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Steric Stabilization of Egg-Phosphatidylcholine Liposomes by Copolymers Bearing Short Blocks of Lipid-Mimetic Units Stanislav Rangelov,*,†,‡ Katarina Edwards,† Mats Almgren,† and Go¨ran Karlsson† Department of Physical Chemistry, University of Uppsala, Box 532, 751 21 Uppsala, Sweden, and Institute of Polymers, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Received July 15, 2002. In Final Form: October 17, 2002 The ability of a number of amphiphilic di- and triblock copolymers bearing short blocks of repeating lipid-mimetic units to sterically stabilize egg-phosphatidylcholine (EPC) liposomes has been studied by means of light scattering, cryogenic transmission electron microscopy (cryo-TEM), and fluorescence spectroscopy. The hydrophobic blocks contain 1-4 lipid-mimetic units, whereas the hydrophilic blocks consist of poly(ethylene glycol) (PEG) of average molecular weights close to 5000 Da. The effect of the chain architecture was investigated as well. The structural changes and membrane permeability were studied as a function of the amount of the incorporated copolymer. The structural investigations, performed by means of cryo-TEM, revealed that the maximum amount of the copolymer that can be incorporated in the EPC membrane without affecting the liposome integrity increases with increasing number of the lipidmimetic anchors per single copolymer chain. This helps the formation of a dense and thick PEG protective layer which, in turn, is expected to enhance the liposome longevity. At the same time, the membrane permeability, investigated using a hydrophilic dye as a probe, was only slightly affected by the incorporation of the copolymers. The structural changes and membrane permeability are discussed in light of the shape of the macromolecules and the phase propensity of the steric stabilizers.

Introduction The syntheses of a number of epoxide monomers and precursors, that is, didodecylglycidylamine, 1,3-didodecyloxy-propane-2-ol (DDP), 1,3-didodecyloxy-2-glycidylglycerol (DDGG), and 1,3-didodecyloxy-glyceryl-2-toluenesulfonate, were described in a series of recent papers.1-3 The common feature of the above compounds is the hydrophobic residue that mimics the lipid anchor of the naturally occurring phospholipids. The above monomers and precursors were used to prepare a variety of amphiphilic copolymers based on poly(ethylene glycol) (PEG).2-5 The aqueous solution properties of these copolymers were investigated by means of viscometry, turbidity measurements, dye solubilization, rheology, fluorescence spectroscopy, light scattering, and cryogenic transmission electron microscopy. In an aqueous environment, they were found to spontaneously form micellar and/or lamellar aggregates4,5 as well as particles of corecorona structure with PEG-water domains randomly distributed in the core.4 Depending on the type and number of the hydrophobic lipid-mimetic anchors and on the chain architecture, the radii and the aggregation numbers of the particles varied from 5 to 70 nm and from 24 to 3600 macromolecules per particle, respectively. These novel copolymers form self-assembled structures that offer many attractive and interesting applications, for example, drug delivery, cellular engineering, surface modification, thickening, and so forth. In the present contribution, their ability to stabilize lipid vesicles, liposomes, is investigated. † ‡

University of Uppsala. Bulgarian Academy of Sciences.

(1) Rangelov, S.; Tsvetanov, Ch. Des. Monomers Polym. 2001, 4, 39. (2) Rangelov, S.; Petrova, E.; Berlinova, I.; Tsvetanov, Ch. Polymer 2001, 42, 4483. (3) Rangelov, S.; Tsvetanov, Ch. Polym. Bull. 2001, 46, 471. (4) Rangelov, S.; Almgren, M.; Tsvetanov, Ch.; Edwards, K. Macromolecules 2002, 35, 4770. (5) Rangelov, S.; Almgren, M.; Tsvetanov, Ch.; Edwards, K. Macromolecules 2002, 35, 7074.

Due to their amphiphatic structure and composition, the liposomes are well fitted to solubilize and encapsulate both hydrophilic and hydrophobic active substances.6 The liposome circulation time in the bloodstream can be prolonged by utilization of dialkyl-substituted glycerols with covalently attached PEG (referred to as PEGlipids).7-15 A critical parameter for these sterically stabilized liposomes is the maximum amount of PEG-lipids that can be incorporated into the phospholipid bilayer without the latter being damaged. This saturation limit (Nsat) is slightly dependent on the bilayer composition,16,17 and from simple shape considerations it is expected to decrease with increasing size of the PEG hydrophilic head. This has been proven both theoretically and experimentally by a number of authors.16-21 With further increase of the PEG-lipid concentration, the performance of the (6) Liposome Technology; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1983. (7) Blume, G.; Cevc, G. Biochim. Biophys. Acta 1990, 1029, 91. (8) Blume, G.; Cevc, G. Biochim. Biophys. Acta 1993, 1146, 157. (9) Klibanov, A.; Maruyama, K.; Torchilin, V. P.; Huang, L. FEBS Lett. 1990, 268, 235. (10) Klibanov, A.; Maruyama, K.; Beckerleg, A. M.; Torchilin, V. P.; Huang, L. Biochim. Biophys. Acta 1991, 1062, 142. (11) Allen, T. M.; Hansen, C.; Martin, F.; Redemann, C.; Yau-Young, A. Biochim. Biophys. Acta 1991, 1066, 29. (12) Allen, T. M.; Hansen, C. Biochim. Biophys. Acta 1991, 1068, 133. (13) Mayhew, E.; Lasic, D.; Babbar, S.; Martin, F. Int. J. Cancer 1992, 51, 302. (14) Chonn, A.; Cullis, P. R. Curr. Opin. Biotechnol. 1995, 6, 698. (15) Lasic, D.; Martin, F. Stealth Liposomes; CRC Press: Boca Raton, FL, 1995. (16) Hristova, K.; Kenworthy, A.; McIntosh, T. J. Macromolecules 1995, 28, 7693. (17) Edwards, K.; Johnsson, M.; Karlsson, G.; Silvander, M. Biophys. J. 1997, 73, 258. (18) Hristova, K.; Needham, D. Macromolecules 1995, 28, 991. (19) Belsito, S.; Bartucci, R.; Montesano, G.; Marsh, D.; Sportelli, L. Biophys. J. 2000, 78, 1420. (20) Kenworthy, A. K.; Simon, S. A.; McIntosh, T. J. Biophys. J. 1995, 68, 1903. (21) Johnsson, M.; Edwards, K. Biophys. J. 2001, 80, 313.

