Effect of chain lengths of PEO-PPO-PEO on small unilamellar liposome morphology and stability: an AFM investigation

The morphology and stability of small unilamellar egg yolk phosphatidylcholine (EggPC) liposomes modified with the Pluronic copolymer (poly (oxyethylene)-poly (oxypropylene)-poly (oxyethylene) (PEO-PPO-PEO)) with different compositions on mica surface have been investigated using atomic force microscopy. Morphology studies reveal significant morphological changes of liposomes upon incorporating the Pluronic copolymer. Bilayers are observed for Pluronic with small hydrophilic (PEO) chain lengths such as L81 [(PEO)2(PPO)40(PEO)2] and L121 [(PEO)4(PPO)60(PEO)4]; bilayer and vesicle coexistence is observed for P85 [(PEO)26(PPO)39.5(PEO)26] and F87 [(PEO)61.1(PPO)39.7(PEO)61.1]; and stable vesicles are observed for F88 [(PEO)103.5(PPO)39.2(PEO)103.5], F127 [(PEO)100(PPO)65(PEO)100], and F108 [(PEO)132.6(PPO)50.3(PEO)132.6]. The micromechanical properties of Pluronic-modified EggPC vesicles were studied by analyzing AFM approaching force curve. The bending modulus (k(c)) of the Pluronic-modified EggPC vesicles increased several-fold compared with that of the pure EggPC vesicles. The significant difference is due to the enhanced rigidity of the EggPC vesicles as a result of the incorporation of PPO molecules and PEO chains. Based on the analysis of onset point by AFM and diameters of vesicles by light scattering, it was concluded that the favorable model to describe the polymer-bilayer interaction is the membrane-spanning model.     

Stability of binary model membranes--prediction of the liposome stability by the Langmuir monolayer study

In this paper, usefulness of the Langmuir monolayer study is demonstrated for predictions of the stability of liposomes composed of dipalmitoyl phosphatidylcholine (DPPC) and cholesterol (Chol). Thermodynamic analysis of the surface pressure (π)-area (A) isotherms of the DPPC/Chol systems was performed, which allowed for concluding on miscibility of the components, their molecular packing, and the interactions between molecules. It was found that the most stable system, in which the strongest interactions between molecules occured, was DPPC/Chol at x(Chol)=0.25. The stability of liposomes of the same composition as that in the Langmuir monolayers was analyzed by determining the size distribution of vesicles and the polydispersity as a function of time. The changes of these parameters confirmed that the system of the greatest stability is that with low Chol content.     

Nanomechanics of phospholipid LB film studied layer by layer with AFM

Background

Phospholipid, a main component of cell membrane, has been explored as a model system of the cell membrane and temporary scaffold materials in recent studies. The mechanical properties of phospholipid layers are essentially interesting since it is involved in several biological processes.

Results

Here, the nanomechanical properties such as indentation force, adhesion force and DMT (Derjaguin-Müller-Toporov) modulus of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) Langmuir-Blodgett (LB) films were analyzed layer by layer with Atomic Force Microscope (AFM) under deionized water condition.

Conclusions

The penetration distances in the indentation force curves are equal to the thicknesses of phospholipid films, and the yield forces of DSPC LB films in deionized water are smaller than that of similar lipid films in buffered solutions due to the influence of ions. Moreover, the DMT modulus of upper layer DSPC LB film is different from that of monolayer DSPC LB film due to the influence of their different substrates. Our results suggest that environment such as surrounding ions and substrate will strongly influence the measured nano-mechanical properties of the lipid bilayer, especially that of the down layer.

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Graphical Abstract A process about the exploration of nanomechanics of DSPC LB film.

     

Nanostructure of a "carpet"-like dense layer/polyelectrolyte brush layer in a block copolymer monolayer at the air-water interface

The "carpet"/brush double layer structure in the polyelectrolyte layer in the amphiphilic diblock copolymer monolayer at the air-water interface was quantitatively studied by in situ neutron reflectometry in addition to X-ray reflectivity measurements. As a result of the higher contrast between polyelectrolyte [poly(methacrylic acid)] and solvent (D(2)O) for the neutron, the brush structure could be estimated more accurately as a function of surface pressure, that is, brush density. The thickness of the carpet layer, which is thought to be formed to reduce the interfacial free energy between water and the hydrophobic layer, was almost constant at 10-20 A at any surface pressure studied. Growth was clearly observed in the whole brush length with increasing surface pressure, and it was estimated to be almost 60% of the full-stretch length of the ionic polymer chain. Furthermore, by the comparison of density profiles by neutron and X-ray reflectometry, an anomalous hydration was suggested.     

