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Langmuir 2007, 23, 10034-10039
Complexation of Polycations to Anionic Liposomes: Composition and Structure of the Interfacial Complexes A. V. Sybachin, A. A. Efimova, E. A. Litmanovich, F. M. Menger,*,† and A. A. Yaroslavov* Department of Chemistry, M. V. LomonosoV Moscow State UniVersity, Leninskie Gory, Moscow 119992, Russia, and Department of Chemistry, Emory UniVersity, Atlanta, Georgia 30322 ReceiVed May 17, 2007. In Final Form: June 20, 2007 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 (ν) 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 (ν) 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 ν ) 0.05; 3 molecules for ν ) 0.1; 4- molecules for ν ) 0.15; and 6 molecules for ν ) 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.
Introduction A suspension of spherical bilayer vesicles (liposomes), composed of natural or synthetic lipids, is a simple and convenient model for quantifying interactions of cells with biologically active polymers and polymer-based constructs. Using vesicles of different lipid composition, size, and surface charge, chemists have examined the composition of resulting polymer/lipid complexes, the reversibility of complexation, the polymer-induced structural rearrangements in the lipid bilayer, and the permeability of polymer-bound bilayers.1-13 The observations and conclusions, arising from multiple experimental approaches, favored better understanding of polymer-induced effects in biological membranes.14,15 * To whom correspondence should be addressed. E-mail: menger@ emory.edu (F.F.M.);
[email protected] (A.A.Y.). † Emory University. (1) Hammes, G. G.; Schullery, S. E. Biochemistry 1970, 9, 2555-2563. (2) Bach, D. J. Membr. Biol. 1973, 14, 57-62. (3) de Kruijff, B.; Rietveld, A.; Telders, N.; Vaandrager, B. Biochim. Biophys. Acta 1985, 820, 295-304. (4) Bordi, F.; Cametti, C.; Gili, T.; Gaudino, D.; Sennato, S. Bioelectrochemistry 2003, 59, 99-106. (5) Kitaeva, M. V.; Melik-Nubarov, N. S.; Menger, F. M.; Yaroslavov, A. A. Langmuir 2004, 20, 6575-6579. (6) Yaroslavov, A. A.; Efimova, A. A.; Lobyshev, V. I.; Kabanov, V. A. Biochim. Biophys. Acta 2002, 1560, 14-24. (7) Simon, J.; Kuehner, M.; Ringsdorf, H.; Sackmann, E. Chem. Phys. Lipids 1995, 76, 241-258. (8) Volodkin, D.; Ball, V.; Schaaf, P.; Voegel, J.-C.; Mohwald, H. Biochim. Biophys. Acta 2007, 1768, 280-290. (9) Bronich, T. K.; Solomatin, S. V.; Yaroslavov, A. A.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Langmuir 2000, 16, 4877-4881. (10) Uchida, D. A.; Irvin, C. G.; Ballowe, C.; Larsen, G.; Cott, G. R. Exp. Lung Res. 1996, 22, 85-99. (11) Silvander, M.; Bergstrand, N.; Edwards, K. Chem. Phys. Lipids 2003, 126, 77-83. (12) Kozlova, N. O.; Bruskovskaya, I. B.; Okuneva, I. B.; Melik-Nubarov, N. S.; Yaroslavov, A. A.; Kabanov, V. A.; Menger, F. M. Biochim. Biophys. Acta 2001, 1514, 139-151.
