Langmuir 1994,10, 403-406
403
Liposomes Containing Amphiphiles Prepared by Using a Lipophilic Chain Transfer Reagent: Responsiveness to External Stimuli Hiromi Kitano' and Yasushi Maeda Department of Chemical and Biochemical Engineering, Toyama University, Toyama 903, Japan
Shigeya Takeuchi and Kaori Ieda Faculty of Education, Toyama University, Toyama 930, Japan
Yasuhiro Aizu Department of Polymer Chemistry, Kyoto University, Kyoto 606-01, Japan Received May 25, 1993. In Final Form: November 1 6 , 1 9 9 P
A lipophilic chain transfer reagent was prepared by coupling of N,N-dioctadecylamine with 3-mercaptopropionic acid, and telomerization of acrylic acid (product, DODA-S-PAA)or N-isopropylacrylamide (product, DODA-S-PIPA) was carried out by using the chain transfer reagent. The amphiphiles obtained formed stable liposomes with lecithin (L-a-dimyristoylphosphatidylcholineor L-a-dipalmitoylphosphatidylcholine),and the liposomes showed a pH-responsive (DODA-S-PAA)or temperature-responsive (DODA-S-PIPA) release of fluorophore from inner water pool. The lipophilic chain transfer reagent was useful to prepare amphiphiles with various functionalities. Hybrid materials, those are compounds consisting of a showed a pH responsiveness and temperature responplural number of functional moieties, are very useful as siveness, respectively. Hachisako et al. recently prepared intelligent materials in many research and application dialkyl L-glutamate amphiphiles with oligo(acry1ic acid) fields. Polymers which are designed to be responsive to head group by using a lipophilic chain transfer reagent external stimuli such as pH,' light? and t e m p e r a t ~ r e , ~ * ~ and examined interaction of the amphiphiles with cationic dyes? The lipophilic chain transfer reagent would be have been very often included into a category of hybrid highly useful to prepare amphiphilic devices with various materials, because various components have to be comfunctionalities. bined to prepare the polymers which are practically useful. Higashi et al., for example, prepared chain-end-anchored Experimental Section poly(methacry1ic acid) by using the catalytic system of halo-terminated lipid and manganese carbonyl ( M n r Materials. NJV-Dioctadecylamine (DODA) was purchased ( C 0 ) d and examined pH-responsiveness of a monolayer from Fluka, Switzerland,and purified by recrystallization from of the amphiphile obtained.lb Previously we prepared acetone. 2,2'-Azobis(isobutyronitrile) (AIBN, Nacalai Tesque, Kyoto, Japan) was purified by recrystallization from methanol. liposome-forming amphiphiles having pH-responsiveness Acrylic acid (NacalaiTesque)was purified by distillationin vacuo. by polymerization of acrylic acid, methacrylic acid, or N-Isopropylacrylamide (IPA) (Kodak, Rochester, NY) was ethacrylic acid by using a lipophilic radical i n i t i a t ~ r . ~ ~ ~ purified by recrystallization from benzene-hexane. L-a-DimyrisPolymers prepared by radical initiators in general, howtoylphosphatidylcholine (DMPC) and L-a-dipalmitoylphosever, have a wide distribution in their molecular weights, phatidylcholine (DPPC)were from Sigma, St. Louis, MO. Other and sometimes have difficulties to be used reproducibly reagents were commercially available. A Milli-Q grade water in practical fields. By use of a chain transfer reagent, on was used for preparation of sample solutions. the other hand, the distribution in molecular weight can Preparation of Lipophilic Chain Transfer Reagent. be made relatively n a r r ~ w e r . ~ DODA (1.36 g) was coupled with 3,3'-dithiobis(propionic acid succinimidyl ester) (0.70g) in dry dichloromethane (70mL) in In this report, we prepared a lipophilic chain transfer the presence of triethylamine (TEA, 95 rL) (Figure 1A). After reagent with a N,N-dioctadecylamino group. With the evaporation of the solvent, the oily product was purified by silica reagent, telomerization of acrylic acid and N-isopropylgel column chromatography (mobile phase, ethyl acetate/ acrylamide was carried out, and the amphiphiles obtained methanol (41)). The elution of the product was checked by a of 5,5'-dithiobis(2-nitobenzoicacid)g(DTNB, 40 mg in 10 * Abstract publishedin Advance ACS Abstracts, January 1,1994. spray mL of 50% (v/v) ethanol-Tris buffer (pH 8.2,0.2M)) and that (1) (a) Kitano, H.; Wolf,H.; Ise, N. Macromolecules 1990,23,195& of Hanes-Isherwood reagent.*O The fraction which contained 1961. (b) Higashi, N.; Shiba, H.; Niwa, M. Macromolecules 1989, 22, DODA-S-S-DODA was collected, and after evaporation of the 46504652. solvent, the oily compound (0.86 g, 0.71 mmol) was reduced by (2) Kitano, H.; Oehmichen, T.; Ise, N. Makromol. Chem. 1991, 192, 1107-1115. - - . - - - -. using 2-mercaptoethanol (1.23 mL, 17.5 mmol) in dichlo(3) Yan, C.; Nakamura, K.; Kitano, H. Makromol. Chem. 1991,192, romethane overnight. After evaporation of the solvent, the oily 291.5-2928. __ __ __ mixture was vigorously stirred with acetonitrile to give slightly (4) Gewehr, M.; Nakamura, K.; Ise, N.; Kitano, H. Makromol. Chem. 1992,193,249-266. (5) Kitano, H.; Akatsuka, Y.; Ise, N. Macromolecules 1991,24,42-46. (6) Kitano, H.; Akatsuka, Y.; Ise, N. Submitted for publication in Macromolecules. (7) Encyclopedia of Polymer Science and Engineering,2nd ed.;John Wiley: New York, 1980; Vol. 3, p 288.