Thermal stability of DNA in DNA-induced DOTAP liposome aggregates

The influence on the melting of calf thymus DNA induced by cationic liposomes, commonly used in gene therapy, was studied by means of ultraviolet spectrophotometry and differential scanning calorimetry. Both the two methods reveal that DNA in DNA-induced liposome complexes undergoes a denaturation process at a much higher temperature than free DNA does. The extent of protection strongly depends on the charge ratio R(+/−) of liposome-DNA complexes. In the case of dioleoyl trimethyl ammonium propane (DOTAP) liposomes, the maximum of the stabilization occurs at R(+/−)=0.7, where the DNA is still native up to temperatures higher than 100°C. This protection against denaturation up to higher temperatures might be of importance for bio-technological applications, such as biomolecular separation, antigene sequencing and for drug design purpose.     

Formation of an intact liposome layer adsorbed on oxidized gold confirmed by three complementary techniques: QCM‐D,AFM and confocal fluorescence microscopy

Supported layers of vesicles of dimyristoyl and dipalmitoyl phosphatidylcholine (DMPC and DPPC) containing cholesterol (CHOL) are adequate models for eukaryotic plasma membranes. Among the possible substrates to support these layers, gold offers the possibility of being used as an electrode for application in sensors. However, the formation of intact liposome layers on gold is not completely understood and several authors use more or less complex strategies to bind the liposomes. In this work we investigate the adsorption of unilamellar vesicles of DMPC, DMPC/CHOL and DMPC/DPPC/CHOL on the surface of oxidized gold using a quartz crystal microbalance with dissipation, atomic force microscopy and laser scanning confocal fluorescence microscopy. The results of all techniques indicate that for lipid concentrations ≥ 0.7 mg?mL^–1 a dense layer of intact liposomes irreversibly adsorbs on the gold surface. Copyright © 2011 John Wiley & Sons, Ltd.     

Dielectric permittivity in a dense layer of the hydration shell of an ion in a strong-electrolyte solution

Based on the experimental data on the dielectric dispersion and the static dielectric permittivity of solutions of strong electrolytes, the effective value of the latter in the dense layer of the hydration shell of an ion has been calculated. The calculations have been carried out in terms of the three-layer model of the hydration complex. The calculations have shown that for the solutions of strong electrolytes the value of the static dielectric permittivity (dielectric constant) in the dense layer of the hydration shell of an ion proves to be close to 2 and is almost independent of the concentration and temperature.     

Effect of amphiphilic drugs on the stability and zeta-potential of their liposome formulations: a study with prednisolone, diazepam, and griseofulvin

Multilamellar liposomes consisting of phosphatidylcholine and incorporating prednisolone (PZ), diazepam (DZ), or griseofulvin (GF) were prepared and characterized. Liposome size, surface charge, and stability (in buffer and serum proteins) were measured for drug-incorporating liposomes and empty liposomes for comparison. The results reveal that for all drugs studied drug incorporation has a substantial effect on the vesicle zeta-potential and stability. Drug-incorporating liposomes have a negative surface charge, while their membrane integrity is significantly higher when compared with that of empty liposomes. Release of DZ from liposomes is induced by dilution. Summarizing, the results of this study demonstrate that the presence of PZ, DZ, or GF in liposome membranes has a significant effect on main vesicle properties and correlates well with those obtained previously for hydrochlorothiazide and chlorothiazide. Thereby, we may conclude that the previously demonstrated effects of the thiazides on liposome properties are not solely related to their structure.     

Two-dimensional arrays of human C-reactive protein formed on a dense aqueoys subphase underneath a lipid layer

A recently developed protein spreading technique, using a subphase of higher density than that of the injected protein solution, was combined with the lipid monolayer approach for two-dimensional crystallization experiments of human C-reactive protein (hCRP) at the air-water interface. Densely packed two-dimensional arrays of hCRP adsorbed on a stearylamine/egg yolk phosphatidylcholine lipid layer have been obtained in the pH range from 5.4 to 5.8. Correct choices of the lipid mixture and of the pH of the subphase were important for the formation of large arrays. Image analysis of transmission electron micrographs showed that the densely packed arrays do not possess crystalline order. The orientational homogeneity of the protein molecules adsorbed on the lipid layer could be used, however, to facilitate single particle averaging techniques. At a resolution of about 1.5 nm a clear handedness of the five-fold symmetric molecule becomes visible, and an asymmetric mass distribution of each subunit is revealed.     

