Biomacromolecules 2005, 6, 2374-2379
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Self-Assembling Bionanoparticles of Poly(E-Lysine) Bearing Cholesterol as a Biomesogen Michiya Matsusaki,†,‡ Takehito Fuchida,‡ Tatsuo Kaneko,†,‡ and Mitsuru Akashi*,†,‡,§ Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita 565-0871, Japan, Department of Nanostructured and Advanced Materials, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi-shi, 332-0012, Japan Received March 16, 2005 Revised Manuscript Received April 28, 2005
Introduction In recent years, amphiphilic polymers have received great interest because they can form self-assembled micro- and nanostructures such as micelles, vesicles, spheres, and nanogels in organic-solvent/water systems and aqueous solutions. Various polymeric micelles, vesicles, and particles have been studied to serve as drug or gene delivery carriers.1-5 Over the past 20 years, we have studied the synthesis, characteristics, and applications of core-corona self-assembling graft copolymer nanoparticles composed of hydrophobic polystyrene and hydrophilic macromonomers.6-10 Core-corona nanoparticles with functional corona layers are successfully conjugated with drugs, peptides, and viruses and acted as controlled-release carriers and nanoparticle-based vaccines.7,8 However, both biodegradability and biocompatibility are required for medical use, which polystyrene-based nanoparticles cannot achieve. Although several biodegradable nanoparticles were recently reported,11,12 they possess low dispersion stability under soap-free aqueous conditions, poor size-control, or weak chemical reactivity on their surface. Soap is undesirable for drug carriers or nanoparticle-based vaccines and a size of at least 100 nm, along with surfacereactivity, are required for protein (size: several tens of nanometers) immobilization on the particle surface. In a previous study, we reported biodegradable nanoparticles (bionanoparticles) consisting of poly(γ-glutamic acid) and L-phenylalanine (Phe).13 Phe-bearing γ-PGA (Pheγ-PGA), which was partly substituted by hydrophobic L-phenylalanine, formed monodispersed nanoparticles (200 nm) using the solvent exchange method. Hydrophobic L-phenylalanine moieties self-aggregated in aqueous solution, and formed a nanoparticle-core. Phe-γ-PGA nanoparticle has an anionic charge and high chemical reactivity corresponding to the carboxyl groups of γ-PGA, which are present on the surface of the nanoparticles. Hydrophobic modification of * Corresponding author. Tel: +81-6-6879-7356. Fax: +81-6-6879-7359. E-mail:
[email protected]. † Osaka University. ‡ Kagoshima University. § JST.
water-soluble polymers is a useful method for preparing a biodegradable particle, micelle, or gel without the use of soap. For example, Akiyoshi et al. reported the preparation of pullulans nanogels by a modification of hydrophobic cholesterol.14 They studied the self-association of cholesterylbearing pullulans and -poly(L-lysine)s to nanogels in water by intermolecular self-aggregation.2 Yoksan et al. reported nanoparticle formation of N-phthaloylchitosan-grafted poly(ethylene glycol) methyl ether (mPEG) in water.15 Nphthaloylchitosan-grafted mPEG showed self-aggregation of sphere-like particles in water following the hydrophobic interactions of its N-phthaloyl groups. However, the dispersion-stability of these nanogels and -particles was low due to the low stability of the core parts. Furthermore, they cannot be redispersed in water after lyophilization. Dispersion stability and freeze-drying are important requirements for applications at the commercial level. We have also reported that biodegradable hollow nanoparticles were prepared by a combination of the silica-template technique and layer-bylayer method.16 Hollow nanoparticles can load many kinds of drugs inside them and have good dispersion-stability and redispersibility characteristics. In this way, we have prepared bionanoparticles