Physicochemical Characterization of Poly (ethylene glycol)-Coated

D. Polo,† I. Haro,† M. A. Alsina,‡ and F. Reig*,† ... 08034 Barcelona, Spain, and Physicochemical Unit, Faculty of Pharmacy, University of ...
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Langmuir 1997, 13, 3953-3958

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Physicochemical Characterization of Poly(ethylene glycol)-Coated Liposomes Loaded with Doxorubicin D. Polo,† I. Haro,† M. A. Alsina,‡ and F. Reig*,† Department of Peptide and Protein Chemistry, CID-CSIC, Jordi Girona 18, 08034 Barcelona, Spain, and Physicochemical Unit, Faculty of Pharmacy, University of Barcelona, Pza. Pius XII, 08028 Barcelona, Spain Received September 23, 1996. In Final Form: May 1, 1997X The influence of PEG in the physicochemical stability of liposomes has been determined. Vesicles composed of PC/PG/Ch/DX (A), PC/PG/Ch/DX/PE-PEG (B), PC/PG/Ch/DX/PE-PEG/NGPE/E(8) (C), and PC/PG/Ch/PE-PEG/NGPE/E(8) (D) were prepared and characterized. The stability of these four liposomal preparations was studied via its ability to spread on air/water interfaces and the microviscosity of its bilayers. Preparations lacking PEG show no tendency to spread on aqueous surfaces, thus indicating a high stability of these types of preparations. Only if liposomes were previously treated with methanol can one observe, as expected, surface activity. On the contrary, liposomes containing 5% of PEG are able to form monomolecular films both after spreading on aqueous surfaces and upon injection into the bulk of a water solution. The contribution of entrapped drug and coating peptide is not important in the range of concentrations under study. Microviscosity studies carried out through fluorescence polarization changes of DPH with temperature show that all the samples are very stable (high polarization values) in the range of temperatures under study. Nevertheless, the presence of PEG has, in general, a slightly fluidifying effect. There is a certain interaction between Doxorubicin and DPH in the sense that this drug reduces the fluorescence intensity of the marker, but it has no influence on the anisotropy values, thus allowing us to carry out the study without interferences or artifacts.

Introduction Liposomes have been extensively used as carriers for drug administration and targeting. Their “in vivo” halflife can be dramatically increased by coating its surface with poly(ethylene glycol) (PEG) chains.1,2 The mechanisms responsible for the recognition and clearance of liposomes from circulation “in vivo” are presently not well-understood. Nevertheless, it is clear that the presence of cholesterol and high Tc phospholipids has a beneficial effect as well as the above cited inclusion of PEG chains on the surface. Both saturated phospholipids and cholesterol have been shown to increase packing densities and reduce bilayer permeability, decreasing in this way the leakage of entrapped compounds.3,4 Moreover, the development of sterically stabilized liposomes by surface modification with PEG linked to phosphatidylethanolamine (PE), represents a new liposome generation.5,6 The role this hydrophilic polymer plays seems to be a reduction in plasma protein binding, which is the first step in the destabilization process of liposomes in blood, although some authors attribute this effect to steric stabilization rather than specific biological interactions.6 Nevertheless, it is logical to assume that not only the main components of the membrane but also the chemical characteristics of entrapped material and eventually the length and physicochemical properties of peptides/proteins linked to immunoliposomes will influence the overall stability both “in vitro” and “in vivo” of liposomal preparations. †

CID-CSIC. University of Barcelona. X Abstract published in Advance ACS Abstracts, June 15, 1997. ‡

(1) Uster, P. S.; Allen, T. M.; Daniel, B. E.; Mendez, C. J.; Newman, M. S.; Zhu, G. Z. FEBS Lett. 1996, 386, 243-246. (2) Gabizon, A.; Chemla, M.; Tzemach, D.; Horowitz, A. T.; Goren, D. J. Drug Targeting 1996, 3, 391-398. (3) Demel, R. A.; De Kruijff, B. Biochim. Biophys. Acta 1976, 457, 109-132. (4) Corvera, E.; Mouritsen, O. G.; Singer, M. A.; Zuckermann, M. J. Biochim. Biophys. Acta 1992, 1107, 261-270. (5) Torchilin, V. P.; Omelyanenko, V. G.; Papisov, I. M.; Bogdanov, A. A., Jr.; Trubetskoy, V. S.; Herron, J. N.; Gentry, C. A. Biochim. Biophys. Acta 1994, 1195, 11-20. (6) Lasic, D. D. Angew. Chem., Int. Ed. Engl. 1994, 33, 1685-1698.

