Anna A. Efimova

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.

Reversibility of structural rearrangements in the negative vesicular membrane upon electrostatic adsorption/desorption of the polycation.

Interaction of small unilamellar vesicles (SUVs), composed of negative diphosphatidylglycerol (cardiolipin, CL(2-)) and neutral dipalmitoylphosphatidylcholine (DPPC), with poly(N-ethyl-4-vinylpyridinium bromide) (PEVP) was studied in water solution above and below the vesicular membrane melting point by means of differential scanning calorimetry, photon correlation spectroscopy, microelectrophoresis, conductometry, and fluorescence techniques. It has been found that CL(2-) species are homogeneously distributed within DPPC-CL(2-) SUV membrane leaflets and between them. Interaction of PEVP with DPPC-CL(2-) SUVs led to drastic structural rearrangements in the membrane if it was in the fluid state (liquid SUVs). Negative CL(2-) molecules migrated from the inner to the outer membrane leaflet and segregated in the vicinity of adsorbed PEVP chains. In addition, PEVP adsorption terminated completely the exchange of lipid molecules between the SUVs. At the same time, the integrity of liquid SUVs contacting PEVP remained unchanged. Since the interaction of PEVP with liquid SUVs was predominantly electrostatic in nature, the polycation could be completely removed from the vesicular membrane by addition of an excess of polyacrylic acid (PAA) polyanions forming a more stable electrostatic complex with PEVP. Removal of PEVP resulted in complete resumption of the original distribution of lipids in lateral and transmembrane directions as well as intervesicular lipid exchange. In contrast, PEVP interacting with DPPC-CL(2-) SUVs formed defects in the vesicular membrane if it was in the gel state (solid SUVs). Such interaction was contributed not only by electrostatic but most likely by hydrophobic interactions involving the defected membrane sites. PEVP kept contacting solid SUVs in the presence of an abundant amount of PAA. The established phenomena may be important for understanding the biological effects of polycations.

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.

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.

Biodegradable multi-liposomal containers

The method of preparing biodegradable multi-liposomal containers via modification of anionic liposomes with a cationic polymer and subsequent adsorption of the obtained liposome-polymer cationic complex on the surfaces of negatively charged polylactide micelles with grafted polyoxyethylene chains is described. Liposomes preserve their integrity in a ternary micelle-polycation-liposome complex, a circumstance that allows the complex to be used as a multi-liposomal container for encapsulation of bioactive compounds.

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.

Effect of anionic-lipid-molecule geometry on the structure and properties of liposome-polycation complexes

The influence of anionic amphiphilic compounds that have different geometric shapes and become incorporated into bilayers on the polycation-mixed liposome interaction and the structure and properties of the resulting complexes is analyzed. Phosphatidylserine, cardiolipin, and sodium dodecyl phosphate are used as anionic lipids, and poly(N-ethyl-4-vinylpyridinium bromide) and polylysine are used as polycations. Polycation adsorption on the surfaces of all examined types of liposomes is accompanied by the neutralization of their charge, an increase in the size of particles of the systems, and quenching of fluorescence labels. Liposomes whose membranes contain incorporated cylindrical phosphatidylserine molecules retain their integrity during contact with polycations. The resulting complexes quantitatively dissociate into initial components during an increase in the salt concentration in the surrounding solution. In the case of liposomes with asymmetric anionic lipids, that is, cardiolipin and sodium dodecyl phosphate, the conditions of retaining the membrane integrity and reversible complexation are fulfilled only at relatively low molar fractions of both lipids. The obtained data witness the decisive effect of the geometry of anionic lipid molecules on the stability of complexes formed from mixed liposomes and polycations.

Polymer migration among phospholipid liposomes.

Complexation of phospholipid lipsomes with a cationic polymer, poly(N-ethyl-4-vinylpyridinium bromide) (PEVP), and subsequent interliposomal migration of the adsorbed macromolecules, have been investigated. Liposomes of two different charge types were examined: (a) a liposomal system, with an overall charge near zero, consisting of zwitterionic phosphatidylcholine (egg lecithin, EL) with added doubly anionic phospholipid, cardiolipin (CL(2-)), and cationic dihexadecyldimethylammonium bromide (HMAB(+)), in a CL(2-)/HMAB(+) charge-to-charge ratio of 1:1; (b) an anionic liposomal system composed of an EL/CL(2-) mixture plus polyoxyethylene monocetyl ether (Brij 58). Both three-component systems were designed specifically to preclude liposomal aggregation upon electrostatic association with the PEVP, a phenomenon that had complicated analysis of data from several two-component liposomes. PEVP macromolecules were found from fluorescence experiments to migrate among the charge-neutral EL/CL(2-)/HMAB(+) liposomes. In the case of anionic EL/CL(2-)/Brij liposomes, a combination of fluorescence and laser microelectrophoresis methods showed that PEVP macromolecules travel from liposome to liposome while being electrostatically associated with anionic lipids.

Synthetic polycations on the surface of negatively charged liposomes

Adsorption of poly-N-alkyl-4-vinylpyridine cations on the surface of negatively charged liquid and solid phosphatidylcholine-cardiolipin liposomes was studied. The poly-n-ethyl-4-vinylpyridine cation adsorbed on the surface of liquid liposomes was found to be completely displaced from the surface into solution by polyacrylate anions, the latter forming an interpolyelectrolyte complex with the polycation. On the contrary, the polycation carrying side-chain cetyl fragments forms a more stable complex with liposomes, probably due to the incorporation of cetyl fragments into the lipid bilayer. In this case, the polyanion cannot displace the polycation from the surface, the liposome-polycation-polyanion ternary complex being formed. Polycation adsorption on the surface of liquid liposomes induces transbilayer migration of the cardiolipin molecules from the inner to outer bilayer leaflet. In solid liposomes, only the outer cardiolipin molecules form a complex with adsorbed polycations.

Synthesis and properties of conjugates involving liposomes, a linear polymer, and the micelle of a polylactide-poly(ethylene glycol) block copolymer

Multiliposome biodegradable conjugates have been obtained via electrostatic binding of a liposome-polylysine complex with micelles formed from a polylactide-poly(ethylene glycol) di- or triblock copolymer. It has been shown that the integrity of liposomes in conjugates is retained. Of special interest is a conjugate based on a polylactide-poly(ethylene glycol) diblock copolymer that exhibits stability in a physiological solution with 0.15 mol/L NaCl, but degrades in the presence of proteolytic enzymes. This fact allows the suggestion that the conjugate will be eliminated from the body after its transport function is completed. The results may be used to create biodegradable liposome containers for drug encapsulation and delivery.