Langmuir 1998, 14, 5999-6004
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Competitive Interactions in Negatively Charged Liposome-Polycation-Polyanion Ternary Systems A. A. Yaroslavov,* V. Ye. Koulkov, E. G. Yaroslavova, M. O. Ignatiev, and V. A. Kabanov Faculty of Chemistry, M. V. Lomonosov Moscow State University, Leninskie Gory, Moscow 119899, Russian Federation
F. M. Menger Department of Chemistry, Emory University, Atlanta, Georgia 30322 Received December 8, 1997. In Final Form: June 19, 1998 This paper involves small unilamellar liposomes (SULs) of phosphatidylcholine (PC) imparted with a negative charge via the incorporation of cardiolipin (CL2-), a lipid with a double-negative charge. Such liposomes in water strongly adsorb polycations such as CP(2) (a 93/7 copolymer of N-ethyl-4-vinylpyridinium bromide and 4-vinylpyridine) or CP(2,16) (a 83/3/14 copolymer of N-ethyl-4-vinylpyridinium bromide, N-cetyl-4-vinylpyridinium bromide, and 4-vinylpyridine). Neither CP(2) nor CP(2,16) disrupts the integrity of the PC-CL2- liposome despite the tight binding. Addition of NaCl is able to totally dissociate CP(2) from the negative SULs, whereas CP(2,16) is only partially dissociated even at high salt concentrations. Thus, only 3% long-chain pendant groups on the polycation is sufficient to immobilize the polycation onto the negative surface of SULs. Similarly, poly(acrylic acid) (PAA) in its anionic state will remove CP(2) from the liposome surface to form a CP(2)-PAA complex. CP(2,16), on the other hand, is more resistant to removal. Evidence is provided that the CP(2,16) associates with PAA but nonetheless remains on the surface of the liposome. Thus, a liposome-CP(2,16)-PAA ternary complex is created. This work makes heavy use of photon correlation spectroscopy, conductometry, electrophoretic mobility, and fluorescent labeling of the liposomes.
Introduction In recent years the study of interactions of native and synthetic polyelectrolytes with cells and artificial biological membranes has gained more and more importance because of the continuously growing application of these polymers in medicine and biology.1-8 Because the surface of cells usually carries a net negative charge, interaction of positively charged polyelectrolytes (e.g., proteins at pH values below their isoelectric points and synthetic polycations) with negative biological membranes has been the main focus of past work. It has been shown that adsorption of polycations on the negatively charged lipid membrane may result in an increase of the gel-to-liquid-crystalline phase transition temperature,9-12 lateral phase sep(1) Anionic Polymeric Drugs; Donaruma, L. D., Ottenbrite, R. M., Vogl, O., Eds.; Wiley: New York, 1980. (2) Heller, J. In Recent Advances in Drug Delivery Systems; Anderson J. M., Kim, S. W., Eds.; Plenum Press: London, 1984; p 101. (3) Kabanov, V. A. Makromol. Chem., Macromol. Sym. 1986, 1, 101. (4) Jozefonvicz, J.; Jozefowicz, M. J. Biomater. Sci. Polym. ed. 1990, 1, 147. (5) Kabanov, V. A. In Macromolecular Complexes in Chemistry and Biology; Dubin, P., Block, J., Davies, R. M., Schulz, D. N., Thies C., Eds.; Springer-Verlag: Berlin-Heidelberg, 1994; Chapter 10, and the references quoted. (6) Bystricky, S.; Malovikova, A. In Macromolecular Complexes in Chemistry and Biology; Dubin, P., Block, J., Davies, R. M., Schulz, D. N., Thies C., Eds.; Springer-Verlag: Berlin-Heidelberg, 1994; Chapter 11. (7) Xia, J.; Dubin, P. L. In Macromolecular Complexes in Chemistry and Biology; Dubin, P., Block, J., Davies, R. M., Schulz, D. N., Thies C., Eds.; Springer-Verlag: Berlin-Heidelberg, 1994; Chapter 15. (8) Kabanov, V. A.; Kabanov, A. V. Makromol. Chem., Macromol. Symp. 1995, 98, 601. (9) Hartmann, W.; Galla, H.-J. Biochim. Biophys. Acta 1978, 509, 474. (10) Carrier, D.; Dufourcq, J.; Faucon, J.-F.; Pezolet, M. Biochim. Biophys. Acta 1985, 820, 131.
