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Langmuir 1999, 15, 6314-6325
Effect of PEO-PPO-PEO Triblock Copolymers on Structure and Stability of Phosphatidylcholine Liposomes Markus Johnsson,* Mats Silvander, Go¨ran Karlsson, and Katarina Edwards Department of Physical Chemistry, Uppsala University, Box 532, S-751 21 Uppsala, Sweden Received March 10, 1999. In Final Form: June 3, 1999
The interactions of five poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) (PEO-PPO-PEO), Pluronic, copolymers and phosphatidylcholine liposomes of varying composition have been studied. Structural studies were performed by means of cryo-transmission electron microscopy (c-TEM) and reveal that inclusion of low amounts (∼2 mol %) of Pluronics gives rise to significant morphological changes of the liposome preparations. Pluronics with large poly(oxyethylene) (PEO) blocks, such as F127, F108, and F87, induce the formation of bilayer disks, whereas those with comparably short PEO blocks, P105 and P85, tend to to promote a reduction in the liposome size. Inclusion of cholesterol in the liposomal preparations reduces the incorporation of copolymers in the lipid bilayer and thus reduces, and in some cases even abolishes, the morphological changes observed in the absence of cholesterol. The effect of the copolymers on liposome permeability was also investigated. All investigated copolymers were found to increase the leakage of carboxyfluorescein from preformed liposomes. This was true also in the case of cholesterol-containing liposomes despite the fact that no change in the liposome structure could be observed by means of c-TEM. The magnitude of the induced leakage was found to correlate well with the hydrophobicity, as measured by the cmc, of the respective Pluronic. By raising the temperature or decreasing the solvency of the copolymer in the solution, the effect of the copolymer on liposome leakage was found to increase significantly.
Introduction The properties of block copolymers have received much attention during the past decade. The molecular architecture of the polymers allows for a wide range of technological applications as well as for fundamental studies of self-assembly. The commericially available PEO-PPO-PEO triblock polymers, where PEO is the hydrophilic poly(ethylene oxide) and PPO is the hydrophobic poly(propylene oxide), are used in a variety of applications as emulsifiers and stabilizers.1 In water solution, many of the triblock polymers have been shown to aggregate in the form of micelles, with the core consisting mainly of the hydrophobic PPO and the corona consisting of the hydrated PEO chains.2,3 The magnitude of the cmc, as well as the phase propensity in general, has been shown to depend on the molecular weight and the segment composition of the particular block copolymer.4-6 Moreover, the formation of micelles (and other aggregates) has been found to be an extremely temperature-dependent process resulting in a decrease in the cmc of several orders of magnitude upon minor increases in the temperature.4,5,7 The properties of the block copolymers in solution are of interest not only from a fundamental point of view. At present, one of the main utilities for these polymers is in applications where the adsorbed/grafted molecules stabilize surfaces toward particle interactions.8-15 Such steric (1) Alexandridis, P. Curr. Opin. Colloid Interface Sci. 1997, 2, 478. (2) Almgren, M.; Brown, W.; Hvidt, S. Colloid Polym. Sci. 1995, 273, 2. (3) Mortensen, K.; Talmon, Y. Macromolecules 1995, 28, 8829. (4) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414. (5) Noolandi, J.; Shi, A.; Linse, P. Macromolecules 1996, 29, 5907. (6) Brown, W.; Schille´n, K.; Hvidt, S. J. Phys. Chem. 1992, 96, 6038. (7) Alexandridis, P.; Athanassiou, V.; Fukuda, S.; Hatton, T. A. Langmuir 1994, 10, 2604. (8) Malmsten, M.; Linse, P.; Cosgrove, T. Macromolecules 1992, 25, 2474. (9) Li, J.; Caldwell, K.; Rapoport, N. Langmuir 1994, 10, 4475. (10) Tadros, Th. F.; Vincent, B. J. Phys. Chem. 1980, 84, 1575.
stabilization can be accomplished in two different ways, by grafting of a polymer to the surface or by adsorbing the polymer at the surface. For effective steric stabilization the amount of the adsorbed or grafted polymer must be relatively high. Furthermore, the nonadsorbing part of the polymer or the grafted polymer should exhibit good solubility in the surrounding solvent and the attachment of the polymers to the surface should be strong enough so as to avoid a fast desorption. Liposomes used, or intended, for drug delivery are often sterically stabilized to prolong the circulation time in the blood stream.16 Normally, the stabilization is achieved via incorporation of an appropriate amount of lipid with a covalently anchored homopolymer, usually PEO chains.16 The lifetime of the PEO-lipid in the membrane depends critically upon the length of the hydrophobic acyl chains.17 Thus, for PEO-lipids with short acyl chains, there will be a gradual decrease of the liposomal surface density of polymer during blood circulation. Eventually, this may lead to a complete loss of the steric stabilization. To minimize the loss of polymer from the lipid membrane, it is necessary to use lipid anchors with long acyl chains such as DSPE (distearoylphosphatidylethanolamine). PEO-PPO-PEO triblock copolymers, incorporated or adsorbed, constitute an interesting alternative to PEOlipids as a stabilizing material for liposomes. Most likely, a membrane-spanning triblock polymer would provide a (11) Moghimi, S. M.; Porter, C. J. H.; Illum, L.; Davis, S. S. Int. J. Pharm. 1991, 68, 121. (12) Tan, J. S.; Butterfield, D. E.; Voycheck, C. L.; Caldwell, K. D.; Li, J. T. Biomaterials 1993, 14, 823. (13) van de Steeg, L. M. A.; Go¨lander, C. Colloids Surf. 1991, 55, 105. (14) Schroe¨n, C. G. P. H.; Cohen Stuart, M. A.; van der Voort Maarschalk, K.; van der Padt, A.; van’t Riet, K. Langmuir 1995, 11, 3068. (15) Quiron, F.; St-Pierre, S. Biophys. Chem. 1991, 40, 129. (16) Papahadjopoulos, D. In Stealth Liposomes; Lasic, D. D., Martin, F., Eds.; CRC Press Inc.: Boca Raton, FL, 1995; Chapter 1. (17) Holland, J. W.; Hui, C.; Cullis, P. R.; Madden, T. D. Biochemistry 1996, 35, 2618.
10.1021/la990288+ CCC: $18.00 © 1999 American Chemical Society Published on Web 07/16/1999
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Table 1. Properties and Critical Micellization Concentrations of the Pluronics in Hepes Buffer (20 mM Hepes, 150 mM NaCl, pH 7.4) Pluronic
composition
P85 P105 F87 F127 F108
(PEO)26(PPO)40(PEO)26 (PEO)37(PPO)56(PEO)37 (PEO)61(PPO)40(PEO)61 (PEO)100(PPO)65(PEO)100 (PEO)132(PPO)50(PEO)132
a
molar cmca (mM), 25 °C mass 4600 6500 7700 12600 14600
2.76 ( 0.25 0.40 ( 0.11 12.09 ( 0.09 0.32 ( 0.08 1.86 ( 0.05
cmca (mM), 37 °C 0.14 ( 0.04 0.023 ( 0.012 0.68 ( 0.08 0.013 ( 0.003 0.18 ( 0.08
Table 2. Critical Micellization Concentrations of the Pluronics in Hepes Buffer Containing CaCl2 (20 mM Hepes, 150 mM NaCl, 200 mM CaCl2, pH 7.4) Pluronic
cmca (mM), 25 °C
cmca (mM), 37 °C
P105 F87 F127 F108
0.22 ( 0.11 8.33 ( 0.25 0.15 ( 0.06 0.45 ( 0.11
0.030 ( 0.009 0.19 ( 0.07 0.015 ( 0.004 0.046 ( 0.006
a
The cmc data are given with estimated experimental errors.