consisting of naturally occurring polymers using various techniques. However, there is no regular structure for the molecular arrangement of any of our bionanoparticles. In nature, various nano constructions such as viruses, bacteria, and organelles have some specific molecular-regulated structures such as capsid proteins or matrixes, and these structures result in functions such as conformation transfer, permeability control, and the recognition of receptors on the cell surface. To develop a new generation of bionanoparticles with high functionalities that can act like viruses, the addition of a molecular-regulated structure into a bionanoparticle would be integral. In this study, we report the synthesis of an amphiphilic biopolymer with a liquid-crystalline structure using a hydrophobic “biomesogen” and self-assembled nanoparticles in water using the solvent exchange method. Liquid-crystal molecules can interact with each other by intermolecularappropriate interactions, and have molecular arrangement such as nematic and smectic phases. We synthesized a novel amphiphilic poly(-lysine-graft-cholesterol hydrogen succinate; poly(-Lys-g-CHS)). -poly-L-lysine {poly(-Lys)} is produced by a streptomycete, Streptomyces albulus 346,17 and has been used as a food additive due to its antimicrobial activities.17,18 Cholesterol hydrogen succinate (CHS) is known to have a smectic-A phase. We conjugated the CHS molecule as a biomesogen to poly(-Lys) using adequate amounts of N,N-dicyclohexyl carbodiimide. Poly(-Lys-gCHS) has a fan-shaped texture corresponding to the smectic-A phase at 210 °C and can retain its texture after cooling to room temperature. This phenomenon suggests that poly(-Lys-g-CHS) can keep its molecular-regulated structure at room temperature. Self-assembled poly(-Lys-g-CHS) created stable nanoparticles in water using the solvent exchange
10.1021/bm050204d CCC: $30.25 © 2005 American Chemical Society Published on Web 05/27/2005
Notes
method from THF to water, and the nanoparticles showed high dispersion stability and redispersibility at the same diameter. Although the evaluation of the applications of the poly(-Lys-g-CHS) nanoparticle has not been completed yet, we believe that cationic nanoparticles with a molecularregulated structure have great potential as cationic drug carriers, with structure-based functions such as anisotropy and thermotropic properties, as well as high biodegradability, chemical reactivity, dispersion-stability and re-dispersibility. This preliminary study suggests that the biomesogenconjugation method would be useful as a novel technology to develop a new generation of bionanoparticles with an ordered structure like a virus for drug delivery system and possesses high functionality based on its molecular-regulated structure.
Biomacromolecules, Vol. 6, No. 4, 2005 2375 Scheme 1. Synthesis of Poly(-Lys-g-CHS)
Experimental Section Materials. Poly(-Lys) (Mw 4,700) was kindly donated from CHISSO Corporation (Tokyo, Japan) and was used as received. Cholesterol hydrogen succinate (CHS), N,N-dicyclohexyl carbodiimide (DCC), sodium dodecyl sulfate (SDS), and 1-hydroxy-1H-benzotriazole (HOBt) were purchased from Wako Pure Chemical Industries (Osaka, Japan). The other chemicals were purchased from Nacalai Tesque (Kyoto, Japan). Synthesis of Poly(E-Lys-g-CHS). Poly(-Lys) cannot be dissolved in organic solvents due to its inter-and intramolecular electrostatic interactions, and this low solubility resists chemical reactions. To improve the low solubility of poly(-Lys), we used sodium dodecyl sulfate (SDS) as a detergent. Detergents such as SDS can inhibit the inter-and intramolecular interactions of poly(-Lys). According to this strategy, we prepared an ionic complex for the first time. Poly(-Lys) (38.2 unit mmol) was stirred in 300 mL of distilled water at room temperature for 12 h in the presence of SDS (1.0 mmol) in order to form an ionic complex. We used about one-40th amount of SDS