Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

siRNA carriers based on carbosilane dendrimers affect zeta potential and size of phospholipid vesicles

One of the major limitations in gene therapy is an inability of naked siRNA to passively diffuse through negatively charged cell membranes. Therefore, the siRNA transport into a cell requires efficient carriers. In this work we analyzed the charge-dependent interaction of the complexes of cationic carbosilane dendrimers (CBD) and anti-HIV siRNA (dendriplexes) with the model membranes - large unilamellar vesicles (LUV). We used the second generation of branched with CBD carbon-silicon bonds (CBD-CS) which are water-stable and that of oxygen-silicon bonds (CBD-OS) which are slowly hydrolyzed in aqueous solutions. The LUVs were composed of zwitterionic dimyristoylphosphatidylcholine (DMPC), negatively charged dipalmitoylphosphatidylglycerol (DPPG) and their mixture (DMPC/DPPG, molar ratio 7:3). The interaction of dendriplexes with LUVs affected both zeta potential and size of the vesicles. The changes of these values were larger for the negatively charged LUV. CBD-CS resulted in the decrease of zeta potential values to more negative ones, whereas an opposite effect took place for CBD-OS suggesting a different kind of interaction between LUVs and the dendriplexes. The results indicate that both CBD-CS and CBD-OS can be used for transport of siRNA into the cells. However, CBD-CS are preferred due to a better stability in water and improved bioavailability of siRNA on their surface.     

Visualizing the Attack of RNase Enzymes on Dendriplexes and Naked RNA Using Atomic Force Microscopy

Cationic polymers such as poly(amidoamine), PAMAM, dendrimers have been used to electrostatically complex siRNA molecules forming dendriplexes for enhancing the cytoplasmic delivery of the encapsulated cargo. However, excess PAMAM dendrimers is typically used to protect the loaded siRNA against enzymatic attack, which results in systemic toxicity that hinders the in vivo use of these particles. In this paper, we evaluate the ability of G4 (flexible) and G5 (rigid) dendrimers to complex model siRNA molecules at low +/− ratio of 2/1 upon incubation for 20 minutes and 24 hours. We examine the ability of the formed G4 and G5 dendriplexes to shield the loaded siRNA molecules and protect them from degradation by RNase V1 enzymes using atomic force microscopy (AFM). Results show that G4 and G5 dendrimers form similar hexagonal complexes upon incubation with siRNA molecules for 20 minutes with average full width of 43±19.3 nm and 62±8.3 at half the maximum height, respectively. AFM images show that these G4 and G5 dendriplexes were attacked by RNase V1 enzyme leading to degradation of the exposed RNA molecules that increased with the increase in incubation time. In comparison, incubating G4 and G5 dendrimers with siRNA for 24 hours led to the formation of large particles with average full width of 263±60 nm and 48.3±2.5 nm at half the maximum height, respectively. Both G4 and G5 dendriplexes had a dense central core that proved to shield the loaded RNA molecules from enzymatic attack for up to 60 minutes. These results show the feasibility of formulating G4 and G5 dendriplexes at a low N/P (+/−) ratio that can resist degradation by RNase enzymes, which reduces the risk of inducing non-specific toxicity when used in vivo.     

Interactions of phosphorus-containing dendrimers with liposomes

The influence of cationic phosphorus-containing dendrimers generation 3 and 4 on model DMPC or DPPC lipid membranes was studied. Measurements of fluorescence anisotropy and differential scanning calorimetry (DSC) were applied to assess changes in lipid bilayer parameters, including fluidity, anisotropy, and phase-transition temperature. Interaction with both hydrophobic and hydrophilic regions of the bilayer was followed by these methods. Dendrimers of both generations influence lipid bilayers by decreasing membrane fluidity. The results suggest that dendrimers can interact both with the hydrophobic part and the polar head-group region of the phospholipid bilayer. Higher generation dendrimers interact more strongly with model membranes, and the concentration, as well as the generation, is of similar importance.     

