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.     

Surface miscibility of EPC/DOTAP/DOPE in binary and ternary mixed monolayers

Surface pressure (π)-molecular area (A) curves were used to characterize the packing of pseudo-ternary mixed Langmuir monolayers of egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and L-α-dioleoyl phosphatidylethanolamine (DOPE). This pseudo-ternary mixture EPC/DOPE/DOTAP has been successfully employed in liposome formulations designed for DNA non-viral vectors. Pseudo-binary mixtures were also studied as a control. Miscibility behavior was inferred from π-A curves applying the additivity rule by calculating the excess free energy of mixture (ΔG(Exc)). The interaction between the lipids was also deduced from the surface compressional modulus (C(s)(-1)). The deviation from ideality shows dependence on the lipid polar head type and monolayer composition. For lower DOPE concentrations, the forces are predominantly attractive. However, if the monolayer is DOPE rich, the DOTAP presence disturbs the PE-PE intermolecular interaction and the net interaction is then repulsive. The ternary monolayer EPC/DOPE/DOTAP presented itself in two configurations, modulated by the DOPE content, in a similar behavior to the DOPE/DOTAP monolayers. These results contribute to the understanding of the lipid interactions and packing in self-assembled systems associated with the in vitro and in vivo stability of liposomes.     

Multicomponent cationic lipid-DNA complex formation: role of lipid mixing

Multicomponent cationic lipid-DNA complexes (lipoplexes) were prepared by adding linear DNA to mixed lipid dispersions containing two populations of binary cationic liposomes and characterized by means of small angle X-ray scattering (SAXS). Four kinds of cationic liposomes were used. The first binary lipid mixture was made of the cationic lipid (3'[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) and the neutral helper lipid dioleoylphosphocholine (DOPC) (DC-Chol/DOPC liposomes), the second one of the cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the neutral dioleoylphosphatidylethanolamine (DOPE) (DOTAP/DOPE liposomes), the third one of DC-Chol and DOPE (DC-Chol/DOPE liposomes), and the fourth one of DOTAP and DOPC (DOTAP/DOPC liposomes). Upon DNA-induced fusion of liposomes, large lipid mixing at the molecular level occurs. As a result, highly organized mixed lipoplexes spontaneously form with membrane properties intermediate between those of starting liposomes. By varying the composition of lipid dispersions, different DNA packing density regimes can also be achieved. Furthermore, occurring lipid mixing was found to induce hexagonal to lamellar phase transition in DOTAP/DOPE membranes. Molecular mechanisms underlying experimental findings are discussed.     

pH-Dependent force spectroscopy of tri(ethylene glycol)- and methyl-terminated self-assembled monolayers adsorbed on gold

Self-assembled monolayers (SAMs) of methoxy-tri(ethylene glycol)- (EG(3)-OMe) and methyl-terminated alkanethiols (C(16)) adsorbed on polycrystalline gold were investigated by chemical force spectroscopy. Measurements were performed in aqueous electrolyte solutions depending on ionic strength and pH value. Charged and hydrophobic tips were employed as probes to mimic local patches of proteins and to study the interaction at the organic/liquid interface in detail. Force-distance curves reveal information about the origin of the observed interaction and the underlying mechanisms. The measurements confirm an effective negative surface charge to be present at the oligo(ethylene glycol) (OEG) and the methyl interface and suggest that the charges are due to the adsorption of hydroxyl ions from aqueous solution. pH-dependent measurements further support the robustness of the established charge associated with the OEG films. Its sign does not change over the whole range of investigated values between pH approximately 3.5 and approximately 10. In contrast, the hydrophobic self-assembled hexadecanethiol films on gold show an isoelectric point (IEP) around pH 4. While the mechanism of charge establishment appears to be similar for both SA films, the strength of hydrogen bonding to interfacial water, which acts as a template for hydroxyl ion adsorption, is likely to be responsible for the observed difference.     