The cmc data are given with estimated experimental errors.
steric stabilization of the liposomes which would suffer very little from depletion during blood circulation. Moreover, the Pluronic copolymers are commercially available at low cost. In a series of papers, the interactions of PEO-PPOPEO block copolymers with phosphatidylcholine liposomes have been investigated.18-21 Kostarelos et al. have used NMR18,21 and dynamic light scattering19-21 in order to probe both the degree of steric stabilization and the effect of the mode of incorporation, that is, if the polymers are just adsorbed at the surface or if the hydrophobic blocks are actually spanning the whole membrane. The results obtained from these studies indicate that it is possible to induce steric stabilization of PC liposomes by use of block copolymers. The largest effect was obtained when the triblock polymer was added to the lipids before liposome preparation.21 Furthermore, in vivo experiments have revealed a limited increase in the blood circulation time of liposomes treated with an aqueous solution of the triblock copolymer Pluronic F108.22 However, other studies have indicated that the effect of the triblock copolymers on liposome permeability is substantial and that the leakage of entrapped water soluble solutes is dramatically increased already upon addition of small amounts of polymers.22,23 The present study involves a systematic investigation of the effect of PEO-PPO-PEO polymers on the morphology and permeability of unilamellar liposomes of varying composition. The structural characterizations were performed by means of cryo-transmission electron microscopy (c-TEM), which constitutes a powerful tool for direct visualization of aggregate morpholgy in dilute aqueous solutions. Experimental Section Materials. The Pluronics F127, F108, F87, P105, and P85 were supplied by BASF Corp., Parsippany, NJ, and used as received. For details about compositions and molecular weights, see Table 1. Egg-phosphatidylcholine (EPC) of grade 1 was purchased from Lipid Products, Nutfield, U.K. Dimyristoylphosphatidylcholine (DMPC) was bought from Avanti Polar Lipids, Alabaster, AL. Cholesterol (Cho) and polyoxyethylene-8-lauryl ether (C12E8) were obtained from Sigma-Aldrich, Stockholm, Sweden. The phospholipids, cholesterol, and C12E8 were used as received. 5(6)-Carboxyfluorescein (CF) was from Kodak Co., Rochester, NY. CF was purified according to standard proce(18) Kostarelos, K.; Kipps, M.; Tadros, Th. F.; Luckham, P. F. Colloids Surf., A 1998, 136, 1. (19) Kostarelos, K.; Luckham, P. F.; Tadros, Th. F. J. Liposome Res. 1995, 5, 117. (20) Kostarelos, K.; Tadros, Th. F.; Luckham, P. F. Langmuir 1999, 15, 369. (21) Kostarelos, K.; Luckham, P. F.; Tadros, Th. F. J. Chem. Soc., Faraday Trans. 1998, 94, 2159. (22) Woodle, M. C.; Newman, M. S.; Martin, F. J. Int. J. Pharm. 1992, 88, 327. (23) Jamshaid, M.; Farr, S. J.; Kearney, P.; Kellaway, I. W. Int. J. Pharm. 1988, 48, 125.
dures24 (involving recrystallization from absolute ethanol for removal of polar impurities and column chromatography through a LH-20 (Pharmacia, Sweden) column for removal of hydrophobic impurities). Sephadex G-50 (fine) was obtained from Pharmacia Biotech, Uppsala, Sweden. 1,6-Diphenyl-1,3,5-hexatriene (DPH) was purchased from Fluka, Stockholm, Sweden, and was used as received. All other salts and reagents were of analytical grade and were used as received. Determination of cmc. Solutions of the block copolymers were prepared by dissolving the polymers in Hepes buffer (20 mM Hepes, 150 mM NaCl, pH 7.4) and diluting to the desired concentrations. In cases where the effect of CaCl2 on the cmc was investigated, CaCl2 was added to the above-mentioned Hepesbuffer at a concentration of 200 mM. To these solutions, an aliquot of DPH in methanol was added from a stock solution of 1 mM DPH in methanol. The solutions thus obtained contained a DPH concentration on the order of 0.006 mM and a final concentration of methanol on the order of 0.6% (v/v). The addition of this amount of DPH and methanol has been shown in other studies to be appropriate for measuring cmc values with the dye solubilization technique.4 The solutions were then thermostated (25 or 37 °C) in the dark for equilibration for at least 12 h before the spectroscopic measurements. The copolymer/DPH/Hepes buffer solutions were put in Teflon-stoppered quartz cuvettes and the absorption spectra were measured using a Hewlett-Packard 8453 UV-visible spectrophotometer connected to a LAUDA RC6 CP thermostat set to 25 or 37 °C. The main absorption peak of DPH was at 356 nm, and the absorbance at 356 nm was used for constructing plots of absorbance versus the logarithm of the concentration of the triblock copolymers. The cmc was determined from the break point in the curve.4 In cases where the estimation of the cmc from such plots yielded large uncertainties, samples with a narrow concentration span around the break point in the curve were prepared. From these samples the cmc was estimated from the first clearly visible absorption spectrum of DPH. The cmc data for the investigated Pluronics are given in Tables 1 and 2. Preparation of Liposomes. Lipid/polymer mixtures were prepared, unless otherwise stated, by codissolving the lipids and the polymers in chloroform, removing the chloroform under a gentle stream of nitrogen, and evaporating the remaining chloroform under vacuum. To the dry lipid/polymer film was added Hepes buffer (20 mM Hepes, 150 mM NaCl, pH 7.4), and the lipid mixtures were then subjected to at least eight freezethaw cycles (including freezing in liquid nitrogen and heating to above 60 °C), whereafter the resulting solutions were extruded (30 times) through polycarbonate filters (pore size 100 nm) mounted in a LiposoFast miniextruder from Avestin, Ottawa, Canada. The extrusion procedure produces liposomes with a mean radius of about 40-60 nm. The final total concentration of lipid and polymer was kept between 6 and 10 mM, and in the following, the amount of polymer added to the lipids will be given in mole percent. The samples were kept in a thermostat at 25 °C, unless otherwise stated, for at least 12 h before the electron microscopy investigations. Small unilamellar liposomes were prepared by ultrasonic irradiation of the lipid/polymer mixtures in Hepes buffer. A Soniprep 150 from MSE Scientific Instruments (Crawley, U.K.) was used for the irradiation. The samples were sonicated for 1 (24) Weinstein, J. N.; Ralston, E.; Leserman, L. D.; Klausner, R. D.; Dragsten, P.; Henkart, P.; Blumenthal, R. In Liposome Technology; Gregoriadis, G., Ed.; CRC Press Inc.: Boca Raton, FL, 1984; Vol. 3, p 183.