Interaction of cationic carbosilane dendrimers and their complexes with siRNA with erythrocytes and red blood cell ghosts

We have investigated the interactions between cationic NN16 and BDBR0011 carbosilane dendrimers with red blood cells or their cell membranes. The carbosilane dendrimers used possess 16 cationic functional groups. Both the dendrimers are made of water-stable carbon–silicon bonds, but NN16 possesses some oxygen–silicon bonds that are unstable in water. The nucleic acid used in the experiments was targeted against GAG-1 gene from the human immunodeficiency virus, HIV-1.By binding to the outer leaflet of the membrane, carbosilane dendrimers decreased the fluidity of the hydrophilic part of the membrane but increased the fluidity of the hydrophobic interior. They induced hemolysis, but did not change the morphology of the cells. Increasing concentrations of dendrimers induced erythrocyte aggregation. Binding of short interfering ribonucleic acid (siRNA) to a dendrimer molecule decreased the availability of cationic groups and diminished their cytotoxicity. siRNA–dendrimer complexes changed neither the fluidity of biological membranes nor caused cell hemolysis. Addition of dendriplexes to red blood cell suspension induced echinocyte formation.     

Gene delivery by dendrimers operates via a cholesterol dependent pathway

Understanding the cellular uptake and intracellular trafficking of dendrimer–DNA complexes is an important prerequisite for improving the transfection efficiency of non-viral vector-mediated gene delivery. Dendrimers are synthetic polymers used for gene transfer. Although these cationic molecules show promise as versatile DNA carriers, very little is known about the mechanism of gene delivery. This paper investigates how the uptake occurs, using an endothelial cell line as model, and evaluates whether the internalization of dendriplexes takes place randomly on the cell surface or at preferential sites such as membrane rafts. Following extraction of plasma membrane cholesterol, the transfection efficiency of the gene delivered by dendrimers was drastically decreased. Replenishment of membrane cholesterol restored the gene expression. The binding and especially internalization of dendriplexes was strongly reduced by cholesterol depletion before transfection. However, cholesterol removal after transfection did not inhibit expression of the delivered gene. Fluorescent dendriplexes co-localize with the ganglioside GM1 present into membrane rafts in both an immunoprecipitation assay and confocal microscopy studies. These data strongly suggest that membrane cholesterol and raft integrity are physiologically relevant for the cellular uptake of dendrimer–DNA complexes. Hence these findings provide evidence that membrane rafts are important for the internalization of non-viral vectors in gene therapy.     

Interaction study between maltose-modified PPI dendrimers and lipidic model membranes

The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers — OS) or completely (dense shell glycodendrimers — DS) modified with maltose residues. As a model membrane, two types of 100 nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D₂O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe³⁺-chelates, which initiated reactions that were dependent on membrane charge: Fe³⁺-EDTA and Fe³⁺-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe³⁺-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe³⁺-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.     

Polyamidoamine dendrimers with different surface charge as carriers in anticancer drug delivery

Second-generation (G2) polyamidoamine (PAMAM) dendrimers are branched polymers containing 16 surface primary amine groups. Due to their structural properties, these polymers can be used as universal carriers in various drug delivery systems. Amine-terminated PAMAM dendrimers are characterized by a high positive surface charge, leading to effective but nonspecific interactions with negatively charged cell plasmatic membranes. To reduce the nonspecific internalization of PAMAM dendrimers, their primary amine groups are often modified by acetic or succinic anhydrides, polyethylene glycol derivatives and other compounds. In this work, the role of primary amine groups, which are localized on the surface of doxorubicin-conjugated (Dox) dendrimers, was studied with regard to their intracellular distribution and internalization rates using SKOV3 human ovarian adenocarcinoma cells. It was demonstrated that all Dox-labeled G2-derivatives containing different numbers of acetamide groups synthesized in this work show high rates of cellular uptake at 37°С. As expected, the conjugate carrying the maximum number of primary amine groups demonstrated the highest rates of binding and endocytosis. At the same time, the G2-Dox conjugate containing the maximum number of acetamide groups showed colocalization with LAMP2, a marker of lysosomes and late endosomes, as well as the highest level of cytotoxic activity against SKOV3 cells. We conclude that second-generation PAMAM dendrimers are characterized by varied pathways of internalization and intracellular distribution due to the number of primary amine groups on their surface and, as a consequence, a different surface charge.     

