ISSN 1061-933X, Colloid Journal, 2009, Vol. 71, No. 1, pp. 55–62. © Pleiades Publishing, Ltd., 2009. Original Russian Text © D.A. Davydov, E.G. Yaroslavova, A.A. Efimova, A.A. Yaroslavov, 2009, published in Kolloidnyi Zhurnal, 2009, Vol. 71, No. 1, pp. 56–63.

Migration of a Cationic Polymer between Lipid Vesicles D. A. Davydov, E. G. Yaroslavova, A. A. Efimova, and A. A. Yaroslavov Department of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 119991 Russia Received December 25, 2007

Abstract—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. DOI: 10.1134/S1061933X09010062

INTRODUCTION

anionic lipid and a cationic lipid-like compound, with the latter two being taken in equal amounts; and (3) anionic liposomes with the surface covalently modified by poly(ethylene oxide) (PEO) molecules. In each case, the system is described in the following order: the formation of a PEVP–liposome complex, its stability in water–salt media, and interliposome migration of the polycation. The study of the behavior of the complexes in the presence of salts made it possible to distinguish the systems, which were stabilized due to the electrostatic contacts between both of the components, the polycation and the liposome. As was previously shown, the electrostatic complexes of two oppositely charged linear polyions are capable of exchanging their components [8]. It was reasonable to assume that the polycation electrostatically adsorbed on the liposome membrane will migrate between the liposomes (electrostatic criterion).

Spherical bilayer lipid vesicles (liposomes) have been known for more than 40 years; they were first prepared and described by Bangham’s group [1, 2]. The stable interest of researchers and practical workers in liposomes is explained by a number of considerations, namely, the good biocompatibility of lipids, versatile methods of their preparation, simple control of lipid composition and size of liposomes, and the ability to prepare mixed lipid/protein vesicles. The aforementioned and other remarkable properties of liposomes are described in some reviews and monographs [3–6]. The ability of liposomes to simulate the behavior of biological membranes was used to analyze the mechanism of the interaction between cells and synthetic water-soluble polymers, particularly polyelectrolytes [7]. A principal question arises as to the reversibility of the polyelectrolyte–liposome contact. The answer is of importance from both theoretical (in connection with the quantitative description of the formation and behavior of polymer–colloid complexes) and practical points of view concerning the development of polymer-based drugs of targeted action. In this work, the migration of a cationic polymer, poly(N-ethyl-4-vinylpyridinium bromide) (PEVP), in suspensions of small unilamellar liposomes was investigated. Liposomes of the following three types were used: (1) traditional liposomes formed from anionic and zwitterionic (neutral) lipids; (2) three-component systems consisting of a zwitterionic lipid plus an

EXPERIMENTAL Materials Cationic polymer, PEVP, was prepared by the quaternization of poly(4-vinylpyridine) with the degree of polymerization of 600 (Aldrich) by ethyl bromide in a 10% alcohol solution [9]. The synthesized polymer was precipitated from the reaction mixture into dry diethyl ether, and the precipitate was washed with ether and dried in vacuum. The degree of alkylation, as determined by IR spectroscopy, was equal to 95%. The polymer concentration is expressed in moles of cationic (quaternized) groups per one liter of solution. 55

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Fig. 1. Dependences of the fluorescence intensity (a) of labeled PC/CL liposomes on PEVP concentration and (b) of the resulting complex on the NaCl concentration. The total concentration of lipids is 1 mg/ml, borate buffer concentration is 10–2 M, pH 9.2, and (b) [PEVP] = 2 × 10–4 M.