in order to form the ionic complex, because the ionic complex can be formed with a small amount of detergent. The precipitate was centrifuged at 10,000 rpm for 10 min, and dried in vacuo for 24 h (yield ) 44%). The ionic complex was composed of poly(-Lys) and SDS and consists of about 1 Poly(-Lys) chain to 1 SDS molecule, dissolved in organic solvents such as methanol, chloroform, dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF). The synthesis of poly(Lys-g-CHS) was carried out in DMF (Scheme 1). Poly(-Lys) and the SDS ionic complex (0.16 mmol) were reacted with cholesterol hydrogen succinate (CHS) (1.8 mmol), various amounts of N,N-dicyclohexyl carbodiimide (DCC), and 1-hydroxy-1H-benzotriazole (HOBt) (280 mg: catalyst) in DMF at 80 °C for 24 h. After the reaction, the reaction solution was purified by dialysis (Spectra/Pore membrane, COMW)50,000) in ethanol and distilled water for 3 days, respectively. We confirmed that SDS molecules were completely removed in dialysis process by 1H NMR. The precipitate was centrifuged at 10 000 rpm for 10 min, and was dried in vacuo for 24 h. The dried precipitate was dissolved in acetone and purified by reprecipitation over chloroform. The yield was increased upon increasing the
amount of DCC. The degree of conversion was measured by the Liebermann-Burchard (LB) method. Structural Analyses of Poly(E-Lys-g-CHS)s. The addition of CHS to poly(-Lys)s was analyzed by 1H NMR with a JEOL JNM-GSX 400 (Tokyo, Japan), and by FT-IR spectroscopy with a Perkin-Elmer SPECTRIM ONE FT-IR SPECTROMETER (Wellesley, USA). The solubility of poly(-Lys), the ionic complex, and poly(-Lys-g-CHS) were investigated with distilled water, methanol, ethanol, acetone, chloroform, DMSO, tetrahydrofuran (THF), DMF, hexane, and toluene. The phase transition of poly(-Lys-g-CHS)s was then observed by crossed-polarizing microscopy. The samples were sandwiched between two glass plates and were heated at a rate of 10 °C/min by a METTLER TOLEDO FP82HT Hot Stage (Greifensee, Switzerland). The Tm and the liquidcrystalline phase were observed directly by microscopy. The liquid-crystalline phase structure of poly(-Lys-g-CHS)s was analyzed by small-angle X-ray diffraction (SAXD) with a RINT InPlane/ultrtaX18SAXS-IP (Rigaku Co., Tokyo, Japan) at scanning angles that ranged from 1.0° to 5.0° at 1.0° min-1. Poly(-Lys-g-CHS)s was cooled from its Tm before the SAXD measurement. Liebermann-Burchard Test. To investigate the degree of conversion of CHS to poly(-Lys), we used the Liebermann-Burchard (LB) method.19 The LB method is used to analyze steroid compounds, and has been used to determine the amount of cholesterol in human blood in biological fields. Briefly, the LB procedure is as follows: A total of 20 mL of acetic anhydride and 1 mL 97% sulfuric acid was gently mixed at 0 °C for 10 min, and 10 mL pf acetic acid was added to this solution at 0 °C. A 2 mL aliquot of this solution (LB reagent) was stirred with 1 mL of a poly(-Lys-g-CHS) chloroform solution at 25 °C for 30 min, and the absorbance at 620 nm was measured by UV-vis spectroscopy with a JASCO V-550 (Tokyo, Japan). The CHS concentration in the poly(-Lys-g-CHS) was calculated from the correlation curve of the CHS molecule. Nanoparticle Formation of poly(E-Lys-g-CHS)s. A total of 10 mg of poly(-Lys-g-CHS)s was self-assembled by the
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Table 1. Synthesis Conditions of Poly(-Lys-g-CHS)sa run Lysb/(mmol) CHSc/(mmol) DCCd/(mmol) yield/(%) Ce/(%) 1 2 3 4 5
0.16 0.16 0.16 0.16 0.16
1.8 1.8 1.8 1.8 1.8
37 18 9.0 1.8 0.92
48 53 48 24 5.8
92 99 99 51 60
a Poly(-Lys-g-CHS)s refers to (-lysine-graft-cholesterol hydrogen succinate). b Lys refers to -poly-l-lysine. c CHS refers to cholesterol hydrogen succinate. d DCC refers to N,N-dicyclohexyl carbodiimide. e Conversion degree was estimated by the Libermann-Burchard method. A 280 mg sample of 1-hydroxy-1H-benzotriazole was used as a esterification reagent.
