E.G. Yaroslavova

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.

MODULATION OF INTERACTION OF POLYCATIONS WITH NEGATIVE UNILAMELLAR LIPID VESICLES

Interactions of small unilamellar negative vesicles composed of diphosphatidylglycerol (cardiolipin, CL2−), 20 mol%, and phosphatidylcholine (egg yolk lecithin, EL), 80 mol%, with various cationic polymers (CP) derived from poly(4-vinylpyridine) (PVP) were studied in water and water–salt solutions by means of photon correlation spectroscopy, microelectrophoresis, conductometry, and fluorescence techniques. The linear charge density and hydrophilic lipophilic balance of CPs were varied by quaternization of PVP with various amounts of different alkyl bromides (ethyl-(2), heptyl-(7), dodecyl-(12), cetyl-(16)). Substantial differences were observed in the behavior of exhaustingly N-ethylated PVP (CP2) and PVP N-ethylated to 50 mol% (CP2(50)) or 30 mol% (CP2(30)). All of them adsorb to the CL2−/EL vesicle membrane, neutralizing the surface negative charge and causing aggregation of the vesicles. However, CP2, a polycation with a maximum linear charge density, strongly enhances transfer of the negative lipid ions from the inner to outer bilayer leaflet, while CP2(50) and CP2(30) do not. Adsorbed CP2 does not disturb integrity of the vesicle membrane and can be completely removed from the surface of aggregated vesicles by adding a simple salt (NaCl) or a negative linear polyelectrolyte (polyacrylic acid (PAA) sodium salt). Such removal is followed by release of the original vesicles. In contrast to that, adsorbed CP2(50) or CP2(30) produce some leak through the lipid bilayer and cannot be completely desorbed either by increasing ionic strength or adding an excess of PAA. The probable reason of these differences is discussed. PVP partially N-alkylated with dodecyl or cetyl bromides (3 mol%) and then completely N-ethylated (CP2,12 and CP2,16), also having a maximum linear charge density, adsorbs to the negative vesicle surface as a result of both electrostatic binding and hydrophobic interaction. Bulky hydrocarbon pendant groups incorporate into the inner bilayer compartment. Similarly to CP2(50) and CP2(30), CP2,12 and CP2,16 cannot be removed from the surface either by adding the simple salt, or an excess of PAA. However, in contrast to CP2(50) and CP2(30), the polycations with the bulky hydrocarbon pendant groups do not cause any leak through the vesicle membrane. Finally, we have succeeded to prepare the ternary vesicles also composed of 20 mol% of CL2−, but partially replacing EL for polyoxyethylene 20 cetyl ether (Brij 58) (up to 30 mol%). The CL2−/EL/Brij vesicle carries a hydrophilic corona formed by polyoxyethylene chains exposed into water, while hydrophobic cetyl radicals are incorporated in the lipid bilayer. The CL2−/EL/Brij vesicles adsorb all studied CPs similar to the binary CL2−/EL vesicles. This means that polyoxyethylene corona is permeable for polycationic species restricting neither electrostatic binding nor incorporation of bulky hydrocarbon groups of CP2,16 into the membrane. However, the corona effectively stabilizes the CP-vesicle complexes against aggregation when the membrane surface is neutralized.

Biomembrane sensitivity to structural changes in bound polymers.

Anionic liposomes containing a 4:1 molar ratio of neutral to anionic phospholipids were treated with an excess of five zwitterionic polymers differing only in the spacer length separating their cationic and anionic moieties. Although the polymers do not disrupt the structural integrity of the liposomes, they can induce spacer-dependent molecular rearrangements within the liposomes. Thus, the following were observed: spacer length = 1, no binding to the liposomes; spacer length = 2, adsorption to the liposomes, but no molecular rearrangement; spacer length = 3, lateral lipid segregation but little or no flip-flop; spacer length = 4 or 5, lateral lipid segregation and flip-flop. These diverse behaviors are relevant to the use of biomedical formulations where polyelectrolytes play a role.

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.

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.

Variable and low-toxic polyampholytes: complexation with biological membranes

In this paper, we describe three series of polyampholytes synthesized via quaternization of poly(4-vinylpyridin) by ω-bromocarboxylic acids and alkyl bromides: (1) with cationic and anionic groups in each unit (polybetaines), (2) with betaine and cationic groups, and (3) with betaine and pendant alkyl groups. The polymers were complexed with anionic mixed lipid membranes, liposomes, and Langmuir monolayers. By varying a length of –(CH2)n– spacer in the betaine group, different behaviors of polybetaines in a suspension of anionic liposomes can be realized: from no interaction to complexation followed by significant structural reorganization in the liposomal membrane. Cytotoxicities of polyampholytes are one to two orders of magnitude less than the cytotoxicity of a pure polycationic polymer with the same degree of polymerization. These results are of importance in designing polyelectrolytes with a higher affinity to the biolodical (cell) membrane and minimum cytotoxicity and demonstrate the potential of polyampholytes in developing biocompatible polymeric structures.

Structure and characteristics of the complexes between polyampholites and anionic liposomes

Polyampholites are synthesized by the alkylation of poly-4-vinylpyridine with ω-bromocarboxylic acids, and their interaction with the negatively charged bilayer lipid vesicles (liposomes) is studied. In the above polymers, quaternized pyridine units are zwitterion (betaine) groups, in which cationic and anionic groups are linked by the -(CH2) n -bridges. Via the methods of fluorescence, laser scattering, and DSC, the length of the ethylene spacer in the betaine group is shown to control the ability of the polymer to interact with anionic liposomes and induce structural rearrangements in the liposomal membrane. At n = 1, polybetaine is not linked to anionic liposomes. At n = 2, polybetaine is sorbed on the membrane, but it causes no dramatic structural rearrangements in the bilayer. At n = 3, the adsorption of polybetaine triggers the lateral segregation of lipids in the outer membrane layer. At n = 5, adsorption of polymer is accompanied by the lateral segregation and flip-flop of lipid molecules; as a result, all anionic membrane lipids are involved in the microphase separation. This evidence is of evident interest for the controlled design of polymers and related complexes and conjugates for biomedical applications.

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.