10.1021/la020632u CCC: $25.00 © 2003 American Chemical Society Published on Web 12/06/2002

Steric Stabilization of Liposomes by Copolymers

Figure 1. Chemical structures of the lipid anchors: DDP (top) and DDGG (bottom). Table 1. Compositions and Nominal Molecular Weights of the Polymer Species composition

nominal molecular weight

(EO)92DDP (EO)115(DDGG)2 (EO)114(DDGG)4 (DDGG)2(EO)136(DDGG)2

4475 6028 6952 7920

stabilized liposomes as drug carriers is gradually deteriorated. It has been assumed earlier2 that copolymers containing short blocks of repeating lipid-mimetic monomer units would provide enhanced anchoring strength of the copolymer macromolecule in the bilayer resulting in an enhancement of the saturation limit. The higher the saturation limit, the denser and thicker the protective PEG layer around the liposome. In contrast to the coneshaped macromolecules of the conventional PEG-lipids for PEG molecular weights higher than 750 Da,20 the copolymers containing short sequences of lipid anchors have large hydrocarbon volumes and hence less coneshaped and more cylindrical macromolecules. In this study, we use light scattering, cryogenic transmission electron microscopy, and fluorescence spectroscopy to determine the effects of the number of lipid anchors and the chain architecture of various copolymers on their ability to sterically stabilize egg-phosphatidylcholine (EPC) liposomes. Four copolymers with PEG moieties of molecular weights close to 5000 were chosen (see Table 1 for the degrees of polymerization of both PEG and polylipid blocks). This particular PEG molecular weight is typically used in the preparation of drug-carrying liposomes.13,22-24 The hydrophobic anchors, abbreviated DDP and DDGG (chemical structures are shown in Figure 1), are structurally similar and allow investigations of the effect of the number of lipid anchors. The bottom two copolymers in Table 1, (EO)114(DDGG)4 and (DDGG)2(EO)136(DDGG)2, represent a suitable pair to probe the effect of the chain architecture. Here, EO denotes ethylene oxide, that is, the monomer unit of the PEG blocks. Experimental Section Materials. Chloroform was of analytical grade and was used as received. Egg-phosphatidylcholine was purchased from Lipid Products, Nutfield, U.K. 5(6)- Carboxyfluorescein (CF) was obtained from Molecular Probes, Leiden, The Netherlands. The copolymers used in the present study were prepared according to procedures described in detail elsewhere.2,4,5 Their compositions and the nominal molecular weights are given in Table 1. Liposome Preparation. Liposomes were prepared by codissolving EPC and the respective copolymer in a chosen copolymer/EPC molar ratio in chloroform. Then the chloroform was evaporated under a gentle stream of nitrogen and the traces (22) Needham, D.; Hristova, K.; McIntosh, T.; Dewhirst, M.; Wu, N.; Lasic, D. J. Liposome Res. 1992, 2, 411. (23) Woodle, M.; Lasic, D. Biochim. Biophys. Acta 1992, 1113, 171. (24) Litzinger, D.; Huang, L. Biochim. Biophys. Acta 1992, 1127, 249.

Langmuir, Vol. 19, No. 1, 2003 173 were removed under vacuum. Purified water (Millipore SuperQ-System) was added to the dry lipid/copolymer film, and the resulting dispersions were subjected to eight freeze-thaw cycles. Afterward, the dispersions were further diluted to about [EPC] ) 1.4 mM and extruded 30 times through polycarbonate filters of pore size 100 nm. The final concentration of EPC was adjusted to 1.0 mM by dilution with water. For the light scattering experiments, to achieve an adequate intensity of the scattered light, the dispersions were additionally diluted with water in a 1/10 ratio. The amount of the copolymer added to EPC is given as a copolymer to EPC molar ratio (r). Methods. Dynamic Light Scattering (DLS). The light scattering (LS) setup consists, as described previously,25 of a 488 nm Ar ion laser and the detector optics with an ITT FW 130 photomultiplier and ALV-PM-PD amplifier-discriminator connected to an ALV-5000 autocorrelator built into a computer. Measurements were made at an angle of 90°, [EPC] ) 0.091 mM, and t ) 25 °C. Analysis of the dynamic data was performed by fitting the experimentally measured g2(t), the normalized intensity autocorrelation function, which is related to the electrical field correlation function g1(t) by the Siegert relationship:26

g2(t) - 1 ) β |g1(t)|2

(1)

where β is a factor accounting for deviation from ideal correlation. For polydisperse samples, g1(t) can be written as the inverse Laplace transform (ILT) of the relaxation time distribution, τA(τ):

g1(t) )

∫ τA(τ) exp(-t/τ) d ln τ

(2)

where t is the lag-time. The relaxation time distribution, τA(τ), is obtained by performing ILT using the constrained regularization algorithm REPES.27 The diffusion coefficients at Θ ) 90°, D90, are calculated as D90 ) Γ90/q902, where Γ90 is the relaxation rate and q90 is the magnitude of the scattering vector q ) (4πn/λ) sin(Θ/2) at Θ ) 90°. Here n is the refractive index of the medium and λ is the wavelength of the light in a vacuum. The hydrodynamic radius at Θ ) 90°, R90, was calculated using the Stokes-Einstein equation:

R90 ) kT/(6πηD90)