arations,9-13 transmembrane migrations of lipid molecules,14-16 increases of permeability of membranes to inorganic ions,17-20 and fusion and disruption of membranes.21-24 Recently it was shown25 that integrity of mixed negative liquid small unilamellar liposomes interacting with a polycation critically depends on their charge (i.e., on the negative lipid content). Much less attention has been paid to the study of polyelectrolyte-membrane interactions in the presence of multicharged negative species which are also able to form rather strong complexes with a polycation. Consider, (11) Laroche, G.; Pezolet, M.; Dufourcq, J.; Dufourc, E. L. Prog. Colloid Polym. Sci. 1989, 79, 38. (12) Takahashi, H.; Matuoka, S.; Kato, S.; Ohki, K.; Natta, I. Biochim. Biophys. Acta 1992, 1110, 29. (13) Mittler-Neher, S.; Knoll, W. Biochem. Biophys. Res. Commun. 1989, 162, 124. (14) Yaroslavov, A. A.; Koulkov, V. Ye.; Polinsky, A. S.; Baibakov, B. A.; Kabanov, V. A. FEBS Lett. 1994, 340, 121. (15) Yaroslavov, A. A.; Efimova, A. A.; Koulkov, V. Ye.; Kabanov, V. A. Polym. Sci. 1994, 36, 215. (16) Kabanov, V. A.; Yaroslavov, A. A.; Sukhishvili, S. A. J. Controlled Release 1996, 39, 173. (17) Gad, A.; Silver, B. L.; Eytan, G. D. Biochim. Biophys. Acta 1982, 690, 124. (18) Maeda, M.; Kumano, A.; Tirrell, D. A. J. Am. Chem. Soc. 1988, 110, 7455. (19) Kato, T.; Lee, S.; Ono, S.; Agawa, Y.; Aoyagi, H.; Ohno, M.; Nishino, N. Biochim. Biophys. Acta 1991, 1063, 191. (20) Lee, S.; Iwata, T.; Oyagi, H.; Aoyagi, H.; Ohno, M.; Anzai, K.; Kirino, Y.; Siguhara, G. Biochim. Biophys. Acta 1993, 1151, 76. (21) Wang, C.-Y.; Huang, L. Biochemistry 1984, 23, 4409. (22) Gad, A. E.; Elyashiv, G.; Rosenberg, N. Biochim. Biophys. Acta 1986, 860, 314. (23) Walter, A.; Steer, C. J.; Blumenthal, R. Biochim. Biophys. Acta 1986, 861, 319. (24) Oku, N.; Shibamoto, S.; Ito, F.; Gondo, H.; Nango, M. Biochemistry 1987, 26, 8145. (25) Yaroslavov, A. A.; Kiseliova, E. A.; Udalykh, O. Yu.; Kabanov, V. A. Langmuir, in press.
