Complexation of polycations to anionic liposomes: composition and structure of the interfacial complexes

Poly(N-ethyl-4-vinylpyridinium bromide) (a polycation with a degree of polymerization of 1100) was adsorbed onto liposomes composed of egg lecithin with a 0.05-0.20 molar fraction (nu) of anionic headgroups provided by cardiolipin (a doubly anionic lipid). According to electrophoretic mobility data, this led to total charge neutralization of the liposomes, whereupon the liposomes adopted a positive charge as additional polymer continued to adsorb. Although the liposomes aggregated at the charge-neutralization point, they disassembled into individual liposomes after becoming positively charged. The degree of polymer adsorption was shown to reach a limit. Thus, by measuring the free polymer content in a liposome suspension, it was possible to determine the polymer concentration at which the liposome surface became saturated with polymer. Beyond this point, an electrostatic/steric barrier at the surface suppressed further adsorption. Dynamic light scattering studies of liposomes with and without adsorbed polymer allowed calculation of the polymer film thickness which ranged from 22 to 35 nm as the molar fraction of cardiolipin (nu) increased from 0.05 to 0.20. The greater the content on the anionic lipid in the bilayer, the thicker the polymer film. The maximum number of polymer molecules adsorbed onto the liposomes was estimated: 1-2 molecules for nu = 0.05; 3 molecules for nu = 0.1; 4- molecules for nu = 0.15; and 6 molecules for nu = 0.2. The polymer appears to lie on the liposome surface, rather than embedding into the bilayer, because addition of NaCl easily dislodges the polymer from the liposome into the bulk water.     

Effect of the phase state of the lipid bilayer on the structure and characteristics of the polycation-(anionic liposome) complex

Interaction of the cationic polymer poly-N-ethyl-4-vinylpyridinium bromide with bilayer vesicles (liposomes) composed of zwitterionic dipalmitoylphosphatidylcholine and anionic cardiolipin (the molar fraction of the negatively charged cardiolipin groups is 0.2) is studied. The composition and characteristics of the polycation-liposome complex are shown to be controlled by the phase state of the lipid membrane. Liposomes whose membranes exist in their LC state (“liquid” liposomes) keep their integrity in the complex with polycation. The adsorbed polycation can be completely removed from the liposomal membrane by the addition excess amounts of a competing polyanion. The adsorption of polycation on the surface of liposomes whose membranes exist the gel state (“solid” liposomes) leads to the formation of defects in the membrane, and the polycation’s adsorption with such liposomes becomes irreversible. The defects that form are also preserved when solid liposomes on whose surface the polycation is sorbed are transformed into the liquid state. Moreover, the reversible contact between polycation and liquid liposomes becomes irreversible once the liposomal membranes bound to the polycation transform into the solid state.     

Polyelectrolyte-coated liposomes: stabilization of the interfacial complexes

Anionic liposomes, composed of egg lecithin (EL) or dipalmitoylphosphatidylcholine (DPPC) with 20 mol% of cardiolipin (CL(2-)), were mixed with cationic polymers, poly(4-vinylpyridine) fully quaternized with ethyl bromide (P2) or poly-l-lysine (PL). Polymer/liposome binding studies were carried out using electrophoretic mobility (EPM), fluorescence, and conductometry as the main analytical tools. Binding was also examined in the presence of added salt and polyacrylic acid (PAA). The following generalizations arose from the experiments: (a) Binding of P2 and PL to small EL/CL(2-) liposomes (60-80 nm in diameter) is electrostatic in nature and completely reversed by addition of salt or PAA. (b) Binding can be enhanced by hydrophobization of the polymer with cetyl groups. (c) Binding can also be enhanced by changing the phase state of the lipid bilayer from liquid to solid (i.e. going from EL to DPPC) or by increasing the size of the liposomes (i.e. going from 60-80 to 300 nm). By far the most promising systems, from the point of view of constructing polyelectrolyte multilayers on liposome cores without disruption of liposome integrity, involve small, liquid, anionic liposomes coated initially with polycations carrying pendant alkyl groups.     