Phosphatidylcholine, PC, diphosphatidylglycerol (cardiolipin, CL), 1,2-dioleoyl-glycero-3-phosphoethanolamine-N-(carboxyfluorescein), (PEA-CF) (all produced at Avanti), dicetyldimethylammonium bromide (DCMAB) (Sigma), and ether of PEO (degree of polymerization of 20) and cetyl alcohol (Briij 58) (Serva), were used as received. Sodium tetraborate Na2B4O7 · 10H2O and sodium chloride (reagent grade) were used as received. Water, which was used for the preparation of solutions of PEVP, liposomes, and low-molecular-mass salts, was distilled twice and passed through a Milli-Q (Millipore) system comprising ion-exchange and adsorption columns for profound purification from organic impurities and through a filter to remove coarse particles. Water thus purified had a specific conductivity of 0.056 µS/cm. Methods of Investigation The hydrodynamic diameters (sizes) of liposomes and their complexes with the polycation were estimated using an Autosizer 2c instrument (Malvern). The fluorescence intensity of liposome solutions was measured with an F-4000 spectrofluorimeter (Hitachi). When preparing liposome suspensions, metallic dust was removed by centrifugation in a J-11 centrifuge (Beckman). The time and speed of rotation were chosen in such a way that the sedimentation of water-soluble components of the systems did not occur during the centrifugation. Solution pH values were measured on a pH-210 instrument (Hanna) equipped with an HI 1131B combined measuring electrode. All measurements were performed at room temperature.

Small unilamellar PC/CL liposomes were prepared by the sonication method [10]. For this purpose, appropriate amounts of solutions of both lipids in chloroform were mixed in a glass flask, and the organic solvent was removed using a Laborota 4000 vacuum rotary evaporator (Heidolph) at 35°ë. The formed thin film of lipids was dispersed in a 10–2 M borate buffer with pH 9.2 (2 ml), and the mixture was subjected to ultrasonic treatment at a frequency of 22 kHz for 400 s (2 × 200 s) with continuous cooling with water. In experiments, a Cole-Parmer 4710 ultrasonic disperser was used. Liposomes were purified from titanium dust in the centrifuge for 5 min at 10000 rpm and used within 24 h. PC/CL liposomes were thus prepared with a molar fraction of negatively charged CL “heads” of v(–) = 2 [CL]/([PC] + 2[CL]) = 0.2 (each CL molecule contains two anionic groups). The total concentration of lipids in the obtained samples was equal to 10 mg/ml. The concentration of lipids in experiments amounted to 1 mg/ml, unless otherwise specified. The above procedure was used to prepare small unilamellar three-component PC/CL/DCMAB and PC/CL/Briij liposomes and liposomes with fluorescent label incorporated into the bilayers. In the latter case, a solution containing PEA-CF (0.01 mg, 0.05 wt % of the total lipid content) was added to the mixture of lipid solutions. The size of liposomes, as determined by quasi-elastic light scattering, ranged from 50 to 60 nm. RESULTS AND DISCUSSION Polycation + PC/CL Liposome System It is well known that PEVP is an efficient fluorescence quencher [11]. Therefore, the formation of a PEVP–PC/CL liposome complex was monitored by fluorescent spectroscopy measuring variations in the relative fluorescence intensity of a labeled lipid (PEA-CF) incorporated into the liposome membrane. The addition of a PEVP solution to a suspension of labeled PC/CL liposomes with v(–) = 0.2 decreased the intensity of the label fluorescence (Fig. 1a), which suggested the adsorption of the polycation on the liposome membrane. The further addition of a salt (NaCl) to the suspension of PEVP–liposome complex was accompanied by an increase in the label fluorescence intensity (Fig. 1b). The complete restoration of the fluorescence intensity was observed at [NaCl] = 0.15 M that was indicative of the complex dissociation into the initial components, liposomes and PEVP molecules. The obtained results unambiguously indicated that the PEVP–PC/CL liposome complex was stabilized via the formation of multiple salt bonds between quaternized pyridine rings of PEVP and phosphate groups of CL. The salt bonds also provided the complexation of the labeled anionic lipid, PEA-CF, with PEVP. An increase in the salt concentration in the system gave rise COLLOID JOURNAL

