Interaction of multicomponent anionic liposomes with cationic pyridylphenylene dendrimer: Does the complex behavior depend on the liposome composition?

Dendrimers are individual macromolecular compounds having a great potential for biomedical application. The key step of the cell penetration by dendrimers is the interaction with lipid bilayer. Here, the interaction between cationic pyridylphenylene dendrimer of third generation (D350+) and multicomponent liquid (CL/POPC), solid (CL/DPPC) and cholesterol-containing (CL/POPC/30% Chol) anionic liposomes was investigated by dynamic light scattering, fluorescence spectroscopy, conductometry, calorimetric studies and molecular dynamic (MD) simulations. Microelectrophoresis and MD simulations revealed the interaction is electrostatic and reversible with only part of pyridinium groups of dendrimers involved in binding with liposomes. The ability of dendrimer molecules to migrate between liposomes was discovered by the labeling liposomes with Rhodamine B. The phase state of the lipid membrane and the incorporation of cholesterol into the lipid bilayer were found to not affect the mechanism of the dendrimer - liposome complex formation. Rigid dendrimer adsorption on liposomal surface does not induce the formation of significant defects in the lipid membrane pave the way for possible biological application of pyridylphenylene dendrimers.

Chitosan-based multi-liposomal complexes: Synthesis, biodegradability and cytotoxicity.

Anionic liposomes were electrostatically adsorbed onto the surface of cationic chitosan particles cross-linked by sulfate anions, forming multi-liposomal containers (MLCs) for encapsulation and delivery of bioactive substances. An increase in molecular mass of chitosan from 30 to 300 kDa results in a size increase of chitosan particles, from 200 to 400 nm. Being saturated by liposomes, chitosan particles give MLCs of 320-540 nm. Each chitosan particle carries between 60 and 200 liposomes. The proteolytic complex Morikrase, a mixture of enzymes with various specificities, induces degradation of MLCs down to particles of size 10-15 nm; the higher the molecular mass of chitosan, the slower the enzyme-induced MLCs' degradation. pH variation within 5.5-7 and cholesterol incorporation into the liposomal membrane both have a minor effect on the rate of MLCs' biodegradation. Both the MLCs and the products of their biodegradation show low cytotoxicity. These results are of interest for constructing biodegradable capacious carriers of bioactive substances.

The one-step synthesis of polymer-based magnetic γ-Fe2O3/carboxymethyl cellulose nanocomposites.

A novel one-step procedure is described for synthesizing water soluble biocompatible nanocomposites from maghemite nanoparticles and carboxymethyl cellulose (CMC). The procedure allows the magneto-sensitive nanocomposites with a controlled content of the inorganic phase. The maghemite formation has been proved by X-ray diffraction analysis and Mossbauer spectroscopy. An average diameter of the maghemite nanoparticles is equal to 11nm according to transmission electron microscopy. As shown by FTIR spectroscopy, the nanoparticles bind to the polymer matrix via electrostatic and coordination interactions. The diameter of the nanocomposites in dilute aqueous solutions vary from 50nm at the iron content of 2-4.3wt.% to 140nm at the iron content of 5.2-8.6wt.%. The study of specific magnetization of the nanocomposites vs. applied magnetic field indicates their ferromagnetic properties. The saturation magnetization and coercive force of the sample with the maximum maghemite content (8.6wt.%) are 11.5emu/g and 30.08Oe, respectively. The nanocomposite motion has been shown to be controlled by an external magnetic field. The biocompartible maghemite-CMC nanocomposites seem to be promising for encapsulation and delivery of biologically active compounds.

Inhibitory Effect of Polyethylene Oxide and Polypropylene Oxide Triblock Copolymers on Aggregation and Fusion of Atherogenic Low Density Lipoproteins.