S0743-7463(96)00930-4 CCC: $14.00

To this end, the present study was undertaken in order to compare the stability of four liposomal preparations covering standard compositions used in biological studies as follows: liposomes loaded with a cytostatic, liposomes loaded with a cytostatic and coated with PEG, liposomes loaded with a cytostatic and coated with PEG and peptide, and finally, liposomes coated with PEG and peptide. Doxorubicin (DX) was used as cytostatic, and to target liposomes to metastasic cells, a peptide fragment corresponding to the E(8) region of Laminin was synthesized and linked via NGPE to the surface of liposomes. Physicochemical studies were carried out using both monomolecular layer techniques and fluorescence anisotropy of diphenylhexatriene. In this way it was possible to determine the influence of the different components on the “in vitro” stability of vesicles. Experimental Section Chemicals. N-[(9-Fluorenylmethoxy)carbonyl] (Fmoc) amino acids and Wang resin were purchased from Novabiochem (Cambridge, England). N,N-Dimethylformamide (DMF) and piperidine were obtained from Scharlau and Aldrich, respectively. Trifluoroacetic acid (TFA), mercaptoethanol, anisol, diphenylhexatriene (DPH), 1-hydroxybenzotriazole (HOBT), N-hydroxysulfosuccinimide sodium salt, and N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) were supplied by Fluka. Phosphatidylcholine (PC) was a preparation of hydrogenated egg lecithin from Asahi. Phosphatidylglycerol (PG) and cholesterol (Ch) were from Sigma. Doxorubicin (DX) was obtained from Farmitalia. Distearoylphosphatidylethanolamine-PEG (DSPE-PEG) of 2000 Da molecular weight was from Shearwater Polymers Europe. N-Glutarylphosphatidylethanolamine (NGPE) was synthesized in our laboratory, following the description given by Weissig.7 Chemical characterization and quantitation were carried out by thin layer chromatography and phosphorus analysis following the Barlett method.8 (7) Weissig, V.; Lasch, J.; Klibanov, A. L.; Torchilin, V. P. FEBS Lett. 1986, 202, 86. (8) Barlett, G. R. J. J. Biol. Chem. 1959, 234, 466-468.

© 1997 American Chemical Society

3954 Langmuir, Vol. 13, No. 15, 1997 Solid Phase Synthesis. Peptide E(8) (CSRARKEAASIKVAVSA) was synthesized by solid phase methodology using the Fmoc/tBu strategy on a Wang type resin. Couplings were carried out with diisopropylcarbodiimide and hydroxybenzotriazol. After completion, peptide was cleaved from resin using mixture of 90% TFA and small percentages of anisol and mercaptoethanol used as scavengers. Crude peptide was purified by HPLC and characterized by amino acid analysis and mass spectrometry (electrospray). Details of synthesis are given elsewhere.9 Liposome Preparation. Stock solutions of the desired lipids (containing in all cases an amount of Ch in 50% molar ratio), in chloroform were dried to a film using a rotatory evaporator, and the residue was further submitted to a vacuum pump for at least 2 h. Lipids were hydrated with MES buffer (pH ) 5.4) or with the standard solution of DX (2 mg/mL) by incubation at 60 °C for 30 min, followed by sonication under nitrogen at a temperature above the Tc of the lipids. These samples were centrifuged to eliminate the remaining MLV and Ti particles. When necessary, the peptide was linked to the vesicle surface through the carboxyl moiety of NGPE in the presence of N-hydroxysulfosuccinimide sodium salt and N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide hydrochloride.10 The liposomal preparation was dialyzed against 0.9% sodium chloride for 24 h. The size of liposomal preparations was measured in a Malvern Autosizer 4700. Physicochemical Studies. Surface activity was determined using a Langmuir balance working with a cylindrical PTFE cuvette of 70 mL. Liposomal suspensions were injected into the bulk of the aqueous subphase (NaCl 0.9%), and surface pressure increases were detected by a platinum plate half-dipped and connected to an electrobalance. All these studies were carried out in triplicate. Compression isotherms were carried out by spreading liposomal suspensions on a subphase of sodium chloride 0.9% contained in a Teflon trough (330 mL) and surface pressure increases, on compression, recorded using a Langmuir balance, as described elsewhere.11 At least 10 min were allowed for liposome spreading and monolayer formation. Films were compressed continuously at a rate of 4.2 cm/min. Changes in the compressibility rate did not alter the shape of the isotherms. The output of the pressure pick up (Sartorious microbalance) was calibrated by recording the well-known isotherm of DPPC. All the compression processes were run at least three times in the direction of increasing pressure with freshly prepared films. Bilayer fluidity was studied in a Perkin-Elmer spectrofluorometer, LS50. Small vesicles prepared as described before were incubated with DPH and transferred to a thermostated cuvette holder in the spectrofluorometer. The polarization values were determined as a function of the temperature, working in the 25-60 °C interval. Details of all these procedures are given elsewhere.12 From these values, bilayer anisotropy was calculated by applying eq 1.