(8) Hachisako, H.; Motozato, Y.; Murakami, R.; Yamada, K. Chem. Lett. 1993, 214-222. (9) Samuel, N. K. P.; Singh, M.; Yamaguchi, K.; Regen, S. L. J. Am. Chem. SOC.1986,107,4247. (10)Hanes, C. S.; Isherwood, F. A. Nature 1949, 164, 1107-1112.
0743-7463/94/2410-0403$04.50/00 1994 American Chemical Society
Kitano et al.
404 Langmuir, Vol. 10,No. 2, 1994
9
H c H:iC{?>N-C -CzHb-SH
HOCzH4SH CH2C12 H37C18 >NH
H37C18 DODA
(C) CH*=$H
+
DODA-SH
HOOC- C2H4 -SH
,.
MPA
AIBN
DODA-SH
k
n37Lls '
R . CONH-CH(CHJ2 COOH
(IPAI
(AA)
Figure 1. Schemeof preparation procedure of amphiphiles: (A) DODA-SH (I); (B) DODA-SH (11); (C) DODA-S-PIPA and DODA-S-PAA. yellow powder, Nfl-diodadecyl-3-mercaptopropionylamide (DODA-SH, 0.50 g, 0.82 mmol, yield 58%)(Rf= 0.94, mobile phase, ethyl acetate/methanol (3:l)). 'H-NMR (CDCh) 6 0.9 (6H, t, Hac-), 1.2-1.5 (64H,m,-CH2-), 2.5-2.6 (2H, m, -C(=O)-CH2-), 2.8-3.0 (2H,m,-CH&-), 3.1-3.4 (4H,m, -CHrN-). Anal. Calcd for CSI-I~~NSO-~H~O C, 74.57; H, 13.00,N, 2.23. Found C, 74.39; H, 12.70; N, 1.84. GC-MS m/e = 608 (M - H), 576 (M - SH), 548 (M - CzHdSH), 520 (M - C(=O)C*H4SH). Another method was also used for a preparation of DODASH, though the yield of the reaction was much smaller than that by the first method (Figure 1B): DODA (14.6 g) was mixed with 3-mercaptopropionic acid (MPA, 4.8 g) and N,"-dicyclohexylcarbodiimide (DCC, 12.8 g) (molar ratio 1:1.6:2.2) in dry dichloromethane (50 mL), and the reaction mixture waS continuously stirred overnight at roam temperature. After filtration of the byproduct, N,"-dicyclohexylurea, the filtrate was evaporated. The oily mixture was purified by a silica gel column (3 X 40 cm; mobile phase, ethyl acetate/methanol/acetic acid (6: 3:l)). The fraction of the product was collected and, after dehydration with Na2SO4, evaporated. The oily mixture was precipitated from acetonitrile and, after filtration, dried in uacuo (3.4 g, yield 12.4%). The Rfvalue of the product obtained was the same as that obtained by the first method. Polymerization of IPA in the Presence of DODA-SH (Figure lC, Table 1). DODA-SH (0.50 g), IPA (0.93 g), and AIBN (0.0135 g) (molar ratio 10100:1) were dissolved in dry 1,4-dioxane. The solution mixture was deoxygenated by passing Nz for 5 min. The test tube with a glass stopper was tightly sealedwith Teflon tape, and the reaction solution was incubated at 70 "C for 17h. The solventwas evaporated and the oily mixture was washed with n-hexane to remove unreacted chain transfer reagent and, subsequently, with a NaCl aqueous solution (1M) to remove the monomer and byproduct polymers without lipophilicchains. After washingwith the NaCl aqueous solution, the polymer product was dissolved in 1,4-dioxaneand filtrated. Thefiltrate was evaporated, and slightlyyellow powder was fiiaUy obtained (DODA-S-PIPA, 0.871 g). The same procedure was used for polymerization of acrylicacid (polymerization of acrylic acid (0.59 g) with AIBN (0.0135 g) and DODA-SH (0.5 g) gave 0.16 g of DODA-S-PAA). Degree of polymerization (DP) of the amphiphiles was determined from elemental analyses (DODA-S-PIPA),or by the conductometric titration (Wayne Kerr autobalance precision bridge B311) of carboxyl groups (DODA-S-PAA).