Structure and properties of complexes formed by cationic polymers and anionic cholesterol-containing liposomes

The adsorption of the synthetic polycation poly(N-ethyl-4-vinylpyridinium bromide) on the surface of three-component lipid vesicles (liposomes) formed from a mixture of anionic cardiolipin, electroneutral egg lecithin, and nonionic cholesterol is studied via laser microelectropheresis, dynamic light scattering, conductometry, fluorescence spectroscopy, and UV spectroscopy. The incorporation of cholesterol into the liposomal membrane increases its microviscosity; however, the membrane remains liquid-crystalline. Simultaneously, an increase in the fraction of cholesterol causes the formation of defects in liposome membranes during their binding with poly(N-ethyl-4-vinylpyridium bromide) and makes complexation irreversible. The results of this study are of interest for predicting the behavior of polyelectrolytes and biologically active structures formed on their basis on the surface of cells and the reaction of the cellular membrane to the adsorbed polymer.     

Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers

The effect of the lipid polar headgroup on melittin-phospholipid interaction was investigated by cryo-TEM, fluorescence spectroscopy, ellipsometry, circular dichroism, electrophoresis and photon correlation spectroscopy. In particular, focus was placed on the effect of the lipid polar headgroup on peptide adsorption to, and penetration into, the lipid bilayer, as well as on resulting colloidal stability effects for large unilamellar liposomes. The effect of phospholipid headgroup properties on melittin-bilayer interaction was addressed by comparing liposomes containing phosphatidylcholine, -acid, and -inositol at varying ionic strength. Increasing the bilayer negative charge leads to an increased liposome tolerance toward melittin which is due to an electrostatic arrest of melittin at the membrane interface. Balancing the electrostatic attraction between the melittin positive charges and the phospholipid negative charges through a hydration repulsion, caused by inositol, reduced this surface arrest and increased liposome susceptibility to the disruptive actions of melittin. Furthermore, melittin was demonstrated to induce liposome structural destabilization on a colloidal scale which coincided with leakage induction for both anionic and zwitterionic systems. The latter findings thus clearly show that coalescence, aggregation, and fragmentation contribute to melittin-induced liposome leakage, and that detailed molecular analyses of melittin pore formation are incomplete without considering also these colloidal aspects.     

Effect of water-soluble polymers on fusion of egg yolk and soybean phospholipid liposomes

The efficiencies of polyelectrolytes, i.e., polycations and polyanions, and several kinds of water-soluble polymers as fusogens on soybean phospholipid liposome (SL) and egg yolk phospholipid liposome (EL) were investigated by the fluorescence quenching method. There were optimal concentrations for the induction of fusion in every system. Polycations induced fusion of liposomes at very low concentration in comparison with other polymers. Poly(carboxylic acid)s induced fusion at relatively high concentration. A strong acidic polyanion with high molecular weight also induced fusion of liposomes. The induction efficiency of poly(ethylene glycol) on fusion was higher than other nonionic polymers. The efficiency of fusion of EL was lower than that of SL in all systems because of the higher stability of EL membrane. It was found that electrostatic interactions, hydrogen bonding and/or hydrophobic interaction between these water-soluble polymers and liposomal membranes played an important role on aggregation and fusion of liposomes.     

The Influence of the Chain Length of Polycations on their Complexation with Anionic Liposomes

A series of strong polycations is synthesized through the anionic polymerization of 2‐vinylpyridine, followed by subsequent quaternization of the resulting polymer. Polycations based on quaternized 2‐vinylpyridine (PVPQs) with degrees of polymerization (DP) from 20 to 440 are adsorbed on the surface of small anionic liposomes. Liposome/PVPQ complexes are characterized by using a number of physicochemical methods. All PVPQs are totally adsorbed onto the liposome surface up to a certain concentration at which saturation is reached (which is specific for each PVPQ). The integrity of the adsorbed liposomes remains intact. Short PVPQs interact with anionic lipids localized on the outer membrane leaflet, whereas long PVPQs extract anionic lipids from the inner to outer leaflet. Complexes tend to aggregate, and the largest aggregates are formed when the initial charge of the liposomes is fully neutralized by the charge of the PVPQ. PVPQs with intermediate DPs demonstrate behavioral features of both short and long PVPQs. These results are important for the interpretation of the biological effects of cationic polymers and the selection of cationic polymers for biomedical applications.     

