Langmuir 2000, 16, 4877-4881
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Steric Stabilization of Negatively Charged Liposomes by Cationic Graft Copolymer Tatiana K. Bronich,† Sergey V. Solomatin,†,‡ Alexander A. Yaroslavov,‡ Adi Eisenberg,§ Victor A. Kabanov,‡ and Alexander V. Kabanov*,† Department of Pharmaceutical Sciences, College of Pharmacy, 986025 Nebraska Medical Center, Omaha, Nebraska 68198-6025; Department of Polymer Sciences, School of Chemistry, M. V. Lomonosov Moscow State University, Leninskie Gory, Moscow V-234, 119899 Russia; and Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, Canada H3A 2K6 Received November 12, 1999. In Final Form: March 7, 2000 This work characterizes interactions between a cationic graft copolymer, PEO-graft-polyethylenimine (PEO-g-PEI) and negatively charged liposomes composed of 80% (wt) dipalmitoylphosphatidylcholine (DPPC) and 20% (wt) diphosphatidylglycerol. Following addition of the copolymer the effects on the size and surface charge of the liposomes were examined by dynamic light scattering and laser electrophoresis techniques. The study concludes that PEO-g-PEI adsorbs on the surface of the liposomes due to electrostatic interactions with the CL phosphate groups exposed at the outer leaflet of the liposome membrane. This results in neutralization of the liposome surface charge and formation of PEO layer at the liposome surface, which sterically stabilizes the particles. Electroneutral PEO-g-PEI/liposome complexes remain stable in dispersed state (∼100 nm) in contrast to the homopolymer PEI/liposome complexes, which aggregate. Binding of PEO-g-PEI did not alter the structure of the lipid bilayer as evaluated by a fluorescence polarization technique, using diphenylhexatriene as a probe, and a microcalorimetry technique.
Introduction Adsorption of polycations at negatively charged liposome membranes has been intensively investigated for the last two decades.1-15 These studies were mainly focused on mimicking interactions of polycations with the cell surface. To a much lesser extent these processes were considered as routes for preparation of self-assembled materials from polycation and liposomes as building blocks. One reason for that is difficulty in control of stability of such materials in the dispersed state. Indeed, as a result of neutralization * Corresponding author: Fax: (402) 559-9543. E-mail: akabanov @unmc.edu. Homepage: www.unmc.edu/PharmSciences/Faculty/ kabanov/index.html. † University of Nebraska Medical Center. ‡ Moscow State University. § McGill University. (1) Chapman, D.; Urbina, J.; Keough, K. M. J. Biol. Chem. 1974, 249, 2512. (2) Verkleij, A. J.; DeKruyff, B.; Ververgaert, P. H. J. Th.; Tocanne, J. F.; Van Deenen, L. L. M. Biochim. Biophys. Acta 1974, 339, 432. (3) Papahadjopoulos, D.; Moscarello, M.; Eylar, E. H.; Isac, T. Biochim. Biophys. Acta 1975, 401, 317. (4) Meers, P.; Daleke, D.; Hong, K.; Papahadjopoulos, D. Biochemistry 1991, 30, 2903. (5) Hartmann, W.; Galla, H. Biochim. Biophys. Acta 1978, 509, 474. (6) Wang, C.-Y.; Huang, L. Biochemistry 1984, 23, 4409. (7) De Kruijff, B.; Rietveld, A.; Telders, N.; Vaandrager, B. Biochim. Biophys. Acta 1985, 820, 295. (8) Gad, A. E.; Elyashiv, G.; Rosenberg, N. Biochim. Biophys. Acta 1986, 860, 314. (9) Kim, J.; Mosior, M.; Chung, L.; Wu, H.; McLaughin, S. Biopys. J. 1991, 60, 135. (10) Oku, N.; Shibamoto, S.; Ito, F.; Gondo, H.; Nango, M. Biochemistry 1987, 26, 8145. (11) Yaroslavov, A. A.; Kul’kov, V. E.; Polinsky, A. S.; Baibakov, B. A.; Kabanov, V. A. FEBS Lett. 1994, 340, 121. (12) Kabanov, V. A.; Yaroslavov, A. A.; Sukhishvili, S. A. J. Control. Release 1996, 39, 173. (13) Yaroslavov, A. A.; Efimova, A. A.; Kul’kov, V. E.; Kabanov, V. A. Polym. Sci. 1994, 36, 215. (14) Yaroslavov, A. A.; Kiseliova, E. A.; Udalykh, O. Yu.; Kabanov, V. A. Langmuir 1998, 14, 5160. (15) Yaroslavov, A. A.; Koulkov, V. Ye.; Yaroslavova, E. G.; Ignatiev, M. O.; Kabanov, V. A. Langmuir 1998, 14, 5999.