solvent exchange method (from 1 mL THF to 1 mL distilled water), which is normally used to form polymeric micelles consisting of diblock copolymers, to yield a clouded solution.20 After the solvent exchange, the THF was removed by dialysis (Spectra/Pore membrane, COMW ) 50 000) in distilled water for 3 days. Nanoparticle formation was confirmed by transmittance electron microscopy (TEM) with a HITACHI H-700H microscope and scanning electron microscopy (SEM) with a HITACHI S-4100H SE microscope. Results and Discussion Syntheses of Poly(E-Lys-g-CHS)s. We observed that the cholesterol hydrogen succinate (CHS) had a smectic-A phase and possessed biodegradability as a biomesogen and exploited this in order to produce a regulated molecular arrangement in a bionanoparticle. Table 1 shows the synthesis conditions of poly(-Lys-g-CHS)s. The ionic complex, CHS, DCC, and HOBt were dissolved in DMF at room temperature. The solution was transparent initially but became translucent and finally a milk-white color after a few hours. A precipitate was formed in the reaction solution after 24 h. The purified products were obtained as a white powder, which was soluble in chloroform and hexane. Figure 1 shows the 1H NMR spectra of poly(-Lys) in D2O, CHS and poly(-Lys-g-CHS) (the synthetic condition is Run 1 from Table 1) in chloroform-d3. The CH peak (a) belonging to poly(-Lys) was shifted from 4.1 to 4.7 ppm (d), indicating that CHS was introduced into poly(-Lys) by an amide linkage. CH2 peaks (b) corresponding to CHS were observed after the reaction; however, all of the CH2 peaks corresponding to poly(-Lys) were hidden behind the peaks of CHS after the reaction. Furthermore, the CH peaks (a and c) belonging to poly(-Lys-g-CHS) were not clearly observed. Estimating the degree of conversion of CHS from the 1H NMR spectra was difficult to perform accurately. Therefore, we tried to estimate the substitution ratio from the Liebermann-Burchard reaction. The LB test is used to analyze steroid compounds, and has been used as a general method in the biological field.19 The degree of conversion of CHS was estimated from the correlation curve of the CHS molecule, and the results indicated that the CHS substitution ratio depended on the amount of DCC. We have previously reported that the substitution ratio of hydrophobic biomolecules to water soluble polymers was controlled by altering condensation regents such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (WSC).13,21 The results of the
Figure 1. 1H NMR spectra of poly(-Lys) in D2O, CHS, and poly(Lys-g-CHS) in chloroform-d3.
LB method should be adequate. Poly(-Lys) was completely modified to poly(-Lys-g-CHS) using a 10-20-fold excess of DCC. Figure 2 shows the FT-IR spectra of poly(-Lys), CHS, and poly(-Lys-g-CHS) (the synthetic condition is Run 1 from Table 1). The IR peaks assigned to the amide I and II groups (νCdO: 1650 and δN-H: 1530 cm-1) in the spectrum for poly(-Lys) appeared in those for poly(-Lys-g-CHS). On the other hand, the IR peaks assigned to the carboxyl group in the spectra for CHS (νCdO: 1700 cm-1) and the amine group in the spectra for poly(-Lys) (δN-H: 1560 cm-1) disappeared in poly(-Lys-g-CHS). This result supports the successful conversion of the carboxyl and amino groups to amide groups. The solubility of poly(-Lys) was changed after the reaction (Table 2). Poly(-Lys-g-CHS)s was dissolved in chloroform and hexane, and solubility change also indicated that CHS was successfully conjugated to poly(-Lys). Thermotropic Properties of Poly(E-Lys-g-CHS)s. The thermotropic properties were investigated by crossed-polarizing microscopic observation (Figure 3). The melting points (Tm) of CHS and poly(-Lys-g-CHS)s were observed at 145170 °C and 200-240 °C, respectively. The Tm of poly(Lys-g-CHS)s was not dependent on the conversion degree of CHS. Crossed-polarizing microscopy showed that CHS and poly(-Lys-g-CHS)s exhibited a strong fan-shaped texture above the Tm. However, conversion degrees under
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Notes
Figure 2. FT-IR spectra of poly(-Lys), CHS, and poly(-Lys-g-CHS). Table 2. Solubility Tests of Poly(-Lys), CHS, and Poly(-Lys-g-CHS) solvent
poly(-Lys)a
CHS
poly(-Lys-g-CHS)a
water methanol ethanol acetone chloroform DMSO DMF THF hexane toluene
+ -
+ + + + + + + +
+ + -
a
Figure 3. Crossed-polarizing microscopic images of CHS at 150 °C (a) and poly(-Lys-g-CHS) (Table, run 1) at 210 °C (b).