Self‐Propagating Association of Zwitterionic Polymers Initiated by Ionene Polymers

A self‐propagating association of zwitterionic polymers is observed when a small amount of x,y‐ionene bromide (x = 3 or 6; y = 3, 4, 6, 10 or 12) polymer is added to aqueous solutions of zwitterionic polymer, poly[3‐dimethyl(methacryloyloxyethyl)ammoniumpropanesulfonate] (PDMAPS), to give large amount of PDMAPS precipitate. The self‐propagating association initiated by ionene polymers is explained in terms of the electrostatic interaction between the ionene polymers and the zwitterionic polymers whereupon the geometry of the charges on the polymer chains plays an important role.     

Stability of anionic liposome-cationic polymer complexes in water-salt media

Laser microelectrophoresis, dynamic light scattering, and fluorescence and UV spectroscopy are employed to study poly-N-ethyl-4-vinylpyridinium bromide adsorption on the surface of bilayer lipid vesicles (liposomes) formed from mixtures of anionic phosphatidyl serine and electroneutral phosphatidylcholine. It is established that polycation adsorption is accompanied by the neutralization of charges on liposomes and their aggregation. The subsequent addition of a low-molecular-weight salt (NaCl) solution to suspensions of complexes causes them to dissociate into their initial components, while the stability of the complexes with respect to the salt action increases with the fraction of the anionic lipid in the liposome membranes. The data obtained are interpreted from the position of the formation-disintegration of a molecular capacitor, the charge of which is generated by spatially separated anionic lipids located in the bilayer membrane and cationic units of the adsorbed polyamine.     

Cationic polymer adsorption on bilayer membrane containing anionic and cationic lipids

Microelectrophoresis, dynamic light scattering, fluorescence, and microcalorimetry are used to study the adsorption of a synthetic polycation, poly-N-ethyl-4-vinylpyridinium bromide, on the surface of three-component liposomes formed from electrically neutral phosphatidylcholine, anionic diphosphatidylglycerol (cardiolipin), and cationic dicetyldimethylammonium bromide, with the two latter being taken in equal amounts. The adsorption of the polycation on the liposomal membrane results in the generation of a positive charge, which provides the polycation-liposome complex with aggregation stability. Increasing salt concentration in the suspension causes the complex to dissociate into its components. According to the microcalorimetry data, the membranes of the initial three-component liposomes consist of two microphases, with one of them being enriched with the neutral lipid and another one, with the ionic components. The polycation adsorption does not lead to noticeable structural rearrangements in the liposomal membranes.     

Competitive Reactions in Three-Component System Cationic Colloid–Anionic Liposome–Protein

The formation of complexes of anionic liposomes (50 nm) and polymer microspheres with grafted polycationic chains with a diameter of 240 nm (spherical polycationic brushes) in a physiological solution at a NaCl concentration of 0.15 mol/L is investigated. Liposomes are quantitatively adsorbed on the surface of brushes; every brush can bind up to 24 intact liposomes. The saturated brush–liposome complex is able to additionally bind negatively charged protein albumin; the excess of protein does not displace liposomes from the complex with brushes. The obtained results are important for understanding the mechanism of formation and functioning of electrostatic multiliposomal containers in biological media containing a high amount of protein.     