The effect of surface charge density on valinomycin-K+ complex formation in model membranes

The model membrane approach was used to investigate the surface charge effect on the ion-antibiotic complexation process. Mixed monolayers of valinomycin and lipids were spread on subphases containing K⁺ or Na⁺. The surface charge density was modified by spreading ionizable valinomycin analogs on aqueous subphases of different pH or by changing the nature of the lipid (neutral, negatively charged) in the mixed film. Surface pressure and surface potential measurements demonstrated that a neutral lipid (phosphatidylcholine) or positively charged valinomycin analogs didn't enhance the antibiotic complexing capacity. However, a maximal complexation is reached for a critical lipid concentration in the valinomycin-phosphatidylserine mixed film. The role of the surface charge on the valinomycin complexing properties was examined in terms of the Gouy-Chapman theory. As a consequence of the negative charge of the lipid monolayer, the K⁺ concentration near the surface is larger than the bulk concentration, by a Boltzmann factor. A good agreement was observed between the experimental results and the theoretical predictions. Conductance measurements of asymmetric bilayers containing a neutral lipid (egg lecithin) on one side and a negatively charged lipid (phosphatidylserine) on the other, confirm the role of the surface charge. Indeed, addition of K⁺ to the neutral side of the bilayer containing valinomycin had no effect on the conductance whereas addition of K⁺ to the charged side of the bilayer caused a 80-fold conductance increase.     

The Effect of PS Content on the Ability of Natural Membranes to Fuse with Positively Charged Liposomes and Lipoplexes

Supramolecular aggregates containing cationic lipids have been widely used as transfection mediators due to their ability to interact with negatively charged DNA molecules and biological membranes. First steps of the process leading to transfection are partly electrostatic, partly hydrophobic interactions of liposomes/lipoplexes with cell and/or endosomal membrane. Negatively charged compounds of biological membranes, namely glycolipids, glycoproteins and phosphatidylserine (PS), are responsible for such events as adsorption, hemifusion, fusion, poration and destabilization of natural membranes upon contact with cationic liposomes/lipoplexes. The present communication describes the dependence of interaction of cationic liposomes with natural and artificial membranes on the negative charge of the target membrane, charges which in most cases were generated by charging the PS content or its exposure. The model for the target membranes were liposomes of variable content of PS or PG (phosphatidylglycerol) and erythrocyte membranes in which the PS and other anionic compound content/exposure was modified in several ways. Membranes of increased anionic phospholipid content displayed increased fusion with DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) liposomes, while erythrocyte membranes partly depleted of glycocalix, its sialic acid, in particular, showed a decreased fusion ability. The role of the anionic component is also supported by the fact that erythrocyte membrane inside-out vesicles fused easily with cationic liposomes. The data obtained on erythrocyte ghosts of normal and disrupted asymmetry, in particular, those obtained in the presence of Ca²⁺, indicate the role of lipid flip-flop movement catalyzed by scramblase. The ATP-depletion of erythrocytes also induced an increased sensitivity to hemoglobin leakage upon interactions with DOTAP liposomes. Calcein leakage from anionic liposomes incubated with DOTAP liposomes was also dependent on surface charge of the target membranes. In all experiments with the asymmetric membranes the fusion level markedly increased with an increase of temperature, which supports the role of membrane lipid mobility. The decrease in positive charge by binding of plasmid DNA and the increase in ionic strength decreased the ability of DOTAP liposomes/lipoplexes to fuse with erythrocyte ghosts. Lower pH promotes fusion between erythrocyte ghosts and DOTAP liposomes and lipoplexes. The obtained results indicate that electrostatic interactions together with increased mobility of membrane lipids and susceptibility to form structures of negative curvature play a major role in the fusion of DOTAP liposomes with natural and artificial membranes.     

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3-pyrene)maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.     

The influence of PAMAM dendrimers surface groups on their interaction with porcine pepsin

In this study the ability of three polyamidoamine (PAMAM) dendrimers with different surface charge (positive, neutral and negative) to interact with a negatively charged protein (porcine pepsin) was examined. It was shown that the dendrimer with a positively charged surface (G4 PAMAM-NH₂), as well as the dendrimer with a neutral surface (G4 PAMAM-OH), were able to inhibit enzymatic activity of pepsin. It was also found that these dendrimers act as mixed partially non-competitive pepsin inhibitors. The negatively charged dendrimer (G3.5 PAMAM-COOH) was not able to inhibit the enzymatic activity of pepsin, probably due to the electrostatic repulsion between this dendrimer and the protein. No correlation between changes in enzymatic activity of pepsin and alterations in CD spectrum of the protein was observed. It indicates that the interactions between dendrimers and porcine pepsin are complex, multidirectional and not dependent only on disturbances of the secondary structure.     

