the electrode surface. Denatured protein was also successfully detected according to the change in the frequency of the liposome-immobilized QCM. The adsorbed mass of denatured carbonic anhydrase from bovine onto immobilized liposomes showed a characteristic peak at a concentration of guanidine hydrochloride that corresponded to a molten globule-like state of the protein, although the mass adsorbed onto deactivated SAM increased monotonously.     

Study on the interaction between nucleic acids and cationic surfactants

The interactions of nucleic acids and cationic surfactants (cetylpyridine bromide (CPB) and cetyltrimethylammonium bromide (CTMAB)) in aqueous solution have been studied using the techniques of resonance light scattering (RLS) spectroscopy, the absorption spectroscopy, zeta potential assay and NMR assignment measurement. It is considered that CPB or CTMAB can assemble on the surface of nucleic acid via electrostatic and hydrophobic forces, which results in the formation of large associate of nucleic acid-cationic surfactant and RLS enhancement of nucleic acid. Besides these forces, the pi-pi stacking force between CPB and nucleic acid also exists in the associate. In comparison with CTMAB, CPB has larger enhancement on RLS of nucleic acid, which is attributed to that the enhancement of the former is only due to the absorption of the bases of nucleic acid, while the enhancement of the latter is own to the synergetic resonance caused by the absorption of both bases of nucleic acid and the pyridyl in CPB. These results have important implication for understanding the influence of surfactants on nucleic acid functionality in life science.     

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.     

The interaction between G-quadruplex-forming oligonucleotide and cationic surfactant monolayer at the air/water interface

We report on the interactions between a 21-mer quadruplex-forming oligonucleotide bearing human telomere sequence of dG(3)(T(2)AG(3))(3) (G4 DNA) and a positively charged dioctadecyldimethylammonium bromide (DODAB) monolayer at the air-aqueous interface, studied by surface film balance measurements. In the presence of G4 DNA, the π-A isotherm of the cationic Langmuir film shifted to lower molecular areas when compared with the reference isotherm recorded on the subphase containing only 50 mM triethylamine-acetate (TEAA) buffer. The presence of quadruplex-stabilizing metal cations (K(+) or Na(+)) further affected profiles of π-A isotherms. Further insight into processes related to the G4 DNA-monolayer interactions was provided by recording time profiles of the surface pressure of monolayer at a constant mean molecular area. In these experiments G4 DNA and/or metal ions were sequentially injected under the monolayer surface. Results indicated that multistranded assemblies of G4 DNA were formed at the monolayer interface even in the absence of metal ions, which suggested that the charged cationic surface of Langmuir monolayer induced aggregation of guanine-rich DNA strands. The presence of sodium and potassium ions inhibited formation of multi-stranded assemblies through the competitive G-quadruplex formation but to different extent that might be related to the differences in stability and topology of both quadruplexes.     

Investigation of the interaction between sodium dodecyl sulfate and cationic polymers

Aggregation properties of sodium dodecyl sulfate (SDS) on a cationic hydroxyethyl cellulose, Polyquaternium-10 (PQ-10), of low charge density were studied by potentiometric and pyrene fluorescence methods and compared with those of poly(diallyldimethylammomium chloride) (PDADMAC) of high charge density. The critical aggregation concentration (cac) was measured with the potentiometric method and further confirmed with the fluorescence method. The former was found to be more accurate. The value of the cac for the SDS/PQ-10 system was measured at 100, 200, and 400 ppm polymer and at 288.2,298.2, and 308.2 K. They showed almost the same cac value, 0.04 mmol dm-3. The I1/I3 value of the pyrene fluorescence spectrum in the SDS/PQ-10 system at higher SDS concentration was smaller than that in SDS/PDADMAC solution and much larger than that of water. From the binding isotherm by the potentiometric method, the free DS- concentration (Cf) and the bound DS- concentration (Cb) could be evaluated with ease over the SDS concentration range above the cac. The aggregation number of DS- aggregates for both the above polymers was evaluated from the fluorescence quenching method using the values of Cf and Cb from the potentiometric method. Because Cf in the SDS/PQ-10 system above the cac did not maintain a constant value contrary to that in the SDS/PDADMAC system but increased quite a lot, Cb should not be regarded as [SDS] - cac above the cac. The aggregation number in the SDS/PQ-10 system increased almost linearly with increasing total concentration of SDS, while that in the SDS/PDADMAC system reached a plateau. With increasing temperature, the aggregation number of the SDS/PDADMAC system decreased more rapidly than that of the SDS/PQ-10 system.     