6316 Langmuir, Vol. 15, No. 19, 1999 h (the temperature was controlled by means of a waterbath held at 20-25 °C), filtered through a 0.2-µm Sartorius Minisart filter, and thereafter stored in a thermostat at 25 °C for at least 12 h before the electron microscopy investigations. Ultrasonic irradiation of lipid mixtures typically produces liposomes with a mean radius of about 10-15 nm. Cryo-Transmission Electron Microscopy. Electron microscopy investigations were performed with a Zeiss 902 A instrument, operating at 80 kV. The procedure has been described elsewhere25,26 but consists, in short, of the following. Thin (10500 nm) sample films were prepared by a blotting procedure performed under controlled temperature (25 °C unless otherwise stated) and humidity conditions within a custom-built environmental chamber. A drop of the sample solution was placed onto a copper EM-grid, and excess solution was thereafter removed by means of a filter paper, leaving a thin film of the solution on the EM-grid. Vitrification of the film was achieved by rapidly plunging the grid into liquid ethane held just above the freezing point. The vitrified sample was then transferred at low temperature to the microscope. The temperature was kept below 108 K during both the transfer and the viewing procedures in order to prevent sample perturbation and the formation of ice crystals. Static Light Scattering. A Spectraphysics He-Ne laser 120 of wavelength 633 nm was used. At an angle of 90° the intensity of the scattered light was measured by use of a Hamamatsu photomultiplier and a Hamamatsu photon counter C 1230. As reference, a toluene standard was used. Small volumes of concentrated polymer solutions were added to preformed liposomal dispersions of 1 mM EPC in Hepes buffer. The lightscattering experiments were performed at 25 °C. Leakage Assays. Liposomes composed of EPC/Cho (40 mol % cholesterol) were prepared as described above with the exception that the purified CF was now dissolved in the hydrating Hepes buffer (with or without 200 mM CaCl2) at a concentration of 100 mM. To get rid of unentrapped dye, the samples were run through a Sephadex G-50 column equlibrated with the appropriate Hepes buffer. The liposome fractions were collected and rapidly diluted with buffer to a lipid concentration of 15 µM. The triblock copolymers were then added to the liposome suspension from stock solutions of the polymers in the appropriate Hepes buffer to achieve the required lipid/polymer molar ratios. The resulting solutions were mixed by turning the vials upside down a couple of times, and aliquots were withdrawn and put into Teflon-stoppered quartz cuvettes. The cuvettes were then transferred to a thermostated cuvette holder, and at this time the clock of the actual leakage measurement was started. Measurement of CF release is a standard method for determining liposome permeability.24 The fluorescence of CF is at 100 mM self-quenched, and release of the dye increases the CF fluorescence because of the dilution-dependent dequenching. We monitored this change using a SPEX-fluorolog 1650 0.22-m double spectrometer from SPEX Industries Inc., Edison, NJ, with the excitation wavelength set to 490 nm and detecting the emitted light at 520 nm. The maximum intensity of the samples was measured after lysis of the liposomes with C12E8 added to the samples from a stock solution in Hepes buffer. The final concentration of C12E8 was 5 mM.
Results Critical Micellization Concentration Data. The compositions, molecular weights, and cmc;s of the Pluronics in Hepes-buffered saline at 25 and 37 °C are given in Table 1. For a given PEO/PPO ratio, the cmc decreases with increasing molecular weight of the Pluronic, and the cmc is strikingly dependent on the temperature. These observations are in accordance with earlier studies. It is interesting to compare the present data with the cmc data obtained by Alexandridis et al.,4 where the same dye solubilization technique was used but the Pluronics were (25) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. Technol. 1988, 10, 87. (26) Dubochet, J.; Adrian, M.; Chang, J. J.; Homo, J. C.; Lepault, J.; McDowall, A. W.; Schultz, P. Q. Rev. Biophys. 1988, 21, 129.
Johnsson et al.
dissolved in pure water. As a general trend, the cmc of the respective Pluronic seems to decrease when the Hepes buffer is used compared to the pure water case. The cmc of F127 in pure water at 25 °C was determined by Alexandridis et al.4 to be 0.555 mM whereas we obtained a value of 0.32 mM in Hepes-buffered saline. The small but significant decrease of the cmc of all the investigated Pluronics when comparing pure water and Hepes-buffered saline can most likely be attributed to the solvated ions in the buffer that decrease the solvency of the polymers. To further investigate the effect of solvency on the micellization behavior of the Pluronics, we performed cmc measurements in the above-mentioned Hepes buffer but with the addition of 200 mM CaCl2. As shown in Table 2, when CaCl2 is added to the Hepes buffer, the cmc at 25 °C is decreased for all Pluronics investigated. The effect is not that obvious at 37 °C, where the cmc data indicate only small differences between the Hepes-buffered saline and the same buffer with added CaCl2. In this case, the effect of the increased temperature seems to be the dominating parameter in determining the cmc of the Pluronics, and the addition of CaCl2 to the Hepes buffer has a comparably small influence on the micelle formation. Structural Characterization of EPC/Pluronic Mixtures. In this section, representative c-TEM micrographs from the respective lipid/polymer systems will be presented. Figure 1 shows micrographs obtained from samples of EPC and triblock polymers at low polymer to lipid molar ratios. As can be seen, the effect of the polymers on the morphology of the liposomes depends critically on the composition and molecular weight of the respective polymer. As shown in Figure 1a, a large number of small bilayer fragments, or disks, could be detected in coexistence with intact liposomes, when 5 mol % F127 was included in the lipid mixture. Moreover, open liposomes were frequently observed at this molar ratio. A similar behavior is displayed by samples of EPC upon inclusion of F108 and F87 (Figure 1b and c). Liposomes with openings or pores through the membrane, as seen in Figure 1d, were observed in samples containing EPC and P105. However, for the sample containing P85, no disk formation or open liposomes could be detected (Figure 1e). It is noteworthy that, in all the samples shown in Figure 1, a large number of small liposomes (smaller than the control pure EPC liposomes (Figure 1f)) are observed. In the case of F87, F127, and F108, we observed disk formation already at very low concentrations (about 2 mol %) of the respective polymer (results not shown). To investigate the ability of the Pluronics to solubilize the EPC bilayers into mixed micelles, the polymer to lipid molar ratios were increased. Upon increasing the concentration of F108 in the preparations, the general appearance of the samples did not differ very much from that of the sample shown in Figure 1b; that is, intact liposomes were observed in coexistence with small disks. For samples with P85, only intact liposomes were observed at intermediate concentrations of the polymer. In the case of the F127 samples, we observed the first signs of spherical micelles at a concentration of F127 of about 10 mol % (results not shown). For F87, the fraction of disklike aggregates increased with polymer concentration. At 20 mol % F87, the small disks were the dominating structures even though occasional liposomes could still be seen (results not shown). At equimolar concentrations of polymer and lipid, some interesting features of the samples were observed, as shown in Figure 2. For F127 (Figure 2a), liposomes can be seen to coexist with spherical micelles. In the case of F108, liposomes, large lamellaes, and spherical micelles
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Figure 1. Cryo-TEM micrographs of extruded EPC/Pluronic samples containing (a) 5.0 mol % F127, (b) 5.3 mol % F108, (c) 5.0 mol % F87, (d) 5.0 mol % P105, and (e) 5.0 mol % P85. Control pure EPC liposomes are shown in (f). Arrows in (a) denote an open liposome (A) and a bilayer disk observed edge-on (B). The arrow in (b) denotes a bilayer disk observed face-on. Arrows in (d) denote an open liposome (A) and an ice crystal deposited on the sample surface after vitrification (B). Note also the disks observed in (c). See text for more information. Bar ) 100 nm (f) (applies to micrographs 2a-f).