(3)

kT is the thermal energy factor, and η is the temperaturedependent viscosity of water. Cryogenic Transmission Electron Microscopy (cryo-TEM). Transmission electron microscopy observations were conducted on a Zeiss EM 902 A instrument operating at 80 kV. The procedure for the sample preparation is described in the following. A drop of the dispersion is deposited on an electron microscopy copper grid coated by a perforated polymer film. The excess of the liquid is blotted by a filter paper, leaving a thin film of the dispersion on the grid. The film on the grid is vitrified by plunging the grid into liquid ethane. The vitrified sample is then transferred to the microscope for observation. Leakage Assays. Fluorescent measurements, with the fluorescent probe 5(6)-carboxyfluorescein, were carried out on a SPEX-fluorolog 1650 0.22-m double spectrometer (SPEX Industries Inc., Edison, NJ). The fluorescence intensity was measured as a function of time at 520 nm with excitation at 490 nm. The measurements were performed at 25 °C. The negligible fluorescence intensity of CF at high concentrations (usually 100 mM) due to self-quenching has been employed for CF leakage measurements. As the dye leaks out from the aqueous compartments of the liposomes, it becomes diluted and the fluorescence intensity increases. Therefore, the liposomes were prepared as described above, however, in a medium containing 100 mM CF and at pH 7.4. The unentrapped dye was removed by gel filtration using a Sephadex G-50 column equilibrated with water. The liposome fractions were collected and rapidly diluted with water to get [EPC] ) 0.25 mM. The (25) Rangelov, S.; Brown, W. Polymer 2000, 41, 4825. (26) Chu, B. Laser Light Scattering: Basic Principles and Practice, 2nd ed.; Academic Press: New York, 1991. (27) Jakes, J. Czech. J. Phys. B 1988, 38, 1305.

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Figure 3. Cryo-TEM micrograph of a sample containing pure EPC liposomes. [EPC] ) 1.0 mM, bar ) 100 nm.

Figure 2. Variations of light scattering intensity (a) and hydrodynamic radii at an angle of 90°, R90 (b), of a series of EPC liposomes stabilized by (EO)92DDP (open squares), (EO)115(DDGG)2 (closed circles), (EO)114(DDGG)4 (open triangles), and (DDGG)2(EO)136(DDGG)2 (closed diamonds) as a function of the copolymer/EPC molar ratio, r. The measurements were performed at 25 °C. The lines through the data points were drawn to guide the eye. above protocol results in liposomes containing CF with a 100 mM concentration dispersed in a medium with no CF. The leakage was followed on the time scale of several days with the first measurements made about 3 min after the gel filtration. Then, the maximum intensity, Imax, was obtained after lysis of the liposomes with a nonionic surfactant, lauryloctaethylene glycol, added to the dispersions from a stock solution in Hepes buffer. The fraction of the released dye at a given time is then defined as (I/Imax)t. The rate constants, k, were determined as slopes of the linear fit to the data from the initial linear parts of the I/Imax versus time curves. Accordingly, the rate constants are expressed as a fraction of the released dye per unit time.

Results LS Experiments. Figure 2 shows the variations of two independent parameters extracted from LS data with the copolymer/EPC molar ratio for each of the copolymers studied. Obviously, the variations of the parameters reflect changes in the aggregate size. The large LS intensity readings and R90 values observed for (EO)114(DDGG)4 could be ascribed to the formation of vesicles of large size, or alternatively they may reflect aggregation of fragments formed as a result of disruption of the liposomes. On the other hand, the marked decrease in the parameters could mean either formation of vesicles of smaller size or solubilization of the bilayers and formation of mixed micelles. Cryo-TEM was employed in order to investigate the nature of the variations and the liposome structure with the copolymer content. Cryo-TEM. The method of liposomal preparation described in the Experimental Section is known to yield

Figure 4. Cryo-TEM micrographs of samples containing EPC liposomes stabilized by (EO)92DDP at r ) 0.021 (a), 0.075 (b), and 0.135 (c). Arrows in (b) denote bilayer fragments. [EPC] ) 1.0 mM, bar ) 100 nm.

nonuniform unilamellar liposomes with a mean hydrodynamic radius of about 50 nm. A micrograph taken from a sample containing EPC liposomes without a steric stabilizer is shown in Figure 3. Figure 4 shows representative micrographs of EPC liposomes stabilized with different amounts of (EO)92DDP. At the lowest (EO)92DDP/ EPC molar ratio (Figure 4a), the effect of this copolymer is marginal although the liposomes look better separated than those in Figure 3. The first open liposomes and bilayer


Figure 5. Cryo-TEM micrographs of samples containing EPC liposomes stabilized by (EO)115(DDGG)2 at r ) 0.030 (a), 0.121 (b), and 0.212 (c). [EPC] ) 1.0 mM, bar ) 100 nm.

fragments appeared at r ) 0.075 (Figure 4b). In addition, the fraction of small liposomes seems to increase. At r ) 0.135, most of the material is in the form of threads, open liposomes, and bilayer fragments (Figure 4c). A fraction of intact liposomes, however, still exists. At higher ratios, the micrographs were quite similar to those at r ) 0.135: intact liposomes coexist with a large fraction of open liposomes and bilayer disks and fragments (not shown). This is consistent with the slight changes in the intensity of the scattered light upon an increase of the (EO)92DDP/ EPC molar ratio (Figure 2a). In DLS experiments, however, the changes in the liposome structure were registered as a decrease in the mean hydrodynamic radius (Figure 2b) and with appearance of a low-intensity fast mode with an increasing amplitude with increasing r (data not shown). The changes in the liposome structure upon addition of (EO)115(DDGG)2, that is, the copolymer that contains an average of 2 lipid anchors per chain, are shown in Figure 5. Up to a ratio r ) 0.1, the micrographs look quite similar: liposomes of a “normal” size coexist with a fraction of small liposomes (Figure 5a). The latter seems to decrease with increasing r. A striking difference to the previous polymer, (EO)92DDP, is that the copolymer content at which the first fragments and open liposomes appeared is shifted toward higher r. In the present case, the bilayer