Thermodynamic and physical interactions between novel polymeric surfactants and lipids: toward designing stable polymer-lipid complexes

Surfactant amphiphilic macromolecules (AMs) were complexed with a 1:1 ratio of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), either by a coevaporation (CE) or postaddition (PA) method, to form AM-lipid complexes with enhanced drug delivery applications. By characterizing the surfactant-lipid interactions, these heterogeneous drug delivery systems can be better controlled and engineered for optimal therapeutic outcomes. In this study, the physical interactions between DOPE:DOTAP liposomes and AM surfactants were investigated. Langmuir film balance and isothermal calorimetry studies showed cooperative intermolecular interactions between pure lipids and AM in monolayers and high thermostability of structure formed by the addition of AM micelles to DOTAP:DOPE vesicles in buffer solution respectively. Increasing the AM weight ratio in the complexes via the CE method led to complete vesicle solubilization--from lamellar aggregates, to a mixture of coexisting vesicles and micelles, to mixed micelles. Isothermal calorimetry evaluation of AM-lipid complexes shows that, at higher AM weight ratios, PA-produced complexes exhibit greater stability than complexes at lower AM weight ratios. Similar studies show that AM-lipid complexes produced by the CE methods display stronger interactions between AM-lipid components than complexes produced by the PA method. The results suggest that the PA method produces vesicles with AM molecules associated with its outer leaflet only (i.e., an AM-coated vesicle), while the CE method produces complexes ranging from mixed vesicles to mixed micelle in which the AM-lipid components are more intimately associated. These results will be helpful in the design of AM-lipid complexes as structurally defined, stable, and effective drug delivery systems.     

Permeability and electrokinetic characterization of poly(ethylene terephthalate) capillary pore membranes with grafted temperature-responsive polymers

Poly(ethylene terephthalate) (PET) track-etched membranes with average pore diameters of 692 and 1629 nm were functionalized using the monomer N-isopropylacrylamide (NIPAAm) and a photoinitiated "grafting-from" approach in which a surface-selective reaction has been most efficiently achieved by combinations of the unmodified PET surface with benzophenone and, alternatively, of an aminated PET surface with benzophenone carboxylic acid. Consistent estimations of the pore diameters of the base PET membranes and of the effective grafted polyNIPAAm layer thicknesses on the PET pore walls were possible only on the basis of the permeabilities measured with aqueous solutions of higher ionic strength (e.g., 0.1 M NaCl). However, the permeabilities measured with ultrapure water indicated that the "electroviscous effect" was significant for both base membranes. The influences of membrane pore diameter, surface charge, and solution ionic strength could be interpreted in the framework of the space-charge model. Functionalized membranes with collapsed grafted polymer hydrogel layer thicknesses of a few nanometers exhibited almost zero values of the zeta potential estimated from the trans-membrane streaming potential measurements. This was caused by a "hydrodynamic screening" of surface charge by the neutral hydrogel. Very pronounced changes in permeability as a function of temperature were measured for PET membranes with grafted polyNIPAAm layers, and the effective layer thickness in the swollen state--here up to approximately 300 nm--correlated well with the degree of functionalization. The subtle additional effects of solution ionic strength on the hydrodynamic layer thickness at 25 degrees C were different from the effects for the base PET membranes and could be explained by a variation in the degree of swelling, resembling a "salting-out" effect. Overall, it had been demonstrated that the functionalized capillary pore membranes are well suited for a detailed and quantitative evaluation of the relationships between the synthesis, the structure, and the function of grafted stimuli-responsive polymer layers.     

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.     