were observed, as shown in Figure 2b. Coexistence of liposomes and micelles was seen also for 50 mol % P105. Importantly, many of the liposomes show stabilized openings in the bilayer structure (Figure 2c). It is noteworthy that despite the large concentration of the micellar-forming Pluronics in the above samples, a large number of liposomes and lamellaes still exist. This indicates that the solubilizing ability of F127, F108, and P105 is not very high, regarding EPC bilayers. Furthermore, it may also indicate that the spherical micelles observed in the samples contain very little EPC and therefore essentially consist of the respective Pluronic.
However, the c-TEM method is not quantitative and the exact distribution of Pluronic molecules between the observed aggregates will have to await further studies. At very high concentrations of P85 and at high temperature (37 °C), spherical micelles were observed in coexistence with intact liposomes (Figure 2d). It is most likely that these micelles consisted purely of P85 polymers or, at least, had very little solubilized EPC. Individual micelles of the Pluronics are difficult to visualize by means of c-TEM due to the low contrast between the polymers and water.3 The PEO chains are unfortunately practically transparent to the electron
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Figure 2. Cryo-TEM micrographs of extruded EPC/Pluronic samples containing (a) 50 mol % F127, (b) 50 mol % F108, (c) 50 mol % P105, and (d) 50 mol % P85. The arrow in (a) denotes a spherical micelle. Note the large lamellae denoted with an arrow in (b). The arrow in (c) denotes an open liposome. Note also the spherical micelles present in all the micrographs 3a-d (observed as dark dots in the background). See text for more information. Bar ) 100 nm (d) (applies to micrographs 3a-d).
beam. However, a number of factors increase the possibility to visualize the micelles in the samples, for example, a high concentration of micelles and the presence of phospholipids in the micelles, that is, mixed micelles. In all cases, it is the hydrophobic core of the micelles that is visualized as dark dots in the micrographs. To further investigate how the addition of the polymers P105 and P85 to EPC liposomes affected the size distribution, static light-scattering measurements were performed. To a preformed dispersion of EPC liposomes, different amounts of polymer were added. As can be seen in Figure 3, even equimolar concentrations of polymer added to pure EPC liposomes did not have any measurable effect. However, heating of the samples to 55 °C resulted in a significant decrease in size. The change in size was supported by c-TEM (Figure 4). Obviously, increasing the temperaure results in a more efficient adsorption of the polymer. Note that the samples were put in a thermostat at 25 °C for at least 12 h after the heating process, before the light-scattering experiments were performed. The liposomal size reduction, induced by the polymers at high temperatures, seems to be an irreversible process, at least on the time scale investigated. To investigate if the mode of liposome preparation affected the structural behavior of the samples, we prepared small unilamellar liposomes, composed of EPC/ F127, by sonication. As shown in Figure 5, the structural changes observed for the liposomes prepared by extrusion (Figure 1a) are reproduced for the sonicated samples. At 2 mol % F127, disks can be seen in coexistence with small
Figure 3. Change in static light scattering after addition of P105 (circles) and P85 (squares) to preformed extruded EPC liposomes. The lipid concentration was 1 mM, and the samples were stored for 20 h at 25 °C before the light-scattering experiments. Filled symbols represent the data obtained without heating. Open symbols represent the intensity readings obtained after heating of the samples to 55 °C for 1 h (the samples were thereafter stored at 25 °C for 16 h before the light-scattering experiments). The experiments were performed at 25 °C.
intact liposomes (Figure 5a). The number of disks increased at the expense of intact liposomes when the concentration of F127 was increased (Figure 5b). Coexistence of small unilamellar liposomes, disks, and spherical micelles was observed at a concentration of 10 mol % F127 (Figure 5c). Besides the structural behavior, it is note-
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worthy that no, or at least no significant, growth of the liposomes can be observed compared with the case of the sonicated control pure EPC liposomes (Figure 5d). This result is in sharp contrast to the observations made by Kostarelos et al.20 They reported a significant increase in the hydrodynamic radius of sonicated liposomes composed of soybean lecithin/F127 at similar polymer to lipid molar ratios. Structural Characterization of EPC/Cho/Pluronic Mixtures. The effect of including cholesterol (40 mol %) in the liposomal membrane on the structural behavior (for low polymer to total lipid molar ratios) is shown in Figure 6. As can be seen, cholesterol completely inhibits the disk formation that was characteristic for most of the samples with EPC liposomes at low polymer content. A predominant feature of the cholesterol-containing preparations is the fact that the liposomes appear to be extremely aggregated and are frequently observed as clusters. Liposomes containing cholesterol normally display a tendency for aggregation when observed by c-TEM.27 However, the addition of the Pluronics to the EPC/Cho
liposomes significantly increases the relative amount and the rate of aggregation. It should be mentioned that the observation of aggregated liposomes can occasionally result due to mechanical stress (liquid flow) on the c-TEM grid during sample preparation. However, pure EPC liposomes rarely aggregate in the fashion observed for EPC/Cho liposomes. Moreover, in a previous study we have shown that cholesterol-containing liposomes, sterically stabilized with PEO lipid, appear well separated in c-TEM micrographs.27 In contrast, as shown in Figure 6, the Pluronics do not seem to bring about any sterical stabilization of the cholesterol-containing EPC liposomes. Nevertheless, the micrograph shown in Figure 7 clearly demonstrate that there is an interaction between F127 and the EPC/Cho liposomes. Small disks can be seen to coexist with intact liposomes. Disks were also observed in samples containing F87 at high (∼30 mol %) polymer concentrations (results not shown). For the sample shown in Figure 7 it should be pointed out that the number of disks was very small and the general feature was the aggregated liposomes, as shown in Figure 6. Note that all the cholesterol-containing samples contained aggregated liposomes, as shown in Figure 6, and increasing the concentration of the respective Pluronic up to about 20 mol % did not change this general feature. Structural Characterization of DMPC/Pluronic Mixtures. The DMPC lipid differs from the EPC lipid in that DMPC consists of saturated acyl chains whereas EPC consists of partly unsaturated acyl chains. Furthermore, the DMPC acyl chains are shorter (C14 chains) than the acyl chains of EPC (C16-C18 chains). The effect of F127, F108, and P85, at low polymer content, on the structure of DMPC liposomes is shown in Figure 8. As can be seen, the effect is dramatically different compared with the EPC case. Figure 8a shows a micrograph of a sample of DMPC with 5 mol % F127, and the dominant feature of this sample is the small bilayered disks in combination with what appears to be spherical micelles. In fact, despite several c-TEM observations of this particular sample, we had difficulties in finding any intact liposomes. However, a number of open liposomes were discovered in addition to the bilayered disks (results not shown). Note that the sample was prepared for TEM observation at 30 °C, that is, safely above the gel to liquid crystalline phase transition temperature of DMPC (the sample was also prepared at 25 °C with the same result as presented above). Also in the case of F108, small bilayer fragments completely dominate the sample, as shown in Figure 8b. The turbidities of the samples shown in Figure 8a and b were extremely low. In the case of DMPC with 5 mol % P85, the turbidity was much higher, and as shown in Figure 8c, the sample consisted of intact liposomes, open liposomes, and relatively large bilayered disks. This micrograph was obtained at a preparation temperature of 25 °C, and the liposomes can be seen to be somewhat uneven, which most likely is an effect of preparing the sample close to the gel to liquid crystalline phase transition temperature of DMPC.28 Again, it is interesting to compare the appearance of this sample with the EPC/P85 sample, where no structural abnormalities could be detected (Figure 1e). The results indicate that the effect of the Pluronics on liposome structure depends to a large degree on the length and/or degree of unsaturation of the acyl chains of the phospholipid. Structural Characterization of DMPC/Cho/Pluronic Mixtures. As shown in Figure 9, inclusion of
(27) Edwards, K.; Johnsson, M.; Karlsson, G.; Silvander, M. Biophys. J. 1997, 73, 258.
