Temperature-Dependent Associating Property of Liposomes Modified

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Bioconjugate Chem. 1998, 9, 382−389

Temperature-Dependent Associating Property of Liposomes Modified with a Thermosensitive Polymer Hiroshi Hayashi,† Kenji Kono,*,‡ and Toru Takagishi‡ Shiga Central Research Laboratories, Noevir, Company, Ltd., Yokaichi, Shiga 527, Japan, and Department of Applied Materials Science, College of Engineering, and Department of Applied Bioscience, Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, Osaka 593, Japan. Received August 6, 1997; Revised Manuscript Received December 1, 1997

Novel temperature-sensitive liposomes having surface properties that can be controlled by temperature were designed as liposomes coated with poly(N-isopropylacrylamide), which exhibits a hydrated coil to dehydrated globule transition at ca. 32 °C. To obtain the polymer with anchoring groups to the liposome, a copolymer of N-isopropylacrylamide and octadecyl acrylate (99:1, mol/mol) was synthesized by radical copolymerization. The copolymer revealed the transition near 30 °C. Liposomes made from various phospholipids were prepared by sonication and coated with the copolymer. When dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine were used as liposome lipids, remarkable aggregation and fusion of the copolymer-modified liposomes took place between the transition temperature of the copolymer and the gel-liquid-crystalline transition temperature (Tc) of the lipid membranes. However, above the Tc, association between the liposomes was much less significant, although the copolymer is still hydrophobic. In the case of the copolymer-modified dilauroylphosphatidylcholine liposome, the membrane of which takes on a liquid-crystalline state under the experimental conditions, association between the liposomes also hardly occurred even when the copolymer became hydrophobic. On the other hand, below the transition temperature of the copolymer, the copolymer-modified liposomes were very stable and aggregation of the liposomes was not observed, irrespective of membrane lipid. Results obtained in this study demonstrate that the copolymer chains fixed on the surface of the liposome with a solid membrane promote aggregation and fusion of the liposomes by hydrophobic interactions between the copolymer chains and/or between the copolymer chains and the liposome membranes above the transition temperature of the copolymer.

INTRODUCTION

Since liposomes made from naturally occurring lipids are biocompatible, their application to drug delivery systems has been extensively attempted (1). To elevate their usefulness, a number of liposomes with various functionalities have been designed. For example, with respect to site specific delivery of drugs, liposomes that release their contents in response to temperature (2, 3), pH (4-6), and light (7-9) have been reported. Also, for cytoplasmic delivery of membrane-impermeable molecules, fusogenic liposomes have been developed (10-14). These liposomes are considered to fuse with the plasma membrane or the endosomal membrane and release their contents into the cytoplasm (14-16). Therefore, fusogenic liposomes are of importance as a carrier of oligonucleotides, nucleic acids, and proteins. While several approaches to the production of liposomes with functionalities have been attempted, one of the most effective methods is their modification with polymers (17, 18). It was shown that coating of liposomes with polysaccharides improves the stability of the liposomes (19). Also, conjugation of synthetic polymers with * Address correspondence to this author at the Department of Applied Materials Science, College of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 593, Japan (telephone +81-722-52-1161; fax +81-722-59-3340; e-mail [email protected]). † Noevir Co. Ltd. ‡ Osaka Prefecture University.

pH sensitivity onto liposomes was shown to provide them with pH-sensitive release and fusion properties (5, 13). Recently, to obtain liposomes with temperature sensitivity, we (3, 20) and other groups (21-23) designed liposomes modified with poly(N-isopropylacrylamide) [poly(NIPAM)].1 It is well-known that poly(NIPAM) is soluble in water and takes on a hydrated coil state below ca. 32 °C, whereas the polymer is insoluble in water and exhibits a dehydrated globule state above this temperature (24). Already, we have demonstrated that release of contents from the poly(NIPAM)-coated liposomes is enhanced above the coil-globule transition temperature of the polymer (3, 20). So far, we have focused on the temperature-sensitive release property of the poly(NIPAM)-coated liposomes. However, fixation of the temperature-sensitive polymer onto the liposome surface is also considered to render the nature of the liposome surface temperature-sensitive. The highly hydrated polymer chains will cover the liposome surface below the transition temperature of the polymer. However, above the polymer transition temperature, the polymer chains become hydrophobic and will adsorb onto 1 Abbreviations: poly(NIPAM), poly(N-isopropylacrylamide); copoly(NIPAM-ODS), copolymer of N-isopropylacrylamide and octadecyl acrylate; DPPC, L-R-dipalmitoylphosphatidylcholine; DLPC, L-R-dilauroylphosphatidylcholine; DSPC, L-R-distearoylphosphatidylcholine; NBD-PE, N-(7-nitrobenz-2-oxa-1,3diazol-4-yl)phosphatidylethanolamine; Rh-PE, N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine; AIBN, azobis(isobutyronitrile).