Dielectric relaxations of ionic thiol-coated noble metal nanoparticles in aqueous solutions: electrical characterization of the interface

The radiowave dielectric properties of organothiol monolayer-protected Au and Ag metallic nanoparticles have been investigated in the frequency range of 10 kHz to 2 GHz, where a dielectric relaxation, due to the polarization of the ionic atmosphere at the aqueous interface, occurs. The simultaneous measurement of the particle size, by means of dynamic light scattering technique, and of the particle electrical charge, by means of laser microelectrophoresis technique, allow us to describe the whole dielectric behavior at the light of the standard electrokinetic model for charged colloidal particles. Au and Ag metallic nanoparticles experience a large charge renormalization, in agreement with the counterion condensation effect for charged spherical colloidal particles. The value of the effective valence Z(eff) of each nanoparticle investigated has been evaluated thanks to the dielectric parameters of the observed relaxation process and further confirmed by direct current electrical conductivity measurements. All in all, these results provide support for the characterization of the electrical interfacial properties of metallic nanoparticles by means of dielectric relaxation measurements.     

The ion sensitivity of surface conductive single crystalline diamond

Charge build-up at the solid/aqueous interface is a ubiquitous phenomenon that determines the properties of interfacial electrical double layers. Due to its unique properties, the surface of diamond offers an attractive platform to investigate charging mechanisms in aqueous solutions. We investigate the surface charge by studying the ion sensitivity of H-terminated single crystalline diamond surface conductive layers. The effect of monovalent and divalent salts has been probed at different pH values. For a pH above 3.5, increasing the ionic strength results in a decrease of the surface conductivity, in contrast to the results obtained for pH below 3.5. Electrokinetic experiments are in good agreement with the surface conductivity measurements, showing an isoelectric point at pH 3.5 for the H-terminated diamond surface. We discuss the results in terms of the Coulombic screening by electrolyte ions of the surface potential, which is induced by a pH-dependent surface charge. The origin of this surface charge is discussed in terms of charge regulation by amphoteric hydroxyl surface groups and unsymmetrical adsorption of hydroxide and hydronium ions induced by the hydrophobic nature of the H-terminated diamond surface. This surface charge can have important consequences for processes governed by the diamond/aqueous interface, such as electron transfer to charged redox molecules, adsorption of charged molecules and proteins, and ion sensitivity.     

Evaluation of different nonspecific binding blocking agents deposited inside poly(methyl methacrylate) microfluidic flow-cells

Poly(methyl methacrylate) (PMMA) flow-cells containing microwells were deposited with different nonspecific binding blocking agents, namely, bovine serum albumin (BSA), cationic lipid (DOTAP:DOPE) and diethylene glycol dimethyl ether (DEGDME). Water contact angle (WCA) and atomic force microscope (AFM) measurements were carried out to confirm the successful depositions of BSA, DOTAP, and DEGDME onto the PMMA surfaces. Fluorescent intensity measurements were performed to evaluate the degree of nonspecific adsorption of Cy5-labeled anti-IgG proteins onto plain and oxygen plasma-treated (PT) PMMA flow-cells as well as PMMA flow-cells deposited with different above-mentioned blocking agents. We then employed a label-free detection method called total internal reflection ellipsometry (TIRE) to evaluate the stability of the deposited blocking agents inside the PMMA flow-cells. It was found that, while DOTAP:DOPE was the best agent for blocking the nonspecific adsorption, it could be removed from the PMMA surfaces of the flow-cells upon rinsing with phosphate buffered saline (PBS) and later deposited back onto the Au-coated glass sensing substrate of the TIRE. The removal of the blocking agents from PMMA surfaces and their deposition onto the sensing substrate were further manifested by measuring the kinetics and the amount of adsorbed anti-α-hCG proteins. Overall, the dry DEGDME coating by plasma-enhanced chemical vapor deposition (PECVD) showed very good blocking and excellent stability for subsequent assay inside the microwells. Our results could be useful when one considers what blocking agents should be used for PMMA-based microfluidic immunosensor or biosensor devices by looking at both the blocking efficiency and the stability of the blocking agent.     