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fragments and disks were not detected until a ratio as high as 0.121 was reached (Figure 5b). Note the abundance of intact liposomes. Their number decreases, however, with further increase of r (Figure 5c). Figure 6 gives the structural changes of EPC liposomes stabilized with increasing amounts of (EO)114(DDGG)4, a copolymer containing twice as many lipid anchors as (EO)115(DDGG)2. The following features deserve special attention: (i) The liposomes are somewhat larger than those observed in the previous cases. (ii) The polydispersity increases with increasing r (Figure 6a,b). The tendency for the formation of large vesicles with increasing (EO)114(DDGG)4 content is also clearly seen in the constant increase of the LS intensity and R90 (Figure 2). (iii) The ratio at which the first fragments appeared is further shifted to higher r values compared to those of (EO)92DDP and (EO)115(DDGG)2: only very few edge-on disks were observed at r ) 0.12, whereas at r ) 0.142 bilayer fragments are seen to coexist with a number of intact liposomes. A fast mode with an amplitude that accounted for about 10% at 90° was detected at this ratio in the relaxation time distribution (not shown). (iv) An anomalous behavior was found at r ) 0.181. This is illustrated in Figure 6d, where surprisingly few fragments and open liposomes were observed. Contrary to the expectation, the liposomes appeared to be intact and well separated. Also, dark spherical objects as that are denoted with an arrow in Figure 6d were found in different places. Such objects were observed in pure (EO)114(DDGG)4/water systems and were attributed to (EO)114(DDGG)4 self-association.4 A most plausible explanation for the anomalous increase of the (EO)114(DDGG)4 efficiency in this interval is that the copolymer prefers to self-associate instead of going into the bilayer membrane. This in turn decreases the actual (EO)114(DDGG)4/EPC ratio. It should be emphasized that dark objects were not found at lower ratios, implying that all the copolymer is intercalated in the liposome membrane. (v) At ratios as high as 0.3, intact liposomes can still be observed (Figure 6e). The effect of the chain architecture on the ability of the copolymers to stabilize EPC liposomes was investigated as well. The composition of (DDGG)2(EO)136(DDGG)2 is similar to that of the previous polymer, (EO)114(DDGG)4. The major difference is the triblock chain architecture with flanking short polylipid blocks and a middle watersoluble PEG block. The sequence of the structural changes of (DDGG)2(EO)136(DDGG)2/EPC liposomes at increasing ratios is presented in Figure 7. Obviously, the stabilizing effect of this copolymer is low, since bilayer disks can be found at a ratio as low as 0.029 (Figure 7a, denoted with arrows A). With increasing r, the vesicles seem to become less polydisperse and bilayer fragments are more frequently observed. At r ) 0.134 (Figure 7b), the latter become dominant, and at r ) 0.208 practically no intact liposomes are present. These structural changes are registered also as a sharp drop in the LS intensity and R90 (Figure 2). In the relaxation time distributions, a fast mode with an amplitude increasing from 1.3 to 65% was observed with increasing copolymer content. Two interesting features illustrating the peculiar interactions of (DDGG)2(EO)136(DDGG)2 should be mentioned: first, dark spherical objects similar to those found in the (EO)114(DDGG)4/EPC preparations only at high (EO)114(DDGG)4 contents and also in pure (EO)114(DDGG)4/ water and (DDGG)2(EO)136(DDGG)2/water systems4 are seen even at very low copolymer contents (Figure 7a, denoted with arrows B); and second, the viscosity of the dispersions, particularly at high (DDGG)2(EO)136(DDGG)2

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Figure 6. Cryo-TEM micrographs of samples containing EPC liposomes stabilized by (EO)114(DDGG)4 at r ) 0.060 (a), 0.120 (b), 0.142 (c), 0.181 (d), and 0.3 (e). Arrows in (c) and (d) denote bilayer fragments and a dark spherical object, respectively. [EPC] ) 1.0 mM, bar ) 100 nm.

content, was visibly higher than that of the dispersions of the rest of the copolymer/EPC and pure EPC systems at comparable ratios and concentrations. Figure 8 shows vials that have just been tilted in order to demonstrate dissimilarities in the flow properties of nonextruded samples of (DDGG)2(EO)136(DDGG)2/EPC and (EO)114(DDGG)4/EPC, both at r ) 0.3, and the control EPC. The different flow properties of the (DDGG)2(EO)136(DDGG)2/ EPC system can be attributed to the triblock chain architecture of (DDGG)2(EO)136(DDGG)2 with a middle PEG block and flanking poly(DDGG) blocks. A part of the (DDGG)2(EO)136(DDGG)2 macromolecules are probably adopting a bridging conformation with the flanking poly(DDGG) blocks anchored in two adjacent membranes. The formation of these physical cross-links most likely explains the enhanced viscosity of the (DDGG)2(EO)136(DDGG)2/ EPC system. In contrast, bridging conformations are not possible for the (EO)114(DDGG)4/EPC system. Therefore, the flow properties of the latter are apparently similar to those of the control EPC. In summary, the results so far indicate that (i) the triblock copolymer architecture with flanking polylipid anchoring blocks and a middle water-soluble PEG block is not favorable as far as the steric stabilization of EPC liposomes is concerned and (ii) the maximum amount of the copolymer that can be incorporated in the liposome

without the latter being damaged increases with increasing number of lipid anchors. The second finding is demonstrated in Figures 9 and 10. Figure 9 shows micrographs taken from samples with the same copolymer/ EPC molar ratio of 0.1. Intact and well-separated liposomes are clearly seen for (EO)115(DDGG)2/EPC and (EO)114(DDGG)4/EPC preparations (Figure 9b,c). In contrast, intact liposomes coexist with an abundance of bilayer fragments and threads in the preparation of liposomes stabilized with (EO)92DDP (Figure 9a). From the cryoTEM study, the saturation limit (Nsat), that is, the ratio at which the first fragments appeared, was estimated. It is plotted as a function of the number of lipid anchors (n) in Figure 10. As clearly seen, Nsat was found to increase with increasing n. Leakage Assay. In the previous section, representative cryo-TEM micrographs from EPC liposomes sterically stabilized by copolymers bearing short polylipid blocks were presented. It was shown how the copolymers affect the structure and stability of the EPC liposomes depending on the copolymer content and the number of lipid-mimetic anchors per single copolymer chain. The cryo-TEM investigation gave detailed information on the structural changes and permitted determination of the maximum amounts of copolymer that could be included in the EPC membrane without inducing a transition into bilayer


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Figure 7. Cryo-TEM micrographs of samples containing EPC liposomes stabilized by (DDGG)2(EO)136(DDGG)2 at r ) 0.029 (a) and 0.134 (b). Arrows A and B in (a) denote bilayer disks and dark spherical objects, respectively. [EPC] ) 1.0 mM, bar ) 100 nm.