S1043-1802(97)00145-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 03/31/1998

Temperature-Sensitive Association of Liposomes

the liposome surface, making the surface hydrophobic. Thus, it is expected that hydrophilicity or hydrophobicity of the liposome surface can be controlled by the ambient temperature. Also, interaction of dehydrated polymer chains with the liposome membrane might reduce the integrity of the membrane structure above the transition temperature of the polymer, which may increase the fusion ability of the liposome. To examine the influence of surface modification of liposomes with the temperature-sensitive polymer on the nature of the liposomes, we have synthesized a copolymer of NIPAM and octadecylacrylate [copoly(NIPAM-ODA)] as poly(NIPAM) having anchors to liposomal membranes and prepared liposomes coated with the copolymer. Temperature-dependent aggregation and fusion of the copolymer-attached liposomes have been reported. EXPERIMENTAL PROCEDURES

Materials. L-R-Dipalmitoylphosphatidylcholine (DPPC), L-R-dilauroylphosphatidylcholine (DLPC), and L-R-distearoylphosphatidylcholine (DSPC) were purchased from Sigma. N-(7-Nitrobenz-2-oxa-1,3-diazol-4yl)phosphatidylethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE) were obtained from Avanti Polar Lipids. NIsopropylacrylamide (NIPAM), octadecylacrylate (ODA), and azobis(isobutyronitrile) (AIBN) were from Tokyo Kasei. NIPAM was purified by recrystallization from cyclohexane/n-hexane (1:1) before use. Synthesis of Copoly(NIPAM-ODA). Copoly(NIPAM-ODA) was synthesized as reported previously (3). In brief, NIPAM (9.9 mmol), ODA (0.1 mmol), and AIBN (2.4 × 10-2 mmol) were dissolved in tetrahydrofuran. The solution was degassed with N2 and kept at 60 °C for 4 h. The copolymer was recovered by precipitation with diethyl ether. The copolymer was dissolved in tetrahydrofuran and reprecipitated with diethyl ether. The copolymer was dissolved in water and lyophilized. Preparation of Liposomes. Liposomes were prepared as follows: a dry thin membrane of each of the phosphatidylcholines (10 mg) was dispersed in 1.0 mL of 10 mM Tris-HCl-buffered solution of pH 7.4 containing 140 mM NaCl. The lipid dispersion was sonicated for 10 min using a probe-type ultrasonic disruptor (Tomy Seiko, UR-20P). To the liposome suspension (1 mL) was added 0.5 mL of an aqueous solution in which 1 or 5 mg of the copolymer was dissolved, and the mixture was then incubated for 4 h at 5 °C with stirring. Free copolymer was removed by gel permeation chromatography on a Sephacryl S-400 column at 10 °C. Estimation of the Amount of Copolymer Bound onto Liposome. The amount of the copolymer bound onto the liposome was estimated by high-performance liquid chromatography (HPLC) analysis on an Asahipak GF-310HQ column using methanol as an effluent, as reported previously (20). The copolymer-coated liposome was dried under reduced pressure and then dissolved in methanol. The solution (20 µL) was injected into the column, and the effluent was monitored by absorbance at 220 nm. For calibration, the copolymer solutions of given concentrations were also injected. From the absorbance of the copolymer separated, the amount of copolymer adsorbed onto the liposome was determined using the calibration curve. Size Change of Liposomes. Liposomes were prepared as mentioned above except that contamination of large multilamellar liposomes was eliminated by cen-