Polo et al. Table 1. Analytical Data of Peptide E(8)a amino acid analysis Ser 1.12 (3) Ile 1.34 (1) Glu 0.92 (1) Lys 2.19 (2) Ala 4.27 (5) Arg 2.38 (2) Val 1.91 (2)

HPLCa

ES-MS [M]+

) 1746.2

K′ ) 1.24

a Analytical reversed-phase HPLC on Kromasyl C column (10 8 mm), using a linear gradient (20 min) of 5-85% CH3CN/H2O (0.1% TFA) with detection at 220 nm.

Table 2. Chemical Composition of Liposomal Preparations PL (mg/mL) DX (mg/mL) E(8) (mg/mL) size (nm)

A

B

C

D

11.7 3.4

10.4 1.46

7.2 1 0.98 175

9

145

120

1.14 130

Peptide Synthesis. The overall yield of the synthesis (E(8): Cys-Ser-Arg-Ala-Arg-Lys-Glu-Ala-Ala-Ser-Ile-LysVal-Ala-Val-Ser-Ala) based on the first amino acid attached to the resin was 80%. The peptide was successfully cleaved in 3 h with a mixture of TFA-methylene chloride-mercaptoethanolanisol (67:3:2:1, v/v). Peptide E(8) was characterized as shown in Table 1 by analytical HPLC, amino acid analysis, and mass spectrometry (electrospray). Liposome Preparation. Liposomes were prepared according to standard procedures given in the literature.13

Final preparations were characterized by size and phospholipid, peptide, and Doxorubicin quantification. Vesicle compositions were (A) PC/PG/Ch/DX, (B) PC/PG/Ch/DX/ PE-PEG, (C) PC/PG/Ch/DX/PE-PEG/NGPE/E(8), and (D) PC/PG/Ch/PE-PEG/NGPE/E(8). Analytical data are summarized in Table 2. Surface Activity. The surface activity of the liposomal preparations as well as those of Doxorubicin and E(8) peptide were determined by measuring surface pressure increases after injecting different volumes of the corresponding solutions/suspensions. Both DX and E(8) gave no activity in the range of concentrations present in liposomes, so that its direct contribution can be discarded. As a common trend, liposomes when injected in an aqueous subphase showed surface activity in a concentration dependent way, with important induction times.14 It is seen that the approach of surface pressure increase (∆π) toward the steady state follows a sigmoidal curve. This delay in the beginning of the increase of surface pressure was dependent on the amount of phospholipid, decreasing as this value increases. This behavior has also been found with hydrophobic peptides and is attributed to the time required for ordered aggregates, liposomes or micelles, to reach the surface and disintegrate, giving monomolecular layers.15 In Figure 1a-c, surface pressure increases versus time are represented for liposomal preparations B, C, and D. Surprisingly, A liposomes gave a very low activity in the same range of phospholipid concentrations assayed for B, C, and D samples. It is not clear why this set of liposomes showed almost no surface activity. Assuming that this pressure increase is due to the adsorption of surface active molecules on the surface, the most plausible explanation is that this vesicle composition is more stable and requires more energy and extended times for the incorporation of lipid molecules to the air/water interface. In our hands, 1 h after injection only small changes in surface pressure could be detected, while other liposomal compositions had already reached saturation. But if A liposomes were previously diluted with 50% methanol, this sample showed surface activity as the rest of liposomal compositions, thus confirming the high stability of this preparation. Besides, the same treatment gave no differences in maxima pressures achieved for B, C, and D liposomes, thus indicating a total incorporation of phospholipids to the interface since the beginning.