S0743-7463(97)01346-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/17/1998
6000 Langmuir, Vol. 14, No. 21, 1998 Chart 1
for example, a polycationic compound introduced into a biological liquid (blood, lymph, etc.). Competitive binding of polycations with multianionic species can restrict or even completely terminate the former’s adsorption and further interactions with cells. One way to enhance the affinity of a polycation toward a cell membrane is to modify a polycation chain with pendant fatty fragments able to incorporate into a hydrophobic part of the membrane. This approach was examined in the present work using negatively charged lipid vesicles (liposomes) as a cell mimic in the presence of a synthetic polyanion. Various synthetic polycations would then compete between the liposomes and the polycations. Experimental Section To prepare a series of cationic polymers (CPs) poly-4-vinylpyridine of degree of polymerization (DP) 1100 was synthesized and then quaternized by various alkyl bromides according to ref 26. The following CPs were prepared (see Chart 1 for structures): N-ethyl-4-vinylpyridinium bromide/4-vinylpyridine (93/ 7) copolymer (I), CP(2); N-ethyl-4-vinylpyridinium bromide/Nheptyl-4-vinylpyridinium bromide/4-vinylpyridine (77/3/20) copolymer (II), CP(2,7); N-ethyl-4-vinylpyridinium bromide/Ndodecyl-4-vinylpyridinium bromide/4-vinylpyridine (74/3/23) copolymer (III), CP(2,12); and N-ethyl-4-vinylpyridinium bromide/ N-cetyl-4-vinylpyridinium bromide/4-vinylpyridine (83/3/14) copolymer (IV), CP(2,16). The compositions of all CPs were determined by infrared spectroscopy using UR-20 spectrophotometer (Karl Zeiss, Germany) as described in ref 27. Poly(acrylic acid) (PAA) of DP ) 70 was obtained from Aldrich and used as received. Phosphatidylcholine (V) (egg yolk lecithin, PC), diphosphatidylglycerol (VI) (cardiolipin, CL2-), and dipalmitoylphospha(26) Fuoss, R. M.; Strauss, U. P. J. Polym. Sci. 1948, 3, 246. (27) Kirsh, Yu. E.; Pluzhnov, S. K.; Shomina, T. S.; Kabanov, V. A.; Kargin, V. A. Vysokomolekulyarnye soedineniya 1970, 12A, 186 (Russ).
Yaroslavov et al. Chart 2
tidylethanolamine, N-fluoresceinisothiocyanyl (VII) (PEA-FITC) were obtained from Sigma and used as received (See Chart 2 for structure). To prepare PC-CL2- small unilamellar liposomes (SULs) with diameter 50-70 nm and the molar content of negatively charged CL2- headgroups ν ) 2[CL2-]/(2[CL2-] + [PC]) ) 0.2 (each CL2molecule carries two negatively charged headgroups) the following procedure was used. First, the corresponding amounts of PC and CL2- solutions in methanol were mixed in a flask. The solvent was then carefully evaporated under vacuum. A thin layer of lipid mixture was dispersed in a borate buffer, pH 9.2, 10-2 M, with a Cole-Parmer 4700 ultrasonic homogenizer under ice cooling. Double-distilled water was used additionally treated by passing it through Milli-Q system for deep purification from organic impurities (Millipore, USA). SUL samples thus obtained were separated from titanium dust by centrifugation and used within 1 day. SULs with a fluorescent PEA-FITC label incorporated into the bilayer were prepared as described above, 0.5 wt % of PEAFITC being added to the lipid mixture. To prepare SULs loaded with 1 M NaCl the PC-CL2- lipid film was suspended and sonificated in 1 M NaCl 10-2 M borate buffer solution. The liposome suspension was then separated from the excess of NaCl by passing it through the column with Sephadex G-50, or dialysis against 10-2 M borate buffer. In the both cases, the purified liposomes retained their size and contained significant amount of NaCl inside. Integrity of NaClloaded SULs was controlled by measuring conductivity of the liposome suspensions with a CDM83 conductometer (Radiometer, Denmark). It is known28-30 that SULs, in contrast to multilamellar and large unilamellar liposomes, are osmotically inert. Although a permeation coefficient of phospholipid bilayer (28) Bangham, A. D.; de Gier, J.; Greville, G. D. Chem. Phys. Lipids 1967, 1, 225. (29) Price, H. D.; Thompson, T. E. J. Mol. Biol. 1969, 41, 443. (30) Fettiplace, R. Biochim. Biophys. Acta 1978, 513, 1.