(28) Andersson, M.; Hammarstro¨m, L.; Edwards, K. J. Phys. Chem. 1995, 99, 14531.
Figure 4. Cryo-TEM micrographs of extruded EPC liposomes to which P85 was added from a concentrated polymer stock solution, giving a final concentration of 78 mol % P85. The micrograph shown in (a) was taken before heating, and the micrograph in (b) was taken from the sample after heating to 55 °C for 1 h (the sample was then allowed to cool back to 25 °C). Note that the polymer was added to preformed EPC liposomes ([EPC] ) 1 mM). Note also the abundance of small liposomes in (b). See text for more information. The arrow in (a) denotes an ice crystal deposited on the sample surface after vitrification. Bar ) 100 nm.
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Figure 5. Cryo-TEM micrographs of sonicated EPC/F127 samples containing (a) 2 mol % F127, (b) 5 mol % F127, and (c) 10 mol % F127. Control sonicated, pure EPC liposomes are shown in (d) ([EPC] ) 8 mM). The arrows in (a) denote bilayer disks as observed edge-on (A) and face-on (B). The arrow in (c) denotes a spherical micelle. Bar ) 100 nm (d) (applies to micrographs 6a-d).
cholesterol in the DMPC bilayer significantly decreases the tendency of the liposomes to break up into bilayer fragments. Figure 9a shows a sample of DMPC with 40 mol % cholesterol and 5 mol % F127, and the tube-shaped aggregated liposomes completely dominate the picture. However, a small number of bilayer disks can also be seen. Similar behavior is displayed by DMPC/Cho systems with the Pluronics F108 and P85, as shown in Figure 9b (from a sample of DMPC/Cho/P85, 55/40/5 mol %). However, in the case of the P85-containing samples, no disks could be observed. As with the EPC/Cho system, the fact that the cholesterol-containing DMPC liposomes appeared aggregated in the micrographs and the fact that the dispersions separated relatively fast into a highly turbid lower phase and a transparent upper phase (observed by visual inspection about 12 h after preparation) together indicate the absence of effective steric stabilization. Effect of the Pluronics on Leakage of CholesterolContaining Liposomes. As already mentioned, for most Pluronics, no visible effect on liposome structure was induced when they were added to lipid mixtures of EPC/ cholesterol. Since it was still possible that some kind of subtle, but important, interaction took place between the polymers and the liposomal membrane, the effect of the block copolymers on the permeability of the liposomeencapsulated carboxyfluorescein (CF) was investigated. To systematically investigate the effect, we performed leakage measurements at two different temperatures (25 and 37 °C) and in the absence and presence of CaCl2. In all the leakage experiments, the initial lipid composition used was EPC/Cho, 60/40 (mol %). Note that, in contrast to the majority of the structural studies, the Pluronics were added to the liposomes after liposome preparation. This procedure was used in order to ensure the appropriate conditions in terms of polymer to lipid molar ratios and to use the exact same preparation of EPC/Cho liposomes to which the different Pluronics were added. (Gel filtration was used to remove unentrapped CF (see Experimental Section). If the polymers are added to the lipid mixture before liposome preparation, Pluronics that are not associated with the liposomes may be removed during the gel filtration and thereby cause uncertainties in the polymer to lipid molar ratios.) The leakage of CF from the various samples is given in terms of relative leakage (∆leakage), that is, the increase in leakage caused by the addition of a precise amount of polymer compared with the leakage from pure EPC/Cho dispersions. (∆-leakage ) L(x mol %, t) - L(0 mol %, t), where L(x mol %, t) is the leakage of EPC/Cho liposomes with added Pluronic at x
mol % and at the time point t (min) and L(0 mol %, t) is the leakage of the pure EPC/Cho liposomes without added Pluronic at the time point t (min).) As shown in Figure 10, the effect on the leakage of CF from the liposomes is strongly dependent on the composition of the polymer. Notably, the Pluronics which induce the largest leakage also display the lowest cmc values (Table 1). Moreover, the increase in leakage is very rapid after addition of the polymer. This implies a fast initial perturbation of the lipid bilayers, resulting in the release of a substantial amount of encapsulated material. The same behavior is observed at 37 °C as shown in Figure 11, but the rapid increase in the ∆-leakage is of larger magnitude than that at 25 °C. Again, the largest increase is observed for the Pluronics with the lowest cmc values (Table 1). In addition, the polymers that did not affect the leakage measureably at 25 °C, such as F87, give a small but significant effect at 37 °C. To examine if PEO as such had any effect on liposome leakage, we performed experiments where the EPC/Cho liposomes were mixed with PEO of molecular weight 3400. As shown in Figure 11, PEO alone, at concentrations e16 mol %, did not induce an increased leakage of CF. Decreasing the solvency of the polymers, via addition of CaCl2 (200 mM) to the Hepes-buffered saline, resulted in an increase in the ∆-leakage at 25 °C, as shown in Figure 12. This is in accordance with the temperature effects presented above, since the induced leakage correlates with the cmc. The cmc of the Pluronics was found to decrease considerably when the temperature was increased or when CaCl2 was added to the Hepes-buffered saline (Tables 1 and 2). The leakage data obtained suggest a correlation between the cmc and the induced leakage of the respective Pluronic. As a general trend, the lower the cmc, the higher the induced leakage. It should be emphasized that due to the low lipid concentration (15 µM) in the leakage experiments the concentration of the respective Pluronic did not, in any case, exceed the cmc. Discussion The use of PEO-PPO-PEO triblock polymers has been suggested as an alternative approach for steric stabilization of phospholipid liposomes. The advantages of using the Pluronics for steric stabilization are several. The Pluronics are commercially available at low cost, and if situated in the bilayer as membrane-spanning objects, they would provide an effective durable steric stabilization.