Model of the dense part of the double layer in the absence of specific adsorption

A model is suggested for the dense part of the double layer in the absence of specific adsorption, which is based on the following assumptions: (1) There are two kinds of adsorbed solvent particles on the electrode surface: associates freely oriented in the double layer field and separate chemisorbed water dipoles; 92) the total number of solvent molecules on the surface is determined by the geometric dimensions of associates and chemisorbed dipoles; (3) account is taken of the electrostatic interaction of associates and chemisorbed dipoles in the surface layer; (4) a discreteness coefficient is introduced, accounting for the degree of influence of the field due to all the other dipoles and associates in the surface layer on the dipole or associate being considered; (5) the dipole moment of a chemisorbed water molecule exceeds the value of μH₂O in the bulk of the solution; (6) account is taken of the decrease with increasing temperature of the average number of water molecules in surface associate.The calculations on the basis of this model describe satisfactorily the shape of the differential capacity curves of the dense layer and their temperature dependence for the system Hg-aqueous NaF solution.     

Are redox probes a useful indicator of film stability? An electrochemical,AFM and XPS study of electrografted amine films on carbon

The electrochemical response of solution-based redox probes is commonly used to detect and monitor the formation and stability of films grafted to conducting substrates. In this work we examine redox probe responses at films grafted to glassy carbon (GC) and pyrolysed photoresist film (PPF) by electrooxidation of aliphatic primary amines. Cyclic voltammograms are obtained before and after soaking the modified surfaces in phosphate buffer and acetonitrile. For conditions where films exhibit large changes in electrochemical blocking properties, AFM measurements of film thickness and surface roughness, and XPS measurements of surface composition, are used to monitor the stability of the films. No changes in film structure or composition can be detected, demonstrating that electrochemical loss of blocking properties cannot be equated with large-scale loss of surface film. The origin of changes in probe response is discussed.     

Influence of the diffuse layer overcharging or undercharging on the stability of charged interfaces: a restricted grand canonical Monte Carlo study

We have applied a restricted grand canonical Monte Carlo procedure to describe, in the framework of the primitive model, the counterion exchange mechanism between diffuse layers of counterions surrounding segregated charged lamellae. The net charge transfer between the dense and dilute domains is shown to vary as a function of the valence of the neutralizing counterions: undercharging of the dense interlayer is detected in the presence of monovalent counterions and overcharging with divalent counterions. Furthermore, no net reduction of the swelling pressure is detected for monovalent counterions, while a large enhancement of the net interlamellar attraction is found for charged lamellae neutralized by divalent counterions.     

Direct observation of the phase transition for a poly(N-isopropylacryamide) layer grafted onto a solid surface by AFM and QCM-D

The temperature-induced structural changes of a thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) layer grafted onto a silica substrate were investigated in aqueous solution using an atomic force microscope (AFM) and a quartz crystal microbalance with dissipation (QCM-D). A PNIPAM layer was grafted onto the silicon wafer surface by free radical polymerization of NIPAM to obtain a high molecular weight polymer layer with low-grafting density overall. By AFM imaging, the transition of the grafted PNIPAM chains from a brush-like to a mushroom-like state was clearly visualized: The surface images of the plate were featureless at temperatures below the LCST commensurate with a brush-like layer, whereas above the LCST, a large number of domain structures with a characteristic size of approximately 100 nm were seen on the surface. Both frequency and dissipation data obtained using QCM-D showed a significant change at the LCST. Analysis of these data confirmed that the observed PNIPAM structural transition was caused by a collapse of the brush-like structure as a result of dehydration of the polymer chains.     

AFM study of mineral wettability with reservoir oils

Wettability plays a key role in determining fluid distributions and consequently the multiphase flow and transport in petroleum reservoirs. Many crude oils have polar organic components that collect at oil-water interfaces and can adsorb onto the mineral surface if the brine film breaks, rendering the medium oil-wet or mixed-wet. Mica and silica surfaces have been aged with brine and crude oils to induce oil component adsorption. Bulk oil is eventually replaced by water in these experiments by washing with common solvents without ever drying the mineral surface. The organic deposit on the mineral surface is studied by atomic force microscopy in the tapping mode under water. Drying the surface during the removal of bulk oil induces artifacts; it is essential to keep the surface wet at all times before atomic force microscopy or contact angle measurement. As the mean thickness of the organic deposit increases, the oil-water contact angle increases. The organic deposits left behind after extraction of oil by common aromatic solvents used in core studies, such as toluene and decalin, are thinner than those left behind by non-aromatic solvents, such as cyclohexane. The force of adhesion with a probe sphere for minerals aged with just the asphaltene fraction is similar to that of the whole oil. The force of adhesion for the minerals aged with just the resin fraction is the highest of all SARA (saturates, aromatics, resins, and asphaltenes) fractions.     