Isolation and physicochemical characterization of highly polymeric cotton-plant nuclear DNA

A highly polymeric fraction of nuclear DNA has been isolated from two-day shoots of the cotton plant. The DNA has been characterized by the methods of circular dichroism (CD), thermal denaturation, and ultracentrifugation. It has been established that the DNA preparation isolated has a molecular mass of 18·10⁶ daltons, melts at 85.5°C in a fairly narrow interval, and reveals no heterogeneity of its intramolecular nucleotide composition. It has been shown by the CD method that in a solution of the cotton-plant DNA with relatively low ionic strength there is a tendency to the transformation of the B form into the C form.V. I. Nikitin Institute of Chemistry, Academy of Sciences of Tadzhik SSR, Dushanbe, Institute of the Physiology and Biophysics of Plants, Academy of Sciences of the Tadzhik SSR, Dushanbe. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 641–644, September–October, 1984.     

Synthesis and physicochemical properties of cationic starches

The reaction kinetics of an aqueous suspension of potato starch, various amounts of NaOH, and cationic 3-chloro-2-hydroxypropyltrimethylammonium (chloride) were studied in detail. It was found that the compositions of the sparsely substituted cationic starch ethers and the efficiency of the reaction depended strongly on the ratio of components in the alkylating mixture. The physicochemical properties of the synthesized samples were studied using chemical analysis, scanning electron microscopy, x-ray structure analysis, and thermogravimetry. It was shown that the temperature regime of the reaction had a determining influence on the thermal stability and structural changes of the cationic starch derivative.     

Compaction process of calf thymus DNA by mixed cationic-zwitterionic liposomes: a physicochemical study

The compaction of calf thymus DNA (CT-DNA) by cationic liposomes constituted by a 1:1 mixture of a cationic lipid, 1,2-distearoyl-3-(trimethylammonio)propane chloride (DSTAP), and a zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE, null net charge at pH = 7.4), has been evaluated in aqueous buffered solution at 298.15 K by means of conductometry, electrophoretic mobility, cryo-TEM, and fluorescence spectroscopy techniques. The results reveal that DSTAP/DOPE liposomes are mostly spherical and unilamelar, with a mean diameter of around 77 +/- 20 nm and a positively charged surface with a charge density of sigmazeta = (21 +/- 1) x 10(-3) C m(-2). When CT-DNA is present, the genosomes DSTAP/DOPE/CT-DNA, formed by means of a surface electrostatic interaction, are generally smaller than the liposomes. Furthermore, they show a tendency to fuse forming cluster-type structures when approaching isoneutrality, which has been determined by the electrochemical methods at around (L/D)phi = 5.6. The analysis of the decrease on the fluorescence emission of the fluorophore ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the genosomes are formed has permitted us to confirm the electrostatic character of the DNA-liposome interaction.     

Effect of cationic surfactants on the interaction between sodium perfluorooctanoate and beta-lactoglobulin

The interaction between the fluorocarbon surfactant, sodium perfluorooctanoate (SPFO), and beta-lactoglobulin (BLG) was studied. In particular, the effects of cationic surfactants, such as alkyltriethylammonium bromide (C(n)NE, n=8, 10, 12), on SPFO-BLG interaction were examined. It was shown that the anionic fluorocarbon surfactant, SPFO, was a strong denaturant of BLG. The ability of SPFO to denature BLG could be weakened by the addition of C(n)NE. The effect of C(n)NE on SPFO-BLG interaction was related to the hydrocarbon chain length of C(n)NE, and also the molar ratio of the added C(n)NE to the SPFO in SPFO-BLG solutions ([C(n)NE]/[SPFO]). Our findings might provide a way to design surfactant systems that are less denaturing to proteins or tailor the ability of surfactant to denature proteins through the appropriate mixing with other surfactants.     