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to the shielding of charges of macromolecules and liposomes and, hence, to the dissociation of the complex. As was shown previously, PEVP is bound to anionic liposomes up to the complete neutralization of their surface charge [12, 13]. For a PC/CL liposome suspension (1 mg/ml, v(–) = 0.2), the neutralization of the surface charge occurs at [PEVP] = 2.8 × 10–4 M [14]. At the same time, in our experiments, the fluorescence intensity of a suspension (1 mg/ml) of labeled PC/CL liposomes with the same composition reached a maximum value at [PEVP] = 1.4 × 10–4 M (Fig. 1a), i.e., at a concentration two times lower than that necessary for the complete neutralization of the surface charge of the liposomes. In other words, the polycation adsorbed on the surface of labeled PC/CL liposomes interacted predominantly with the negatively charged lipid containing the fluorescent label. The size of PC/CL liposomes in the presence of PEVP was estimated by quasi-elastic light scattering. Figure 2 shows the dependence of the hydrodynamic diameter of PEVP–liposome complex on the polycation concentration. It can be seen that an initial increase in the particle size was followed by its reduction with a further rise in PEVP concentration. However, the particle size noticeably exceeded the size of the initial PC/CL liposomes throughout the considered concentration range of the polycation. As was previously shown [15], the aggregation of PC/CL liposomes could only be completely suppressed in the presence of a fivefold excess of polycation, i.e., under the conditions when the majority of the incorporated polycation remained free (unbound with liposomes). Thus, PEVP is adsorbed quantitatively on the surface of PC/CL liposomes; however, at small amounts of added polycation ([PEVP]/2[CL] < 1), binding is accompanied by an increase in particle size. COLLOID JOURNAL

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Fig. 3. Fluorescence intensity in a three-component system containing PEVP and labeled and nonlabeled PC/CL liposomes. Curve 1 was obtained by adding nonlabeled liposomes to a PEVP–labeled liposome complex. The complex was prepared by mixing a 1 × 10–4 M PEVP solution with a suspension of labeled liposomes (1 mg/ml). Curve 2 was obtained by adding labeled liposomes to a PEVP–nonlabeled liposome complex. The complex was prepared by mixing a 1 × 10–4 M PEVP solution with a suspension of nonlabeled liposomes (1 mg/ml); 10–2 M borate buffer, pH 9.2.

The question arises as to whether PEVP molecules migrate between liposomes in such aggregates. To answer this question, the following experiment (which seemed to be reasonable) was carried out. The polycation was added to labeled PC/CL liposomes that resulted in the quenching of label fluorescence and liposome aggregation. The total concentration of lipids was equal to 1 mg/ml and the polycation concentration was 10–4 M. As was mentioned above, under such conditions, the added polycation was completely adsorbed on the liposome membrane. Label-free PC/CL liposomes were added to the resulting suspension and, 10 min after mixing, the fluorescence intensity and the average hydrodynamic diameter of the particles in the system were measured. The results obtained for different concentrations of the nonlabeled PC/CL liposomes are presented by curves 1 in Figs. 3a and 3b. It is evident that the label fluorescence intensity increased with the concentration of nonlabeled PC/CL liposomes and reached the ultimate value at the equimolar ratio between the lipids introduced into the system with labeled and nonlabeled liposomes. At the same time, the average particle size in the system decreased more than twofold, i.e., from 420 to 200 nm. At first glance, the obtained result indicates that some fraction of PEVP units initially bonded to the surface of labeled PC/CL liposomes was transferred to the surface of nonlabeled liposomes added at the latter stage. However, this result can be interpreted in another way. An approximately 0.2-rel. unit increase in the fluorescence intensity recorded in the experiment can

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ecules as a whole between individual liposomes. We could do no more than state that the liposomes incorporated into aggregates exchanged more or less long fragments (segments) of adsorbed PEVP molecules.