Temperature Responsiveness of Liposomes Which Contain DODA-S-PIPA. DODA-S-PEA (5 mg) was dissolved in CHCls (5 mL) and mixed with L-a-dimyristoylphosphatidylcholine (DMPC,5 mg) in "all round-bottomed flask. The midure was evaporated to form a thin membrane. Toavoid an adsorption of DMPC molecules on the segment of PIPA chains (the adsorption induces association of liposomes),the mixed lipids were dispersed in 5 mL of Eosin Y solution (0.1 M)dissolved in HEPES buffer (5 mM, pH 7.0, 5 mL) at 28 "C (between the phase transition of DMPC (gel- liquid crystal)and the transition of poly(N4sopropylacrylamide)chain (coil globule)). Eosin Y is known to show a self-quenching phenomenon, and after dilution, the Eosin Y solution shows a significant fluorescence.6.6 The suspension mixture was passed through a Sephadex G-10 column (2 X 30 cm), and the liposome fraction (7 mL) was collected. The liposome suspension (0.1 mL) was mixed with a HEPES buffer (5 mM, pH 7.0,2.9 mL) at various temperatures in a quartz cell in a fluorescence spectrophotometer (F-401, Hitachi, Tokyo, Japan). Changes in fluorescence at 555 nm (excitation at 305 nm) due to a release of Eosin Y from the liposome were followed. A similar procedure was used to follow changes in turbidity of the liposome suspension at 400 nm by using a UV-visible spectrophotometer (U-2OO0, Hitachi). pH Responsiveness of Liposomes Containing DODA-SPAA. DODA-S-PA4 (2.5 mg) and DPPC (5 mg) were dissolved in 5 mL of CHC13 in a s m d round-bottomed flask. After evaporation of the solvent the mixed lipids were dispersed in a HEPES buffer (pH 8.0, 5 mM, 5 mL, 0.1 M of Eosin Y was contained) by vortexing and sonication (Astrason W-385, Heat Systems-Ultrasonics, Inc., NY) for 6 min at 50 "C. At this pH, carboxyl groups in PAA chains are deprotonated, which diminishesan adsorption of DPPC moleculesto the segment of polymer chains. The liposome was separated from free fluorophore by a gel permeation chromatography (Sephadex G-10, 2 X 30 cm). The liposomesuspensionwas quickly mixed with an equalvolume of HEPES buffer of various pH's, and the release of Eosin Y from the liposome was followed by a fluorescencestopped-flow spectrophotometer (excitation 305nm, emission >460 nm) (RA401, Otsuka Electronics, Hirakata, Japan). Dynamic Light Scattering (DLS) Measurements. The hydrodynamicdiameter of the DODA-S-PIPA/DMPCliposomes was estimated by a dynamic light scattering technique (at a scattering angle of 90")using a ELS-800 (Otsuka Electronics) at various temperatures. Differential Scanning Calorimetry (DSC). The phase transition temperature of the liposomes was estimated by a differential scanning calorimetry by using a SSC580 (Seiko E&I Co., Tokyo, Japan). The raising rate of the temperature was 2 "C/min.
-
Results and Discussion A. Temperature Responsiveness of DODA-S-PIPA Liposome. The presence of liposomal structure in the suspension mixture of DODA-S-PIPA (DP = 34) and DMPC (1:lin weight) after passing through a GPC column was confirmed by the increase in fluorescence of the suspension after sonication for 3 min at 60 "C. The DSC data of the suspension showed two broad peaks at around 24 and 32 "C corresponding to the gel-liquid crystal transition of DMPC (T,= 24.0 oC)ll and the coil-globule transition of PIPA chains, respectively. The presence of transition at 24 "C suggested that DODA-S-PIPA and DMPC moleculesmade a phase separation on the liposome surface. On the other hand, the transition of N,Ndioctadecylamino group of DODA-S-PIPA could not be distinctly observed, probably because the amount of N,Ndioctadecylamino group in the liposome was very small (14 mol %). By the increase in temperature the turbidity of the liposome suspension was observed to increase above 32 (11) B l u e , A. Biochemistry 1983,22,6436-5442.