Study on the interaction between anionic and cationic latex particles and Portland cement

The interaction between organic latex polymers and the surface of hydrating cement was investigated by measuring the zeta potential and adsorbed amount of polymer on cement. First, differently charged model latex particles were synthesized in aqueous media by well-known emulsion polymerization technique. The latex polymers were characterized by differential scanning calorimetry (DSC), dynamic light scattering (DLS) and environmental scanning electron microscopy (ESEM). Electrokinetic latex surface properties were investigated by means of streaming potential measurements using a particle charge detector (PCD). It is shown that the anionic latexes adsorb a considerable amount of Ca²⁺ from the cement pore solution. Next, adsorption of the latex polymers on the surface of hydrating cement was confirmed by zeta potential measurements using the electroacoustic method. A water to cement ratio in the cement paste as low as 0.5 was studied, representing actual conditions in mortar and concrete. Additionally, adsorption isotherms were determined in a sedimentation test using the depletion method. For all latex polymers, Langmuir type adsorption isotherms were found. The latex dosages required to achieve saturated adsorption on the cement surface obtained from zeta potential measurements correspond well with those determined in the sedimentation test. Electron microscopy photographs confirm that the charged latex polymers adsorb selectively on surface areas of hydrating cement showing opposite charge. This way, domains of organic latex polymers exist on the cement surface. They provide adhesion between the inorganic cement matrix and the organic polymer film formed later on by particle coalescence as a result of cement hydration and drying.     

Micellization of cationic gemini surfactant and its interaction with DNA in dilute brine

The micellization of cationic gemini surfactant trimethylene-1,3-bis (dodecyldimethylammonium bromide) (12-3-12·2Br) was investigated and critical micelle concentrations (CMC) and thermodynamic parameters were evaluated as functions of ionic strength and temperature. The micellization of 12-3-12·2Br is entropically driven and thermodynamically favored. Raising the temperature slightly increases the CMC, while increasing the ionic strength lowers the CMC. A multi-technique study of the 12-3-12·2Br/DNA interaction and its dependence on ionic strength, temperature and DNA concentration were presented. DNA with loose coil conformation, necklace-like structure, highly ordered toroidal aggregates and coexisting of large aggregates and small structures in DNA/12-3-12·2Br system were observed. Critical aggregation concentrations (CAC), interaction saturation concentrations (C(2)), and associated thermodynamic parameters were determined. The screening effect of salt decreases the DNA/12-3-12·2Br electrostatic attraction, but favors the formation of free 12-3-12·2Br micelles or aggregates on the DNA chain. DNA acts as a separate phase contacting with the surfactant molecules and therefore CAC is independent of DNA concentration. Increasing DNA concentration postpones the appearance of free micelle in bulk phase, consequently increases the C(2). Finally an interaction mechanism between 12-3-12·2Br and DNA was proposed.     

Electrophoretic properties of complexes between DNA and the cationic surfactant cetyltrimethylammonium bromide

We use agarose gel electrophoresis to characterize how the monovalent catioinic surfactant cetyltrimethylammonium bromide (CTAB) compacts double-stranded DNA, which is detected as a reduction in electrophoretic DNA velocity. The velocity reaches a plateau at a ratio R = 1.8 of CTAB to DNA-phosphate charges, i.e., above the neutralization point, and the complexes retain a net negative charge at least up to R = 200. Condensation experiments on a mixture of two DNA sizes show that the complexes formed contain only one condensed DNA molecule each. These CTAB-DNA globules were further characterized by time-resolved measurements of their velocity inside the gel, which showed that CTAB does not dissociate during the migration but possibly upon entry into the gel. Using the Ogston-model for electrophoresis of spherical particles, the measured in-gel velocity of the globule is quantitatively consistent with CTAB having two opposite effects, reduction of both the electrophoretic charge and DNA coil size. In the case of CTAB the two effects nearly cancel, which can explain why opposite velocity shifts (globule faster than uncomplexed DNA) have been observed with some catioinic condensation agents. Dissociation of the complexes by addition of anionic surfactants was also studied. The DNA release from the globule was complete at a mixing ratio between anionic and cationic surfactants equal to 1, in agreement with equilibrium studies. Circular DNA retained its supercoiling, and this demonstrates a lack of DNA nicking in the compaction-release cycle which is important in DNA transfection and purification applications.