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Fig. 4. Dependences of the fluorescence intensity (a) of labeled PC/CL/DCMAB liposomes on PEVP concentration and (b) of the resulting complex on NaCl concentration. The total concentration of lipids is 1 mg/ml, borate buffer concatenation is 10–2 M, pH 9.2, and (b) [PEVP] = 2.5 × 10–5 M.

result from a decrease in the turbidity of the system because of a reduction in the particle size of the suspension. The above uncertainty demanded additional experiments. In a subsequent series of experiments, the reverse procedure of component mixing was used; labeled PC/CL liposomes (taken always in the same concentration of 1 mg/ml) were added to a PEVP complex preformed with nonlabeled PC/CL liposomes. The concentration of the latter was varied from 0 to 2 mg/ml, and the PEVP concentration amounted to 10–4 M. Figure 3a (curve 2) exhibits of the fluorescence intensity (in relative units) established in the system 10 min after the components were mixed. It is seen that, in all cases, the fluorescence intensity proved to be lower than that for the initial labeled PC/CL liposomes. In other words, the addition of labeled liposomes to a complex of nonlabeled liposomes and PEVP gave rise to the quenching of the label fluorescence. This process developed in parallel to a decrease in the suspension particle size (curve 2, Fig. 3b). The decline in the fluorescence intensity recorded in the aforementioned experiment unambiguously indicates that a fraction of the PEVP units was transferred from nonlabeled liposomes to labeled ones. It is of interest that the fluorescence level, which was attained upon the reverse mixing of the components (PEVP–nonlabeled liposome complex + labeled liposomes), nearly coincided with the fluorescence magnitude obtained at the direct mixing (PEVP– labeled liposome complex + nonlabeled liposomes) (compare curves 1 and 2 in Fig. 3a); i.e., it was independent of the method used for the preparation of the final three-component system. However, the presence of rather coarse aggregates (several times larger than the original liposomes) in the system did not allow us to relate the fluorescence quenching with the migration of polycation macromol-

Polycation + PC/CL/DCMAB Liposome System As was shown above, the development of the aggregation in the liposome + polycation systems complicates the experiments on the interliposome migration of macromolecules and the interpretation of the obtained results. This leads us to analyze methods for improving the aggregation stability of liposomes, especially their complexes with polycations. One of these methods is the incorporation of a third component, a cationic lipidlike surfactant (“artificial lipid”), into the membranes of two-component negatively charged liposomes [16]. Polycation adsorption on the surface of such threecomponent liposomes results in the neutralization of the charge of the anionic lipid and simultaneous release of the cationic surfactant, whose positive charge provides the electrostatic stabilization of the polycation– liposome complex. Following this approach, three-component liposomes containing electroneutral PC, anionic CL, and cationic DCMAB with the molar fraction of negatively charged CL “heads” v(–) = 2[CL]/([PC] + 2[CL] + [DCMAB]) = 0.2 and the molar fraction of positively charged DCMAB “heads” v(+) = [DCMAB]/([PC] + 2[CL] + [DCMAB]) = 0.17 were prepared. The addition of PEVP to a suspension of PC/CL/DCMAB liposomes labeled by PEA-CF resulted in the quenching of the label fluorescence (Fig. 4a), i.e., in the adsorption of PEVP molecules on the liposome membrane. As can be seen from the figure, at [PEVP] ≤ 2.5 × 10–5 M, the fluorescence intensity decreased linearly with the concentration of the polycation, i.e., in this concentration range, all added PEVP was adsorbed on the surface of liposomes. The parallel measurement of the particle size showed that the adsorption of the polycation is not accompanied by the aggregation of the liposomes. Upon the addition of a low-molecular-mass salt solution, the PEVP–liposome complex dissociated into the initial components that was demonstrated by the complete restoration of the fluorescence of the label incorporated into the bilayer (Fig. 4b). The ability of adsorbed PEVP to migrate between PC/CL/DCMAB liposomes was estimated by the fluorescence method. The polycation (2.5 × 10–5 M) was added to labeled PC/CL/DCMAB liposomes, and the label fluorescence decreased to its minimum level (Fig. 4a). Then, label-free liposomes of the same composition were added to the obtained suspension. Figure 5a shows the fluorescence intensity established in the system 10 min after mixing as a function of the nonlabeled liposome concentration. It is seen that the intensity of label fluorescence increased with the liposome concentration and reached the ultimate value at the equimolar ratio between the lipids introduced into COLLOID JOURNAL