Langmuir, Vol. 10, No. 2, 1994 405
Liposomes Containing Amphiphiles
Table 1. Premration of AmvhiDhilee monomer (g) AIBN (9) dioxane (mL) ~
amphiphile
DODA-SH (9)
DODA-S-PIPA
0.500 0.587 0.300 0.200 0.200
~~
~
~~
DPC'
yield (mg)
IPA 0.929 1.005 0.280 0.250 0.190
0.0135 0.0168 0.0040 0.0038 0.0030
10 16 15 15 10
87 1
346 28b 2Bb 6b 3b
23 6.4 87 18
AA
DODA-S-PAA 0.500 0.590 0.0135 0 Degree of polymerization. b By elemental analysis. By conductometric titration.
3
-
10
1
0.159
1OC
20 -
15 -
10 -
10
20
Temperature
51b
40
30
pc)
Figure 2. Temperature effect on the turbidity changes of liposome suspension: B,DP = 3; 0,DP = 6;0 , DP = 26; 0 , DP = 28. [DMPC/DODA-S-PIPA] = 0.42 mg/3 mL of pH 7.0 HEPES buffer (5 mM) DMPC:DODA-S-PIPA = 3:2. "C (Figure 2). The larger the value of DP, the larger the increase in turbidityI2 (the weight percents of the lipid mixture in the suspensions were kept constant). As for DODA-S-PIPAof DP = 3, the turbidity even below 30 "C was significant, because the amount of lipophilic N A dioctadecylamino group in the 'suspension of DODAS-PIPA (DP = 3) was the largest. There was, however, a distinct increase in turbidity at about 32 "C. It was previously shown that the PIPA chain (M > 50 0oO) makes a coil-globule transition at about 32 "C, because of the collapse of the balance between hydrophobicity and hydrophilicity of the PIPA chain induced by the dehydration of polar amide groups in the polymer.13-16 The increase in turbidity observed here is, therefore, due to the increase in hydrophobicity of the liposomal surfaces, which might induce an association of the destabilized liposomes. By the DLS measurements we could observe an increase in hydrodynamic diameter (DM)of the liposomes with an increase in temperature (Figure 3), which strongly supports the association of DODA-S-PIPA/DMPCliposomes suggested by the turbidity measurements. When the fluorescence of the liposome suspension was followed, a significant release of Eosin Y from the liposome was detected above 30 "C (Figure 4). This is probably because, by the transition of PIPA chains on the liposome surface, the liposomes made a self-association and, at the same time, the liposomal structure was destabilized to release the inner content, which resulted in an increase in fluorescence intensity above 30 "C. However, we cannot exclude the probability that the liposomes were destroyed (12)(a) Takeuchi, S.;Oike, M.; Kowitz, C.; Shimasaki, C.; Hasegawa, K.; Kitano, H. Makromol. Chem. 1993,194,651-558.(b) Takeuchi, S.; Omodaka, I.; Maeda, Y.;Hasegawa, K.; Kitano, H. Makromol. Chem. 1993,194,1991-1999. (13)Fujishige, S.;Kubota, K.; Ando, I. J . Phye. Chem. 1989,93,33113314. (14)Kubota, K.; Fujishige, S.;Ando, I. J. Phys. Chem. 1990,94,51545157. Prep. . Jpn. 1990,39,4007-4009. (15)Matsuyama, A. P O ~ Y M
2b
3b
4b
("c) Figure 3. Temperature effect on the diameter of the liposome suspension. [DMPC/DODA-S-PIPA (1:1)] = 0.02 mg/3 mL of pH 7.0 HEPES buffer (5 mM). DP of DODA-S-PIPA = 34. Temperature
I 10
20
30 Temperature
40 ("C)
Figure 4. Temperature effect on the release of Eosin Y from
various liposomes in 60 s: (X) DMPC liposome; (0) DMPC/ DODA-S-PIPA liposome (DP = 34, DMPC:DODA-S-PIPA = 1:l); (0) DMPC/DODA-S-PIPA liposome (DP = 3, DMPC: DODA-S-PIPA = 32). [lipid] = 0.02 mg/3 mL of pH 7.0 HEPES buffer (5 mM).