Triblock copolymers of poly(ethylene oxide) and poly(propylene oxide) (so-called pluronics) were shown to influence the aggregation and fusion of atherogenic low density lipoproteins (atLDL) and be able to inhibit these processes. The character of the influence and the degree of the stabilizing effect depended on the structure, relative hydrophobicity, and concentration of the copolymer. Pluronics L61, P85, and L64 characterized by the hydrophilic-lipophilic balance (HLB) value from 3 to 16 had the greatest ability to suppress the aggregation of atLDL. Pluronic L81 with the higher hydrophobicity (HLB = 2) partially inhibited atLDL aggregation at low concentrations but stimulated it at high concentrations. The influence of pluronics did not have a direct connection with their ability for micelle formation, but it was realized through individual macromolecules. We suppose that effects of pluronics could be due to their interaction with the lipid component of LDL and to a possible influence of these copolymers on the structure and hydrophilic-lipophilic characteristics of lipoproteins.

Multi-liposomal containers.

Small unilamellar liposomes, 40-60 nm in diameter, composed of anionic diphosphatidylglycerol (cardiolipin, CL(2-)) or phosphatidylcerine (PS(1-)) and zwitter-ionic egg yolk lecithin (EL) or dipalmitoylphosphatidylcholine (DPPC), electrostatically complex with polystyrene microspheres, ca. 100 nm in diameter, grafted by polycationic chains ("spherical polycationic brushes", SPBs). Polymer/liposome binding studies were carried out using electrophoretic mobility (EPM), dynamic light scattering (DLS), fluorescence, conductometry, differential scanning calorimetry (DSC), and cryogenic transmission electron microscopy (cryo-TEM) as the main analytical tools. By these means a remarkably detailed picture emerges of molecular events inside a membrane. The following are among the most important conclusions that arose from the experiments: (a) binding of liposomes to SPBs is accompanied by flip-flop of anionic lipids from the inner to the outer leaflet of the liposomal membrane along with lateral lipid segregation into "islands". (b) The SPB-induced structural reorganization of the liposomal membrane, together with the geometry of anionic lipid molecules, determines the maximum molar fraction of anionic lipid (a key parameter designated as ν) that ensures the structural integrity of liposomes upon complexation: ν=0.3 for liposomes with conically-shaped CL(2-) and ν=0.5 for liposomes with anionic cylindrically-shaped PS(1-). (c) The number of intact liposomes per SPB particle varies from 40 for (ν=0.1) to 13 (ν=0.5). (d) By using a mixture of liposomes with variety of encapsulated substances, multi-liposomal complexes can be prepared with a high loading capacity and a controlled ratio of the contents. (e) In order to make the mixed anionic liposomes pH-sensitive, they are additionally modified by 30 mol% of a morpholinocyclohexanol-based lipid that undergoes a conformational flip when changing pH. Being complexed with SPBs, such liposomes rapidly release their contents when the pH is reduced from 7.0 to 5.0. The results allow loaded liposomes to be concentrated within a rather small volume and, thereby, the preparation of multi-liposomal containers of promise in the drug delivery field.

Polymeric stabilizers for protection of soil and ground against wind and water erosion.

The article is devoted to the design, development and application of a new generation of binders for various dispersed systems, including soil, ground, sand, waste rock and others. The binders are formed by interaction of oppositely charged polyelectrolytes, both chemically stable and (bio)degradable. The fundamental aspects of interpolyelectrolyte reactions are discussed; the IPC structure and properties of the resulting interpolyelectrolyte complexes (IPCs) allow considering them as unique and universal binders. Numerous results of laboratory experiments and field trials of the IPC formulations are presented. In particular, large-scale tests have been done in the Chernobyl accident zone where the IPC binders were shown to be effective means to suppress water and wind erosion thereby preventing a spread of radioactive particles (radionuclides) from contaminated sites. Ecologically friendly IPC compositions are described, including those based on commercially available polymers; prospects for improving their efficiency and extending the range of their possible use are discussed.

Pluronics suppress association of low-density lipoproteins inducing atherogenesis.