Physicochemical and biological properties of polyampholytes: Quaternized derivatives of poly(4-vinylpyridine)

The modification of poly(4-vinylpyridine) with ω-bromocarboxylic acids and alkyl bromides yields three types of polyampholytes: polyampholytes containing both cationic and anionic groups in each monomer unit (polybetaines), polyampholytes containing betaine and cationic units, and polyampholytes containing betaine units and side cetyl radicals. Their complex formation with liposomes formed from zwitterionic (electroneutral) phosphatidylcholine and anionic diphosphatidylglycerol (cardiolipin) is investigated. The method for fixation of polymers on the liposomal membrane and the stability of the formed complexes are determined by the chemical structure of macromolecules. For the most part, polyelectrolytes are electrostatically adsorbed on the membrane and are fully removed from it with an increase in the salt concentration in the surrounding solution. An exception is the polybetaine obtained through the modification of poly(4-vinylpyridine) with ω-bromobutyric acid, which irreversibly binds to liposomes probably owing to the incorporation of macromolecular fragments into the hydrophobic part of the lipid bilayer. The insertion of side cetyl radicals into polybetaine molecules stabilizes their complexes with liposomes in the presence of salts. The cytotoxicity of the synthesized polyampholytes is one to two orders of magnitude lower than that of a cationic polymer with the same degree of polymerization.     

pH sensitivities of egg phosphatidylcholine liposomes and dioleoylphosphatidylethanolamine liposomes triggered by poly(<Emphasis Type="Italic">N</Emphasis>-isopropylacrylamide-co-methacrylic acid-co-octadecylacrylate)

Egg phosphatidylcholine (PC) liposomes bearing pH-sensitive polymers and dioleoylphosphatidylethanolamine (DOPE) liposomes including the same polymers were prepared by a sonication method. As pH-sensitive polymers, copolymers of N-isopropylacrylamide, methacrylic acid, and octadecylacrylate were used. The liposomes were stable in neutral pH ranges in terms of release. But the release became marked at pH 5.5, and it was accelerated as pH further decreased. For example, the degree of release from egg PC liposomes (polymer/lipid ratio is 3:10, w/w) for 120 s increased from 2% to 63% as pH decreased from 7.5 to 4.5. Under the same condition, the degree of release from DOPE liposomes increased from 4% to 80%. These results indicate that DOPE liposome is more pH-sensitive than egg PC liposome.     

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.     

Stabilization of small unilamellar DMPC-liposomes by uncharged polymers

 Polymer-free and polymer-bearing small unilamellar (SUV) liposomes from dimyristoyl-phosphatidylcholine (DMPC) were prepared under standardized conditions. Polymer-bearing liposomes were formed by incorporating an uncharged polymer [hydrolyzed poly(vinyl alcohol) (PVA), poly(vinyl alcohol-co-vinylacetal) (PVA-Al), poly(vinyl alcohol-co-vinyl propional) (PVA-Prol) poly(vinyl alcohol-co-vinyl butiral) (PVA-Bul) copolymer or poly(vinyl pyrrolidone) (PVP)] into the membrane bilayer of vesicles. The kinetic (long-term) stability of the liposome dispersions stored in distilled water, in physiological NaCl solution and at various pH values, respectively, were studied. The physical stability of vesicles was tested by measuring the size and the zeta potential of liposomes by means of a Malvern Zetasizer 4 apparatus. It was shown that most of these polymers are effective steric stabilizers for the DMPC-liposomes. Among the polymers, the PVA-Bul and PVA-Prol copolymers and the PVP of high molecular mass exhibited the most efficient stabilizing effect at each pH studied, indicating that the formation of a relatively thick polymer layer around the lipid bilayers ensures an enhanced and prolonged physical stability of liposomes. Also, the butiral or propional side chain in the PVA-based copolymers presumably promotes the anchoring of macromolecules to the vesicles. Using these macromolecules, the colloidal interactions between vesicles can be modified and so the physical stability of liposomes and the kinetic stability of liposome dispersions can also be controlled. Received: 20 May 1997 Accepted: 03 September 1997     

Stabilized liposomes with phospholipid polymers and their interactions with blood cells