Liposome-Polycation-Polyanion Ternary Systems membranes with respect to water is high (10-3-10-2 cm/s28,29,31,32), SULs are rather stable and do not undergo an osmotic shock. Permeation coefficients of SULs with respect to Na+ (10-1410-13 cm/s33-35) and Cl- [10-12-10-11 cm/s35) are very small. According to our estimation, for the time of preparation and separation from outside salt (about 2 h) SULs could lose less than 1% of incorporated NaCl. Indeed, we did not observe any conductivity change of newly prepared and purified SUL suspensions at least during 2 h. However, SULs prepared were completely destroyed by addition of nonionic surfactant Triton X-100 in 10-fold molar amount with respect to the overall lipid content. It was followed by an increase of conductivity of the dispersion which corresponded to about 0.5-1.0 M concentration of NaCl in the original SULs. It means that conductivity measurement could be actually used as a direct method to control SUL integrity. To measure the amount of CPs unbound to the liposomes, the following procedure was used. Liposome-CP mixtures were centrifuged for 50 min at 18 000 rpm using a J-21 centrifuge (Beckman). The absorbance at λ ) 257 nm was then determined in the clear supernatant using a 150/20 UV/vis spectrophotometer (Hitachi, Japan). The concentrations of CPs were calculated using the calibration curve. Mean hydrodynamic diameter, D, of liposomes and their complexes with CPs were determined by photon correlation spectroscopy in a thermostatic cell with a fixed 90° scattering angle using an Autosizer 2c (Malvern, UK) equipped with a HeNe-laser. A Malvern K7032090 autocorrelator was used. The software provided by the manufacturer was employed to calculate D values. An average value over 10 consecutive measurements is reported. Electrophoretic mobility (EPM) of liposomes and their complexes with CPs was measured by laser microelectrophoresis in a thermostatic cell with using a Zetasizer 2c (Malvern, UK) equipped with a He-Ne laser. A Malvern K7032090 autocorrelator was also used. To determine EPM values, the software provided by the manufacturer was employed. Fluorescence intensity of suspensions of FITC-labeled liposomes was measured using a F-4000 spectrofluorimeter (Hitachi, Japan) at λem ) 525 nm (λex ) 495 nm). pH measurements were done using a PHM83 potentiometer with standard glass electrode 2040C (Radiometer, Denmark). Bromide ion concentrations were measured using a CDM83 conductometer with bromoselective electrode F1022Br (Radiometer, Denmark). All experiments were performed in borate buffer 10-2 M, pH 9.2 at 20 °C.
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Figure 1. Dependence of concentration of CP unbound to PCCL2- SULs on the total CP concentration. CP(2) (1) and CP(2,16) (2). SUL concentration 1 mg/mL; pH 9.2; borate buffer 10-2 M; 20 °C.
Figure 2. Dependence of EPM of PC-CL2- SULs on CP concentration. CP(2) (1); CP(2,7) (2); CP(2,12) (3); and CP(2,16) (4). SUL concentration 1 mg/mL; pH 9.2; borate buffer 10-2 M; 20 °C.
Results and Discussion To clarify the competitive interactions in ternary SULspolycation-polyanion systems, it is reasonable to start studying the behavior of the corresponding binary systems. Liposomes-Polyions. Negative PC-CL2- SULs strongly adsorb polycations from aqueous solutions. Figure 1 demonstrates the tipical binding isotherms for CP(2) and CP(2,16) showing that no polycation remains in solution up to a liposome/CP ratio at which saturation occurs. Adsorption of all studied CPs on PC-CL2- SULs leads to a decrease of the negative surface charge and therefore to a decrease in EPM of the liposomes (Figure 2). EPM reaches a zero value at the charge neutralization point and then becomes positive due to adsorption of an excess of the polycations. As can be seen from Figure 3, polycation-liposome interaction is accompanied by enlargement of the particles (31) Huang, C.; Thompson, T. E. J. Mol. Biol. 1966, 15, 539. (32) Reeves, J. P.; Doulen, R. M. J. Membrane Biol. 1970, 3, 123. (33) Brunner, J.; Graham, D. E.; Hauser, H.; Semenza, G. J. Membrane Biol. 1980, 57, 133. (34) Papahadjopoulos, D.; Nir, S.; Ohki, S. Biochim. Biophys. Acta 1972, 266, 561. (35) Hauser, H.; Barratt, M. D. Biochem. Biophys. Res. Commun. 1973, 53, 399.