Figure 9. Cryo-TEM micrographs of samples containing EPC liposomes stabilized by (EO)92DDP (a), (EO)115(DDGG)2 (b), and (EO)114(DDGG)4 (c). [EPC] ) 1.0 mM, r ) 0.1, bar ) 100 nm.

Figure 8. Vials containing nonextruded samples of [(EO)115(DDGG)2]/[EPC] at r ) 0.3 (left), [(DDGG)2(EO)136(DDGG)2]/ [EPC] at r ) 0.3 (right), and the control EPC sample (middle) tilted at an angle of 45° to demonstrate the different flow properties. [EPC] ) 12.6 mM.

fragments. However, there may be effects caused by the inclusion of copolymers that are not detected by cryoTEM. Indeed, the resolution of the electron microscope is approximately 4 nm, which is the thickness of the lipid bilayer. Therefore, defects that are smaller than 4 nm and supposedly transient in nature are not observable using cryo-TEM. On the other hand, the liposomes are useless as carriers of hydrophilic drugs if they are not able to retain the encapsulated substance. To evaluate the extent to which the permeability of the membrane is affected by the incorporation of PEG-polylipid copolymers, a leakage assay using CF was carried out. Measurement of CF release is a standard method for determination of liposome permeability.7,8,28 The molecule of (28) 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: Boca Raton, FL, 1984; Vol. 3, p 183.

Figure 10. Saturation limit, Nsat, as a function of the number of lipid anchors, n.

CF is large and at pH 7.4 hydrophilic. Substances such as CF are believed to be released from the internal water pools mainly through transient pores and defects that are spontaneously formed in the bilayer.29-32 (29) Kaschiev, D.; Exerowa, D. Biochim. Biophys. Acta 1983, 732, 133.

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Figure 12. Fraction of the released dye at t ) 24 h, (I/Imax)24, as a function of the copolymer/EPC molar ratio, r: (EO)92DDP (squares), (EO)115(DDGG)2 (triangles), (EO)114(DDGG)4 (circles), and (DDGG)2(EO)136(DDGG)2 (diamonds). Table 2. 5(6)-Carboxyfluorescein Release Rate Constants as a Function of the Liposome Composition and Copolymer Content liposome composition EPC DDP(EO)92/EPC (EO)115(DDGG)2/EPC (EO)114(DDGG)4/EPC (DDGG)2(EO)136(DDGG)2/EPC

Figure 11. Leakage of CF from EPC liposomes stabilized by (EO)92DDP (a), (EO)115(DDGG)2 (b), (EO)114(DDGG)4 (c), and (DDGG)2(EO)136(DDGG)2 (d) at various [copolymer]/[EPC] ratios: open triangles, r ) 0.01; open squares, r ) 0.05; open circles, r ) 0.068 (a), r ) 0.11 (b), r ) 0.127 (c), r ) 0.1 (d). The leakage from pure EPC liposomes is given by closed squares in (a)-(d).

The effect of the copolymers on the membrane permeability of EPC liposomes was investigated at 25 °C. The leakage of CF is given as a fraction of the released dye, I/Imax (see Experimental Section). The results are presented in Figure 11 as plots of I/Imax versus time for each copolymer at various copolymer/EPC molar ratios. The leakage was followed on the time scale of several days. For better presentation, however, the results up to the 500th minute are shown. The leakage profiles at three different copolymer/EPC ratios for each of the copolymers studied are presented in Figure 11a-d. Two of the ratios, 0.01 and 0.05, are common for all of the copolymers, whereas the highest ratio was copolymer dependent. It was selected as 0.9Nsat. The triblock copolymer (DDGG)2(EO)136(DDGG)2 is the only exception (see below). The (30) Paula, S.; Volkov, A. G.; Deamer, D. W. Biophys. J. 1994, 4, 725. (31) Edwards, K.; Almgren, M. Prog. Colloid Polym. Sci. 1990, 82, 190. (32) Edwards, K.; Almgren, M. Langmuir 1992, 8, 824.

[copolymer]/[EPC] ratio

104 × k (s-1)

0.0 0.01 0.05 0.068 0.01 0.05 0.11 0.01 0.05 0.127 0.01 0.05

6.77 11.69 15.66 18.35 7.25 8.40 11.81 11.28 22.71 28.00 8.04 11.39

leakage profile of pure, unmodified EPC liposomes is given in each of the figures as well. The common feature of all leakage profiles is that their initial slopes change after approximately 100 min. After that, the fluorescence intensity increases with a continuously decreasing rate. Generally, the leakage profiles of the stabilized liposomes have a more pronounced Γ-shape than that of the reference EPC liposomes. Other parameters that were extracted from the data were the fraction of the released dye at t ) 24 h, (I/Imax)24, and the rate constants. They are given in Figure 12 and Table 2, respectively. The leakage profiles of EPC liposomes stabilized with increasing amounts of (EO)92DDP are shown in Figure 11a. The fractions of the released CF from the stabilized liposomes were invariably higher than that of the reference EPC liposomes; only at r ) 0.05 and zero time was I/Imax of the stabilized probe lower than that of the pure EPC liposomes. The leakage profiles of the stabilized liposomes and the magnitude of the fraction of the released dye are practically independent of the (EO)92DDP content incorporated in the liposomes. This is most evident in Figure 12 where (I/Imax)24 is plotted against the (EO)92DDP/EPC molar ratio. The rate constants, however, are seen to increase with increasing ratio (Table 2). Not only was the bilayer permeability little affected by the incorporation of (EO)114(DDGG)2 (Figures 11b and 12), but in the initial periods even I/Imax values of the stabilized liposomes were lower than that of the EPC liposomes (note that 21 points lay below the EPC leakage curve in Figure 11b). The leakage rate constants were found to increase