+, soluble; -, insoluble.
60% (Table 1, runs 4 and 5) did not result in any birefringence. The texture was easily transformed into another morphology, including a dark-field view by sliding the coverglass. These results indicated that poly(-Lys-gCHS)s with conversion degrees over 60% exhibited a smectic-A phase, where CHS molecules were autonomously oriented to a lamellar structure. The texture was retained without recrystallization after cooling from 210 °C to room temperature. This result demonstrates the key point that poly(-Lys-g-CHS)s has a molecular-regulated structure at room temperature. Thus, a new generation of bionanoparticles such as viruses and organelles, which have high functionalities (for example, conformation transfer, permeability control, recognition of receptors on the cell surface, and so on) due to the regulated molecular arrangement, can be prepared using a biomesogen-conjugation method. The fan-shaped structure of poly(-Lys-g-CHS)s seems different from that of CHS, and suggests that poly(-Lysg-CHS)s and CHS have different lamellar structures. We
Figure 4. SAXD diagrams of CHS and poly(-Lys-g-CHS)s (Table, runs 1 and 2) recorded at 25 °C. The samples were cooled from a liquid-crystalline state.
investigated the intermolecular structures of CHS and poly(-Lys-g-CHS)s by SAXD. Structural Analyses of CHS and Poly(E-Lys-g-CHS)s. Figure 4 shows SAXD diagrams of CHS and poly(-Lysg-CHS)s (Table 1, runs 1 and 2) recorded at 25 °C. The samples were cooled from the liquid crystalline state. The SAXD patterns of CHS and poly(-Lys-g-CHS)s showed one
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Figure 5. Schematic illustrations of the lamellar structure of CHS and poly(-Lys-g-CHS) (Table 1, Run 1). The intermolecular distances were calculated by the CPK model. The measured phase distances of CHS (d ) 27.9 Å) and poly(-Lys-g-CHS) (d ) 49.4 Å) were calculated from the results of SAXD.