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trifugation (18 000 rpm, 40 min) before incubation with the copolymer. The liposomes were suspended in 10 mM Tris-HCl and 140 mM NaCl solution (0.1 mg of lipid/mL) at pH 7.4 at desired temperatures for 3 min. Mean diameter of the liposome was measured immediately after the incubation or after an additional 10 min of incubation at 20 °C using a laser particle analyzing system (Otsuka Electronics, LPA 3100S). Fusion of Liposomes. Fusion of liposomes was detected as reported previously (13, 25) by measuring resonance energy transfer between NBD-PE and Rh-PE as reported by Struck et al. (26). Liposomes containing 0.3 mol % of NBD-PE and Rh-PE were prepared according to the above method. The liposomes containing the fluorescent lipids were mixed in a 1:1 mole ratio with probe-free liposomes. The suspension (0.1 mg of lipid/ mL) was excited at 475 nm, and fluorescence emission spectra were measured at various time intervals. The R value, the ratio of fluorescence intensity at 532 nm to that at 591 nm, was calculated. The ratio was converted into apparent concentration of the fluorophores in the membrane using a standard curve of the reciprocal of the concentration of the fluorophores vs R value. The percent fusion was defined as

% fusion ) (C0 - Ct)/(C0 - Cf) where C0 and Ct are the initial and intermediate concentrations of the fluorophores, respectively. Cf, which equals 0.15 mol % of each fluorophore, represents the concentration at which complete fusion of the liposomes occurs. Electron Microscopy. The copolymer-modified and the bare DPPC liposomes were suspended in 10 mM TrisHCl and 140 mM NaCl solution (pH 7.4). The liposome samples were heated at 39 °C for 3 min and returned to room temperature (ca. 20 °C). A small drop of the liposome sample was placed on collodion-coated grids and drawn off with filter paper. A drop of 2% (w/v) phosphotungstic acid was applied to the grid, drawn off with filter paper, and then allowed to dry. The grids were viewed under an electron microscope (JEOL, JEM-2000FEX II). Other Methods. Nuclear magnetic resonance (NMR) spectra were taken with a JEOL JNM-GX 270 MHz instrument. Differential scanning calorimetry (DSC) was performed with a Seiko Electronics DSC 120 microcalorimeter. The samples were analyzed at a heating rate of 0.5 °C/min. Phospholipid concentration was measured by an assay using Phospholipids B-Test Wako purchased from Wako Pure Chemical Industries. The molecular weight of the copolymer was estimated by HPLC analysis on a Shodex KD-803 column (Showa Denko) using N,N-dimethylformamide. RESULTS AND DISCUSSION

Characterization of Copoly(NIPAM-ODA). To obtain poly(NIPAM) with anchors to liposomal membranes, we synthesized the copolymer of NIPAM and ODA, because a number of studies have shown that hydrophilic polymers with a few hydrophobic groups are fixed onto the liposome membranes through hydrophobic interactions (13, 17, 27). We have previously demonstrated that the NIPAM-ODA copolymer binds onto DPPC and egg yolk phosphatidylcholine liposomes (3). The copolymer used here was evaluated to contain NIPAM and ODA units in the ratio of 99:1 (mol/mol) by 1H NMR from the ratio of isopropyl groups in the NIPAM unit (δ 1.2 ppm) to n-alkyl groups in the ODA unit (δ 1.3

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Figure 1. Microcalorimetric endotherms for aqueous copolymer samples in the absence (A) and in the presence (B) of egg yolk phosphatidylcholine liposome: (A) copolymer solution (0.4 wt %); (B) copolymer solution containing egg yolk phosphatidylcholine liposome (0.4 wt %). The samples (70 µL) were heated at 0.5 °C/min. ∆H for curves (A) and (B) are 4.2 and 4.1 cal/g of the copolymer, respectively.

ppm). The weight-average molecular weight, the numberaverage molecular weight, and the heterogeneity of the copolymer were determined by gel permeation chromatography to be 12200, 5600, and 2.2, respectively. It was shown by Schild and Tirrell (28) that the coilglobule transition of poly(NIPAM) can be detected by DSC. Therefore, a DSC measurement was performed to estimate the coil-globule transition temperature of the copolymer (Figure 1). DSC curves of the copolymer in water and in the presence of egg yolk phosphatidylcholine liposomes displayed endotherms with a peak centered at 29.4 °C and with a peak centered at 29.7 °C, respectively. These values are somewhat lower than the transition temperature of homopolymer of NIPAM, possibly due to the existence of ODA units in the copolymer. It has been previously reported that incorporation of hydrophobic groups into poly(NIPAM) lowers the transition temperature of poly(NIPAM) in water (29). Fixation of Copoly(NIPAM-ODA) onto Liposomes. In this study, DPPC, DSPC, and DLPC were used as liposomal lipids because membranes of these lipids exhibit the gel-liquid-crystalline phase transition at different temperatures. Liposomes were prepared by sonication and coated with copoly(NIPAM-ODA) by incubation of the liposomes with the copolymer solutions and subsequent gel filtration on a Sephacryl S-400 column. Typical examples of the elution profiles for a DPPC liposome dispersion (A), for the copolymer solution (B), and for DPPC liposomes suspended in the copolymer solution (C) are shown in Figure 2. As is apparent in Figure 2A,B, the liposomes passed through the column more rapidly than the copolymer. Therefore, free copolymer can be removed from the copolymer-attached liposomes by using this column. We collected the effluent of the elution band shown in Figure 2C and used it for experiments. The amount of the copolymer attached onto the liposome after the gel filtration was quantified by HPLC analysis on an Asahipak column for gel permeation chromatography using methanol as the eluent. Figure 3 shows typical examples of the chromatograms for the copolymer (A), DPPC (B), and copolymer-fixed DPPC liposomes (C). When an aliquot of the copolymer solution was injected into the column, two peaks (7.8 and 13.7