Figure 3. Dependence of size of PC-CL2- SULs on CP concentration. CP(2) (1); CP(2,7) (2); CP(2,12) (3); and CP(2,16) (4). SUL concentration 1 mg/mL; pH 9.2; borate buffer 10-2 M; 20 °C.
in the system studied, apparently caused by CP-induced interaction of the original SULs. The largest size of the scattering species, measured by photon correlation spectroscopy, is observed at EPM ) 0. However, further increase of CP concentration results in decrease of the particle size. In this CPs concentration range, the dispersed microscopic phase is stabilized against aggregation by the abundant positive charge of adsorbed CPs.
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Figure 4. Dependence of concentration of Br- ions in a solution after precipitation of PC-CL2- SULs with adsorbed CP on the total Br- ion concentration. CP(2) (1) and CP(2,16) (2). SUL concentration 1 mg/mL; pH 9.2; borate buffer 10-2 M; 20 °C.
As mentioned above, adsorption of polycations on the surface of negatively charged liposomes can lead to drastic reorganizations of the lipid bilayer, including irreversible disruption of the liposomal membrane.21-24 In our experiments, integrity of PC-CL2- SULs after mixing with polycations was monitored conductometrically. The two polycations, CP(2) and CP(2,16), were added to the SULs loaded with 1 M NaCl. Any disruption of the liposome membrane should be accompanied by a leakage of the electrolyte and an increase of solution conductivity. However, neither CP(2) nor CP(2,16) affected solution conductivity. This means that in the large particles resulting from interaction of PC-CL2- SULs with CPs integrity of the individual liposomes is preserved. It was shown earlier15 that in the complexes formed by CP(2) and liquid liposomes composed of CL2- and dipalmitoylphosphatidylcholine, about 90% of quaternized polycation units were involved in ionic contacts with negatively charged CL2- headgroups. The approach was as follows. CP(2) was added to liposomes in salt-free aqueous solution. Liposome-CP(2) aggregates were then isolated from solution by centrifugation, and the concentration of Br- anions was determined in the supernatant using a bromoselective electrode. This concentration was obviously equal to the concentration of quaternized polycation units formed ionic bonds with CL2- headgroups. The same approach was used in the present work to find the amount of CP(2) and CP(2,16) positive units bound to PC-CL2- SUL negative headgroups. The results are given in Figure 4. One can see that also in this case about 90% of CP positive units form ionic pairs with the negative liposomes. The above results allowed us to calculate the composition of the liposome-CP complexes following the procedure first described in ref 15. Obviously, at the point EPM ) 0 the charge introduced by adsorbed CPs equals the surface charge of the liposomes. In other words, at EPM ) 0 the concentration of the positive CP units contacting CL2headgroups, [CP]c, equals the concentration of CL2headgroups exposed on the outer leaflet of the liposomal membrane, [CL2-]out. The fraction of CL2- molecules complexed with CP, γ, can then be determined as γ ) [CL2-]out/[CL2-]t ) [CP]c/[CL2-]t ) [CP]t × 0.9/[CL2-]t, where [CP]t and [CL2-]t are the total concentrations of positive CP units and CL2- headgroups in solution, respectively. The calculation according to this scheme gives a γ value equal to nearly 1 for all liposome-CP complexes studied.
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Figure 5. Dependence of relative fluorescence intensity of labeled PC-CL2- SULs on CP concentration. CP(2) (1); CP(2,7) (2); CP(2,12) (3); and CP(2,16) (4). SUL concentration 1 mg/mL; pH 9.2; borate buffer 10-2 M; 20 °C.