Electrostatic interactions of colloidal particles at vanishing ionic strength

Electrostatic interactions of colloidal particles are typically screened by mobile ions in the solvent. We measure the forces between isolated pairs of colloidal polymer microspheres as the density of bulk ions vanishes. The ionic strength is controlled by varying the concentration of surfactant (NaAOT) in a nonpolar solvent (hexadecane). While interactions are well-described by the familiar screened-Coulomb form at high surfactant concentrations, they are experimentally indistinguishable from bare Coulomb interactions at low surfactant concentration. Interactions are strongest just above the critical micelle concentration, where particles can obtain high surface potentials without significant screening, kappaa < 1. Exploiting the absence of significant charge renormalization, we are able to construct a simple thermodynamic model capturing the role of reverse micelles in charging the particle surface. These measurements provide novel access to electrostatic forces in the limit where the particle size is much less than the screening length, which is relevant not just to the nonpolar suspensions described here, but also to aqueous suspensions of nanoparticles.     

Properties of mixed DOTAP-DPPC bilayer membranes as reported by differential scanning calorimetry and dynamic light scattering measurements

We have investigated the effect of a cationic lipid [DOTAP] on both the thermotropic phase behavior and the structural organization of aqueous dispersions of dipalmitoyl-phosphatidylcholine [DPPC] by means of high-sensitivity differential scanning calorimetry and dynamic light scattering measurements. We find that the incorporation of increasing quantities of DOTAP progressively reduces the temperature and the enthalpy of the gel-to-liquid crystalline transition. We are further showing that, in mixed DOTAP-DPPC systems, the reduction of the phase transition temperature is accompanied by a reduction of the average size of the structures present in the aqueous mixtures, whatever the DOTAP concentration is. These results, which extend a previous investigation by Campbell et al. (Campbell, R. B.; Balasubramanian, S. V.; Straubinger, R. M.; Biochim. Biosphys. Acta 2001, 27, 1512.) limited to a DOTAP concentration below 20 mol %, confirm that the insertion of cationic head groups in zwitterionic phosphatidylcholine bilayers facilitates the formation of stable, relatively small, unilamellar vesicles. This self-assembling restructuring from an aqueous multilamellar structure toward a liposomal phase is favored by decreasing the phospholipid phase transition temperature and by increasing the temperature of the system. This reduction of the average size and the appearance of a stable liposomal phase is also promoted by a heating and cooling thermal treatment.     

Self-organization of polymer particles on hydrophobic solid substrates in aqueous media. II. Self-organization of cationic polymer particles on polymer films and wettability control with particle monolayers

Self-organization of cationic polymer particles through hydrophobic interaction on polymer films in aqueous system and characteristic properties of the resulting particle monolayers were investigated. Cationic polymer particles bearing quaternary ammonium groups on their surfaces effectively self-organized on polymer films. With an increase of the particle surface charge density, the surface coverage and average aggregate size (N _a) decreased. The surface coverage control was accomplished by tuning the ionic strength of the media. The wettability of polymer films for water was imparted by the formation of particle monolayers on them. Annealing of the particle monolayers resulted in the increase of the adhesive strength, while the wettability for water was lost. Further improvements of both wettability and adhesive strength of particle monolayers were achieved by the immobilization of silica colloids on the particle monolayers. This method would be effective for the hydrophilization of polymer films.     

Adsorption of poly(vinyl formamide-co-vinyl amine) onto silica particle surfaces and stability of the formed hybrid materials