Hayashi et al.

Figure 2. Typical elution profiles through a Sephacryl S-400 column for DPPC liposomes (6.7 mg/mL) (A), copolymer (3.3 mg/ mL) (B), and DPPC liposomes incubated in copolymer solution (lipid ) 6.7 mg/mL; copolymer ) 3.3 mg/mL). Optical density at 220 nm was monitored. The samples (0.5 mL) were loaded onto the column. Flow rate of the effluent was 1 mL/min.

Figure 3. Typical elution profiles through an Asahipak column for copolymer (0.1 mg/mL) (A), DPPC (0.7 mg/mL) (B), and copolymer-fixed DPPC liposome (0.3 mg DPPC/mL) (C). Sample solutions (20 µL) were injected into the column and eluted with methanol at 0.8 mL/min.

mL) were observed in the chromatogram as shown in Figure 3A. The first and second peaks are attributable to the copolymer and solvent, respectively. It was confirmed that area of the first peak is proportional to the concentration of the copolymer solution under the experimental conditions. When an aliquot of the lipid

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Table 1. Preparation of Copoly(NIPAM-ODA)-Modified Liposomes

liposome

copoly(NIPAMODA) added (mg/mg of lipid)

copoly(NIPAMODA) bound (mg/mg of lipd)

NIPAM unit lipid (mol/mol)

DPPC 1 DPPC 2 DLPC DSPC

0.1 0.5 0.5 0.5

0.06 0.16 0.20 0.15

0.38 1.01 1.07 1.02

solution was applied into the column, a new peak at 11.4 mL was seen in addition to the solvent peak (Figure 3B). As shown in Figure 3C, the chromatogram for the copolymer-fixed liposomes exhibited three peaks near 7.8, 11.4, and 13.7 mL, corresponding to the copolymer, lipids, and solvent, respectively. The amount of copolymer fixed on the liposomes was evaluated from the chromatograms using a calibration curve that shows the relationship between the area of the copolymer peak and the amount of copolymer. When DPPC liposomes were incubated with the copolymer in the ratio 0.1:1 (copolymer/lipid, w/w), 0.06 mg of the copolymer was adsorbed onto 1 mg of the liposomal lipid. By increasing the copolymer/lipid ratio to 0.5:1 (w/ w) during the incubation, the amount of copolymer adsorbed onto the DPPC liposomes increased to 0.16 mg/ mg of lipid. The amounts of copolymer adsorbed on various liposomes are listed in Table 1. When these liposomes are incubated with the copolymer in the ratio 0.5:1 (copolymer/lipid, w/w), the numbers of NIPAM units adsorbed per lipid molecule for DLPC, DPPC, and DSPC liposomes are 1.07, 1.01, and 1.02, respectively, indicating that density of the copolymer on the liposome surface is nearly identical among these liposomes, despite the difference in their membrane fluidities. Temperature-Dependent Association of Copoly(NIPAM-ODA)-Fixed Liposomes. To investigate the effect of temperature on association of the copolymermodified liposomes, we first examined size change of liposomes depending on temperature. Liposomes were kept at various temperatures for 3 min, and then the temperature was changed to 20 °C. After 10 min of standing at 20 °C, the diameter of the liposomes was measured. The result is shown in Figure 4. The sizes of the bare DPPC and the bare DSPC liposomes hardly change throughout the range of temperatures we investigated. Also, the copolymer-modified DPPC and the copolymer-modified DSPC liposomes do not change their mean diameters below 30 °C, at which the copolymer bound on the liposomes is hydrophilic. However, above this temperature when the copolymer is dehydrated and becomes hydrophobic, the size of these liposomes increases greatly. For the copolymer-modified DPPC liposome, a substantial increase in diameter was observed around 39 °C. This temperature appears to be slightly lower than the gel-liquid-crystalline phase transition temperature of DPPC membranes (ca. 41-42 °C) (30). However, it is known that DPPC liposomes made by sonication exhibit the phase transition in a temperature region lower than 41 °C because the membranes of the sonicated liposomes contain packing inhomogeneities caused by their high curvature (31). Thus, it is considered that aggregation of the copolymer-fixed DPPC liposomes is induced most intensively at their phase transition temperature. At the phase transition temperature, the gel and liquid-crystalline phases coexist in the membrane, and hence the membrane contains structural defects. Interaction of the copolymer with the liposome membrane should promote further generation