This means that all CL2- molecules both from the outer and inner leaflets of the liposomal membrane are involved in complexation with adsorbed CPs. Taking into account that PC-CL2- SULs contacting CPs retain their integrity, this result can be explained only if CPs can induce migration of CL2- molecules from the inner to outer leaflet of the lipid bilayer (polycation-induced flip-flop). This conclusion made earlier for PC-CL2- SUL-CP(2) system now can be extended to other systems studied here. One can assume that complexation of negative SULs with CPs is contributed both by electrostatic and hydrophobic interactions. To distinguish between them we studied the effect of a low molecular salt on stability of the liposome-CP complexes. SULs with a fluorescent labeled lipid, PEA-FITC, incorporated into the membrane were used. It is known that quaternized CP units are effective fluorescence quenchers. Therefore, adsorption and desorption of CPs on the liposome surface should be followed by changing of fluorescence intensity of the label. Actually, adsorption of all CPs is accompanied by decrease of the fluorescence intensity. Such a decrease, being caused by nonradiative energy transfer from exited FITC labels to quaternized pyridine rings of adsorbed CPs, is described by the same curve as expected (Figure 5). Addition of NaCl to the liposome-CPs complexes results in fluorescence recovery, indicating complex dissociation due to electrostatic shielding of the liposome and CP charged groups (Figure 6). However, the extent of recovery is different for various CPs. In the cases of CP(2) and CP(2,7), fluorescence intensity increases up to the initial level (Figure 6, curves 1 and 2) that is evidence of the complete dissociation of corresponding complexes. Importantly, this process develops in rather narrow NaCl concentration range (from 0.15 to 0.25 M). This means that adsorption of CP(2) and CP(2,7) on the surface of PC-CL2- SULs is mainly of electrostatic nature. At the same time, dissociation of the obtained complexes proceeds in a rather narrow range of the salt concentration similar to that of other electrostatic complexes formed by linear polyelectrolytes.5,36-39 On the contrary, in the case of the complexes formed by CP(2,12) and CP(2,16), only partial recovery of fluores(36) Abe, K.; Tsuchida, E. Adv. Polym. Sci. 1982, 45, 1 and the references quoted. (37) Kabanov, V. A.; Zezin, A. B. Makromol. Chem., Suppl. 1984, 6, 259 and the references quoted. (38) Philipp, B.; Dautzenberg, H.; Linow, K.-J.; Kotz, J.; Dawydoff, W. Prog. Polym. Sci. 1989, 14, 91 and the references quoted.
Liposome-Polycation-Polyanion Ternary Systems
Figure 6. Dependence of relative fluorescence intensity of labeled PC-CL2- SULs contacting CP on NaCl concentration. CP(2) (1); CP(2,7) (2); CP(2,12) (3); and CP(2,16) (4). SUL concentration 1 mg/mL; [CP] 1.5 × 10-4 M; pH 9.2; borate buffer 10-2 M; 20 °C.
Figure 7. Dependence of size of PC-CL2- SULs contacting CP on NaCl concentration. CP(2) (1); CP(2,7) (2); CP(2,12) (3); and CP(2,16) (4). SUL concentration 1 mg/mL; [CP] 2 × 10-4 M; pH 9.2; borate buffer 10-2 M; 20 °C.