In spite of impressive achievements, however, a number of questions remain in doubt. In particular, the structure of a polymer layer at the water/lipid membrane interface is not understood. This problem is exacerbated by a tendency of the liposomes to aggregate when binding to the polymers.8,16,17 An uncontrollable increase in the size of complex particles does not allow, for example, estimation of the thickness of the adsorbed polymer layer, a parameter responsible for the stabilization and fusion of polymer-covered liposomes. In the present work, we analyze the complexation of a cationic polymer, namely, quaternized poly(4-vinylpyridine) or PEVP, drawn below as structure I, to small anionic liposomes that serve as a mimic for cell membranes. Our main goal was to quantify the adsorbed polycation layer, particularly with regard to the amount of polycation and the thickness of its layer, as a function of the anionic charge on the liposomes. Such information might be useful for controlling polymer-mediated events in actual biological membranes. Experimental Section Poly-4-vinylpyridine (PVP) was synthesized by radical polymerization of 4-vinylpyridine in 30% methanol solution using azoisobutyronitrile as the initiator and then fractionated according to a sequential sedimentation procedure. Ethylacetate was used as a soft precipitator.18 Twelve PVP fractions with different molecular masses (MM) were thus obtained. The fourth fraction with MM ) 110 000 (obtained by light scattering), or a degree of polymerization (13) Glazunova, O. O.; Korepanova, E. A.; Efimov, V. S.; Smirnov, A. I.; Vladimirov, Yu. A. Membr. Cell Biol. 1998, 12, 401-409. (14) Kabanov, A. V. AdV. Drug DeliVery ReV. 2006, 58, 1597-1621. (15) Yaroslavov, A. A.; Melik-Nubarov, N. S.; Menger, F. M. Acc. Chem. Res. 2006, 39, 702-710. (16) Kozlova, N. O.; Bruskovskaya, I. B.; Okuneva, I. B.; Melik-Nubarov, N. S.; Yaroslavov, A. A.; Kabanov, V. A.; Menger, F. M. Biochim. Biophys. Acta 2001, 1514, 139-151. (17) Ohki, S.; Duax, J. Biochim. Biophys. Acta 1986, 861, 177-186.
10.1021/la701411y CCC: $37.00 © 2007 American Chemical Society Published on Web 08/24/2007
Complexation of Polycations to Anionic Liposomes (DP) equal to 1100, was modified with ethyl bromide following a technique described elsewhere.18 The polydispersity index was roughly estimated to be 1.2-1.3. As determined by IR spectroscopy, the resulting product was a copolymer containing 93 mol % vinyl pyridinium and 7% residual 4-vinylpyridine units (I). Concentrations of PEVP are given in moles of quaternized units per liter.
Zwitterionic egg lecithin (phosphatidylcholine, PC) (II), doubly anionic diphosphatidylglycerol (cardiolipin, CL2-) (III), and Nfluorescein-isothiocyanyldipalmitoylphosphatidylethanolamine (FITCDPPE) (IV) from Sigma were used as received.
Langmuir, Vol. 23, No. 20, 2007 10035 The fluorescence intensity of FITC-labeled liposome suspensions was measured at λem ) 525 nm (λex)495 nm) using a F-4000 Hitachi fluorescence spectrophotometer. The pH measurements were carried out with a PHM83 potentiometer coupled to a standard glass electrode 2040C (Radiometer). To measure the amount of PEVP unbound to the liposomes, PEVP-liposome mixtures were centrifuged for 40 min at 18 500 rpm using a J-21 Beckman centrifuge. Absorbances of the clear supernatants were then measured at λ ) 257 nm with a 150/20 UV/vis Hitachi spectrophotometer using ) 3350 M-1 cm-1.20 PEVP concentrations were calculated from a calibration curve. The electrophoretic mobility (EPM) of the liposomes and their complexes with PEVP was measured by laser microelectrophoresis in a thermostatic cell using a Zetasizer IIc instrument (Malvern) equipped with a He-Ne-laser and a K7032090 Malvern autocorrelator. Software provided by the manufacturer generated the EPM values directly. Dynamic light scattering measurements were carried out using a Photocor Complex laser light goniometer (Photocor Instruments) equipped with a He-Ne laser. Light intensity fluctuations (g2(τ)) were monitored using a Photocor FC digital real-time 288-channel correlator (Photocor Instruments). Data processing was performed using DynaLS software. This allowed the calculation of diffusion coefficients for the liposomes, PEVP macromolecules, and liposome-PEVP complexes. The results were converted into mean hydrodynamic diameters with the aid of the Stokes-Einstein equation assuming sphericity for all scattered species. Experiments were performed in a 10-2 M borate buffer (pH 9.2). To prepare the solutions, double-distilled water was used, which was additionally treated by passing it through Milli-Q Millipore system composed of ion-exchange and adsorption columns as well as a filter to remove large particles. Polymer-to-liposome binding was examined at 20 °C. Under these conditions, the membranes of PC/CL2- liposomes were in the fluid (liquid-crystalline) state.21
Results and Discussion
Small unilamellar anionic liposomes 40-60 nm in diameter were prepared by the following procedure. Appropriate amounts of PC and CL2- solutions in methanol were mixed in a flask, after which the solvent was evaporated under vacuum. A thin film of the lipid mixture was dispersed in a borate buffer (pH 9.2, 10-2 M) with a 4700 Cole-Parmer ultrasonic homogenizer for 400 s. Liposome samples thus obtained, separated from titanium dust by centrifugation for 5 min at 10 000 rpm, were used within 1 day. EL/CL2- liposomes with the FITC-DPPE label incorporated into the bilayer were prepared by the same procedure except that 0.5 wt % FITC-DPPE was added to the lipid mixture solution before solvent evaporation. (18) Fuoss, R. M.; Strauss, U. P. J. Polym. Sci. 1948, 3, 602-603. (19) Starodubtsev, S. G.; Kirsh, Yu. E.; Kabanov, V. A. Eur. Polym. J 1977, 10, 739-744.