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Figure 7. Cryo-TEM micrograph of an extruded EPC sample containing 40 mol % cholesterol and 10.4 mol % F127. The arrow denotes a bilayer disk. Bar ) 100 nm.
Figure 6. Cryo-TEM micrographs of extruded EPC samples containing 40 mol % cholesterol and Pluronics in concentrations of (a) 5.3 mol % F127, (b) 6.0 mol % F87, and (c) 6.0 mol % P85. Note the extremely aggregated liposomes present in all the micrographs. Bar ) 100 nm (c) (applies to micrographs 7a-c).
Moreover, since the end groups of the Pluronics consist of hydroxyl groups, they provide a platform, similar to the PEO-lipids, for conjugation of various targeting agents such as antibodies or antibody fragments. However, the results obtained in the present study, regarding structural effects and the effect on the permeability, show that the Pluronics have considerable drawbacks when used for the purpose of steric stabilization of phospholipid liposomes. In a recent study we presented evidence of disk formation in preparations of liposomes sterically stabilized with PEO-lipid.27 The effect was observed at a critical concentration of about 10 mol % PEO-lipid in the bilayer.
Below this concentration, the liposomes appeared intact and well separated when observed by c-TEM. Moreover, the samples constituted very stable dispersions where the liposomes did not sediment, due to flocculation, on the time scale of several months. Many of the Pluronics investigated in the present work also induce formation of bilayer disks and thus show some resemblance to the PEO-lipids. However, the concentration needed for the disk formation is shifted toward lower concentrations in the case of the Pluronics (Figure 1). Bilayer disks are normally expected to be short-lived due to the thermodynamically unfavorable exposure of hydrocarbons to the aqueous environment at the edges of the disks. However, if micellar-forming surfactants are present, these molecules can act as “edgeactants”, that is, adsorb to the highly curved edges and thereby reduce the hydrocarbon exposure.29-32 Many of the Pluronics investigated in the present study, as well as the PEO-lipids, appear to function as such “edgeactants”. The formation of bilayer disks has been observed in a number of phospholipid liposome/surfactant systems,32,33 and the disks may in these systems be regarded as an intermediate structure occurring during solubilization of liposomes into mixed micelles. However, in most cases this intermediate structure does not appear until a considerable concentration of the surfactant is reached within the bilayer.32,33 In the present study, the appearance of bilayer disks was observed already at very low concentrations (about 2 mol %) of the Pluronics F127, F108, and F87. To understand the mechanism behind the disk formation in the present system, it is necessary to consider possible models for the Pluronic-liposome interactions. The polymers may be incorporated within the liposomal membrane in several ways. Assuming that it is the hydrophobic PPO residue that anchors the Pluronic molecule to the membrane, the more hydrophilic PEO chains have different possibilities, as shown schematically in Figure 13. Either both PEO chains stick out on the same side of the membrane or they reside on either side of it, that is, a membrane-spanning configuration. The latter configuration is only likely to occur when the polymers are added to the lipid mixture before liposome (29) Lasic, D. D. Biochim. Biophys. Acta 1982, 692, 501. (30) Fromherz, P. Chem. Phys. Lett. 1983, 94, 259. (31) Edwards, K.; Almgren, M. Langmuir 1992, 8, 824. (32) Edwards, K.; Gustafsson, J.; Almgren, M.; Karlsson, G. J. Colloid Interface Sci. 1993, 161, 299. (33) Silvander, M.; Karlsson, G.; Edwards, K. J. Colloid Interface Sci. 1996, 179, 104.
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Figure 9. Cryo-TEM micrographs of extruded DMPC samples containing 40 mol % cholesterol and Pluronics in concentrations of (a) 5 mol % F127 and (b) 5 mol % P85. Arrows in (a) denote bilayer disks, as observed nearly edge-on (A) and face-on (B). Note also the extremely aggregated liposomes in both micrographs. Bar ) 100 nm (b) (applies to micrographs 10a-b).
Figure 8. Cryo-TEM micrographs of extruded DMPC/Pluronic samples containing (a) 5 mol % F127, (b) 5 mol % F108, and (c) 5 mol % P85. Arrows in (c) denote polymer support on a c-TEM grid (A) and relatively large bilayer disks, as observed edge-on (B) and face-on (C). The samples shown in (a) and (b) were prepared at 30 °C whereas the sample shown in (c) was prepared at 25 °C. Note disks and spherical micelles in (a) and disks in (b). See text for more information. Bar ) 100 nm (c) (applies to micrographs 9a-c).
preparation. The preferred position of the polymers is, at present, unknown. It is possible that both alternatives occur. In addition, in the case where both PEO chains reside on the same side of the membrane, the degree of PPO block penetration into the lipid bilayer may vary depending on the conditions. At a certain surface density, the PEO chains will start to interact laterally and there will be a transition of the
Figure 10. Pluronic-induced leakage of CF from preformed EPC/Cho (60/40) liposomes. The leakage measurements were performed in Hepes buffer at 25 °C. The induced leakage is given in terms of relative leakage, that is, the excess leakage caused by the addition of Pluronics compared with the leakage from the pure EPC/Cho liposomes (see text). 2 mol % F127 (filled circles); 2 mol % P105 (small open circles); 8 mol % P105 (large open circles); 2 mol % F108 (small open squares); 8 mol % F108 (large open squares); 8 mol % P85 (diamonds); 2 mol % F87 (small filled squares); 8 mol % F87 (large filled squares). The data represent the mean of at least two measurements. For clarity reasons, no error bars are shown in the figure. Typically, the standard deviation was about 0.05-0.10.
polymer layer from a relaxed “mushroom” regime to a “brush” regime, in which the chains are more extended.
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Figure 11. Pluronic-induced leakage of CF from preformed EPC/Cho (60/40) liposomes. The leakage measurements were performed in Hepes buffer at 37 °C. (For more information, see legend of Figure 12 and text.) 2 mol % F127 (filled circles); 2 mol % P105 (small open circles); 8 mol % P105 (large open circles); 2 mol % F108 (open squares); 2 mol % P85 (small diamonds); 8 mol % P85 (large diamonds); 2 mol % F87 (small filled squares); 8 mol % F87 (large filled squares); 16 mol % PEO(3400) (crosses). The data represent the mean of at least two measurements. For clarity reasons, no error bars are shown in the figure. Typically, the standard deviation was about 0.050.10. Figure 13. Schematic models of possible interactions between a Pluronic molecule and a lipid membrane. Two configurations of the Pluronic molecule are depicted: a membrane spanning configuration (top) and a configuration where both PEO chains reside on the same side of the membrane (middle). The hydrophobic PPO block is shaded in gray. As a comparison, a membrane stabilized by PEO-lipids is shown at the bottom. The lipid anchor is shaded in gray.