cence intensity is observed (Figure 6, curves 3 and 4). This means that CP(2,12) and CP(2,16) are not completely desorbed from the liposome surface even at [NaCl] > 0.25 M when all ionic pairs originally formed by polycation units and CL2- anions should be completely destroyed if other attractive force is not involved. Apparently, such a force is produced by hydrophobic interaction of CP sidechain alkyl radicals incorporated into the liposomal membrane. Importantly, 3 mol % of dodecyl or cetyl pendant groups is already enough to prevent dissociation of the complexes in aqueous salt solutions. The above-mentioned data are in consistence with the results of measurement of the size of SUL-CP complex species in aqueous salt solutions (Figure 7). It is seen that addition of NaCl resulting in complete dissociation of SUL-CP(2) and SUL-CP(2,7) complexes is followed by complete recovery of their size to that of the original SULs (Figure 7, curves 1 and 2). This is not the case of the complexes formed by CP(2,12) and CP(2,16) when addition of NaCl also results in a certain decrease of the complex species size, but not in its complete recovery (Figure 7, curves 3 and 4). (39) Dautzenberg, H.; Koetz, J.; Linow, K.-J.; Philipp, B.; Rother, G. In Macromolecular Complexes in Chemistry and Biology; Dubin, P., Block, J., Davies, R. M., Schulz, D. N., Thies C., Eds.; Springer-Verlag: Berlin-Heidelberg, 1994; Chapter 8 and the references quoted.
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Figure 8. Dependence of EPM of CP-PAA complex particles on [CP]/[PAA] ratio. CP(2) (1); CP(2,7) (2); CP(2,12) (3); and CP(2,16) (4). [PAA] 10-4 M; pH 9.2; borate buffer 10-2 M; 20 °C.
Figure 9. Dependence of size of CP-PAA complex particles on [CP]/[PAA] ratio. CP(2) (1); CP(2,7) (2); CP(2,12) (3); and CP(2,16) (4); [PAA] 10-4 M; pH 9.2; borate buffer 10-2 M; 20 °C.
In contrast to studied CPs, addition of PAA to the negative PC-CL2- SULs does not affect either their EPM or their size. The same is true also for the neutral PC SULs. In other words, polyacrylate anions apparently are not adsorbed on the liposome surface. Now, we have enough information to switch over to interactions in the ternary system. Liposome-Polycations-Polyacrylate Anions. It is known that oppositely charged linear polyelectrolytes form interpolyelectrolyte complexes (IPECs) in aqueous solutions.5,36,38 Therefore, in the ternary system containing PC-CL2- SULs, CPs, and PAA one should expect a competition of the negative SULs and PAA for binding with CPs. Addition of CP solutions to PAA solution in the absence of liposomes results in neutralization of the charge of the polyanion chains (Figure 8) and arising of rather large IPEC particles (Figure 9). Zero EPM value and the maximum particle size are observed at the stoichiometric ratio of the polyelectrolyte components, [CP]/[PAA] ) 1. This is an evidence of quantitative binding of CPs in the strong complex with PAA. At [CP]/[PAA] > 1, IPEC particles acquire a positive charge and their size decreases. As shown above, the four examined polycations, as regards their interaction with SULs, can be divided into the two groups. The first includes CP(2) and CP(2,7)
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Figure 10. Changing of relative fluorescence intensity of labeled PC-CL2- SULs after addition of CPs and PAA. SULs + CP(2) (1); SULs + CP(2,16) (2); SULs with adsorbed CP(2) + PAA (3); SULs with adsorbed CP(2,16) + PAA (4); SULs + PAA-CP(2) complex (5); and SULs + PAA-CP(2,16) complex (6). To obtain curve 3 and 4, SUL-CP complexes of different SUL/CP ratios were first prepared, and then PAA was added to each of them so that [CP]/[PAA] ) 1/3. To obtain curves 5 and 6, different concentrations of CP-PAA complex (1/3) were added to SULs. SUL concentration 1 mg/mL; pH 9.2; borate buffer 10-2 M; 20 °C.