of the charges, adsorption of polycations often leads to aggregation of liposomes. Similarly, complexes of polyelectrolytes with oppositely charged surfactants (“polymersurfactant complexes”) usually precipitate from aqueous solutions.16 There is a narrow range of conditions when stable dispersions can be formed, which usually requires the use of a relatively long polycation added to the liposomes at sufficient excess. Recently possibilities for the control of the stability in the dispersion and morphology of the polymer-surfactant complexes have been greatly extended by utilizing block and graft copolymers having ionic and nonionic watersoluble polymer chains, for example, poly(ethylene oxide) (PEO).17-19 In such systems, ionic headgroups of the surfactant form salt bonds with the units of the polyion segment while surfactant alkyl radicals segregate into hydrophobic domains. In contrast to regular polymersurfactant complexes, which precipitate, these materials remain stable in aqueous dispersions even upon complete neutralization of charges due to the effect of the nonionic segment. Depending on the chemical structures of the polymer and surfactant components these complexes selfassemble into small vesicles18 or micelle-like aggregates.19 In this work, we explore a similar route for preparation of stable dispersions from cationic copolymer, PEO-graftpolyethylenimine (PEO-g-PEI) and negatively charged liposomes composed of dipalmitoylphosphatidylcholine (16) (a) Macromolecular Complexes in Chemistry and Biology; Dubin, P., Block, J.;, Davies, R. M., Schulz, D. N., Thies, C., Eds.; SpringerVerlag: Berlin-Heidelberg, 1994. (b) Goddard, E. D. Colloid Surf. 1986, 19, 255. (c) Ibragimova, Z. K.; Kasaikin, V. A.; Zezin, A. B.; Kabanov, V. A. Polym. Sci. USSR 1986, 28, 1826. (d) Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Ed.; CRC Press: Boca Raton, FL, 1993. (17) Bronich, T. K.; Kabanov, A. V.; Kabanov, V. A.; Yu, K.; Eisenberg, A. Macromolecules 1997, 30, 3519. (18) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. J. Am. Chem. Soc. 1998, 120, 9941. (19) Bronich, T. K.; Cherry, T.; Vinogradov, S. V.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Langmuir 1998, 14, 6101.
10.1021/la991484j CCC: $19.00 © 2000 American Chemical Society Published on Web 04/21/2000
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Figure 1. Cationic copolymer PEO-g-PEI.
(DPPC) and diphosphatidylglycerol (cardiolipin, CL). In contrast to the previous work described in ref 18 in which the formation of the vesicles was induced by the block copolymer, this paper proposes to cover the charged surface of already preformed liposomes by adsorption of the oppositely charged copolymer. Such composite materials have potential applications in controlled drug delivery, radioimaging, and other areas, which might benefit from utilization of self-assembling systems of nanoscale size. Experimental Section Materials. Randomly branched PEI samples with Mw ≈ 2000 and Pw ≈ 46 (PEI46) and Mw ≈ 25000 and Pw ≈ 580 (PEI580) were purchased from Aldrich Co. PEO-g-PEI copolymer was synthesized by conjugation of poly(ethylene oxide) (PEO), Mn ≈ 8000, with PEI46 following the procedure described in ref 20. The schematic structure of PEO-g-PEI copolymer is presented in Figure 1. The molecular weight of PEO-g-PEI is Mw ) 16 600 g/mol as determined by static light scattering. The concentration of the total nitrogen in the PEO-g-PEI sample determined by the element analysis was 2.16 µmol/mg. The data on the molecular weight and concentration of total nitrogen corresponded to ∼1.8 and ∼2.4 PEO segments per PEI chain, respectively. This was in a reasonable agreement with an average of PEO/PEI ratio of 1.7 as determined by NMR. Poly(methacrylic acid) (PMA) with Mw ≈ 185 000 (Pw ) 2150) was obtained by radical polymerization21 and then labeled with 1-pyrenyldiazomethane as previously reported.22 Labeled PMA* contained one pyrenylmethyl