We studied the effect of pluronics P85, L61, and F68 with different hydrophilic-lipophilic characteristics on association of LDL. It was found that pluronics with pronounced hydrophobic properties (P85 and L61) in concentrations close to or surpassing the critical concentration of micelle formation inhibited LDL association, while hydrophilic pluronic F68 in all concentrations had no effect on LDL association.

Complexes between anionic liposomes and spherical polycationic brushes. An assembly of assemblies.

This paper has at its objective the assembling of liposomal assemblies onto nanoparticles. In this manner, one generates nanoparticles with a high loading capacity. Thus, spherical spherical polycationic "brushes" (SPBs) were synthesized by graft polymerizing a cationic monomer, (trimethylammonium)ethylmethacrylate chloride, onto the surface of monodisperse polystyrene particles, ca. 100 nm in diameter. These particles were complexed with small unilamellar anionic liposomes, 40-60 nm in diameter, composed of egg lecithin (EL) and anionic phosphatidylserine (PS(1-)) in PS(1-)/EL ratios from 0.10 to 0.54, a key parameter designated as ν. These complexes were then characterized according to electrophoretic mobility, dynamic light scattering, conductivity, fluorescence, and cryogenic transmission electron microscopy, with the following main conclusions: (a) All added liposomes are totally associated with SPBs up to a certain saturation concentration (specific for each ν value). (b) The number of liposomes per SPB particle varies from 40 (ν = 0.1) to 14 (ν = 0.5). (c) At sufficiently high liposome concentrations, the SPBs experience an overall change from positive to negative charge. (d) SPB complexes tend to aggregate when their initial positive charge has been precisely neutralized by the anionic liposomes. Aggregation is impeded by either positive charge at lower lipid concentrations, or negative charge at higher lipid concentrations. (e) The liposomes remain intact (i.e., do not leak) when associated with SPBs, at ν ≤ 0.5. (f) Complete SPB/liposome dissociation occurs at external [NaCl] = 0.3 M for ν = 0.1 and at 0.6 M for ν = 0.5. Liposomes with ν = 0.54 do not dissociate from the SPBs even in NaCl solutions up to 1.0 M. (g) Complexation of the PS(1-)/EL liposomes to the SPBs induces flip-flop of PS(1-) from the inner leaflet to the outer leaflet. (h) The differences in the ability of PS(1-) (a cylindrical lipid) and CL(2-) (a conical lipid) to create membranes defects are attributed to geometric factors.

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.

Contrasting behavior of zwitterionic and cationic polymers bound to anionic liposomes.

Zwitterionic polymers were prepared by quaternizing polyvinylpyridine (DP = 1100) with bromoacids (Br(CH2)nCOOH, where n = 1, 2, 3, and 5). The resulting polymers were then added to unilamellar liposomes composed of egg lecithin or dipalmitoylphosphatidylcholine admixed with 20 mol % of cardiolipin (a phospholipid with two negative charges). These systems were compared (along with polyethylvinylpyridinium chloride, a polycation) by light scattering, electrophoretic mobility, fluorescence, and high-sensitivity differential scanning calorimetry. The external zwitterionic polymers induce no flip-flop of cardiolipin from the inner leaflet to the outer leaflet as does the polycation. Aside from this similarity, the four zwitterionic polymers all behave differently from each other toward the anionic liposomes: (a) For n = 1, there is no detectable interaction between the polymer and the liposomes. (b) For n = 2, electrostatic attraction induces polymer-liposome association (reversed by the addition of NaCl) that maintains the original negative charge on the liposome. Aggregation of the liposomes accompanies polymer adsorption. (c) For n = 3, electrostatic binding also occurs along with aggregation. However, the binding is so strong that NaCl is unable to induce polymer/liposome dissociation. (d) For n = 5, there is polymer binding and NaCl-promoted dissociation but no substantial aggregation. These differences among the closely related polymers are discussed and analyzed in molecular terms.

Doxorubicin-poly(acrylic acid) complexes: interaction with liposomes.