interacting with SULs mainly due to electrostatic coupling of polycation units and CL2- headgroups. The two other polycations, CP(2,12) and CP(2,16), in addition to ionic interaction, are bound to the liposome surface apparently due to incorporation of long side-chain alkyl groups into hydrophobic part of the lipid bilayer. Therefore, when studying the competition, we used one polycation from each group, namely CP(2) and CP(2,16). First, the complexes containing fluorescent-labeled SULs and various amounts of CP(2) or CP(2,16) were prepared, and the equilibrium level of fluorescence quenching in all the systems was achieved (ca. 1 min after mixing). PAA was then added to each sample of SULCP(2) and SUL-CP(2,16) complexes so that [CP]/[PAA] ) 1/3. Removal of CPs from the liposome surface due to recomplexation with PAA was followed by increase of fluorescence intensity. Figure 10 represents the equilibrium levels of fluorescence quenching caused by adsorption of CP(2) and CP(2,16) on the liposome surface (Figure 10, curves 1 and 2) and the limit equilibrium levels of fluorescence recovery after addition of the excess of PAA to SUL-CP(2) and SUL-CP(2,16) complexes (Figure 10, curves 3 and 4). One can see that addition of PAA to SUL-CP(2) complex leads to recovery of fluorescence intensity up to the initial level. This is evidence of complete removal of CP(2) from the liposome surface due to formation of CP(2)-PAA complex. Removal of the polycation leads to desegregation of the SUL-CP(2) complex particles and to a decrease of their size down to that of the initial SULs (cf. curves 1 and 3 in Figure 11). Addition of PAA to SUL-CP(2,16) complex results only in partial increase of fluorescence intensity (cf. curves 2 and 4 in Figure 10). At the same time, the particle size does not reach the initial (cf. curves 2 and 4 in Figure 11). The partial fluorescence recovery can be explained either by removal of some CP(2,16) chains as a whole from the
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Figure 11. Changing of size of labeled PC-CL2- SULs after addition of CPs and PAA. SULs + CP(2) (1); SULs + CP(2,16) (2); SULs with adsorbed CP(2) + PAA (3); SULs with adsorbed CP(2,16) + PAA (4). To obtain curve 3 and 4, SUL-CP complexes of different SUL/CP ratios were first prepared, and then PAA was added to each of them so that [CP]/[PAA] ) 1/3. SUL concentration 1 mg/mL; pH 9.2; borate buffer 10-2 M; 20 °C.
liposome surface or partial replacement of their contacts with CL headgroups by the contacts with added PAA. To distinguish between these two cases, the particles formed after addition of PAA to SUL-CP(2,16) complex were separated from aqueous solution by centrifugation. No P(2,16) was then found in the supernatant by UV spectroscopy. It shows that CP(2,16) macromolecules remain bound to the liposome surface. Most likely, CP(2,16)-PAA IPEC is formed in this case as well; however, it keeps contacting the liposomes due to incorporation of CP(2,16) cetyl groups into the liposomal membrane. In the other series of experiments, negatively charged CP(2)-PAA and CP(2,16)-PAA IPECs with [CP]/[PAA] ) 1/3 were added to fluorescent labeled PC-CL2- SULs and fluorescence intensity was monitored. As it follows from Figure 11 (curve 5), addition of CP(2)-PAA IPEC to SUL suspension has no effect on fluorescence that indicates no interaction of this complex with the liposomes. However, when CP(2,16)-PAA IPEC is added, fluorescence intensity decreased (Figure 11, curve 6), indicating adsorption of negatively charged CP(2,16)-PAA IPEC on the liposome surface apparently because of incorporation of cetyl pendant groups into the hydrophobic part of the liposomal membrane. In other words, the SUL-CP-PAA ternary complex is formed. Thus, polycations adsorbed on the negatively charged liposome surface due to only electrostatic interactions can be completely desorbed by addition of a low molecular salt, or removed by a negatively charged polymeric competitor. Modification of a polycation with dodecyl or cetyl pendant groups provides additional stability to polycation-liposome complex in aqueous-salt solutions as well as in the presence of a negatively charged polymeric competitor. Acknowledgment. The authors highly appreciate the support of some parts of this research by the Russian Foundation for Fundamental Research (grant 96-0333725) and the National Institutes of Health. LA971346Z