by the association. Furthermore, there was a drop of the release of Eosin Y at about 30 "C for the liposome DMPC: DODA-S-PIPA= 3:2 (DP = 3). Thispeculiar phenomenon cannot be simply ascribed to a transition of DMPC and PIPA chains in the liposome. We only guess that a rearrangement of phase-separated DMPC molecules (which make a transition a t 24 "C) and DODA-S-PIPAmolecules (which makes a transition above 30 "C) on the liposome surface might induce such a phenomenon. In addition, at about 20 "C there was a small increase in fluorescence intensity due to the phase transition of DMPC molecules which destabilize the liposomal structure (Figure 4).6 The degree of increase in fluorescence intensity at the transition of PIPA chains (above 30 "C) was, however, much larger than that at the transition of DMPC molecules (above 20 "C). According to the light scattering measurements, PIPA molecules (M,= 8.4 X 106) gradually decrease both hydrodynamic radii and root-mean-square radii of gyration with temperature up to 30 OC,and drastically shrink at the transition region (30-32 "C),l3 which suggests that
406 Langmuir,
Kitano et al.
Vol. 10,No. 2, 1994
even below the transition region PIPA chains gradually increase their hydrophobicity. As for the DMPC liposome, on the other hand, the release of Eosin Y was observed above 10 "C which might correspond to the pretransition of the DMPC liposomes (T,= 15.3 OC).11 The percent of release from the DMPC liposome arrived a t its maximum around 25 "C, which is different from the release behavior of the DODA-S-PIPA liposome. The release of Eosin Y from a DPPC liposome below the main transition temperature (T, = 41.5 OC)ll was previously reported.e In that case the pretransition temperature of the DPPC liposome is 35.5 OCll and consistent with the temperature region where the release of Eosin Y was observed. Previously we examined the micellar structure of an amphiphilic PIPA prepared by the polymerization of IPA in the presence of lipophilic radical initiator (DODA-501).16 Above the transition temperature we could detect the destruction of micellar structure due to the partial penetration of PIPA chains into micellar cores by using pyrene as a probe. The present results that the liposomal structure was largely perturbed above the transition temperature of PIPA chains are not inconsistent with the previous report. Recently Ringsdorf et al. reported the temperature effect on the liposome covered with PIPA chains which have lipophilic anchor groups.17 They reported that the liposomal structure was retained during the temperature changes in spite of an accordion-like movement of the PIPA chains on the liposome surface. The difference in the thermal behavior of the PIPA-carrying liposomes examined by us and that by Ringsdorf et al. might be due to the difference in the chemical structure of the PIPA derivatives examined (with a lipophilic group a t its end (present work) or with many pendant lipophilic groups (by Ringsdorf et ala)).
B. pH Responsiveness of DODA-S-PAALiposome. (16) Winnik, F.; Davidson, A. R.; Hamer, G. K.; Kitano, H. Mucromolecules 1992,25,1876-1880. (17) Ringsdorf,H.: Venzmer,J.; Winnik, F. M. Angew. Chem.,lnt. Ed. Engl. 1991,-30, 315-318.
a o ;
d
i
i
PH
Figure 5. Effect of pH (final) on the percent of release of Eosin Y from the liposomes in 50 s at 35 O C : 0,DODA-S-PUDPPC = 1:2 (DP of DODA-S-PAA= 10). 0;DPPC. [liposome1 = 0.02 mg/3 mL. Initial pH 8.0, HEPES buffer (5 mM). Next, a pH responsiveness of the DODA-S-PAA liposome was examined as exemplified in Figure 5. Similar to the liposomes which contain amphiphiles prepared by the polymerization of acrylic acid with a lipophilic radical initiator,ba the liposomes which contained DODA-S-PAA showed a clear pH responsiveness due to the perturbation of packing of lipid molecules on the liposome surface (a further destruction of liposomal structure cannot be excluded). This phenomenon might be induced by the hydrogen bonding between a phosphate group of DPPC and protonated carboxyl groups of DODA-S-PAAbteJ8as confirmed previously by Raman spectroscopy.6 In conclusion, the lipophilic chain transfer reagent used here would be useful to prepare various amphiphiles with a variety of functionalities by using various kinds of monomers.
Acknowledgment. We wish to thank Professor Norio Ise, Department of Polymer Chemistry, Kyoto University, for his encouragement and helpful suggestions throughout this work. We are grateful to Dr. Akira Tsuchida and Professor Masahide Yamamoto, Department of Polymer Chemistry, for allowing us to use the fluorescence spectrophotometer. This work was supported by the Izumi Foundation for the Promotion of Science and Technology, Tokyo, Japan. (18) Seki,K.; Tirrell, D. A. Mucromolecdes 1984,17,1692-1698.
