8308
Langmuir 2007, 23, 8308-8315
Articles Direct Formation of Giant Vesicles from Synthetic Polypeptides Jing Sun,†,‡ Xuesi Chen,† Chao Deng,†,‡ Haijun Yu,†,‡ Zhigang Xie,†,‡ and Xiabin Jing*,† State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China, and Graduate School of Chinese Academy of Sciences, Beijing, 100039, P. R. China ReceiVed April 15, 2007. In Final Form: May 29, 2007 This report describes direct formation of giant vesicles from a series of poly(L-lysine)-block-poly(L-phenylalanine) (PLL-b-PPA) block copolymers from their water solution. These polymers are prepared by successive ring-opening polymerization (ROP) of the two R-amino acid N-carboxyanhydrides and then removing the side chain protecting groups by acidolysis. The structures of the copolymers are confirmed by nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and size exclusion chromatography (SEC). The vesicles are studied by atomic force microscopy (AFM), field emission scanning electron microscopy (ESEM), and confocal laser scanning microscopy (CLSM). Rhodamine B is used as a fluorescent probe to confirm the existence of the vesicle with an aqueous interior. The vesicle size is in the range 0.55-6 µm, depending on the absolute and relative lengths of the two blocks, on initial polymer concentration, and on solution pH. The vesicles are still stable in water for 2 months after preparation. Addition of the copolymer to DNA solution results in complex formation with it. The complex assumes the morphology of irregular particles of less than 2 µm. It is expected to be used in drug and gene delivery.
1. Introduction During the past three decades, biodegradable polymers have been widely investigated and used in pharmaceutical and medical applications.1-6 Of them, polypeptides receive increasing research interest not only in improving the natural proteins but also in the development of medicine and biotechnology.7-12 In comparison to natural proteins, synthetic polypeptides offer more advantages in stability and processability. They tend to adopt stable conformations and their structure and properties can be tailored with ease, and therefore, they can find important applications. For example, recently, Akashi and co-workers reported a onestep preparation of poly(ethylene glycol)-poly(L-phenylalanine) (PEG-PPA) nanospheres with dual initiators in a solvent mixture of water/DMSO.13 Lecommandoux et al. studied stimuli* To whom correspondence should be addressed. Telephone: +86-43185262775. Fax: +86-431-85685653. E-mail:
[email protected]. † Chinese Academy of Sciences. ‡ Graduate School of Chinese Academy of Sciences. (1) Caillol, S.; Lecommandoux, S.; Mingotaud, A. F.; Schappacher, M.; Soum, A.; Bryson, N.; Meyrueix, R. Macromolecules 2003, 36, 1118. (2) Gadzinowski, M.; Sosnowski, S. J. Polym. Sci., Part A: Polym. Chem. 2003, 41, 3750. (3) Holland, S. J.; Tighe, B. J.; Gould, P. L. J. Controlled Release 1986, 4, 155. (4) Gilding, D. K.; Reed, A. M. Polymer 1979, 20, 1459. (5) Albertsson, A. C. Biomacromolecules 2005, 6, 2. (6) Meng, F. H.; Hiemstra, C.; Engbers, G. H. M.; Feijen, J. Macromolecules 2003, 36, 3004. (7) Grombotz, W. R.; Pettit, D. K. Bioconjugate Chem. 1995, 6, 332. (8) Klok, H. A. Angew. Chem., Int. Ed. 2002, 41, 1509. (9) Hrkach, J. S.; Ou, J.; Lotan, N.; Langer, R. Macromolecules 1995, 28, 4736. (10) Deng, M. X.; Wang, R.; Rong, G. Z.; Sun, J. R.; Zhang, X. F.; Chen, X. S.; Jing, X. B. Biomaterials 2004, 25, 3553. (11) Guan, H. L.; Xie, Z. G.; Zhang, P. B.; Deng, C.; Chen, X. S.; Jing, X. B. Biomacromolecules 2005, 6, 1954. (12) Yuan, M. L.; Wang, Y. H.; Xiong, C. D.; Deng, X. M. Macromolecules 2000, 33, 1613. (13) Matsusaki, M.; Waku, T.; Kaneko, T.; Kida, T.; Akashi, M. Langmuir 2006, 22, 1396.