DNA complexing with polyamidoamine dendrimers: implications for transfection.

DNA and polyamidamine (PAMAM) dendrimers form complexes on the basis of the electrostatic interactions between negatively charged phosphate groups of the nucleic acid and protonated (positively charged) amino groups of the polymers. Charge neutralization of both components and subsequent increases of the net positive charge of the complex result in changes in the physicochemistry and biological properties of the complexes. The formation of soluble, low-density and insoluble, high-density complexes was analyzed using UV light absorption and measurements of radioactive labeled DNA. Formation of high molecular weight and high-density complexes depended mainly on the DNA concentration and was enhanced by increasing the dendrimer-DNA charge ratio. Electrostatic charge related effects (attraction or repulsion of charged particles) appeared to be modulated by the generation of dendrimer (size of the polymer). With the progressive increases in the dendrimer-DNA charge ratio (above 20), an increase in the amount of low-density, soluble complexes was observed. Functional analysis revealed that the great majority (>90%) of transfection is carried by low-density, soluble, complexes which only represent approximately 10-20% of total complexed DNA. The ability of the dendrimer to complex and form aggregates with DNA is crucial for efficient transfection and the function of the complexed DNA.     

Effects of verbascoside,a phenylpropanoid glycoside from lemon verbena,on phospholipid model membranes

Phenylpropanoid glycosides are water-soluble compounds widely distributed, most of them deriving from medicinal herbs. Among them, verbascoside or acteoside has exhibited a wide biological activity, being free radical scavenging the most representative one. Moreover, antitumor, antimicrobial, anti-inflammatory, anti-thrombotic and wound healing properties have been previously described. Herein, the interaction of verbascoside with phospholipid membranes has been studied by means of differential scanning calorimetry, fluorescence anisotropy and dynamic light scattering. Verbascoside showed stronger affinity for negatively charged membranes composed of phosphatidylglycerol (PG) than for phosphatidylcholine (PC) membranes. This compound promoted phase separation of lipid domains in PC membranes and formed a stable lipid complex with and approximate phospholipid/verbascoside ratio of 4:1. Despite its hydrophilic character, verbascoside's caffeoyl moiety was located deep into the hydrophobic core of PC membranes and was almost inaccessible to spin probes located at different depths in PG membranes. This compound affected the ionization behavior of the PG phosphate group and most likely interacted with the vesicles surface. The presence of verbascoside decreased the particle size in PG unilamellar vesicles through the increase of the phospholipid head group area. A localization of verbascoside filling the upper region of PG bilayers close to the phospholipid/water interface is proposed. These effects on membranes may help to understand the mechanism of the biological activity of verbascoside and other similar phenylpropanoid glycosides.     

Interaction of cationic antimicrobial peptides with model membranes

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including alpha-helical, beta-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two alpha-helical peptides V681(n) and V25(p,) the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the beta-sheet peptide polyphemusin, intermediate for alpha-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes.     

A test of discreteness-of-charge effects in phospholipid vesicles: measurements using paramagnetic amphiphiles

A new series of negatively charged, paramagnetic alkylsulfonate probes was synthesized and can be used to measure both the internal and the external surface potentials of model membrane systems. We tested for discreteness-of-charge effects in lipid membranes by comparing the surface potentials, estimated by use of these negatively charged amphiphiles, with that of a series of positively charged alkylammonium nitroxides in charged membranes. From the partitioning of these probes, the membrane surface potential was estimated in phosphatidylcholine membranes containing either phosphatidylserine or didodecyldimethylammonium bromide. The surface potentials, estimated with either positive or negative probes, were identical, within experimental error, in either positive or negative membranes, and they were well accounted for by a simple Gouy-Chapman-Stern theory. This symmetry, with respect to the sign of the charge, indicates that discreteness-of-charge effects are not significant in determining the potential-sensitive phase partitioning of these probes in model membranes. Thus, despite the fact that charge on membranes is discrete, models that assume a uniform density of charge in the plane of the membrane adequately account for the potentials measured by these amphiphilic probes.     