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the system with labeled and nonlabeled liposomes. It is of importance that, at any change in the fluorescence, the average diameter of particles in the system was only slightly larger than the diameter of the initial liposomes (Fig. 5b). Some increase in particle size was related to the formation of the surface layer of adsorbed PEVP. The above result suggested that the addition of nonlabeled three-component PC/CL/DCMAB liposomes to labeled liposomes of the same composition but containing adsorbed PEVP was followed by the redistribution of the polycation between all liposomes in the system. The absence of the aggregation enabled us to state that polycations migrated as a whole between individual liposomes in the course of this process. Polycation + PC/CL/Briij Liposome System The other method for enhancing the aggregation stability of liposomes is the formation of a hydrophilic layer on their surface, for example, by the covalent modification of liposome membranes with PEO molecules [17, 18]. Anionic liposomes with hydrophilic “corona” retain the ability to efficiently bind cationic polymers, and, in this case, the aggregation does not occur even at the complete neutralization of their surface charge by adsorbed polycations [19, 20]. In this work, the surface of PC/CL liposomes was hydrophilized by incorporating molecules of a nonionic surfactant Briij 58 [ether of PEO (degree of polymerization of 20) and cetyl alcohol] into their bilayers. Three-component PC/CL/Briij liposomes with the molar fraction of negatively charged CL heads ν(–) = 2[CL]/([PC] + 2[CL] + [Briij]) = 0.2 and the molar fraction of PEO heads v(neutr) = [Briij]/([PC] + 2[CL] + [Briij]) = 0.3 were prepared. COLLOID JOURNAL

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Fig. 6. Dependences of the fluorescence intensity of (a) labeled PC/CL/Briij liposomes on PEVP concentration and (b) of the resulting complex on NaCl concentration. The total concentration of lipids is 1 mg/ml, borate buffer concentration is 10–2 M, pH 9.2, and (b) [PEVP] = 1 × 10–4 M.

The addition of a PEVP solution to a suspension of three-component PC/CL/Briij liposomes was accompanied by the quenching of the fluorescence of the label incorporated into the liposome membrane (Fig. 6a); in this case, only a slight increase in particle size was observed. Taken together, these results are indicative of the formation of a PEVP complex with individual (nonaggregated) liposomes. A small increase in the liposome sizes in the presence of PEVP was related to the formation of a surface layer of adsorbed macromolecules similar to the case of PEVP attachment to threecomponent PC/CL/DCMAB liposomes (see above). A further addition of a low-molecular-mass salt (NaCl) solution to the suspension of PEVP–liposome complex resulted in the fluorescence restoration to the initial level (Fig. 6b), i.e., in the quantitative desorption of polycation molecules from the liposome membrane. This, in turn, was suggestive of the electrostatic nature of PEVP binding on the surface of hydrophilized anionic liposomes. As in the aforementioned cases, the migration of PEVP molecules between three-component PC/CL/Briij liposomes was monitored by the fluorescence method. As follows from Fig. 6a, a linear decrease in the intensity of the label fluorescence was observed in the range of [PEVP] ≤ 10–4 M. In this concentration range, the polycation was quantitatively adsorbed on the liposome membrane. Based on these considerations, PEVP (10–4 M) was added to a suspension of labeled PC/CL/Briij liposomes that provided the complete binding of PEVP and the maximum quenching of the label. To the resulting suspension, nonlabeled PC/CL/Briij liposomes were added in increasing amounts; however, no rise in the fluores-

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Fig. 7. Fluorescence intensity in a three-component system containing PEVP and labeled and nonlabeled PC/CL/Briij liposomes vs. concentration of liposomes. Curve 1 was obtained by adding nonlabeled liposomes to a PEVP–labeled liposome complex. The complex was prepared by mixing a 1 × 10–4 M PEVP solution with a suspension of labeled liposomes (1 mg/ml). Curve 2 was obtained by adding labeled liposomes to a PEVP–nonlabeled liposome complex. The complex was prepared by mixing a 1 × 10–4 M PEVP solution with a suspension of nonlabeled liposomes (1 mg/ml); 10–2 M borate buffer, pH 9.2.

cence intensity of the system was observed (Fig. 7a, curve 1).

ble to the formation of ionic contacts (salt bonds) with PEVP units.