Binding of cationic PEVP to anionic liposomes is accompanied by neutralization of their surface charges that can be detected by measuring the electrophoretic mobility (EPM) of PEVP/ liposome complex particles using the laser microelectrophoresis technique. EPM values as a function of PEVP concentration are presented in Figure 1 for PC/CL2- liposomes with four different molar fractions of anionic cardiolipin headgroups (ν) where ν ) 2[CL2-]/(2[CL2-] + [PC]) ) 0.05 (curve 1); 0.1 (curve 2); 0.15 (curve 3); and 0.2 (curve 4). Each experimental point on curves 1-4 was obtained by adding a corresponding amount of PEVP solution to a fresh sample of the original liposome dispersion. In all cases, the EPM of the particles decreased down to zero as the negative charge on the liposomes was totally neutralized by adsorbed PEVP. The overall surface charge then became positive when the charge supplied by PEVP was in excess over that of CL2-. Concentrations of polycation required for complete neutralization of the liposome surface charges ([PEVP]EPM)0) appeared to be linear in ν (see inset in Figure 1). It had been shown earlier that in the liposomal membranes of PC/CL2- (ν ) 0.2) the CL2molecules are uniformly and equally distributed between both membrane leaflets.22 Adsorption of PEVP induces the migration of CL2- molecules from the inner to outer leaflet (flip-flop). The linearity of [PEVP]EPM)0 with respect to ν definitely shows that (20) Kirsh, Yu. E.; Pluzhnov, S. K.; Shomina, T. S.; Kabanov, V. A.; Kargin, V. A. Vysokomol. Soedin. 1970, 12, 186-193. (21) New, R. R. C. In Liposomes: a practical approach; Rickwood, D., Hames, B. D., Eds.; Practical Approach Series 301; Oxford University Press: New York, 1990. (22) Yaroslavov, A. A.; Kuchenkova, O. Y.; Okuneva, I. B.; Melik-Nubarov, N. S.; Kozlova, N. O.; Lobyshev, V. I.; Menger, F. M.; Kabanov, V. A. Biochim. Biophys. Acta 2003, 1611, 44-54.
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Figure 1. EPM of PC/CL2- liposomes versus PEVP concentration: ν ) 0.05 (1), 0.1 (2), 0.15 (3), and 0.2 (4). Inset: PEVP concentration, required for complete neutralization of the liposome surface charge, versus ν. Total lipid concentration ) 1 mg/mL; 10-2 M borate buffer, pH 9.
Figure 2. Relative fluorescence intensity of labeled PC/CL2- (ν ) 0.2) liposomes versus PEVP concentration. Inset: Changes in the fluorescence intensity of labeled EL/CL2- liposomes, complexed with PEVP, after NaCl addition. Total lipid concentration ) 1 mg/ mL; 10-2 M borate buffer, pH 9.
PEVP, being bound to PC/CL2- liposomes with ν e 0.2, also electrostatically interacts with the entire CL2- population. This means that PEVP induces the CL2- flip-flop in all studied PC/ CL2- liposomes with ν e 0.2. Electrostatic PEVP-to-liposome complexation was, as expected, sensitive to the concentration of salt in the surrounding solution. PEVP, an effective fluorescence quencher, was added to a suspension of fluorescent-labeled PC/CL2- (ν ) 0.2) liposomes, whereupon a decrease in the tag’s fluorescence resulting from complexation was observed (Figure 2). Further injection of NaCl solution into the PEVP/liposome complex suspension led to a recovery of the fluorescence intensity up to its initial level at [NaCl] ) 0.25 M (see inset in Figure 2), indicating a complete complex dissociation into the component vesicles and PEVP. Complexation of PEVP to PC/CL2- liposomes with lower ν values also demonstrated sensitivity to the salt concentration (data not shown). As mentioned, the liposomes were able adsorb an excess of polycation over that needed for complete neutralization of their surface charge. However, the exact amount of adsorbed PEVP
Sybachin et al.