methacrylate group per 450 repeating units. DPPC and CL were obtained from Sigma Chemical Co. and used without further purification. Aldrich Co. supplied diphenylhexatriene (DPH). Preparation of Liposomes. CL/DPPC liposomes (20/80 w/w%) were prepared using the following procedure. Corresponding amounts of DPPC and CL solutions in ethanol or chloroform were mixed in a flask, and solvents were thoroughly evaporated under vacuum to obtain a thin layer of the lipid mixture. After that, lipids were dispersed in phosphate buffer (10 mM, pH 7.0) using Sonicator Ultrasonic Processor XL (Misonix Inc.) for 2 min (room temperature). This results in the formation of small unilamellar liposomes.13 Titanium dust (from the sonicator) was separated from the liposomes by centrifugation (15 min, 13 000 rpm). Freshly prepared liposome dispersions were used in further experiments. Fluorescence Labeling of Liposomes. Stock solution of DPH in tetrahydrofuran (2 mM) was added to a suspension of CL/DPPC liposomes to obtain final concentration of DPH 2 µM. Samples were incubated in the dark for 1 h at room temperature prior to further experiments. ζ-Potential and Sizing Measurements. Electrophoretic mobility measurements were performed using “ZetaPlus” ζ-potential analyzer (Brookhaven Instrument Co.) with 15 mV solidstate laser operated at a laser wavelength of 635 nm. ζ-Potential of the particles was calculated from the electrophoretic mobility values using Smoluchowski equation. Effective hydrodynamic diameters (Deff) were measured by photon correlation spectroscopy in a thermostatic cell at a scattering angle of 90° using the same instrument equipped with the Multi Angle Option. Software provided by the manufacturer was employed to calculate Deff (20) Vinogradov, S. V.; Bronich, T. K.; Kabanov, A. V. Bioconjugate Chem. 1998, 9, 805. (21) Lipatov, Yu. S.; Zubov, P. N. Vysokomol. Soedin., Ser. A 1959, 1, 88. (22) Krakovyak, M. G.; Anufrieva, E. V.; Skorokhodov, S. S. Vysokomol. Soedin., Ser. A 1969, 11, 2499.
Bronich et al. values. All solutions were prepared using double distilled water and were filtered repeatedly through the Millipore membrane with pore size 0.45 µM. Fluorescence Measurements. All fluorescence measurements were performed using a Shimadzu P5000 spectrofluorophotometer. The excitation and emission wavelengths were set at 346 and 396 nm, respectively, for pyrenyl-labeled PMA* (both slit widths were 5 nm). The data obtained are presented in terms of relative fluorescence intensity I/Io, where Io is the fluorescence intensity of PMA* solution under the same conditions. For the fluorescence polarization studies, the spectrofluorophotometer was equipped with polarizers in the right-angle configuration. The excitation and emission wavelengths for DPH were 365 and 425 nm respectively (both slit widths were 10 nm). The polarization was calculated using the relationship
P ) (IVV - GIVH)/(IVV + GIVH) where G ) IHV/IHH is an instrumental correction factor and IVV, IVH, IHV, and IHH refer to fluorescence intensity polarized in vertical and horizontal detection planes (second subscript index) upon excitation with either vertically or horizontally polarized light (first subscript index). Differential Scanning Calorimetry. Phase transitions in free and copolymer-bound liposomes were studied microcalorimetrically using a Microcal MC-2 (Northampton, MA) differential scanning calorimeter (DSC). Typically, the liposome samples with lipid concentration of 1 mg/mL vs buffer were scanned from 15 to 60 °C at a heating rate of 0.75 °C/min. A buffer versus buffer scan was subtracted from the sample scan and normalized for the heating rate; i.e., each data point is divided by the corresponding heating rate. The area of the resulting curve is proportional to the transition heat, which when normalized for the number of moles used is equal to the transition enthalpy (∆Hcal). The instrument was calibrated with a standard electrical pulse.