AFM study of defect-induced depressions of the smectic-A/air interface

The smectic-A/air interface of liquid-crystal droplets with antagonistic boundary conditions is studied by atomic force microscopy (AFM). The droplets are prepared on coated silicon wafers on which a planar alignment is preferred in contrast to the homeotropic alignment at the air interface. As a result, focal conic defects appear in the smectic-A phase causing a characteristic pattern of depressions in the droplet surface. The dimensions of the defect-induced depressions are measured by AFM as a function of temperature for two different compounds possessing a smectic-A-isotropic and a smectic-A-nematic transition. Whereas the results are independent of temperature in the smectic-A-isotropic case, reflecting the first-order nature of the transition, a pronounced temperature dependence is observed for the second compound, where the depth of the defect-induced depressions decreases continuously with increasing temperature and vanishes at the second-order transition to the nematic phase. These observations can be qualitatively explained through the behavior of the layer compressional elastic constant at the smectic-A-nematic transition.     

An atomic force microscope (AFM) and tapping mode AFM study of the solvent-induced crystallization of polycarbonate thin films

Thin films of bisphenol-A polycarbonate (PC) were progressively crystallized in a controllable manner under the action of an appropriate plasticizing agent. No conducting layer was applied to the polymer surface, so that imaging with the atomic force microscope (AFM) left the sample exposed for additional plasticizer treatment. The PC sample was observed many times throughout the crystallization process using the AFM in both the contact and tapping modes. Data regarding the growth process for spherulites in addition to high-resolution morphology studies were achieved. © 1996 John Wiley & Sons, Inc.     

Real-time observation of lamellar branching induced by an AFM tip and the stability of induced nuclei

Branching of edge-on lamellae of poly(bisphenol A octane ether) (BA-C8) was studied at room temperature (21 +/- 1 degrees C) using real-time tapping mode atomic force microscopy (AFM). Lamellar branches were shown to develop from induced nuclei that had originated from lamellar defects, namely protruding cilia and loose loops, of parent lamellae. Induced nuclei appeared and also disappeared-a phenomenon observed first time by AFM. Induced nuclei are one of the possible origins for the branching of lamellae. Branching of edge-on lamellae can be induced by an AFM tip though the adjustment of the set-point amplitude ratio (r(sp)). Using an r(sp) smaller than 0.65, defects could be created on the lamellar surface, which gave rise to lamellar branches.     

Structural stability against disintegration by anionic lipids rationalizes the efficiency of cationic liposome/DNA complexes

Reported here is the correlation between the transfection efficiency of cationic liposome/DNA complexes (lipoplexes) and the structural evolution that they undergo when interacting with anionic membrane lipids. Multicomponent lipoplexes, incorporating from three to six lipid species simultaneously, presented a much higher transfection efficiency than binary lipoplexes, which are more commonly used for gene-delivery purposes. The discovery that a high transfection efficiency can be achieved by employing multicomponent complexes at a lower-than-ever-before membrane charge density of lipoplexes was of primary significance. Synchrotron small-angle X-ray diffraction (SAXD) experiments showed that anionic liposomes made of dioleoylphosphatidylglycerol (DOPG) disintegrated the lamellar phase of lipoplexes. DNA unbinding was measured by electrophoresis on agarose gels. Most importantly, structural changes induced by anionic lipids strictly depended on the lipid composition of lipoplexes. We found evidence of the existence of three different regimes of stability related to the interaction between complexes and anionic membranes. Both unstable (with low membrane charge density, sigmaM) and highly stable lipoplexes (with high sigmaM) exhibited low transfection efficiency whereas highly efficient multicomponent lipoplexes exhibited an "optimal stability". This intermediate regime reflects a compromise between two opposing constraints: protection of DNA in the cytosol and endosomal escape. Here we advance the concept that structural stability, upon interaction with cellular anionic lipids, is a key factor governing the transfection efficiency of lipoplexes. Possible molecular mechanisms underlying experimental observations are also discussed.