distinct diffraction at 2θ ) 3.16 (CHS), 1.79 (run 1, C ) 92%), and 1.78 (run 2, C ) 99%) (θ: diffraction angle), corresponding to spacings of 27.9, 49.4, and 49.7 Å, respectively. This spacing distance might represent the interphase distances of the smectic-A phase. The interphase distances of run 1 and 2 were almost the same, but were about 2 times larger than that of CHS. To clarify the lamellar structures of CHS and poly(-Lys-g-CHS), we calculated the intermolecular distances using a CPK model. Figure 5 shows schematic illustrations of CHS and poly(-Lys-g-CHS) (Table 1, Run 1). The succinate and steroid moieties of CHS are 7.2 and 15.2 Å in size, respectively. The whole length of CHS is 22.4 Å. The interphase distance of CHS calculated by SAXD is 27.9 Å, and this length is almost the same as the whole length of CHS, which contains another succinate moiety, from the CPK model (29.6 Å). These results suggest that CHS should have a monosteroidal lamellar structure like the upper panel illustration in Figure 5. On the other hand, the main chain of poly(-Lys-g-CHS), poly(-Lys) is about 2.0 Å from the CPK model. The interphase distance of poly(-Lys-g-CHS) calculated by SAXD is 49.4 Å, and this is almost the same as 2 times the whole length of poly(-Lysg-CHS) from the CPK model (48.8 Å). These results suggest that poly(-Lys-g-CHS) should have a bisteroidal lamellar structure like that in the lower panel illustration in Figure 5. We can speculate about the reasons why poly(-Lys-g-CHS) has a different layered structure from CHS. CHS has
hydrophilic succinate and hydrophobic cholesterol moieties. To form the most stable conformation, the succinate moieties should interact with each other at the intermolecular level. According to this reason, CHS forms monosteroidal lamellar structure, whereas the succinate moieties of poly(-Lys-gCHS) conjugated to poly(-Lys) by covalent binding. To form stable intermolecular interactions in poly(-Lys), the CHS moiety has to be oriented in the same direction. Accordingly, the intermolecular structure of poly(-Lys-gCHS) must be a bisteroidal lamellar structure. Nanoparticle Formation of Poly(E-Lys-g-CHS)s. Poly(-Lys-g-CHS) nanoparticles were prepared by the solvent exchange method. A total of 10 mg/mL of poly(-Lys-gCHS) in THF was gently added to 1 mL of distilled water, and the solution color changed from transparent to milkywhite. Nanoparticles were successfully prepared in all conditions of Table 1. Figure 6 shows TEM and SEM images of the poly(-Lys-g-CHS) nanoparticles (Table 1, run 1). The diameter of the poly(-Lys-g-CHS) nanoparticles was approximately 150-200 nm as determined by TEM observation (average of 50 particles). The diameters of nanoparticles were almost the same as that of run 1, although the conversion degrees were different. It is not well understood why all nanoparticles have approximately same diameter. The selfassembling mechanism may be as follows. CHS molecule is hydrophobicity, so CHS molecules began to shrink at the first step of nanoparticle formation in water by hydrophobic
Notes
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Conclusions In conclusion, we demonstrated the biomesogen-conjugating method as a novel technology to develop a new generation of bionanoparticles such as viruses, which have high functionalities due to the molecular-regulated structure. High-performance functionalities for bionanoparticles are required in drug delivery systems and in the biomedical and tissue engineering fields, and we believe that our method will be one of the key technologies to create a new generation of bionanoparticles. An evaluation of the functionalities of poly(-Lys-g-CHS) nanoparticles by altering the molecularregulated structure is now in progress. Acknowledgment. This study was supported by Industrial Technology Research Grant Program in 03A44014c from New Energy and Industrial Technology Development Organization (NEDO) of Japan. The authors are grateful to Associate Professor Dr. T. Serizawa of Research Center of Advanced Science and Technology, The University of Tokyo, for his advice and suggestions. References and Notes Figure 6. TEM (a) and SEM (b) images of poly(-Lys-g-CHS) nanoparticles (Table 1, Run 1).