Results and Discussion It is well-known that negatively charged liposomes strongly adsorb cationic polyelectrolytes (proteins,1-4 polypeptides,5-9 and synthetic polycations10-15) from aqueous solutions. Recently adsorption of poly(N-ethyl-4vinylpyridinium bromide) (PEVP) on the surface of negatively charged small unilamellar liposomes from CL/ DPPC mixtures was extensively studied.11-15 It was shown that interaction of PEVP chains with the liposomes leads to neutralization of the surface charge followed by increase in particle size and aggregation of liposomes. In this work we studied interaction of PEI homopolymer and PEO-g-PEI copolymers with small unilamellar CL/ DPPC liposomes. All experiments were performed at ambient temperature, 22 °C. Under these conditions CL/ DPPC liposomes are in the solid state (gel to liquid crystalline phase transition in this system is above 30 °C). In solid liposomes lateral and transbilayer mobility of lipids is restricted and both lipid components are practically uniformly distributed between inner and outer leaflets of the bilayer.13,14 Polycation solutions were added to liposome dispersion to obtain mixtures of various compositions. In this work we express the composition of the mixture, Z+/-, as a ratio of concentration of charged amino groups of the polycation (pH 7.0) to total concentration of CL phosphate groups in the liposomes (each CL molecule contains two phosphate groups). Figure 2 presents ζ-potential of particles formed in the mixtures as a function of Z+/-. In all cases addition of polycations to liposomes resulted in increase of ζ-potential. This provides evidence of adsorption of polycations on the liposome surface leading to neutralization of the negative charge of the liposomes. There was practically no difference in the ζ-potential values observed with all polycations studied at Z+/- < 0.45. In the vicinity of Z+/-
Stabilization of Negatively Charged Liposomes
Figure 2. ζ-Potential of particles formed in polycation/ liposomes systems at various compositions of the mixture, Z+/-: (O) PEO-g-PEI/liposomes, (9) PEI46/liposomes, and (2)PEI580/ liposomes. Vertical arrow indicates the point of precipitation. Lipid concentration is 1 mg/mL; CL/DPPC, 20/80 w/w %; pH, 7.0; T, 22 °C. (Here and in other figures Z+/-, is determined as a ratio of concentration of charged amino groups of the polycation to total concentration of CL phosphate groups in the liposomes.)
) 0.5 phase separation was observed in the mixtures of long PEI580 and liposomes. In contrast, short PEI46 and its PEO-modified analogue, PEO-g-PEI, formed stable dispersions under these conditions. At Z+/- ) 0.5, ζ-potential of the particles formed in these dispersions approximated zero. This point corresponds to neutralization of a half of phosphate groups of CL. This suggests that there is no transbilayer migration of CL (“flip-flop”) during interaction of liposomes with polycations and polycations bind only with CL molecules located at the outer leaflet of the liposome membrane. A similar result was reported previously for adsorption of PEVP on the surface of solid CL/ DPPC liposomes.11,12 It appears that the absence of “flipflop” is attributable to a relatively rigid structure of the lipid bilayer present in the solid liposomes that cannot be perturbed by the studied polycations. On the other hand the correspondence of the electroneutrality point to the neutralization of only a half of phosphate groups of CL indirectly suggests that the integrity of the lipid bilayer is preserved upon binding of the polycation. At Z+/- > 0.5, the mixtures of long PEI580 and liposomes remained phase separated independent of the order of mixing of the components. ζ-Potential of particles formed in the mixtures of liposomes with short PEI46 or PEOb-PEI leveled-off at zero at Z+/- > 0.5. Therefore, no excess of these polycations bind to the liposome surface at Z+/> 0.5. ζ-Potential study suggests that grafting of PEI46 with extended PEO chains does not prevent adsorption of the polycation on the liposome. Figure 3 presents the dependence of the effective diameters of CL/DPPC dispersions in the presence of polycations as a function of Z+/-. The size of initial CL/ DPPC liposomes was ∼80 nm. Addition of long PEI580 in the range 0.25 < Z+/- < 0.45 resulted in the profound increase in the particle size compared to the polycation free system. Under these conditions, polycation chains neutralize liposome charges, which leads to formation of hydrophobic sites and aggregation of the liposomes. It is also possible that long polycation chains join several liposome species in the aggregates formed, which promotes precipitation at Z+/- > 0.45. Addition of short polycation PEI46 also results in formation of aggregates with an effective diameter of ∼500 nm at Z+/- > 0.3 (Figure 3). The size reaches maximum at the charge neutralization point at Z+/- ) 0.5 and then
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Figure 3. Effective diameter (Deff) of particles formed in polycation/liposomes systems at various compositions of the mixture, Z+/-: (O) PEO-g-PEI/liposomes, (9) PEI46/liposomes, and (2)PEI580/liposomes. Vertical arrow indicates the point of precipitation. Lipid concentration is 1 mg/mL; CL/DPPC, 20/ 80 w/w %; pH, 7.0; T, 22 °C.