To stabilize a phospholipid liposome, addition of various water-soluble polymers into a liposomal aqueous suspension was investigated. The water-soluble polymers were poly(ethylene oxide) (PEO), poly(N-vinyl pyrrolidone) (PVPy) and poly[2-methacryloyloxyethyl phosphorylcholine(MPC)], and poly[MPC-co-n-butyl methacrylate(BMA)]. The gel–liquid crystal transition temperature (T_c) of the diparmitoylphosphatidylcholine (DPPC) liposome was not changed by addition of these polymers significantly. However, membrane fluidity of DPPC liposome treated with water-soluble polymers, which was measured with fluorescence probe, depended on the chemical structure of the water-soluble polymers. In the case of PEO and PVPy, the temperature dependence of membrane fluidity was the same as that of the original DPPC liposome, on the other hand, poly(MPC) and poly(MPC-co-BMA) induced a rise in the temperature where an increase in the membrane fluidity was observed. The release of carboxy fluorescein from the DPPC liposome was suppressed by the addition of the MPC polymers. The liposomes in the MPC polymer solution were stable compared with those in water when plasma was added into the suspension. Interactions with stabilized liposome with blood cells such as platelets and erythrocytes were evaluated. Activation of platelets in contact with liposome covered with poly(MPC) or poly(MPC-co-BMA) was less than PEO-stabilized liposome. On the other hand, no hemolysis of erythrocytes was observed when every polymer-treated liposome was added in the suspension of erythrocytes. Based on these results, the MPC polymers could interact with the liposome surface, adsorb on the liposomes and stabilize them, and had no adverse effect to the blood cells even when they were in a physiological environment.     

Polymethylmethacrylate derivatives Eudragit E100 and L100: Interactions and complexation with surfactants

Precipitation or coprecipitation of polyelectrolytes has been largely investigated. However, the precipitation of polyelectrolytes via addition of charged and non‐charged surfactants has not been systematically studied and reported. Consequently, the aim of this work is to investigate the effect of different surfactants (anionic, cationic, non‐charged and zwitterionic) on the precipitation of cationic and anionic polymethylmethacrylate polymers (Eudragit). The surfactants effect has been investigated as a function of their concentration. Special attention has been dedicated to the CMC range and to the colloidal characterization of the formed dispersions. Moreover, the effect of salt (NaCl) and pH was also addressed. It is pointed out that non‐ionic and zwitterionic surfactants do not interact with charged Eudragit E100 and L100. For oppositely charged Eudragit E100/SDS and Eudragit L100/CTAB, precipitation occurs, and the obtained dispersions have been characterized in terms of particle size distribution and zeta potential. It was established that the binding of SDS molecules to Eudragit E100 polymer chains is made through the negative charges of the surfactant heads under the CMC value whereas binding of CTAB to Eudragit L100 chains is made at a CTAB concentration 5 times above its CMC. For Eudragit E100/SDS system, a more acidic medium induces aggregation. A same result was observed for the Eudragit L100/CTAB at a more basic pH. Moreover, it was observed that increasing salt concentration (higher than 100 mM) led to aggregation as generally observed for polycations/anionic surfactant systems.     

Migration of a cationic polymer between lipid vesicles

The adsorption of a synthetic polycation, poly(N-ethyl-4-vinylpyridinium bromide) (PEVP), on the surface of bilayer lipid vesicles (liposomes) and the migration of adsorbed macromolecules between the liposomes are studied. Liposomes of three types are used, including (1) traditional two-component liposomes composed of neutral phosphatidylcholine (PC) and anionic cardiolipin (CL); (2) three-component liposomes consisting of PC, CL, and cationic dicetyldimethylammonium bromide (DCMAB); and (3) anionic PC/CL liposomes with a nonionic surfactant, poly(ethylene oxide)-cetyl alcohol ether (Briij 58), incorporated into their bilayers. The adsorption of PEVP on the surface of PC/CL liposomes is accompanied by their aggregation. Using the fluorescence method, it is shown that the units (segments) of the polycation undergo partial redistribution between the liposomes inside the aggregates formed from PC/CL liposomes (with and without a fluorescent label) and PEVP. On the contrary, three-component PC/CL/DCMAB and PC/CL/Briij liposomes are not aggregated, even with the complete neutralization of their charges by adsorbed PEVP. In both cases, the migration of PEVP molecules between individual (nonaggregated) liposomes is observed. Possible reasons for the aggregative stability of the three-component PC/CL/DCMAB and PC/CL/Briij liposomes and the mechanism of interliposome migration of PEVP in such systems are discussed.     