Figure 3. PEVP concentration in the supernatant after centrifugation of the PEVP/liposome (ν ) 0.2) complex versus total PEVP concentration. PC/CL2- (ν ) 0.2) liposomes; total lipid concentration ) 1 mg/mL; 10-2 M borate buffer, pH 9.
Figure 4. Concentration of liposome-tied PEVP, over neutralizing the liposome surface charge, versus ν. PC/CL2- (ν ) 0.2) liposomes; total lipid concentration ) 1 mg/mL; 10-2 M borate buffer, pH 9.
could not be determined merely from EPM plots in Figure 1. To obtain these values, PEVP/liposome complex particles were separated from water-salt solutions by centrifugation, and the concentrations of PEVP in the supernatants were then measured spectrophotometrically. The results are given in Figure 3 for the case of the PC/CL2- (ν ) 0.2) liposomes. As follows from the figure, complete PEVP binding to our standard liposome preparation was observed at [PEVP]max ) 3.7 × 10-4 M. At higher PEVP concentrations, free polymer appeared in solution. The same procedure was applied to the analysis of PEVP binding to PC/CL2- liposomes with lower CL2- molar fractions, and [PEVP]max concentrations were similarly determined. In all cases, [PEVP]max values exceeded the neutralizing PEVP concentrations ([PEVP]EPM)0) by the same amount given by ∆P ) [PEVP]max - [PEVP]EPM)0 as shown in Figure 4. This means that PEVPto-liposome binding ceased at an identical excess of polycation, ∆P ) 0.7 × 10-4 M, an excess that imparted to the complex particles an EPM of approximately +2 (µm/s)/(V/cm) (see Figure 1). At this EPM value, the positive electrostatic barrier generated on the liposome surface sufficed to suppress all further polycation adsorption.
Complexation of Polycations to Anionic Liposomes
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Figure 5. Size of PC/CL2- liposomes versus PEVP concentration: ν ) 0.05 (1), 0.1 (2), 0.15 (3), and 0.2 (4). Total lipid concentration ) 1 mg/mL; 10-2 M borate buffer, pH 9.
Neutralization of the liposome surface charge resulted in enlargement of the particles in the system (Figure 5), an observation in agreement with earlier published data.6,23 The largest particles were observed at EPM ) 0 (cf. data of Figures 1 and 5). However, a further increase in PEVP concentration caused the particle radius to decrease. In this latter PEVP concentration range, the complex particles were obviously stabilized against aggregation by the positive charge provided by the “overadsorbed” polycation. Our plan was, therefore, to preclude unwanted aggregation of PEVP-coated liposomes by increasing the concentration of polycation. In the absence of aggregation, the thickness of the adsorbed polycation monolayer on the surface of a single liposome could be estimated with less interference. Sizes of initial liposomes with and without adsorbed PEVP were carefully monitored by dynamic light scattering. The procedure was as follows: First, diffusion coefficients for PC/ CL2- liposomes were measured at several liposome concentrations and then reformulated into hydrodynamic radii using the Stokes equation. As shown in Figure 6 (curve 1), where the results for PC/CL2- (ν ) 0.2) liposomes are represented, the radius of the liposomes hardly changed with their concentration, with the mean radius (Rh,lip) being equal to 30 nm. The experiment was then repeated for PC/CL2- liposomes with ν ) 0.05, 0.1, and 0.15, and an independence of liposome radii on liposome concentration for these three systems was also observed. The mean radii fluctuated slightly from sample to sample but always remained within the 30-35 nm range. The same approach was used to determine a hydrodynamic radius of the PEVP macromolecule versus PEVP concentration (Figure 6, curve 2) in the buffer solution. A constant radius was also registered with a mean value (Rh,pol) equal to 35 nm. This proves that the concentration of the buffer solution used in our experiments (10-2 M) was sufficient to suppress any possible swelling effects of polyelectrolyte coil. Finally, PEVP was added to a series of PC/CL2- liposome suspensions with different liposome concentrations, where [PEVP]/[CL2-] ) 5 in every sample, and the hydrodynamic radii for the liposomes complexed with PEVP (Rh,comp) were (23) Kabanov, V. A.; Yaroslavov, A. A. J. Controlled Release 2002, 78, 267271.