Investigation of the interaction between modified ISCOMs and stratum corneum lipid model systems

The modified ISCOMs, so-called Posintro™ nanoparticles, provide an opportunity for altering the surface charge of the particles, which influences their affinity for the negatively charged antigen sites, cell membranes and lipids in the skin. Hypothetically, this increases the passage of the ISCOMs (or their components) and their load through the stratum corneum. The subsequent increase in the uptake by the antigen-presenting cells results in enhanced transcutaneous immunization. To understand the nature of penetration of Posintro™ nanoparticles into the intercorneocyte space of the stratum corneum, the interaction between the nanoparticles and lipid model systems in form of liposomes and/or supported lipid bilayer was studied. As a lipid model we used Stratum Corneum Lipid (SCL), a mixture similar in composition to the lipids of the intercorneocyte space. By Förster Resonance Energy Transfer (FRET), Atomic Force Microscopy (AFM), Electrochemical Impedance Spectroscopy (EIS) and cryo-Transmission Electron Microscopy (cryo-TEM) it was shown that application of nanoparticles to the SCL bilayers results in lipid disturbance. Investigation of this interaction by means of Isothermal Titration Calorimetry (ITC) confirmed existence of an enthalpically unfavorable reaction. All these methods demonstrated that the strength of electrostatic repulsion between the negatively charged SCL and the nanoparticles affected their interaction, as decreasing the negative charge of the Posintro™ nanoparticles leads to enhanced disruption of lipid organization.     

Adhesion of Positively Charged Liposomes to Mucosal Tissues

Abstract

A series of positively charged phospholipid and cholesterol derivatives was synthesized and evaluated as membrane components for liposomes. Small unilamellar liposomes containing up to 40 mole% of the synthetic lipids were prepared by sonication. Selected liposome preparations containing these synthetic lipid materials were found to be noncytotoxic in vitro by using a cell growth inhibition assay, whereas liposomes containing more classic positively charged components (stearylamine and cetyltrimethylammonium bromide) showed considerable cytotoxicity. Using an unanesthetized rabbit eye model, we have found that inclusion of the positively charged lipid derivatives into the liposomes significantly enhanced the ocular retention compared to neutral or negatively charged liposomes, presumably by molecular association with poly anionic corneal and conjunctival surface mucoglycoproteins. the increased retention was dependent on charge density and rigidity of the lipid bilayer. An assay for primary amino groups in these liposomes suggested that the distribution of the charged molecules between the inner and outer leaflets of the bilayer could be manipulated by lipid composition. Studies of liposomes containing cholesteryl esters of amino acids of various carbon chain lengths indicated that the charged amino groups need to extend from the surface of the lipid bilayers for better adhesion and retention. the ocular surface was saturable with respect to applied liposomes, which were cleared slowly from the eye with a half-time of clearance of about 2 hr. these data suggest a specific adhesion of the cationic liposomes to the surface of mucosal tissues.     

Lipid bilayers cushioned with polyelectrolyte-based films on doped silicon surfaces

Silicon semiconductors with a thin surface layer of silica were first modified with polyelectrolytes (polyethyleneimine, polystyrene sulfonate and poly(allylamine)) via a facile layer-by-layer deposition approach. Subsequently, lipid vesicles were added to the preformed polymeric cushion, resulting in the adsorption of intact vesicles or fusion and lipid bilayer formation. To study involved interactions we employed optical reflectometry, electrochemical impedance spectroscopy and fluorescent recovery after photobleaching. Three phospholipids with different charge of polar head groups, i.e. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used to prepare vesicles with varying surface charge. We observed that only lipid vesicles composed from 1:1 (mole:mole) mixture of DOPC/DOPS have the ability to fuse onto an oppositely charged terminal layer of polyelectrolyte giving a lipid bilayer with a resistance of >100 kΩ. With optical reflectometry we found that the vesicle surface charge is directly related to the amount of mass adsorbed onto the surface. An interesting observation was that zwitterionic polar head groups of DOPC allow the adsorption on both positively and negatively charged surfaces. As found with fluorescent recovery after photobleaching, positively charged surface governed by the presence of poly(allylamine) as the terminal layer resulted in intact DOPC lipid vesicles adsorption whereas in the case of a negatively charged silica surface formation of lipid bilayers was observed, as expected from literature.