Figure 4. Mean diameter of various liposomes: (b) copolymermodified DPPC liposome; (O) bare DPPC liposome; (9) copolymermodified DSPC liposome; (0) bare DSPC liposome. The liposomes were kept at various temperatures for 3 min and followed by incubation at 20 °C for 10 min, and then diameters of the liposomes were measured at 20 °C. The copolymer-modified DPPC liposome 2 was used (see Table 1).

of defects in the membrane and destabilize the liposome. In addition to the hydrophobic interactions between the copolymers fixed on the liposomes, instability of the defective liposomes might enhance their aggregation, resulting in a significant increase in vesicle size. In contrast, the increase of the vesicle size was much less significant above 42 °C. It is well-known that liposomes are more stable above the phase transition temperature than below it, where fusion of small vesicles takes place more readily (32). On the other hand, since the copolymers fixed on the liposome are laterally mobile on the membrane surface above the gel-liquid-crystalline phase transition temperature, they will form aggregates through hydrophobic interactions within the membrane. In addition to the stability of liposomes with a fluid membrane, the difference in mobility of the copolymer on the membrane may also affect the potential of the liposome for association, as mentioned below. A similar trend can be seen in the case of the copolymermodified DSPC liposome, except that the most intensive increase in vesicle size occurred near 55 °C, which corresponds to the phase transition temperature of DSPC membrane. These results clearly demonstrate that the size increases of copolymer-modified liposomes are influenced by the fluidity of the liposome membrane as well as the hydrophobicity of the copolymer. Because the copolymer alters its nature between hydrophobic and hydrophilic states reversibly, responding to the ambient temperature, it is expected that association and dissociation of the copolymer-modified liposomes occur reversibly. Therefore, the reversibility of this process for the copolymer-modified DPPC liposomes was examined. We measured the size of the liposomes immediately after a 3 min incubation at various temperatures, as well as after an additional incubation for 10 min at 20 °C. The results are shown in Figure 5. While in both cases the diameter of the liposome increases significantly near 40 °C, a much greater diameter was observed for the liposomes without additional incubation at 20 °C. This result indicates that liposomes which are associated through hydrophobic

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Figure 5. Mean diameter of the copolymer-modified DPPC liposomes. Diameters of the liposomes were measured immediately after 3 min of incubation at various temperatures (b) or after an additional 10 min of incubation at 20 °C (9). The copolymer-modified DPPC liposome 1 was used (see Table 1).

Figure 6. Time courses of fusion of the copolymer-modified DPPC liposomes in 10 mM Tris-HCl-buffered solution containing 140 mM NaCl at various temperatures: (2) 20 °C; (1) 30 °C; (b) 39 °C; (9) 45 °C. The copolymer-modified DPPC liposome 1 was used.

interactions between the copolymers can be dissociated when the ambient temperature is lowered below the transition temperature of the copolymer. However, the liposome does not return to its original size, ca. 60 nm, during the 10 min cooling at 20 °C, suggesting that irreversible association of the liposomes also occurs at the same time. Temperature-Dependent Fusion of Copoly(NIPAM-ODA)-Fixed Liposomes. As mentioned above, irreversible association between the copolymermodified liposomes was observed. As such association, fusion of the liposomes is most likely. Thus, we examined the occurrence of fusion by the resonance energy transfer method (26). Figure 6 shows time courses of fusion of the copolymermodified DPPC liposomes at various temperatures. Apparently, fusion of the liposomes depends on temperature. The liposomes hardly fuse at 20 °C, whereas intensive fusion takes place at 39 °C, at which a significant increase in vesicle size occurs (Figure 4). Fusion of the liposomes is quite fast and is complete within 1 min.