interaction. In the second step, CHS molecules formed hydrophobic core part, and then this hydrophobic core was stabilized by hydrophilic poly(-lysine) main chain. Approximately the same self-assembling mechanism was reported to other amphiphilic polymers such as phenylalanine bearing poly(γ-glutamic acid),13 cholesteryl-bearing pullulans2 and N-phthaloylchitosan-grafted mPEG.15 The nanoparticles were stably dispersed in distilled water for several weeks. Furthermore, freeze-dried samples could be redispersed into aqueous solution and could reproduce almost the same particle size (data not shown). The biological preliminary experiment of poly(-Lys-g-CHS) nanoparticles about polyplex formation with plasmid DNA was evaluated. Polyplex formation of poly(-Lys-g-CHS) with plasmid DNA (φX174 RF DNA, Promega Co. Ltd.) was evaluated by intercalation with ethidium bromide. Plasmid DNA seemed to interact with amine groups of poly(-Lys-g-CHS) by electrostatic interaction. However, polyplex could not disperse well in water because of decrement of hydrophilic amine group in poly(-Lys-g-CHS). To solve this problem, we conjugated poly(ethylene glycol) (PEG) to poly(-Lysg-CHS). Polyplex of PEG conjugated poly(-Lys-g-CHS) and plasmid DNA was stably dispersed in water for several weeks. The detailed studies are now in progress, and we will report it in the next paper. We believe that our novel nanoparticle formation method, which used liquid-crystalline expression as a driving force, may be useful in preparing stable nanoparticles for drug and gene delivery applications.
(1) Itaka, K.; Kanayama, N.; Nishiyama, N.; Yamasaki, Y.; Nakamura, N.; Kawaguchi, H.; Kataoka, K. J. Am. Chem. Soc. 2004, 126, 13612-13613. (2) Akiyoshi, K.; Ueminami, A.; Kurumada, S.: Nomura, Y. Macromolecules 2000, 33, 6752-6756. (3) Kukula, H.; Schlaad, H.; Antonietti, M.; Fo¨rster, S. J. Am. Chem. Soc. 2002, 124, 1658-1663. (4) Savic, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Science 2003, 300, 615-618. (5) Arimura, H.; Ohya, Y.; Ouchi, T.; Yamada, H. Macromol. Biosci. 2003, 3, 18-25. (6) Akashi, M.; Kirikihira, I.; Miyauchi, N. Angew. Makromol. Chem. 1985, 132, 81-89. (7) Sakuma, S.; Suzuki, N.; Kikuchi, H.; Hiwatari, K-i.; Arikawa, K.; Kishida, A.; Akashi M. Int. J. Pharm. 1997, 149, 93-106. (8) M. Akashi, Niikawa, T.; Serizawa, T.; Hayakawa, T.; Baba, M. Bioconjugate Chem. 1998, 9, 50-53. (9) Serizawa, T.; Takehara, S.; Akashi, M. Macromolecules 2000, 33, 1759-1764. (10) Kaneko, T.; Hamada, K.; Chen, M.-Q.; Akashi M. Macromolecules 2004, 37, 501-506. (11) Ruan, G.; Feng, S.-S. Biomaterials 2003, 24, 5037-5044. (12) Rouzes, C.; Leonard, M.; Durand, A.; Dellacherie, E. Colloids Surf. B 2003, 32, 125-135. (13) Matsusaki, M.; Hiwatari, K-i.; Higashi, M.; Kaneko, T.; Akashi, M. Chem. Lett. 2004, 33, 398-399. (14) Akiyoshi, K.; Deguchi, S.; Moriguchi, N.; Yamaguchi, S.; Sunamoto, J. Macromolecules 1993, 26, 3062-3068. (15) Yoksan, R.; Matsusaki, M.; Akashi, M.; Chirachanchai S. Colloid Polym. Sci. 2004, 282, 337-342. (16) Itoh, Y.; Matsusaki, M.; Kida, T.; Akashi, M. Chem. Lett. 2004, 33, 1552-1553. (17) Shima, S.; Sakai, H. Agric. Biol. Chem. 1977, 41, 1807-1809. (18) Shima, S.; Fukuhara, Y.; Sakai, H. Agric. Biol. Chem. 1982, 46, 1917-1919. (19) Coleman, D. L.; Wells, W. W.; Baumann, C. A. Arch. Biochem. Biophys. 1956, 60, 412-418. (20) Desbaumes, L.; Eisenberg, A. Langmuir 1999, 15, 36-38. (21) Matsusaki, M.; Serizawa, T.; Kishida, A.; Endo, T.; Akashi, M. Bioconjugate Chem. 2002, 13, 23-28.
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