with increasing ratio, but even at a ratio as high as 0.11 the rate constant is close to that of the reference liposomes (Table 2). The leakage from the liposomes stabilized by (EO)115(DDGG)4 at r ) 0.01 is quite similar to that from the liposomes stabilized by (EO)114(DDGG)2: lower leakage at the initial periods, approximately equal (I/Imax)24 values, and similar rate constants (Figures 11c and 12, Table 2). At the ratios 0.05 and 0.127, however, the leakage profiles changed and had the most pronounced Γ-shape. This was reflected in the values of the rate constants which substantially increased with increasing r (Table 2). Figure 11d shows the leakage profiles of liposomes stabilized by (DDGG)2(EO)136(DDGG)2. Somewhat surprisingly, the curves at ratios 0.01 and 0.05 are found to lie just a little above the EPC curve and the (I/Imax)24 values as well as the leakage rate constants (Figure 12, Table 2) seem to be only slightly affected by the incorporation of (DDGG)2(EO)136(DDGG)2. It was shown above that the latter copolymer is able to adopt a bridging conformation with polylipid blocks anchored in two adjacent membranes. During the extrusion, the high shear forces can cause disruption of the integrity of the membranes. This was registered by cryo-TEM even at a ratio as low as 0.029 (Figure 7a). However, after the gel filtration, used in the preparation of liposomes for leakage experiments, only intact liposomes are collected. The leakage from these liposomes that are presumably stabilized by (DDGG)2(EO)136(DDGG)2 in a looping conformation is comparable to that from liposomes stabilized by (EO)115(DDGG)4, for example. However, the total amount of CF entrapped in the liposomes stabilized by (DDGG)2(EO)136(DDGG)2 was clearly lower than that of the pure EPC liposomes or liposomes stabilized by the rest of the copolymers studied. This was evidenced by Imax values which were typically 1.5-1.7-fold lower for the compositions with (DDGG)2(EO)136(DDGG)2. The leakage profile at r ) 0.1 is very peculiar (Figure 11d). The curve pattern can be explained assuming that this is a collective effect of leakage from vesicles of different compositions. As noted in the previous section, at a ratio up to 0.134 a population of intact liposomes still exists. On the other hand, (DDGG)2(EO)136(DDGG)2 showed a strong tendency to self-associate rather than to be incorporated in the EPC membrane: particles supposedly consisting of (DDGG)2(EO)136(DDGG)2 only are identified in the cryo-TEM micrographs at a ratio as low as 0.029 (Figure 7a, arrow B). As shown elsewhere,4 the size of these particles is comparable to the size of liposomes prepared by extrusion through a 100 nm pore size filter. The particles have been shown to have a core/corona structure with water compartments, in which CF can be entrapped, randomly distributed in the core.4 In the gel filtration, these particles, together with (DDGG)2(EO)136(DDGG)2/EPC liposomes of different compositions, were collected. They have different leakage behaviors, and each type has a contribution to the curve at r ) 0.1 depicted in Figure 11d. Discussion In contrast to the micelle-forming surfactants that have been shown to induce a considerable increase in permeability for hydrophilic substances upon interaction with liposomes,31,33-35 the CF release is just slightly influenced by the incorporation of the present copolymers in the liposome membrane. For some of the copolymers, (EO)115(DDGG)2 and (EO)115(DDGG)4, initially the leakage of CF was even reduced compared to that of the pure EPC

Langmuir, Vol. 19, No. 1, 2003 179

Figure 13. Fraction of the released dye at t ) 24 h, (I/Imax)24, as a function of the number of lipid anchors, n: r ) 0.01 (squares), r ) 0.05 (circles), r ) 0.9Nsat (diamonds).

liposomes. Assuming a mechanism of CF leakage through spontaneously formed transient pores or defects in the membrane,29,31,32,36 similar findings have been interpreted earlier in terms of reducing the probability of pore formation upon PEG-lipid inclusion.37 Although the results show that the CF release is not strongly influenced by the incorporation of the investigated copolymers, detectable differences in the membrane permeability were observed depending on the type and quantity of the copolymer. The effects of the copolymer composition, as represented by the number of lipid-mimetic anchors per polymer chain, are shown in Figure 13, where (I/Imax)24 is plotted against the number of lipid anchors, n. The curves go through a maximum at n ) 1, that corresponds to (EO)92DDP. The (I/Imax)24 values at n ) 2 and 4, corresponding to (EO)115(DDGG)2 and (EO)114(DDGG)4, respectively, at r ) 0.01 and 0.05 are not much different from that of the unmodified EPC liposomes. The presence of maxima at n ) 1 in the curves depicted in Figure 13 could be related to the shape of the copolymer macromolecules. PEG-lipids with attached PEGs of molecular weights greater than 750 have been shown to be cone shaped.20 The introduction of more lipid anchors in the macromolecules of (EO)115(DDGG)2 and (EO)114(DDGG)4 leads to a more cylindrical shape. The above geometrical considerations are consistent with the phase propensity of the copolymers. According to the shape concept,38-40 the cone-shaped molecules self-aggregate into micelles, whereas lamellae or bilayers are the preferred aggregate structures of molecules with a cylindrical shape. Indeed, the cone-shaped (EO)92DDP was found to spontaneously form micelles in water, whereas the less conical (EO)115(DDGG)2 and (EO)114(DDGG)4 form large lamellar aggregates.4,5 The conical molecules have been found to possess a greater solubilization power toward bilayer membranes. This has been nicely demonstrated for a series of distearoylphosphatidylcholine-poly(ethylene glycol)s (DSPC-PEGs) with PEGs of average molecular weights from 350 to 5000.20 The authors argue (33) Edwards, K.; Almgren, M. J. Colloid Interface Sci. 1991, 147, 1. (34) Ruiz, J.; Goni, F. M.; Alonso, A. Biochim. Biophys. Acta 1988, 937, 127. (35) Ueno, M. Biochemistry 1989, 28, 5631. (36) Paula, S.; Volkov, A. G.; Van Hoek, A. N.; Haines, T. H.; Deamer, D. W. Biophys. J. 1996, 70, 339. (37) Silvander, M.; Johnsson, M.; Edwards, K. Chem. Phys. Lipids 1998, 97, 15. (38) Tartar, H. V. J. Phys. Chem. 1955, 59, 1195. (39) Tanford, C. J. Phys. Chem. 1972, 76, 3020. (40) Israelachvili, J. N.; Mitchel, J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525.

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Rangelov et al.