Hayashi et al.

Figure 7. Percent fusion of various liposomes after 5 min of incubation as a function of temperature: (b) copolymer-modified DPPC liposome 1; (9) copolymer-modified DPPC liposome 2; (O) bare DPPC liposome; (2) copolymer-modified DLPC liposome; (4) bare DLPC liposome.

The temperature dependence of fusion of various liposomes is shown in Figure 7. For the copolymermodified DPPC liposomes, the liposomes hardly fuse below 30 °C, whereas above this temperature fusion occurs remarkably. Maximum fusion is seen near 39 °C, followed by a gradual decrease at temperature above 41 °C. This temperature dependence of fusion is similar to that of vesicle size shown in Figure 4. Because the bare DPPC liposomes exhibit a low percent of fusion throughout the experimental temperatures, this fusion property of the copolymer-modified liposomes is apparently controlled by the copolymer. Since the liposome membrane contains structural defects near the gel-liquid-crystalline phase transition temperature, the liposomes will be strongly destabilized by interactions with the hydrophobic copolymer. Moreover, the copolymers fixed on the liposomes facilitate aggregation of the liposomes by their hydrophobic interactions. Thus, fusion of the liposomes is promoted strongly near the transition temperature of the membrane. When DLPC was used as the liposome lipid, enhancement of fusion hardly occurs above 30 °C. Because a DLPC membrane takes on a liquid-crystalline phase under the experimental conditions, it is suggested that the copolymer does not promote fusion of liposome with a fluid membrane. To confirm the occurrence of fusion of the copolymerfixed liposomes, the liposomes were observed by TEM. The copolymer-fixed and the bare DPPC liposomes were incubated at room temperature (ca. 20 °C) or at 39 °C for 3 min and then returned to room temperature and kept for 30 min. Typical TEM images of these liposomes are shown in Figure 8. For the bare DPPC liposomes, diameters of 20-70 nm were observed (Figure 8A). No apparent difference in vesicle size was seen between the liposomes incubated at room temperature and those incubated at 39 °C (result not shown). The size distribution of the liposomes is roughly consistent with the mean diameter (60-70 nm) estimated using dynamic light scattering (Figure 4). In contrast, a significant difference in diameter was seen between the copolymer-fixed liposomes kept at room temperature and those incubated at 39 °C. When the copolymer-fixed liposomes were incubated at room temperature, liposomes with diameter of ∼60 nm were

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Figure 8. Negative stain electron micrographs of liposome preparations: bare DPPC liposomes incubated at room temperature (A) and copolymer-fixed DPPC liposomes incubated at room temperature (B) and at 39 °C (C). The bars shown in the (A), (B), and (C) represent 50, 50, and 100 nm, respectively.

observed as shown in Figure 8B. This diameter is similar to that of the bare DPPC liposomes. However, the size of the liposomes treated at 39 °C was quite different. The diameter of a large fraction of the liposomes was much >100 nm (Figure 8C). These results clearly indicate that fusion of the copolymer-fixed liposomes occurred during the incubation at 39 °C. Temperature-Dependent Associating Property of the Copolymer-Modified Liposomes. As mentioned above, association of the copolymer-modified liposomes was induced above the copolymer transition temperature where the copolymer is hydrophobic. Therefore, it is considered that hydrophobic interactions play an important role in this association. However, association of the copolymer-modified liposomes is affected by the membrane fluidity of the liposomes. This association occurred only at and below the gel-liquid-crystalline transition temperature of the liposome membrane. When the copolymer is bound on the solid membrane, it may exist on the surface of the liposomes even above the transition temperature of the copolymer. However, when the copolymer is bound on the fluid membranes, it should penetrate more easily into the membrane. Therefore, the dehydrated copolymer is considered to be buried partly in the lipid membrane and may not interact with other liposomes effectively. Ringsdorf et al. showed that hydrophobically modified poly(NIPAM) fixed on a liposome shrinks more efficiently on the surface of the liposome with a liquid-crystalline membrane than on the surface of the liposomes with a gel membrane, because the lateral diffusion of the hydrophobic groups inserted into the membrane is precluded in the gel membrane (33). Similarly, in this study, when the copolymer is fixed on the liposome with a fluid membrane, the copolymer will take a compact conformation above its transition temperature and, as a result, the bare lipid surface will be exposed. However, when the copolymer is fixed on the liposome with a solid membrane, the liposome surface will be covered more effectively by the hydrophobic copolymer chains. The position of the copolymer might also affect the association of the copolymer-modified liposomes, as indicated schematically in Figure 9.