Figure 4. Schematic showing interaction of PEO-g-PEI with the liposome membrane. PEI chains bind electrostatically with the CL molecules (white headgroups) at the outer leaflet of the liposome membrane. PEO chains become “grafted” to the liposome outer surface. The inner leaflet of the liposome membrane contains free DPPC (gray headgroups) and CL molecules.
practically does not change in the presence of polycation excess. This result is consistent with the conclusion of the ζ-potential study that PEI46 added to the liposomes at excess does not incorporate into the complex. The behavior of the systems on the basis of the graft copolymer, PEO-g-PEI, is totally different from that of the above-described homopolymer systems. Following addition of the PEO-g-PEI copolymer to CL/DPPC liposomes the particle size slightly increases and levels off at ∼100 nm at Z+/- > 0.5 (Figure 3). Consequent measurements with these samples showed that the size of the particles remained unchanged for at least several weeks. Thus, upon adsorption of PEO-g-PEI onto liposomes PEI chains form ionic bonds with the phosphate groups of CL on the surface of the lipid membrane, while PEO chains grafted to PEI become attached to the liposome surface as schematically shown in Figure 4. Since the size of the liposomes changes only slightly upon addition of PEO-g-PEI, it is unlikely that the copolymer bridges several liposomes together. Indeed, the polycation chain is relatively short (∼46 repeating units) which further reinforces the suggestion about the absence of the bridging. On the basis of the experiments conducted in this work
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it is not clear what conformations the chain segments of the graft copolymer adopt upon interaction with the liposome. While the uncharged, nonpolar parts of PEI chains can possibly penetrate into some hydrophobic regions of the lipid bilayer, the interaction of PEO chains with the lipids is highly unlikely on the basis of the previous studies on sterically stabilized the liposomes.23-28 It is more probable that as a result of the adsorption of the copolymer onto the liposomes, PEO chains form a nonionic hydrophilic layer, which shields the sites of attachment of PEI to liposome surface and sterically stabilizes the dispersion. Under the conditions of complete neutralization of phosphate groups at the outer leaflet of the bilayers, the number of PEO chains grafted to the liposome surface is determined by the content of negatively charged lipid in the mixture. In our case (i.e., CL/DPPC 20/80) the estimated average content of PEO is one polymer chain per ∼60 total lipid molecules (i.e., ∼1.7 mol %). According to the theoretical estimation by Torchilin et al.23 this amount of PEO chains is sufficient for the complete shielding of the surface of small unilamellar liposomes. Furthermore, on the basis of the previous studies on regular PEO-grafted “Stealth” liposomes this content of PEO chains corresponds to the range of compositions of in which steric stabilization of the liposomes is observed.24-28 Adsorption of polycations on the surface of liposome can be accompanied by structural changes in the lipid membrane particularly by (a) change in lipid packing and (b) lateral segregation of the lipids.4,5,29-31 To evaluate possible changes in the lipid packing as a result of adsorption of PEO-g-PEI at CL/DPPC liposomes we characterized membrane fluidity using DPH entrapped in the membrane as a probe. It is generally known that the DPH probe tends to localize in the most hydrophobic part of a lipid bilayer.32 Changes in lipid packing alter