Binding of sodium dodecyl sulfate to linear and star homopolymers of the nonionic poly(methoxyhexa(ethylene glycol) methacrylate) and the polycation poly(2-(dimethylamino)ethyl methacrylate): electromotive force, isothermal titration calorimetry, surface tension, and small-angle neutron scattering measurements

We investigated the binding of sodium dodecyl sulfate (SDS) to various linear and star polymers of the nonionic methoxyhexa(ethylene glycol) methacrylate (PMHEGMA) and the ionic 2-(dimethylamino)ethyl methacrylate (PDMAEMA), the latter being a polycation at low pH. The dodecyl sulfate ion selective electrode (EMF), isothermal titration calorimetry (ITC), and surface tension (ST) were applied to gain detailed information about interactions. In all cases there is evidence of significant binding of SDS over an extensive SDS concentration range spanning from ca. 10(-6) to 0.1 mol dm(-3). At pH 3, the polymer PDMAEMA is a strong polycation and here the binding is dominated by electrostatic 1:1 charge neutralization with the anionic surfactant. At their natural pH of 8.6, PMHEGMA and PDMAEMA polymers are essentially nonionic and bind SDS in the form of polymer-bound aggregates in the concentration range of ca. 1 x 10(-3) to 3 x 10(-2) mol dm(-3). All the polymers also bind SDS to a lesser extent at concentrations below 1 x 10(-3) mol dm(-3) reaching as low as 10(-7) mol dm(-3). This low concentration binding process involves the polymer and nonassociated SDS monomers. As far as we are aware, this is the first example that such a low concentration noncooperative binding process could be observed in SDS/neutral polymer systems by EMF and ST. We also showed that the nonionic surfactant hexa(ethylene glycol) mono-n-dodecyl ether (C12EO6) and the cationic cetyltrimethylammonium bromide (C16TAB) interact with star PDMAEMA. We believe that the interaction of C12EO6 and CTAB is of similar noncooperative type as the first SDS binding process in the range from ca. 10(-5) to 0.3 x 10(-3) mol dm(-3). At the high concentration binding limit Csat of SDS, the above polymers become fully saturated with bound SDS micelles. We applied small angle neutron scattering (SANS) to determine the structure and aggregation numbers of the star polymer/bound SDS micelles and calculated the stoichiometry of such supramolecular complexes. The SANS data on PDMAEMA star polymers in the presence of C12EO6 showed only a limited monomer binding in contrast to linear PDMAEMA, which showed monomer C12EO6 binding at low concentrations but micellar aggregates at 6 x 10(-3) mol dm(-3).     

Stable polymeric nanoballoons: lyophilization and rehydration of cross-linked liposomes

The cross-linking of supramolecular assemblies of hydrated lipids is an effective method to stabilize these assemblies to disruption by surfactants or aqueous alcohol. The heterobifunctional lipids, Acryl/DenPC(16,18) and Sorb/DenPC(18,21), are examples of a new class of polymerizable lipid designed for the creation of cross-linked lipid structures. The robust nature of cross-linked liposomes was demonstrated by lyophilization of the liposomes followed by their essentially complete redispersion in water. The resulting liposomes were compared to the original sample by quasi-elastic light scattering and transmission electron microscopy. There was no major change in the size or structure of the cross-linked liposomes after rehydration of the freeze-dried powder of liposomes. Moreover, the rehydrated cross-linked liposomes continued to be resistant to surfactant solubilization. Neutral cross-linked liposomes were predominantly redispersed after freeze-drying with the aid of bath sonication. The small amount of residual liposome aggregation observed with neutral liposomes could be prevented by incorporating a surface charge into the liposome or attaching hydrophilic polymers, for example, poly(ethylene glycol), onto the liposome.