Figure 9. Schematic illustration of temperature-dependent associating property of the copolymer-modified liposomes. Below the copolymer transition temperature, the highly hydrated copolymers cover the liposome surface and stabilize liposome (A). Above this temperature, the liposome surface is covered with the dehydrated copolymers, which enhance association of the liposomes when the liposome has a solid membrane (B). When the copolymer is fixed on a liposome with a fluid membrane, the copolymers shrink effectively and the bare surface is exposed (C).

In addition, it is well-known that the gel phase and liquid-crystalline phase coexist in lipid membranes at the phase transition temperature and that, as a consequence, these membranes contain structural defects. Therefore, it is likely that the dehydrated copolymer destabilized

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the liposomes at this temperature and promotes association of liposomes strongly. CONCLUDING REMARKS

It was found that surface modification of liposomes with copoly(NIPAM-ODA) provided liposomes with a temperature-sensitive associating property: both the aggregation and the fusion of the copolymer-modified DPPC and DSPC liposomes were remarkably enhanced above the transition temperature of the copolymer when these liposomes had a solid membrane. The copolymer-modified liposomes prepared in this study exhibited temperature response above 30 °C, corresponding to the transition temperature of the copolymer. Since the transition temperature of this type of polymer is controllable by copolymerization with monomers with varying hydrophilicity (34), it is possible to obtain temperature-sensitive polymers with the transition temperature at a desired temperature. Kim et al. recently showed that the transition temperature of copolymers of NIPAM and acrylic acid was elevated as the content of acrylic acid in the copolymers increased. They prepared the copolymer-coated liposomes and showed that release of calcein loaded within the liposomes is enhanced above the transition temperature of the copolymers to some extent (35). In addition, several polymers are known to exhibit transition temperatures slightly higher than physiological temperature (28, 36). If such polymers are fixed on the liposomes, the liposomes are expected to reveal a temperature response near the physiological temperature. When the liposomes with the temperature-sensitive surface property are injected into the blood stream, their interaction with the blood components, such as plasma proteins and various kinds of cells, should be affected by temperature. Therefore, the distribution of the liposomes in the body may be controlled by temperature. Moreover, the liposomes may deliver their contents into the cytoplasm by fusion with the cellular membranes. Interactions of the liposomes with proteins and cells are currently under investigation. LITERATURE CITED (1) Lasic, D. D. (1993) Liposomes: From Physics to Applications, Elsevier, Amsterdam. (2) Yatvin, M. B., Weinstein, J. N., Dennis, W. H., and Blumenthal, R. (1978) Design of liposomes for enhanced local release of drugs by hyperthermia. Science 202, 1290-1293. (3) Kono, K., Hayashi, H., and Takagishi, T. (1994) Temperature-sensitive liposomes: liposomes bearing poly(N-isopropylacrylamide). J. Controlled Release 30, 69-75. (4) Yatvin, M. B., Kretz, W., Horwitz, B. A., and Shinitzky, M. (1980) pH-Sensitive liposomes: possible clinical implications. Science 210, 1253-1255. (5) Maeda, M., Kumano, A., and Tirrell, D. A. (1988) H+-induced release of contents of phosphatidylcholine vesicles bearing surface-bound polyelectrolyte chains. J. Am. Chem. Soc. 110, 7455-7459. (6) Nayar, R., and Schriot, A. J. (1985) Generation of pHsensitive liposomes: use of large unilamellar vesicles containing N-succinyldioleoylphosphatidylethanolamine. Biochemistry 24, 5967-5971. (7) Pidgeon, C., and Hunt, C. A. (1983) Light sensitive liposomes. Photochem. Photobiol. 37, 491-494. (8) Frankel D. A., Lamparski, H., Liman, U., and O’Brien, D. F. (1989) Photoinduced destabilization of bilayer vesicles. J. Am. Chem. Soc. 111, 9262-9263. (9) Anderson, V. C., and Thompson, D. H. (1992) Triggered release of hydrophilic agents from plasmalogen liposomes using visible light or acid. Biochim. Biophys. Acta 1109, 3342.

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