(9) Polo, D.; Haro, I.; Reig, F. Manuscript in preparation. (10) Mori, A.; Huang, L. In Liposome Technology, 2nd ed.; CRC Press: Boca Raton, FL, 1994; Vol. 3, pp 153-162. (11) Verger, R.; Da Haas, G. H. Chem. Phys. Lipids 1973, 10, 127. (12) Cajal, Y.; Rabanal, F.; Alsina, M. A.; Reig, F. Biopolymers 1996, 38 (5), 607-618.

(13) Nagayasu, A.; Shimooka, T.; Kinouchi, Y.; Uchiyama, K.; Takeichi, Y.; Kiwada, H. Biol. Pharm. Bull. 1994, 17, (7), 935-939. (14) Garcı´a, M.; Pujol, M.; Reig, F.; Alsina, M. A.; Haro, I. Analyst 1996, 121, 1583-1588. (15) Alsina, M. A.; Sole´, N.; Mestres, C.; Busquets, M. A.; Haro, I.; Garcı´a-Anto´n, J. M. Int. J. Pharm. 1991, 70, 111-117.

r ) 2P/3 - P

Results

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Figure 2. Compression isotherm of liposomes A spread on air/water interfaces after a previous disruption with 50% methanol.

Figure 1. Time course of pressure increases for several liposomal concentrations in the subphase: (a) vesicles type B; (b) vesicles type C; (c) vesicles type D. Inset Figure 1a: Surface pressure concentration dependence measured after 1 h of liposome injection.

When chemical compositions of these samples are compared, all of them have PEG as a common component, except A; thus it seems logical to assume that this component is responsible for this lower stability of liposomes. The physicochemical properties of pure PEG as well as PEG-lipid derivatives have been studied in detail.16,17 It is clear that these polymeric molecules are able to spread at an air/water interface and form stable monolayers. Moreover, when the molecules are mixed with phospholipids, the shape and slope of compression isotherms suggest the lack of a real miscibility at low lateral pressures, especially at PEG contents above 10%.18 Results obtained in the adsorption kinetics of different liposomal compositions here described can be attributed to the high spreadability of PEG molecules. The energy involved in the process can be supplied according to the literture17 by the increment in entropy associated with the transfer of a molecule from an ordered aggregate to a monomeric form, and the increase in motion of the polymer chain linked to the bilayer compared to its freedom in solution. According to the commonly accepted conformations of PEG chains (pancake, mushroom, brush), in our system (16) Winterhalter, M.; Bu¨rner, S.; Marzinka, S.; Kasianowicz. Biophys. J. 1995, 69, 1372-1381. (17) Silvius, J. R.; Zuckermann, M. J. Biochemistry 1993, 32, 31533161. (18) Baekmark, T. R.; Elender, G.; Lasic, D. D.; Sackmann, E. Langmuir 1995, 11 (10), 3975-3987.

due to the low PEG content, it seems more likely that PEG chains located at the external surface of liposomes should be in an extended (pancake) conformation. This state allows the chains to move freely in the bulk of the aqueous phase and spread at the air/water interface. Probably, the decrease of energy involved in the higher degree of disorder will help the disorganization of the bilayers. Comparing the maximum values of ∆π/c calculated for B, C, and D preparations after 60 min, one can appreciate that all of them are in the same range: 0.68, 0.86, and 0.67, respectively. Although differences are very small, one can speculate that the higher activity shown by C liposomes can be due to the contribution of Doxorubicin and peptide present in this composition, in spite of the fact that these two components do not show surface activity by themselves in this range of concentrations. In some cases, the presence of a monolayer spread at the air/water interface can have an enhancing effect on the surface activity of some mildly active molecules.15 For this reason the above described experiments were also carried out by injecting the liposomal solutions under a DPPC monolayer spread at 5 mN‚m-1 of surface pressure. In these experiments surface pressures achieved by A, B, and D liposomes were 7.2 and that of C liposomes was 9.6 mN‚m-1. Here we can appreciate a common behavior for all the preparations, C samples showing a slightly higher activity, as was already described before. Nevertheless, the kinetics of the process was slowest for A composition. It seems that the A composition renders more stable liposomes that need some source of extra energy to disperse its lipid components on the surface. Compression Isotherms. Monolayers were prepared from liposomal solutions A, B, C, and D by spreading fixed volumes on an aqueous sodium chloride subphase at the same pH of liposomal preparations. Compressions were carried out at 4.2 cm/min, and pressure increases were recorded until collapse or disruption of the monolayer. The area/molecule was calculated from initial lipid concentrations and the area avaliable at different compression times. A liposomes gave no isotherm on compression no matter the amount of lipids spread, thus indicating that vesicles dilute in the bulk of solution or remain as such on the surface instead of fusing the bilayers at the water/air interface. In order to check this assumption, A liposomes were previously broken with 50% methanol and solutions spread as usual. In this case, on compression, pressure increased, as was to be expected for a stable monolayer (Figure 2). The maximum pressure