Figure 6. Hydrodynamic radii of the liposomes (ν ) 0.2) (1) and the PEVP/liposome complex (3) versus liposome concentration (lower axis), and hydrodynamic radius of the PEVP macromolecule versus PEVP concentration (2) (upper axis). [PEVP]/[CL2-] ) 5 (for curve 3). 10-2 M borate buffer, pH 9.
measured. Light scattering from the complexes was several orders of magnitude more intense in comparison with that induced by the PEVP macromolecule alone. Therefore, the latter produces negligible error in our measurements and their interpretation. The results for the PEVP/liposome (ν ) 0.2) system are given in Figure 6 (curve 3). Similar to free (unbound) liposomes, the radius of complex particles remained nearly constant within the entire liposome concentration range. The same tendency was found for complexes of PEVP with the other studied liposomes. A change in radius, ∆R ) Rh,comp - Rh,lip, represents, therefore, the thickness of the polymer film on the surface of the liposome. The parameter is given as a function of the molar fraction of anionic CL2- headgroups (ν) in Figure 7. As seen, ∆R increases from 22 to 35 nm as ν increases from 0.05 to 0.2. Intuitively, this is reasonable; the more CL2- in the liposome, the more PEVP binds to it, the larger the radius. The maximum number of PEVP macromolecules adsorbed on the surface of each liposome (Nmax) was calculated based on
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Figure 8. Liposomes covered with 3 (A) and 6 (B) PEVP macromolecules and hydrodynamic radii of the corresponding complex species (dashed lines). Schematic presentation only.
Figure 7. Thickness of the PEVP layer on the liposome surface versus ν (lower axis) and Nmax (upper axis). 10-2 M borate buffer, pH 9.
the following simple considerations. First, the concentration of PEVP macromolecules, ultimately bound to one liposome (PEVPlim), was found by dividing the corresponding [PEVP]max into the degree of polymerization. An amount of lipid molecules involved in one liposome was then estimated, taking into account a 30-35 nm radius of PC/CL2- liposomes and a 70 Å2 area per one lipid molecule.24 By dividing the lipid concentration (taken as moles of lipids per liter) into the amount of the liposomeforming lipids, the “molar” concentration of liposome (L) was obtained. Finally, a PEVPlim-to-L ratio gave Nmax values for liposomes with different ν values: 1-2 molecules (ν ) 0.05), 3 molecules (ν ) 0.1), 4-5 molecules (ν ) 0.15), and 6 molecules (ν ) 0.2). This allowed a correlation between ∆R and Nmax, with its profile being reproduced on the top scale of Figure 7. We know from previous work that the liposomal integrity remains intact unless the ν value exceeds 0.3; at higher ν values, polycation binding results in irreversible membrane disruption.25 Since all our work was carried out at ν e 0.3, we can be secure in believing that membrane destruction was not complicating our data. The fact that the polymer can be completely removed from the liposomes by increasing the salt concentration (e0.4 M) suggests that PEVP macromolecules lie on the liposome surface and are not actually incorporated into the membrane. Thus, the ∆R values given in Figure 7 can be attributed to the thickness of the PEVP layer electrostatically adsorbed onto the anionic liposomal membrane. An “S-shape” of the ∆R versus Nmax profile (Figure 7) could, in principle, result from a conformational change of the adsorbed macromolecules when the fraction of polymer-occupied surface is increased. Although such an effect was described previously for the adsorption of polymers on solid supports, the systems involve rather dense polymer layers in close contact with each other.26-29 For this reason, such a model is hardly suitable for our case, because in our experiments the fraction of CL2- in the (24) Huang, C.; Mason, J. T. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 308-310. (25) Yaroslavov, A. A.; Kiseleva, E. A.; Udalykh, O. Yu.; Kabanov, V. A. Langmuir 1998, 14, 5160-5163. (26) Robb, I. D.; Smith, R. Eur. Polym. J. 1974, 10, 1005-1010. (27) Koopal, L. K.; Lyklema, J. J. Electroanal. Chem. 1979, 100, 895-912. (28) Meadows, J.; Williams, P. A.; Garvey, M. J.; Harrop, R.; Phillips, G. O. J. Colloid Interface Sci. 1989, 132, 319-328. (29) Samoshina, Y.; Nylander, T.; Shubin, V.; Bauer, R.; Eskilsson, K. Langmuir 2005, 21, 4490-4502.