Figure 12. Effect of adding CaCl2 to the Hepes buffer on Pluronic-induced leakage of CF from preformed EPC/Cho (60/ 40) liposomes. The measurements were performed in Hepes buffer with or without 200 mM CaCl2 at 25 °C. 2 mol % F127 in buffer + 200 mM CaCl2 (filled squares); 2 mol % F127 in plain buffer (open squares); 2 mol % P105 in buffer + 200 mM CaCl2 (filled diamonds); 2 mol % P105 in plain buffer (open diamonds); 2 mol % F108 in buffer + 200 mM CaCl2 (filled circles); 2 mol % F108 in plain buffer (open circles).
Increasing the concentration of the polymer even further will lead to a high lateral tension in the polymer/head group region. This tension can be relaxed by curving the surface, and this may be accomplished by, for example, a transition of the liposomes into mixed micelles.34 However, a transition into bilayer disks also offers a relaxation of the lateral tension, since the polymers now can be situated at the highly curved rim of the disk. We propose that the disks observed in the present study are formed as a result of the stress imposed by the repulsion between the hydrophilic flexible polymer chains. Several parameters may influence the extent of disk formation. Among these are the bilayer cohesive strength, the degree of phospholipid unsaturation, and the length of the acyl chains. Inclusion of cholesterol is known to increase the cohesive strength of PC bilayers.35 However, (34) Hristova, K.; Kenworthy, A.; McIntosh, T. Macromolecules 1995, 28, 7693. (35) Needham, D.; Nunn, R. S. Biophys. J. 1990, 58, 997.
in a previous study, we concluded that for PC liposomes the critical concentration of PEO(2000)-lipid needed to induce disks was independent of cholesterol content.27 It is therefore reasonable to expect that it is the packing properties of the phospholipids in the bilayer, and not the bilayer cohesive strength, that determine the maximum amount of polymer that can be incorporated (or adsorbed) before any structural transitions of the liposomes occur. Even though the hydrophobic anchors of PEO-lipids and the Pluronics (the PPO-residue) are structurally different, the effect of either molecule on the lipid bilayer depends strikingly on the packing properties of the host lipid. Thus, the tendency of the liposomes to break up into disks under the influence of the Pluronics is significantly enhanced when using DMPC liposomes compared with EPC liposomes. In other words, it is easier to accommodate the DMPC lipid than the EPC lipid in an aggregate of higher curvature. The concentration of polymer in the bilayer at which the chains start to interact laterally (assuming an even distribution of polymer in the bilayer and neglecting interactions arising from freely diffusing polymer mushrooms) depends on the length of the PEO chains. In this context it is interesting to calculate the concentration needed in the bilayer of, for example, F127 in order to enter the so-called brush regime. Assuming that the water medium acts as a good solvent for the PEO chains, the Flory radius can be calculated according to the following:
RF ) aN3/5
(1)
where a is the effective length of the monomeric unit and N is the number of monomers in the chain. With a ) 3.5 Å (CH2CH2O) and N ) 100, we obtain a Flory radius of about 55.5 Å. According to the Alexander-de Gennes
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Figure 14. Schematic picture showing the definition of the Flory radius RF of the grafted polymer and the distance between the grafting points s.
scaling theory,36,37 the transition from the mushroom regime to the brush regime occurs when the chains start to overlap; that is, the distance between the grafting points s must be equal to or less than the Flory radius. This is shown schematically in Figure 14. In our case this means that s e 55.5 Å. The number of PEO chains per unit area Γ is approximately given by
Γ ) 1/s2
(2)
Taking s ) 55.5 Å gives Γ ) 0.0325 chains/nm2. Using an average area per lipid molecule of 0.65 nm2,38 the transition from the mushroom regime to the brush regime may be expected to take place when the concentration of F127 exceeds 2 mol %. The calculated concentration pertains to a flat surface. Simple geometrical considerations show that the overlap concentration is dependent on the curvature of the surface. For a liposome of radius 50 nm, the difference from the flat surface is very small. However, for a liposome of radius 15 nm, the effect of the increased curvature becomes significant. Thus, the overlap concentration of F127 is in this case calculated to be about 3 mol %. In the above calculation it is assumed that the polymer is actually spanning the membrane. Other studies have implicated the presence of membrane-spanning F127,18 but it cannot be ruled out that the two different configurations, shown in Figure 13, may coexist. A model that takes into account both configurations would be more complex, since the length of the hydrophobic PPO chains, the depth of incorporation, and other parameters would have to be considered. However, the calculated concentration of 2 mol % coincides well with the appearance of bilayer fragments, as observed by c-TEM. The corresponding surface concentrations calculated for the other Pluronics are as follows: F108, 1.5%; F87, 4%; P105, 7%; P85, 11%. For F108 and F87 the values agree well with the observation of disks in the EPC system. In the case of P105 and P85, however, no disks were found even at concentrations higher than those calculated for the mushroom to brush transition. Nevertheless, the effect of adding increasing amounts of P85 or P105 to preformed EPC liposomes was a decrease in liposome size, as observed by both c-TEM and static light scattering. This may indicate that, in these preparations, the higher curvature needed to decrease the repulsion at a high surface concentration of polymer is achieved by a liposomal size reduction. Note that the decrease in liposomal size was not significant until the temperature of the solutions was raised to 55 °C for 1 h (and was then allowed to cool back to 25 °C) (Figures 3 and 4). Clearly, the heating resulted in an increased interaction of the lipid bilayer with the Pluronics. (36) Alexander, S. J. Phys. (Paris) 1977, 38, 983. (37) de Gennes, P. G. Macromolecules 1980, 13, 1069. (38) McIntosh, T. J.; Magid, A. D.; Simon, S. A. Biochemistry 1989, 28 (8), 7904.