the fluorescence polarization (P) of DPH molecule. Figure 5 presents the dependence of DPH fluorescence polarization on Z+/- in the presence of PEO-g-PEI. As is seen in the figure, the values of P slightly increased with increase in Z+/- and leveled off at Z+/- ) 0.5. This suggests that the rotational mobility of the probe incorporated into the lipid membrane decreases upon binding of the cationic copolymer, indicating some increase in the lipid order in the hydrocarbon interior of the membrane. It is noteworthy that the changes in polarization are relatively small and correspond to those observed during incorporation of PEOmodified lipids into the liposomes33 or adsorption of hydrophobic polyelectrolytes on the surface of bilayer membrane.34 It is possible that interactions of the head(23) Torchilin, V. P.; Papisov, M. I.; Bogdanov, A. A.; Trubetskoy, V. S.; Omelyanenko, V. G. In Stealth Liposomes; Lasic, D., Martin, F., Eds.; CRC Press: Boca Raton, 1995; p 51. (24) Torchilin, V. P.; Klibanov, A. L.; Huang, L.; O’Donell, S.; Nosiff, N. D.; Khaw, B. A. FASEB J. 1992, 6, 2716. (25) Allen, T. M.; Hansen, C. Biochim. Biophys. Acta 1991, 1068, 133. (26) Kenworthy, A. K.; Simon, S. A.; McIntosh, T. J. Biophys. J. 1995, 68, 1903. (27) Hristova, K.; Needham, D. Macromolecules 1995, 28, 991. (28) Szleifer, I.; Gerasimov, O. V.; Thompson, D. H. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1032. (29) Lentz, B. R.; Moore, B. M.; Kirkman, C.; Meissner, G. Biophys. J. 1982, 37, 30. (30) Mittler-Neher, S.; Kholl, W. Biochem. Biophys. Res. Commun. 1989, 162, 124. (31) Yaroslavov, A. A.; Efimova, A. A.; Lobyshev, V. I.; Ermakov, Yu. A.; Kabanov, V. A. Membr. Cell Biol. 1997, 10, 683. (32) Davenport, L.; Dale, R. E.; Bisby, R. H.; Cundall, R. B. Biochemistry 1985, 24, 4097. (33) Yoshida, A.; Hashizaki, K.; Yamauchi, H.; Sakai, H.; Yokoyama, S.; Abe, M. Langmuir 1999, 15, 2333.
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Figure 5. Dependence of DPH fluorescence polarization in PEO-g-PEI/liposomes mixtures on composition of the mixture, Z+/-. Lipid concentration is 1 mg/mL; CL/DPPC, 20/80 w/w %, pH, 7.0; T, 22 °C.
Figure 6. Calorimetric curves of the phase transition in (1) CL/DPPC liposomes and (2) PEO-g-PEI/liposomes complex (Z+/) 0.5). Lipid concentration is 1 mg/mL; CL/DPPC, 20/80 w/w %, pH, 7.0; T, 22 °C.
groups of CL molecule exposed on the liposome surface with PEI chains lead to more tight packing arrangements of the CL hydrocarbon tails. At the same time the fluorescence signal of DPH is averaged for the probes located in the vicinity of CL molecules (presumably stronger affected by polycation) and in the vicinity of DPPC molecules (less affected by polycation). To evaluate possible lateral segregation of CL and DPPC molecules as a result of binding of the polycation, thermotropic behavior of the liposomes was characterized using microcalorimetry technique. Figure 6 presents the calorimetric curves obtained for free and PEO-g-PEI bound CL/DPPC liposomes. As is seen in the figure, gel to liquid crystalline phase transition occurs in these systems in the range of ∼30 °C to ∼40 °C. In both cases, a broad and asymmetric peak is observed, which possibly corresponds to melting of membrane regions with altering CL content. It is seen that binding of the polycation does not affect position of the maximum of the peak (∼39 °C). However, enthalpy of the phase transition (∆Hcal) changes from 4.6 kcal/mol for free liposomes to 13.2 kcal/mol for PEO-gPEI/liposome complex. This result is in marked contrast (34) Thomas, J. L.; Borden, K. A.; Tirrell, D. A. Macromolecules 1996, 29, 2570.