Complexation of antitumor drug, doxorubicin (DOX), with poly(acrylic acid) (PAA) in buffer solutions was examined. The DOX-to-PAA binding was governed by electrostatic and stacking interactions resulting in a complex of characteristic composition with a PAA/DOX = 1.6 molar ratio. Sizes of the complex particles were found to lie in 600-900-nm range. However, the particles were able to interact with small neutral egg yolk lecithin liposomes (80-100 nm in diameter), a ternary DOX/PAA/liposome complex being formed. The observations and conclusions we made may be useful for interpreting biological effects of polymer-based bioactive constructs.

Membrane transport of a polyacid-tied doxorubicin.

A model, based on fluorescence data, is developed for the poly(acrylic acid)-assisted transport of doxorubicin, a cationic antitumor drug, through a bilayer membrane. Accordingly, the doxorubicin binds to the poly(acrylic acid) via electrostatic polymer-drug interactions plus drug-drug stacking within the complex. This complex associates with neutral egg lecithin vesicles by means of hydrophobic attraction between the doxorubicin and the vesicle bilayers. In the process, the doxorubicin "destacks", providing a fluorescence change that can be monitored. Finally, the doxorubicin enters the vesicle interior which has been imparted with an acidic pH to protonate the doxorubicin and thus, in a second stage, yield an additional fluorescence change that can also be detected. A portion of the poly(acrylic acid), now devoid of doxorubicin, then leaves the outer vesicle surface and enters to external solution.

Effect of polylysine on transformations and permeability of negative vesicular membranes.

Small (40-60 nm in diameter) and large (300-350 nm) negative vesicles were complexed with a cationic polypeptide, poly-L-lysine (PL). Laser microelectrophoresis experiments showed that in small vesicles rendered anionic with the addition of cardiolipin (CL(2-)), only the CL(2-) in the outer leaflet is involved in the complexation with PL. Calorimetric and other data demonstrate that the binding of PL to the membrane surface causes domains ("rafts") of CL(2-) to form in the outer leaflet, and it is these domains that electrostatically bind the polymer. The kinetics of transmembrane permeation of doxorubicin (Dox, a fluorescent anti-tumor drug) was monitored with and without PL binding to the outer surface of the vesicles. It was found that PL mediates the permeation of Dox into the vesicle interior. In the absence of PL, the Dox molecule (possessing an amino group of pK(a)=8.6) binds to the anionic vesicles in the protonated form and, consequently, suffers an impaired mobility through the membrane. On the other hand, when the PL covers the vesicle surface, Dox passes though the membrane with greater ease. The effects of salt and polyanion on the stability of PL-vesicle complexes and the PL-mediated Dox permeation are also discussed.

Kinetically different populations of O-pyromellityl-gramicidin channels induced by poly-L-lysines in lipid bilayers.

Clustering of membrane proteins, in particular of ion channels, plays an important role in their functioning. To further elucidate the mechanism of such ion channel activity regulation, we performed experiments with a model system comprising the negatively-charged gramicidin analog, O-pyromellitylgramicidin (OPg) that forms ion channels in bilayer lipid membrane (BLM), and polycations. The effect of polylysines on the kinetics of OPg channels in BLM was studied by the method of sensitized photoinactivation. As found in our previous work, the interaction of polylysine with OPg led to the deceleration of the OPg photoinactivation kinetics, i.e., to the increase in the characteristic time of OPg photoinactivation. It was shown here that in a certain range of polylysine concentrations the photoinactivation kinetics displayed systematic deviations from a monoexponential curve and was well described by a sum of two exponentials. The deviations from the monoexponential approximation were more pronounced with polylysines having a lower degree of polymerization. These deviations increased also upon the elevation of the ionic strength of the bathing solution and the addition of calcium ions. A theoretical model is presented that relates the OPg photoinactivation kinetics at different concentration ratios of OPg and polylysine to the distribution of OPg molecules among OPg-polylysine clusters of different stoichiometry. This model is shown to explain qualitatively the experimental results, although the quantitative description of the whole body of evidence requires further development, assuming that the interaction of polylysine with OPg causes segregation of membrane domains enriched in OPg channels. The single-channel data, which revealed the insensitivity of the single-channel lifetime of OPg to the addition of polylysine, are in good agreement with the theoretical model.