C-TEM micrographs obtained from samples of EPC/ P85 or EPC/P105, where the polymers were added to the lipids before liposome preparation, showed a relatively high abundance of liposomes smaller than the control pure EPC liposomes (besides the defective liposomes observed for EPC/P105 (Figure 1d)). It therefore seems likely that a liposomal size decrease occurs whatever the mode of lipid/Pluronic mixing, given that the temperature is high enough during the addition of P85 or P105 to preformed EPC liposomes. It should again be noted, however, that c-TEM is not quantitative, and it is a difficult task to estimate small differences in liposome size by means of c-TEM micrographs. Interestingly, Li et al.9 investigated the adsorption of a number of Pluronics on a series of differently sized polystyrene colloids. The mobility of the PEO chains was found to be higher when the Pluronics were adsorbed on particles of higher curvature, indicating a decrease in the lateral interactions between the PEO chains. This is in qualitative agreement with the results presented above, where the decrease in liposomal size most likely results in decreased lateral interactions between the PEO chains of the adsorbed Pluronic molecules. When EPC was exchanged for DMPC, we observed the formation of open liposomes and relatively large bilayer disks at about 5 mol % P85 (Figure 8c). This is much lower than the predicted polymer regime transition, which, as mentioned before, occurs at about 11 mol %. However, in the analysis above, we have assumed that the polymers are situated in the bilayer as membrane-spanning objects.This may be inaccurate, as mentioned earlier, especially for Pluronics with short hydrophobic blocks such as P85. By including cholesterol in the bilayers of EPC or DMPC, the formation of disks was effectively reduced and in some cases even abolished. This is an important difference compared to the case of the PEO-lipid bilayers previously referred to, where cholesterol had no effect on the levels of disk formation.27 In the present case, it is likely that cholesterol inhibits the partitioning of the respective Pluronics in the lipid bilayer. This may be due to the ability of cholesterol to increase the strength of the lateral interactions between the hydrocarbon chains of the lipids and thereby increase the cohesive strength of the membrane.35 A polymer molecule spanning the membrane would most likely perturb the condensing effect of cholesterol on the bilayer and presumably also increase the amount of hydrocarbons exposed to the aqueous environment compared with the case of pure PC/Cho bilayers. Thus, it would be energetically unfavorable to accommodate the polymer within the bilayer. In contrast, the pure EPC or DMPC bilayers are much more fluid (at 25 °C) and a polymer spanning these bilayers would not affect the packing of the phospholipids to the same extent compared with the case of the cholesterol-containing bilayers. As a general feature of the cholesterol-containing samples, we observed heavily aggregated liposomes, which indicates the absence of steric stabilization and therefore a negligible amount of polymer incorporated in the lipid bilayers. Nevertheless, in some cases we did observe a small number of disks also with cholesterol present, implying an interaction of some kind also in these systems. Kostarelos et al. have in a number of publications reported on successful incorporation of Pluronics into phospholipid liposomes.18-21 In addition, they call attention to the suitability of such Pluronic/liposome mixtures for drug delivery applications. The studies reported in refs 18-21 contain a number of interesting data, but in
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light of the present study, we feel that some of the conclusions may have to be reconsidered. On the basis of data obtained by dynamic light scattering, the authors claim that the addition of Pluronic typically results in a significant increase in liposomal size. It is possible that the assumption of an increase in liposomal size is correct for their systems, which differ from ours in that they are based on bath-sonicated liposomes composed of soybean lecithin. However, the increase in hydrodynamic radius may have other origins. As described above (Figures 6 and 9), the addition of polymer can increase the formation of liposome clusters. This was especially obvious for compositions containing cholesterol but may well occur also for other compositions. Even a small degree of increased interliposomal interaction would lead to an increase in apparent liposome mean radius, as measured by dynamic light scattering. Importantly, we could not observe any growth in either the EPC system or the DMPC system. C-TEM investigations of small liposomes prepared by sonication confirmed the lack of growth in the EPC/ F127 system (Figure 5). Kostarelos and co-workers, furthermore, suggest that the increased diffusion coefficients obtained at high polymer concentrations are due to scattering from free polymer in solution.20 It is more likely, in our view, that the increase in diffusion coefficients, interpreted by Kostarelos et al. as scattering from free polymers, instead represents the breakdown of the liposomal structure. The leakage data obtained in the present study reveal that all investigated Pluronics do affect the leakage of encapsulated CF. The magnitude of the polymer-induced leakage is strongly dependent on temperature, molecular composition, and the solvency of the polymers in the aqueous medium. It is noteworthy that large increases in leakage were observed for many of the Pluronics at concentrations where no structural defects of the liposomes could be observed by c-TEM. Note that, in the leakage experiments, the Pluronics were added to preformed liposomes, and therefore, the membrane-spanning configuration is very unlikely to occur (Figure 13). However, as shown above, no indications of steric stabilization of cholesterol-containing EPC liposomes could be observed when the Pluronics were added to the lipid mixture before liposome preparation. It is therefore likely that none, or at least very little, of the Pluronics are situated in the bilayer as membrane-spanning objects in the preparations used to examine the structural effects. One of the most interesting findings in this study, regarding the permeability effects, is that the increase in the leakage occurs very rapidly after mixing of liposomes and Pluronics. This implies a fast initial perturbation of the lipid bilayer. This effect levels off after about 60 min (Figures 10-12), with no or very little further increase in the relative leakage (∆-leakage). In fact, the leakage profiles obtained for most of the Pluronics show similarities to the induced leakage observed by the addition of more conventional surfactants to liposomes.31 At present, the precise mechanism for the induced leakage is not known and further investigations have to be performed to accurately explain the leakage profiles. Nevertheless, it is likely that the initial perturbation of the lipid bilayer is caused by the adsorption of Pluronic molecules at the membrane surface (with possible PPO block penetration into the lipid bilayer). It may be speculated that the adsorbing Pluronics cause serious
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packing disturbances resulting in a fast, significant release of encapsulated material from part or all of the liposomes. This packing disorder is then relaxed, and the following release proceeds with the same kinetics as those for the release from pure EPC/Cho liposomes. Increasing the temperature or adding CaCl2 to the buffer resulted in an increased effect of the Pluronics on liposome leakage. This is an interesting observation because, in both cases, the cmc of the respective Pluronic is seen to decrease (Tables 1 and 2). Accordingly, there exists a correlation between the cmc of the Pluronic and the magnitude of the induced leakage. Assuming, as above, that the perturbation of the lipid bilayer, resulting in an elevated leakage, is caused by adsorbing Pluronics, then it is clear that the lower the cmc (at a given temperature and solvent composition) of the Pluronic, the higher the adsorbed amount at the membrane surface. In fact, this behavior is in qualitative agreement with the results obtained by Tadros and Vincent,10 who investigated the adsorption of the Pluronic P75 (50 wt % PPO) on latex particles with a mean particle radius of about 120 nm. Also in this case, decreasing the solvency of the polymer in the aqueous medium by increasing the temperature or adding electrolytes such as CaCl2 resulted in higher adsorption of the Pluronic. Accordingly, the trends observed in the present study do correlate with those observed for Pluronic adsorption on latex particles. Conclusions The present study has shown that the Pluronics affect the structure of phospholipid liposomes at varying degrees depending on polymer composition, type of phospholipid, and cholesterol content in the bilayer. The most obvious effect is the formation of small bilayer fragments, or bilayer disks, observed at low polymer to lipid molar ratios. In cases where no disk formation could be observed, the effect of increasing Pluronics concentration was instead a reduction of the liposome size. The effect of the Pluronics on the permeability of liposome-encapsulated carboxyfluorescein was also investigated. It was shown that the magnitude of the induced leakage depends on the composition of the polymer, the temperature, and the solvency of the respective Pluronic in the aqueous medium. The enhanced leakage observed when the Pluronics were added to preformed EPC/Cho liposomes was interpreted in terms of packing disturbances in the lipid bilayer, caused by adsorbing polymers. The magnitude of the induced leakage was found to correlate with the cmc of the respective Pluronic; that is, the lower the cmc of the polymer (at a given temperature and solvent composition), the higher the magnitude of the induced leakage. Regarding the use of Pluronics as stabilizing material for phospholipid liposomes, we conclude that it is doubtful if effective steric stabilization can be achieved without serious structural changes, as well as increased permeability, of the liposomes. Acknowledgment. Financial support from the Swedish Foundation for Strategical Research and the Swedish Research Council for Engineering Sciences is gratefully acknowledged. LA990288+