membrane, and hence the fraction of PEVP-occupied liposome surface, never exceeded 20% (40% following PEVP-induced lipid flip-flop). More likely, our ∆R versus Nmax profile reflects a distribution of adsorbed PEVP macromolecules over the liposome surface. Binding of PEVP to liquid PC/CL2- liposomes is accompanied by lateral lipid segregation, that is, the formation of CL2- rafts due to ionic contacts of CL2- headgroups with adsorbed polycation units.6,30,31 Adsorbed PEVP imparts an overall positive charge to PEVP/CL2- interfacial clusters, as was seen from the electrophoresis data (Figure 1). Since, in our experiments, PEVP with a degree of polymerization (DP) equal to 1100 was used, it is reasonable to assume that each adsorbed PEVP polymer chain retains several hundred CL2- molecules in its proximity. Because of electrostatic repulsion, PEVP/CL2- clusters, having an overall positive charge and being presumably capable of traveling along the membrane surface, should likely separate from each other by the maximum distance possible. As shown above, at low CL2- content (ν e 0.1), the number of adsorbed PEVP macromolecules per liposome does not exceed 3. If, under these conditions, PEVP/CL2- clusters move more or less independently, cluster distribution over the membrane could be asymmetric. Such asymmetry would lead to underestimates of Rh,comp and, hence, of the ∆R values. At higher ν, that is, at a higher content of clusters, the clusters are likely distributed more uniformly (more symmetrically) over the external membrane surface to minimize cluster/cluster repulsion. According to this construct, a gain in ∆R with increasing ν should be expected, and this was confirmed by light scattering data (see Figure 7). The maximum ∆R value, equal to 35 nm and representing 6 adsorbed PEVP macromolecules per liposome, was attained at ν ) 0.2. Structures of the PEVP/CL2- clusters and the relative positions of the clusters on the liposomal membrane are schematically represented in Figure 8. Although the numbers of polymer molecules per liposome are only estimates (with uncertainties being associated with departures from spherical shapes and with conformations of adsorbed polymer), the trend is clear: the radius increases as the CL2- content of the bilayer, and hence the number of polymer molecules per liposome, increases. To our knowledge, “counting” polymer adsorption onto liposomes as a function of liposome composition has until now not been attempted. To summarize our experiments and conclusions, poly(N-ethyl4-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 (ν) of anionic headgroups provided by cardiolipin (a doubly anionic lipid). According to electrophoretic mobility data, this led to total charge (30) Ikeda, T.; Lee, B.; Yamaguchi, Y.; Tazuke, S. Biochim. Biophys. Acta 1990, 1021, 56-62. (31) Yaroslavov, A. A.; Kuchenkova, O. Ye.; Okuneva, I. B.; Melik-Nubarov, N. S.; Kozlova, N. O.; Lobyshev, V. I.; Kabanov, V. A.; Menger, F. M. Biochim. Biophys. Acta 2003, 1611, 44-54.
Complexation of Polycations to Anionic Liposomes
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 (ν) increased from 0.05 to 0.20. The greater the content on the anionic lipid
Langmuir, Vol. 23, No. 20, 2007 10039
in the bilayer, the thicker the polymer film. The maximum number of polymer molecules adsorbed onto the liposomes was estimated: 1-2 molecules for ν ) 0.05; 3 molecules for ν ) 0.1; 4-5 molecules for ν ) 0.15; and 6 molecules for ν ) 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. Acknowledgment. The authors thank the following for financial support: The Russian Foundation for Basic Research (RFBR), Grant 06-03-32907; The Fogarty International Research Cooperative Award (FIRCA), Grant TW05555; and the National Institutes of Health, Grant GM021457. LA701411Y