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achieved was 48 mN‚m-1 without reaching collapse. The area/molecule at this pressure was 1.2 nm2/molecule. No further compression was possible due to the geometry of the apparatus, but the lack of collapse and the type of monolayer liquid-condensed as well as the high area/ molecule suggest that compression could still continue to lower areas and higher surface pressures. Liposomes B, C, and D gave, after spreading, compression isotherms similar in shape and slope. Two phase changes can be clearly appreciated. The first one, corresponding to the gas versus liquid-expanded state, located between 11 and 14 mN‚m-1 and a second one due to the transition to liquid condensed. As A liposomes, devoid of PEG, gave no changes all over their compression, it seems likely that these discontinuities in the slope of the isotherms should be attributed to the presence of this polymer. According to the data given in the literature,18 the pancake to mushroom transition for PEG chains in mixtures with phospholipids at similar PEG concentrations and lengths are located around 7.4 mN‚m-1 of surface pressure and 1.25 nm2/molecule. Although the presence of cholesterol in our samples clearly reduces the area/ molecule, the values determined for the first transition, 14-11.5 mN‚m-1 and 0.75-1 nm2/molecule, still remain of the same order of magnitude as those mentioned above. The second transition located around 21-17 mN‚m-1 could be attributed to the transition mushroom-brush conformation. In this last state PEG heads consist of chains of lightly packed blobs with diameters slightly larger than the section area ocuppied by two alkyl chains in a phospholipid molecule. In our system, the area/molecule at this second transition is around 0.6 nm2/molecule, lower than that described for pure DSPE-PEG (MW 2000) monolayers, but there is good agreement with the corresponding values of DMPE/5% DSPE-PEG mixtures, as indicated in ref 18. Maximum compression pressures varied according to chemical composition, B liposomes being the most stable monolayers (maximum surface 78 mN‚m-1 with collapse). Up to 15 mN‚m-1 monolayers were almost in the solid state; this could be due to the rigidifying and condensing effect of cholesterol, which is a common component in all of them19 (Figure 3a-c). Although monolayers were shown to be stable and the compression process very reproducible, the same experiment was repeated with samples previously treated with 50% methanol. The new set of isotherms obtained was similar in shape and area/molecule values to those previously obtained, confirming in this way the complete incorporation of liposome lipids to the air/water interface. In all cases, the condensing effect of cholesterol was clearly evident in the area/molecule values. Small differences in area/molecule values among the three monolayer compositions indicated that at low surface pressures liposomes C gave more expanded monolayers than those of types B and D. This soft expansion of monolayers can be attributed to the presence of small amounts of both peptide and Doxorubicin. Microviscosity Studies. Membrane fluidity of phospholipid vesicles was determined using the membrane interior probe DPH. These types of experiments have been carried out usually with pure phospholipids or at least liposomes with two components; for this reason, first of all, the possible interference of Doxorubicin or E(8) peptide with DPH fluorescence, either by transference of energy or by quenching effects was studied. To this end, (19) Zerouga, M.; Jenski, L. J.; Stillwell, W. Biochim. Biophys. Acta 1995, 1236, 266-2726.

Polo et al.

a

b

c

Figure 3. Compression isotherms of liposomes spread on air/ water interfaces: (a) liposomes B; (b) liposomes C; (c) liposomes D.

standard liposomes prepared with DPPC were saturated with DPH and incubated with different amounts of DX or E(8). The results indicate that Doxorubicin increases very softly the polarization of liposomes all over the range of temperatures studied (23-50 °C) but has no influence on the transition temperature. Moreover, the maximum wavelength is not affected by this drug, but the fluorescence decreases in a concentration dependent way (Figure 4a,b). As λexcitation of Doxorubicin is far from λemission of DPH it seems that fluorescence decreases must be most likely due to a quenching effect. Peptide E(8) has influence in none of these parameters. From these previous results, it seems that Doxorubicin is able to interact with bilayers by inducing a soft