In the next series of the experiments, the reverse order of component mixing was used. A PEVP solution (10–4 M) was first mixed with different volumes of the suspension of nonlabeled liposomes. To the resulting PEVP–liposome complexes, the labeled liposomes were added (always in the same concentration of 1 mg/ml) and 10 min later the established fluorescence was recorded. Figure 7a (curve 2) shows the dependence of the fluorescence intensity on the concentration of nonlabeled liposomes. It is noteworthy that this curve is nearly coincident with curve 1, which describes variations in the fluorescence intensity upon direct mixing of the components (labeled liposomes + PEVP + nonlabeled liposomes). At all component ratios (for both direct and reverse mixing), the particle size in the system was only slightly larger than the size of the initial liposomes.

The above structure of the interfacial complex reflects a lower relative contribution of PEVP/CL contacts and a higher contribution of PEVP/PEA-CF contacts into the stabilization of PEVP–liposome complex. At a high content of Briij molecules in a membrane, the density of the PEO layer on the surface of liposomes may be sufficient for the shift of the equilibrium to the formation of PEVP–label complex and hence to unidirectional migration of PEVP molecules from nonlabeled PC/CL/Briij liposomes to labeled ones.

The obtained results can be considered proof of the unidirectional migration of PEVP molecules in suspensions of hydrophilized liposomes, that is, from nonlabeled PC/CL/Briij liposomes to labeled ones. The high affinity of PEVP for PC/CL/Briij liposomes containing the labeled lipid (PEA-CF) incorporated into the membrane can be explained as follows. As was noted above, the polycation adsorbed on the surface of CF-labeled PC/CL liposomes deprived of the external hydrophilic PEO layer interacts predominantly with negatively charged PEA-CF. The formation of a PEO layer on the liposome surface must be accompanied by the spatial separation of cationic units of adsorbed PEVP and anionic groups of CL, i.e., by a decrease in the efficiency of the PEVP–CL electrostatic contact. In this case, the negative charge of the label, PEA-CF, exposed into the external aqueous solution can remain accessi-

The results obtained enable us to describe the behavior of the cationic polymer, PEVP, in the liposome suspension as follows. The adsorption of the polycation on the surface of PC/CL liposomes is accompanied by their aggregation. The particle size increases with the degree of neutralization of the surface charge of liposomes by the adsorbed polycation. Three-component PC/CL/Briij and PC/CL/DCMAB liposomes are not aggregated, even at the complete neutralization of the liposome charge by the adsorbed PEVP. The migration of the adsorbed polycation between particles was monitored by the fluorescence method taking advantage of the ability of the polycation to quench the fluorescence of the label incorporated into the liposome membrane. In the interior of the aggregates formed from PC/CL liposomes and PEVP, the units (segments) of the polycation are redistributed between fluorescently labeled and nonlabeled liposomes (Fig. 8a). Adsorbed PEVP molecules are capable of migration between individual (nonaggregated) three-component PC/CL/DCMAB liposomes. As a result, the polycation COLLOID JOURNAL

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Fig. 8. Migration of PEVP between (a) PC/CL, (b) PC/CL/DCMAB, and (c) PC/CL/Briij liposomes (schematic representation).

molecules are distributed rather uniformly between labeled and nonlabeled liposomes (Fig. 8b). PEVP molecules are transferred quantitatively from the surface of PC/CL/Briij liposomes to fluorescently labeled liposomes of the same lipid composition (Fig. 8c). ACKNOWLEDGMENTS The authors are grateful to Prof. A.Yu. Ermakov (Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences) for his participation in the discussion and interpretation of the results of migration experiments. This work was supported by the Russian Foundation for Basic Research (projects nos. 08-03-00744 and 06-03-32907). COLLOID JOURNAL

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COLLOID JOURNAL

Vol. 71

No. 1

2009