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.

What happens to negatively charged lipid vesicles upon interacting with polycation species?

Complexation of synthetic polycations with negative lipid vesicles as cell-mimetic species was studied. It was found that such interaction could be accompanied by lateral lipid segregation, highly accelerated transmembrane migration of lipid molecules (polycation-induced flip-flop), incorporation of adsorbed polycations into vesicular membrane as well as aggregation and disruption of vesicles. A polycation adsorbed on the surface of liquid vesicles due to electrostatic attraction could be completely removed from the membrane by increase in simple salt concentration or by recomplexation with polyanions. In contrast, adsorption of a polycation carrying pendant hydrophobic groups was irreversible apparently due to incorporation of these groups into the hydrophobic part of the vesicular membrane. The above mentioned phenomena were examined depending on the polycation structure, fraction of charged lipids in the membrane, vesicle phase state and ionic strength of solution.

Amphiphilic poly-N-vinylpyrrolidones: synthesis, properties and liposome surface modification.

Certain amphiphilic water-soluble polymers including amphiphilic derivatives of polyvinyl pyrrolidone (PVP) were found to be efficient steric protectors for liposomes in vivo. In this study, we have tried to develop synthetic pathways for preparing amphiphilic PVP and to investigate the influence of the hydrophilic/hydrophobic blocks on some properties of resulting polymers and polymer-coated liposomes. To prepare amphiphilic PVP with the end stearyl (S) or palmityl (P) residues, amino- and carboxy-terminated PVP derivatives were first synthesized by the free-radical polymerization of vinyl pyrrolidone in the presence of amino- or carboxy-mercaptans as chain transfer agents, and then modified by interaction of amino-PVP with stearoyl chloride or palmitoyl chloride, or by dicyclohexyl carbodiimide coupling of stearylamine with carboxy-PVP. ESR-spectra of the hydrophobic spin-probe, nitroxyl radical N-oxyl-2-hexyl-2-(10-methoxycarbonyl)decyl-4,4'-dimethyl oxazoline, in the presence of amphiphilic PVP demonstrated good accessibility of terminal P- and S-groups for the interaction with other hydrophobic ligands. Spontaneous micellization and low CMC values (in a low micromolar range) were found for amphiphilic PVP derivatives using the pyrene method. In general, S-PVP forms more stable micelles than P-PVP (at similar MW, CMC values for S-PVP are lower than for P-PVP). It was found that amphiphilic PVP incorporated into negatively charged liposomes effectively prevents polycation(poly-ethylpyridinium-4-vinylchloride)-induced liposome aggregation, completely abolishing it at ca. 10 mol% polymer content in liposomes. Additionally, the liposome-incorporated PVP prevents the fluorescence quenching of the membrane-incorporated hydrophobic fluorescent label [N-(4-fluoresceinthiocarbamoyl)dipalmitoyl-PE] by the free polycation. PVP-modified liposomes were loaded with a self-quenching concentration of carboxyfluorescein, and their destabilization in the presence of mouse serum was investigated following the release of free dye. Amphiphilic PVP with MW between 1,500 and 8,000 provides good steric protection for liposomes. The degree of this protection depends on both polymer concentration and molecular size of the PVP block.

Interaction of a cationic polymer with negatively charged proteoliposomes.

Proteoliposomes were prepared by making bilayer vesicles from neutral egg yolk lecithin and negatively charged alpha-chymotrypsin that had been previously stearoylated. Interaction of these proteoliposomes with a cationic polymer, poly-(N-ethyl-4-vinylpryidinium bromide) (PEVP) was examined. For comparison purposes, interaction of PEVP with egg lecithin vesicles containing an anionic phospholipid, cardiolipin, was also examined. Binding of PEVP to both types of vesicles was electrostatic in nature with the polymer manifesting a higher affinity to the cardiolipin relative to the enzyme. PEVP had no effect on the permeability of the bilayer membranes to sodium chloride. On the other hand, PEVP increased the transmembrane permeability of the nonionic anti-tumor drug, doxorubicin. The greater the negatively charged component in the membrane, the greater the PEVP effect. Polycation binding to the vesicles was accompanied by clustering of the stearoylated chymotrypsin (sCT) molecules within the membrane. This protein clustering is most likely responsible for the increase in the doxorubicin permeation. Enzymatic activity of the membrane-associated sCT remained unchanged upon PEVP binding. These findings seem relevant to the effects of polyelectrolytes on cellular membranes.