Table 3. Values of Hydrodynamic Radii at an Angle of 90° (R90), Total Bilayer Area, Aggregation Number (Nagg), Number of PEG Chains per Vesicle, and Grafting Density (σ) at Copolymer to EPC Molar Ratios Close to but below the Saturation Limitsa copolymer

molar ratio

R90 (nm)

total bilayer area (nm2)

Nagg

number of PEG chains per vesicle

σ (nm-2)

σtr (nm-2)

(EO)92DDP (EO)115(DDGG)2 (EO)114(DDGG)4 PEG(2000)-lipid

0.070 0.098 0.135 0.100

41.2 51.7 66.0 50.0

42 661 67 177 109 478 62 832

65 632 103 349 168 428 96 665

4594 10 128 22 738 9667

0.108 0.151 0.208 0.154

0.036 0.028 0.028 0.085

a The grafting density at the mushroom to brush transition (σ ) does not depend on the copolymer to EPC molar ratio (see the text for tr more information).

that the shape of the DSPC-PEGs is the key factor in the differences in the observed phase behavior of DSPC/ DSPC-PEG suspensions: thus the cone-shaped DSPCPEG 2000 and DSPC-PEG 5000 convert DSPC bilayers to micelles, whereas DSPC-PEG 350 is not able to induce micelle formation even at considerably high lipid to PEGlipid ratios. This behavior is in accordance with our cryoTEM data showing an increase of the saturation limit with increasing number of lipid anchors, that is, with the change of the shape of the macromolecules from conical to less conical and possibly cylindrical. Consequently, the higher membrane permeability for DDP(EO)92 than for (EO)115(DDGG)2 and (EO)114(DDGG)4 at the same ratio was not a surprise (Figure 13). Since the molecules of (EO)115(DDGG)2 and (EO)115(DDGG)4 have the propensity to form aggregates with a zero surface curvature, one may expect that they fit better at less curved surfaces than the cone-shaped molecules of (EO)92DDP. That may be why the liposomes stabilized by (EO)115(DDGG)2 and particularly by (EO)114(DDGG)4 are somewhat larger, that is, of a lower curvature. This effect is most pronounced at high copolymer to EPC ratios. In contrast, (EO)92DDP, which spontaneously forms micelles in aqueous solution, converts the EPC membrane into bilayer fragments and mixed micelles at considerably lower ratios. As seen from Figure 13, the cone-shaped (EO)92DDP induces invariably higher leakage than the less conical (EO)114(DDGG)2. This is valid also at the ratio defined as 0.9Nsat although the quantity of (EO)92DDP incorporated in the liposome membrane is 1.6-fold lower than that of (EO)115(DDGG)2, However, the curve at r ) 0.9Nsat depicted in Figure 13 is also peculiar with the sudden increase of (I/Imax)24 values at n ) 4. Such an increase is not observed at r ) 0.01 and 0.05 where the leakage from (EO)114(DDGG)4/EPC liposomes is comparable to that from the pure EPC liposomes. Since no structural defects of the liposomes were detected by cryo-TEM even at higher (EO)114(DDGG)4/EPC, it is possible that the incorporation of high quantities of (EO)114(DDGG)4 introduces a packing disorder. The packing disorder increases the probability for pore or defect formation in the membrane which results in an enhanced leakage. The introduction of a packing disorder upon the incorporation of the present copolymers in the membrane can be correlated with the more pronounced Γ-shape pattern of I/Imax versus time curves clearly seen in Figure 11 and quantified in the higher rate constants, k (Table 2). After ca. 100 min, the packing disorder is relaxed and the leakage follows the same release kinetics as the leakage from unmodified EPC liposomes. As an alternative explanation for the pronounced Γ-shape pattern, one can speculate on the existence of small liposomes of relatively short lifetime that are not detected by cryo-TEM but able to influence the leakage. Note that cryo-TEM was carried out several hours or even a couple of days after the initial preparation

of the liposomes, in contrast to the leakage measurements that were performed immediately. Another aspect of the systems to be considered is the grafting density, σ, of the PEG chains in the bilayer. It was calculated for each copolymer/EPC system using the results from DLS at ratios close to but still below the saturation limits determined from the cryo-TEM data (Table 3). An average area of 0.65 nm2 41 per both a lipid molecule and a lipid-mimetic monomer unit was used for the calculation of the aggregation number, Nagg. Also, an even distribution of the PEG chains on the outer and inner monolayers of the bilayer membrane was assumed. Indeed, the surface of liposomes of radius ca. 50 nm can be considered flat; that is, the outer and inner surfaces are equivalent, and consequently the probability for grafting is the same. In reality, however, due to the negative surface curvature, fewer PEG chains are grafted on the inner monolayer. This is particularly true for grafted molecules of higher molecular weights. Although not precise due to the above assumptions, the data in Table 3 give reasonable estimates of the aggregation numbers, the number of PEG chains per vesicle, and the grafting density. The latter is especially valuable when compared to the grafting density at which the PEG chains start to interact laterally. At low surface coverage, the grafted chains are separated with a size given by the Flory radius (eq 4):

RF ) RN3/5

(4)

where N is the degree of polymerization and R is the size of the monomer unit. The polymer chains are in the socalled mushroom regime. With increasing surface coverage, the polymer chains start to overlap and enter the so-called brush regime.42,43 Using calculations presented elsewhere,44 it was possible to calculate the grafting density at which the mushroom to brush transition occurs, σtr. The calculations were done only for the diblock copolymers and for a commercially available PEG-lipid with a PEG molecular weight of 2000 Da, PEG(2000)lipid. The values of σtr are given in the last column of Table 3. As seen from Table 3, in all cases the grafting densities at ratios close to Nsat are above σtr, indicating that the PEG chains are in a brush regime. With σ values from 3 to 7.4-fold higher than σtr, the PEG chains of the copolymers are considered to be in a much more stretched conformation compared to those of PEG(2000)-lipid having a value of only 1.8σtr. Furthermore, the increasing number of lipid anchors allows an enhancement of Nsat, and consequently high grafting density and an extent of stretching, as reflected by σ/σtr, can be achieved. Accord(41) McIntosh, T. J.; Magid, A. D.; Simon, S. A. Biochemistry 1989, 28, 7904. (42) Alexander, S. J. Phys. (Paris) 1977, 38, 983. (43) de Gennes, P. G. Macromolecules 1980, 13, 1069. (44) Johnsson, M.; Silvander, M.; Karlsson, G.; Edwards, K. Langmuir 1999, 15, 6314.