In order to produce silica/polyelectrolyte hybrid materials the adsorption of the polyelectrolyte poly(vinyl formamide-co-vinyl amine), P(VFA-co-VAm) was investigated. The adsorption of the P(VFA-co-VAm) from an aqueous solution onto silica surface is strongly influenced by the pH value and ionic strength of the aqueous solution, as well as the concentration of polyelectrolyte. The adsorption of the positively charged P(VFA-co-VAm) molecules on the negatively charged silica particles offers a way to control the surface charge properties of the formed hybrid material. Changes in surface charges during the polyelectrolyte adsorption were studied by potentiometric titration and electrokinetic measurements. X-ray photoelectron spectroscopy (XPS) was employed to obtain information about the amount of the adsorbed polyelectrolyte and its chemical structure. The stability of the adsorbed P(VFA-co-VAm) was investigated by extraction experiments and streaming potential measurements. It was shown, that polyelectrolyte layer is instable in an acidic environment. At a low pH value a high number of amino groups are protonated that increases the solubility of the polyelectrolyte chains. The solvatation process is able to overcompensate the attractive electrostatic forces fixing the polyelectrolyte molecules on the substrate material surface. Hence, the polyelectrolyte layer partially undergoes dissolving process.     

Charge renormalization of charged spheres based on thermodynamic properties

At strong electrostatic coupling, counterions are accumulated in the vicinity of the surface of the charged particle with intrinsic charge Z. In order to explain the behavior of highly charged particles, effective charge Z(*) is therefore invoked in the models based on Debye-Huckel approximation, such as the Derjaguin-Landau-Verwey-Overbeek potential. For a salt-free colloidal suspension, we perform Monte Carlo simulations to obtain various thermodynamic properties omega in a spherical Wigner-Seitz cell. The effect of dielectric discontinuity is examined. We show that at the same particle volume fraction, counterions around a highly charged sphere with Z may display the same value of omega as those around a weakly charged sphere with Z(*), i.e., omega(Z)=omega(Z(*)). There exists a maximally attainable value of omega at which Z=Z(*). Defining Z(*) as the effective charge, we find that the effective charge passes through a maximum and declines again due to ion-ion correlation as the number of counterions is increased. The effective charge is even smaller if one adopts the Debye-Huckel expression omega(DH). Our results suggest that charge renormalization can be performed by chemical potential, which may be observed in osmotic pressure measurements.     

Dielectric properties of aqueous zwitterionic liposome suspensions

The dielectric spectra of aqueous suspensions of unilamellar liposomial vesicles built up by zwitterionic phospholipids (dipalmitoylphosphatidyl-choline, DPPC) were measured over the frequency range extending from 1 kHz to 10 MHz, where the interfacial polarization effects, due to the highly heterogeneous properties of the system, prevail. The dielectric parameters, i.e., the permittivity epsilon'(omega) and the electrical conductivity sigma(omega), have been analyzed in terms of dielectric models based on the effective medium approximation theory, considering the contribution associated with the bulk ion diffusion on both sides of the aqueous interfaces. The zwitterionic character of the lipidic bilayer has been modeled by introducing an "apparent" surface charge density at both the inner and outer aqueous interface, which causes a tangential ion diffusion similar to the one occurring in charged colloidal particle suspensions. A good agreement with the experimental results has been found for all the liposomes investigated, with size ranging from 100 to 1000 nm in diameter, and the most relevant parameters have briefly discussed in the light of the effective medium approximation theory.     

Viscosity, Surface Tension, and Refractive Index Measurements of Mixtures of Isomeric Butanediols with Water

Solution properties of aqueous mixtures of isomeric butanediols have been investigated employing viscosity, surface tension, and index of refraction measurements as functions of temperature. The deviation of viscosity, surface tension, and molar refraction from ideal solution behavior is evaluated from the experimental data. The deviation from ideality is discussed in terms of molecular interactions between the components. Surface activity of the diols is evident from the surface tension measurements. It is found that the degree of hydrophobicity of the diols varies in the order 1,2 > 2,3 > 1,3 > 1,4. The strength of interaction of diols with the water varies in the order 2,3 > 1,4 1,3 > 1,2.     