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a

b

Figure 5. Fluorescence intensity (a) and fluorescence anisotropy (b) of (9) A, (0) B, (2) C, and (4) D liposomes saturated with DPH recorded as a function of temperature. Figure 4. Fluorescence polarization (a) and fluorescence intensity (b) of DPPC liposomes saturated with DPH versus temperature and Doxorubicin concentration, respectively.

rigidification of them without changing the hydrophobicity of DPH molecules environment. It will be possible thus to apply this technique to our samples without expecting artifacts. Fluorescence polarization studies were carried out at different temperatures and the parameters measured were maximum wavelength, fluorescence intensity, and polarization (Figure 5a,b). Maximum fluorescence was obtained with sample D in such a way that it was out of range. Only when reducing the slit width was it possible to get values lower than 1000. Nevertheless, as the rest of the measurements were made with different slits in Figure 5a component D is not included. Moreover, as can be expected, fluorescence decreased with increasing temperature. Looking for some explanation for this strong fluorescence associated with liposomes D, one can appreciate that this is the only sample that does not contain Doxorubicin; this is in agreement with the results described above in the sense that the presence of this drug has a quenching effect on DPH quantum yield. Concerning anisotropy, the data displayed in Figure 5b show that the influence of temperature is very weak. This stability can be attributed to the well-known rigidifying and stabilizing effects of cholesterol. Moreover, as we observed previously, the presence of Doxorubicin has a rigidifying effect, as can be observed by the lowest values of sample D, the only one that does not contain this drug in its composition. Discussion The stability of liposomes can be studied in different ways and by applying a great diversity of techniques;

among them, leakage of entrapped molecules and packing of bilayers are the most interesting from a practical point of view. In our system, the first one was not significant due to the high affinity of Doxorubicin for phosphatidylglycerol, one of the components of the bilayers. Nevertheless, the influence of PEG chains and peptide on the overall characteristics of the vesicle was a key point in the potential uses of our formulation. Monolayer techniques are a suitable and simple approach to determine the maintenance of bilayer integrity when exposed to the air/water interface, while anisotropy studies give information about the degrees of packing of bilayers. The results we have already described indicate that the presence of PEG chains on the external surface of liposomes increase its spreadability and ability to incorporate into the air/water interface, this fact being indicative of a certain degree of instability. To have a reference with conventional liposomes, the same property was studied for egg PC and hydrogenated egg PC liposomes either with or without cholesterol (1:1) (data not shown). One could observe a gradation between unsaturated liposomes and saturated ones as well as the beneficial effects of cholesterol, liposomes composed of natural phosphatidylcholine being those with the highest and fastest spreading capacity, as expected. These values are in agreement with the well-known lower stability of unsaturated liposomes, and are thus indicative that the technique was adequate to compare the stability of liposomal samples and that, according to the kinetics of the processes, the effect of PEG was soft. The same conclusion was achieved from the behavior observed with compression isotherms. No differences in shape and slope between liposomal samples added as such or previously treated with methanol were observed. This is indicative that although these

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vesicles are stable in size and retention of Doxorubicin (data not shown), the energy required to pass from bilayer to monolayers is supplied by interaction of hydrophilic PEG chains with water molecules when they are spread on the water/air interface. Anisotropy studies were also carried out with the standard curve of DPPC for reference. In this case, differences among samples were higher than before but, as a general trend, the microviscosity of bilayers is high and, what is more important, its dependence temperature was clearly shown in Figure 5b. As a summary, the presence of PEG in a liposomal composition increases the spreading ability of these preparations but at the same time improves the packing characteristics of bilayers, thus increasing the microviscosity, as can be deduced from higher anisotropy values

Polo et al.

as obtained in comparison with DPPC liposomes. These results are in agreement with the idea that the stabilizing effect of PEG chains is mainly due to a reduction of opsonin binding to the surface of liposomes rather than to the increase of bilayer packing. Acknowledgment. We gratefully acknowledge Mrs. Maria Osuna for excellent technical assistance in peptide synthesis and Mrs. Helena Carvajal and Mr. David Mur in polarization studies. This work was supported by a research grant SAF93-0063 from CICYT, Spain. D. Polo was a recipient of a grant from the Ministerio de Ciencia (Spain). LA960930P