Conventional and gemini surfactants embedded within bilayer membranes: contrasting behavior.

Laser microelectrophoresis (coupled with conductance, fluorescence, and dynamic light scattering) is shown to be a highly instructive tool in comparing the dynamics of conventional and gemini surfactants embedded within vesicle bilayers. The following can be listed among the more important observations and conclusions: a) Cationic conventional surfactant, added to a "solid" (gel) lipid vesicle containing an anionic phospholipid, charge-neutralizes only half the anionic charge. With a "liquid" (liquid crystalline) vesicle, however, the entire negative charge is neutralized. Thus, the cationic conventional surfactant can "flip-flop" readily only in the liquid membrane. b) A cationic gemini surfactant charge-neutralizes only the anionic lipid in the outer membrane leaflet of either solid or liquid membranes, thus indicating an inability to flip-flop regardless of the phase-state of the bilayer. c) Mixed population experiments show that surfactants can hop from one vesicle to another in liquid but not solid membranes. d) In liquid, but not solid, bilayers, a surface-adsorbed cationic polymer can electrostatically "drag" anionic surfactant from the inner leaflet to the outer leaflet where the polymer resides. e) Peripheral fluorescence quenching experiments show that a cationic polymer, adhered to anionic vesicles, can be forced to dissociate in the presence of high concentrations of salt or an anionic polymer. f) Adsorbed polymer, of opposite charge to that imparted to vesicles by a gemini surfactant, is unable to dislocate surfactant even in a liquid membrane. g) In our systems, ionic polymers will not bind to neutral vesicles made solely of zwitterionic phospholipid. On the other hand, ionic polymers bind to neutral vesicles if charge neutrality is obtained by virtue of the membrane containing equimolar amounts of cationic and anionic surfactant. This is attributable to surfactant segregation within the bilayer. h) Experiments prove that polymer migration can occur among a population of neutral ternary vesicles.

Stabilization of O-pyromellitylgramicidin channels in bilayer lipid membranes through electrostatic interaction with polylysines of different chain lengths.

Functioning of membrane proteins, in particular ionic channels, can be modulated by alteration of their arrangement in membranes. We addressed this issue by studying the effect of different chain length polylysines on the kinetics of ionic channels formed in a bilayer lipid membrane (BLM) by O-pyromellitylgramicidin carrying three negative charges at the C-terminus. The method of sensitized photoinactivation was applied to the analysis of the channel association-dissociation kinetics (characterized by the exponential factor of the curve describing the time course of the flash-induced decrease in the transmembrane current, tau). Addition of polylysine to the bathing solutions of BLM led to the deceleration of the photoinactivation kinetics, i.e. to the increase in tau. It was shown here that for a series of polylysines differing in their chain lengths, the value of tau grew as their concentration increased above a threshold level until at a certain concentration of each polylysine tau reached maximum. At higher polylysine concentrations tau began to decrease and finally became close to the control level observed in the absence of polylysine. With lengthening of the polylysine chain the maximum value of tau increased, the concentration dependence became steeper, and the threshold concentration decreased. The increase in the ionic strength of the medium shifted the concentration dependence of tau to higher polylysine concentrations and decreased the maximum value of tau. It was concluded that the increase in tau was caused by the formation of domains of O-pyromellitylgramicidin molecules induced by binding of polylysines. This can be related to functional aspects of polycation-induced sequestering of negatively charged transmembrane peptides in neutral membranes.