Effect of salt on the hybridization of DNA by sequential immobilization of oligonucleotides at the air-water interface in the presence of ODA/DOTAP monolayers

The effect of low ionic strength on the binding of preformed DNA duplexes and the hybridization of single-stranded oligonucleotides at the air-water interface in the presence of cationic Langmuir monolayers of octadecylamine (ODA), as well as 1,2-dioleoyloxytrimethylammonium propane (DOTAP), is investigated. The complexation of the single-stranded DNA molecules and preformed duplexes with NaCl in solution with ODA/DOTAP Langmuir monolayers was followed in time by monitoring the pressure-area isotherms, wherein a very large and rapid expansion of the ODA/DOTAP monolayer was observed. In the case of sequential immobilization of complementary oligonucleotides, after addition of the complementary strand and intercalator, there was not much expansion, indicative of the fact that equilibrium had been rapidly achieved. Langmuir-Blodgett (LB) films of the ODA/DOTAP-DNA complex were formed on different substrates and characterized using quartz-crystal microgravimetry (QCM), fluorescence spectroscopy, and thermal melting studies. These measurements clearly showed that the preformed duplexes retained their native form as double helices and further, hybridization of the complementary single-stranded DNA molecules had occurred at the air-water interface, leading to the characteristic double-helical structure.     

Polyadenylic acid binding on cationic liposomes doped with the non-ionic nucleolipid Lauroyl Uridine

In this work unilamellar liposomes doped with a novel non-ionic 5′-Uridine-head nucleolipid, Lauroyl Uridine (LU), were prepared and characterized for their ability to interact with the polynucleotide polyadenylic acid (poly-A). Vesicles, were made up of the cationic lipid DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propane), the zwitterionic lipid DOPE (1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine), and the novel amphiphile Lauroyl Uridine. The influence of the non-ionic nucleolipid on essential liposomes properties, such as the structure and net charge was first investigated by a comparative analysis performed on the different lipoplex preparations by means of ζ-potential and size measurements. Both structure and net charge of liposomes were shown to be not modified by the presence of the non-ionic nucleolipid.The role of the synthetic lipid inserted as anchor in the liposome bilayer in the condensation process between vesicles and the polynucleotide poly-A was then analyzed by UV–vis, Circular Dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The data presented comparative UV–vis analyses that evidenced the occurrence of staking interactions in the poly-A only in LU containing lipoplexes. CD and NMR studies indicated the presence of H-bonding interaction between Lauroyl Uridine containing vesicles and the polynucleotide poly-A. The results presented in this work support a role for Lauroyl Uridine in A-U molecular recognition, thus, suggesting that cationic liposomes doped with the non-ionic nucleolipid Lauroyl Uridine could represent a model system to study molecular interactions among single stranded polynucleotides and lipid anchor bearing the complementary bases.     

Phase behavior of DOPE/TritonX100 (reduced) in dilute aqueous solution: aggregate structure and pH-dependence

The phase behavior of dilute mixtures of dioleoylphosphatidylethanolamine (DOPE) and reduced TritonX100 (TX100(r)) has been investigated at pH 7.4 and 10. Using simple turbidity measurements and optical observations, together with cryo-transmission electron microscopy (cryo-TEM), we estimate the phase boundaries. We show that at both pH 7.4 and 10, a very large amount of surfactant is needed for the onset of micelle formation (X(TX100(r)) approximately 0.60-0.70) as well as for a complete solubilization of DOPE into mixed micelles (X(TX100(r)) > 0.94). We find that the micelles that are formed at high TX100(r) concentrations are of spherical shape. Increasing the pH from 7.4 to 10 has a comparably small effect on the transition from a lamellar (Lalpha) to a micellar (L1) phase. However, the reversed hexagonal phase (H(II)) that is present at low surfactant content at pH 7.4 is absent at pH 10. This is due to the partial negative charge of DOPE at pH 10. We determine the fraction of charged DOPE (alpha = 0.34) at pH 10 in a 150 mM NaCl buffer using zeta-potential (zeta-potential) measurements in combination with a Poisson-Boltzmann (PB) model. The intrinsic pK(a) of the primary amino group of DOPE, in a pure DOPE membrane, is estimated to 9.15 +/- 0.2.