Stabilization of Negatively Charged Liposomes
Figure 7. Relative fluorescence intensity of PMA* (I/Io) in the presence of (1) PEO-g-PEI and (2) PEI-g-PEI/liposomes complex (Z+/- ) 0.5) as a function of the ratio of concentrations of charged amino groups of the polycation and carboxylate groups of PMA*, R. Lipid concentration is 1 mg/mL; CL/DPPC, 20/80 w/w %, pH, 7.0; T, 22 °C.
with previous study of PEVP interaction with CL/DPPC liposomes.31 In that study significant changes in the phase transition temperatures were observed. Those changes have been attributed to the lateral segregation of the CL and DPPC and microphase separation induced by the polycation binding. In our case even if CL molecules laterally segregate to form PEI-bound domains, the sizes of these domains are possibly too small to cause microphase separation and significant change in phase transition profile. One possible reason for this is the lateral repulsion of PEO chains grafted to PEI, preventing assembly of the polycation-bound CL molecules into continuous microphase regions within the liposome membrane. Increase in ∆Hcal can be explained by possible changes in hydration and/or some ordering of lipid bilayer as a result of binding of PEO-g-PEI to the liposome surface. In fact, fluorescence polarization data using DPH is consistent with the increase in the solid membrane order upon interaction with the polycation. In any case this result is very similar to the effects protein adsorption on thermotropic behavior in lipid membranes observed in the case of electrostatic binding of protein to the membrane surface.3 One potential application of the described cationic copolymer/liposome complexes is in drug delivery as a special type of sterically stabilized drug carrier. In view of this application, the possibility of removal of PEO-gPEI from the liposome surface as a result of competitive interactions with polyanions that are present in biological fluids (i.e., proteins, polysaccharides, DNA) is of interest. In this respect we evaluated polyion exchange reaction involving polycation/liposome complex using pyrenyllabeled PMA* as a model. It is known that aromatic and aliphatic amines are efficient quenchers of fluorescence of pyrenyl groups.35 Therefore, by monitoring fluorescence quenching one can study interactions between PMA* and
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PEO-g-PEI. Figure 7 presents relative fluorescence intensity of PMA* as a function of the ratio of concentrations of charged amino groups of the polycation and carboxylate groups of PMA* (R). As is seen in the figure addition of PEO-g-PEI to PMA* resulted in the decrease of the fluorescence intensity (curve 1), indicating that the PEOg-PEI/PMA* complex is formed. In contrast, no change in the fluorescence intensity of PMA* was observed in the presence of the PEI-g-PEI/liposome complex (Z+/- ) 0.5) (curve 2). This suggests that the cationic copolymer absorbed at the liposome surface is not removed from it and does not form a complex with the PMA*. Therefore, the PEO-g-PEI complex with liposomes remains stable in the presence of the polyanions. This result is consistent with the absence of change in the size of PEO-g-PEI/ liposome particles after addition of PMA* (data not presented). Conclusions This study demonstrates that cationic graft copolymer (PEO-g-PEI) adsorbs on the surface of negatively charged solid CL/DPPC liposomes as a result of electrostatic interactions with the CL phosphate groups exposed at the outer leaflet of the liposome membrane. This leads to attachment of PEO chains grafted to PEI to the liposome surface. PEO chains form a nonionic hydrophilic layer, which streically stabilizes the particles. As a result, electroneutral PEO-g-PEI/liposome complexes remain stable in dispersed state in contrast to the homopolymer PEI/liposome complexes, which aggregate. Binding of the cationic copolymer does not significantly alter the structure of the lipid bilayer. Potential applications of liposomes covered with PEO-g-PEI and similar copolymer materials are in pharmaceutics. They may be used in controlled drug delivery, radioimaging, and other areas, which might benefit from utilization of self-assembling systems of nanoscale size. Acknowledgment. This work was supported by USA National Science Foundation grants DMR-9502807 and DMR-9617837 (international collaboration). Studies of the group in Moscow State University are supported by Russian Foundation for Fundamental Research (grant 99-03-33460). A.E.’s research in this field is supported by a grant from the Natural Sciences and Engineering Research Council, Canada (STR-0181003). We are grateful to Professor L. Marky (UNMC) for assistance in the calorimetry studies and very fruitful discussions. Finally, we acknowledge Dr. S. V. Vinogradov (UNMC) who prepared the cationic copolymer sample in connection with another project. LA991484J (35) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983; p 258.