ingly, the density and the thickness of the protective layers are expected to increase with increasing number of lipid anchors. In the regime of lateral interactions between the PEG chains, there is energy stored in the brush which is proportional to the molecular weight and grafting density.45 The stored energy is usually expressed in the lateral pressure between the chains. The latter for liposomes stabilized by the present copolymers is higher than that of liposomes stabilized by PEG(2000)-lipid due to the higher either molecular weight [(EO)92DDP and (EO)115(DDGG)2] or both molecular weight and grafting density [(EO)114(DDGG)4]. At certain conditions, the bilayer packing is no longer energetically favorable and the liposome membrane is disintegrated. There must be, however, mechanisms by which the lateral pressure can be relaxed before disruption of the liposome. Since the brush regime is entered at a ratio as low as 0.02 and Nsat is reached at several-fold higher ratios and assuming that the size of the vesicles does not change in this concentration range, one can expect that the lateral pressure continuously increases. In fact, as documented in the previous section the liposomes were found to vary in size depending on the copolymer to EPC molar ratio. Thus, for (EO)92DDP the mechanism by which the lateral pressure is relaxed probably involves curving of the surface through formation of vesicles with decreasing size as the grafting density increases. Just opposite is the mechanism for vesicles stabilized by (EO)115(DDGG)2 and (EO)114(DDGG)4 which were found to increase in size with increasing σ. The increasing vesicle size means that the curvature of the inner monolayer becomes less negative which in turn favors grafting of more PEG chains and consequently leads to an increase of the lateral pressure between the chains grafted on this monolayer. This helps to maintain the balance between the lateral pressures of the brushes grafted on the outer and inner monolayers and the bilayer cohesion and to achieve high saturation limits. The two different mechanisms are determined by the different phase propensities of the copolymers. The results are in agreement with earlier findings of Hristova et al.16 showing that it is the phase propensity of the stabilizing agent that determines the maximum amount of the latter that can be incorporated in the bilayers. Last but not least, a question to be clarified is the partition of the copolymers between the vesicles and solution. The partition coefficients have not been determined; however, as shown elsewhere,4,5 the critical aggregation concentrations of the copolymers are in the low micromolar range, that is, practically they do not exist as unimers. This suggests that the solubility of the copolymers is not high enough to favor such partition and the copolymer macromolecules are incorporated in the bilayer. Coexistence of liposomal aggregates and copolymer selfassembled structures was only very rarely observed, implying that the saturation limits were correctly determined. Implications for Development of Liposomal Drug Delivering Systems Nowadays, it is generally recognized that the longevity in the bloodstream of liposomes sterically stabilized by PEG-lipids depends strongly on both the molecular weight of the PEG and the amount of the PEG-lipid incorporated in the liposome membrane. PEG molecular weights and PEG-lipid concentrations up to 5000 Da and 10 mol %, (45) Milner, S.; Witten, T.; Cates, M. Macromolecules 1988, 21, 2610.

Langmuir, Vol. 19, No. 1, 2003 181

respectively, are typically used in the formulations. Larger molecular weights and/or molar concentrations are detrimental to drug delivery, since they lead to a phase transition from bilayers to a micellar phase. In the present paper, we demonstrate for the first time that the saturation limit can be increased by increasing the number of lipid anchors per copolymer chain. Our results, summarized in Figure 10, show that the introduction of more than one lipid anchor is beneficial as far as the increasing of the saturation limit is concerned. The enhanced saturation limit is expected to favor the formation of a dense and thick protective layer and hence to increase the liposome longevity. The enhanced longevity is meaningless if the sterically stabilized liposomes are not able to retain the encapsulated substance. The membrane permeability was only slightly affected by the incorporation of the present copolymers in the EPC liposomes. As a matter of fact, (EO)115(DDGG)2 and (EO)114(DDGG)4 were found to initially reduce the CF release. The latter copolymers are able to fulfill requirements that always cause a dilemma, that is, that high concentrations are needed to achieve prolonged circulation time and at the same time the membrane permeability should not be affected. From the pharmacological point of view, the diblock architecture is of particular interest. The triblock architecture with a middle PEG block and flanking poly(DDGG) blocks is not favorable since the triblock copolymer was shown to adopt a bridging conformation between adjacent membranes and to act as a cross-linking agent. When such cross-linked material is subjected to high shear forces during extrusion, part of it is converted to bilayer fragments instead of closed liposomes. Apparently, interactions leading to cross-linking can be avoided using diblock copolymers. In addition, specific ligands can be attached to the water-exposed hydrophilic end of the PEG chain, thus making targeting liposomes. “Inverted” triblock copolymers, that is, with a middle poly(DDGG) block and flanking PEG blocks, are expected to have the same advantages. Their efficiency as steric stabilizers is still to be established, however. The lack of charge and the ether linkage are also advantages worth mentioning. The carbamate linkage through which the PEG chains are usually connected to the lipid anchors in the commercially available PEG-lipids introduces a net negative charge in the membrane surface which in turn may provoke undesirable changes of the membrane properties.37,46 By replacement of the carbamate linkage with an ether bond, this disadvantage is avoided. Furthermore, the ether linkage provides excellent stability against hydrolysis which is also beneficial as far as the longevity of the stabilized liposomes is concerned. In conclusion, the copolymers bearing blocks of lipidmimetic aliphatic double-chain units can be considered as promising materials for the generation of new sterically stabilized liposomes. A final feature that makes them attractive for purposes of steric stabilization of liposomes is that their anchoring strength can be varied using a number of controlling parameters, that is, the length and the type of the aliphatic chains, the molecular weights of both PEG and polylipid moieties, and the ratio between them. Acknowledgment. Financial support from the Swedish Technical Research Council and the Swedish Cancer Foundation is gratefully acknowledged. LA020632U (46) Flewelling, R. F.; Hubbell, W. L. Biophys. J. 1986, 49, 531.

Steric Stabilization of Egg-Phosphatidylcholine Liposomes by

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