responsive vesicles from a peptide-based diblock copolymer polybutadiene-block-poly(L-glutamic acid) (PB-b-PGA).14 Deming et al. synthesized a series of diblock copolypeptides via a living polymerization of several N-carboxy anhydrides (NCA). These copolypeptides were used in the synthesis of ordered silica structures and inorganic hollow spheres.15,16 Shantz et al. obtained a series of inorganic nanospheres in the presence of poly(Llysine)-block-poly(L/rac-phenylalanine) because of the unique opportunities offered by this kind of copolypeptides.17 In biology, lipids and proteins can spontaneously assemble into vesicles or vesicle-like structures, because of the driving forces such as electrostatics and hydrophobicity.18 Vesicles can also be formed from synthetic amphiphilic polymers,19-23 such as polystyrene-block-poly(acrylic acid) and polypeptide-containing copolymers. Because polypeptides may be either hydrophilic or hydrophobic, depending on which kind of amino acid is used as the building block, they usually serve as the hydrophilic or hydrophobic segment in these amphiphilic copolymers.13,14 In special cases, both hydrophilic and hydrophobic segments are made of polypeptides.15,16,22,23 In comparison with those block copolymers without polypeptide blocks, polypeptide-containing (14) Che´cot, F.; Lecommandoux, S.; Gnanou, Y.; Klok, H. A. Angew. Chem., Int. Ed. 2002, 41, 1339. (15) Wong, M. S.; Cha, J. N.; Choi, K. S.; Deming, T. J.; Stucky, G. D. Nano Lett. 2002, 2, 583. (16) Cha, J. N.; Stucky, G. D.; Morse, D. E.; Deming, T. J. Nature (London) 2000, 403, 289. (17) Jan, J. S.; Lee, S.; Carr, S.; Shantz, D. F. Chem. Mater. 2005, 17, 4310. (18) Ruysschaert, T.; Sonnen, A. F. P.; Haefele, T.; Meier, W.; Winterhalter, M.; Fournier, D. J. Am. Chem. Soc. 2005, 127, 6242. Wang, Q.; Lin, T. W.; Tang, L.; Johnson, J. E.; Finn, M. G. Angew. Chem., Int. Ed. 2002, 41, 459. (19) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967. (20) Ranqoin, A.; Verse´es, W.; Meier, W.; Steyaert, J.; Van, Gelder, P. Nano Lett. 2005, 5, 2220. (21) Choucair, A.; Lavigueur, C.; Eisenberg, A. Langmuir 2004, 20, 3894. (22) Holowka, E. P.; Pochan, D. J.; Deming, T. J. J. Am. Chem. Soc. 2005, 127, 12423. (23) Rodriguez-Herna´ndez, J.; Lecommandoux, S. J. Am. Chem. Soc. 2005, 127, 2026.
10.1021/la701038f CCC: $37.00 © 2007 American Chemical Society Published on Web 07/06/2007
Formation of Giant Vesicles from Polypeptides
ones can simulate not only the shape of natural vesicles or micelles, but also their biological performances. Up to the present date, the laboratory preparation of polymer vesicles and micelles is usually carried out in two steps, i.e., dissolving in a “common solvent” (common in hydrophilic and hydrophobic segments) and then aggregating upon replacement of the common solvent with a “selective solvent”. Usually, the common solvent is an organic solvent, and the selective solvent is water. For polypeptide-containing copolymers, sometimes, it is difficult to find such an organic common solvent, and sometimes, proteins loaded in the vesicles may lose their bioactivity upon contact with the organic solvent. Furthermore, the complete removal of the organic solvent is often difficult, so the stability of the vesicle system is limited. Therefore, it is desirable to prepare an aqueous vesicle solution without the usage of any organic solvents.24-28 Tirelli et al. obtained oxidation-responsive vesicles in aqueous solution by film swelling.24 Kataoka et al. prepared aqueous vesicles from a pair of oppositely charged copolymers, PEG-poly(2aminoethyl-R,β-aspartamide) (PEG-P(Asp-AE)) and PEG-poly(5-aminopentyl-R,β-aspartamide) (PEG-P(Asp-AP)) by virtue of electrostatic interaction.25 On the basis of the pH sensitivity of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-blockpoly(2-(diisopropylamino)ethyl methacrylate) (PMPC-b-PDPA), Armes et al. converted an aqueous solution into aqueous vesicles by changing the solution pH from 2 to 6.26 Herein, we synthesized a series of diblock copolypeptides, poly(L-lysine)-block-poly(L-phenylalanine) (PLL-b-PPA). They consist of a hydrophilic PLL block and a hydrophobic PPA block so that they can self-assemble into micelles or vesicles. Each L-lysine residue carries an amino group which can be protonated, and thus, can interact with negatively charged proteins or DNAs. Each L-phenylalanine residue carries a phenyl group that may result in intra- and intermolecular hydrophobic interactions like those in some biological systems.29 Sure enough, micrometerscale vesicles can be formed spontaneously from this simple block copolymer in aqueous media under proper conditions. To our knowledge, it is the first time that giant vesicles were directly obtained from an aqueous polypeptide solution. The factors which govern the vesicle size have been examined, such as pH value of the solution and copolymer concentration. Finally, the interaction between PLL-b-PPA and deoxyribonucleic acid (DNA) is investigated by UV and fluorescence spectroscopy. 2. Experimental Section 2.1. Materials and Methods. 2.1.1. Materials. -Benzyloxycarbonyl L-lysine (ZLL) and l-phenylalanine were purchased from GL Biochem (Shanghai) Ltd. A 33 wt % solution of HBr in HAc was supplied by Acros. n-Hexylamine (99.9%) from Aldrich was used without further purification. Dimethyl formamide (DMF) was dried over CaH2 and distilled under vacuum before use. Tetrahydrofuran (THF) was dried and distilled in the presence of sodium before use. Calf thymus DNA was purchased from Dingguo Ltd. (China). 2.1.2. Synthesis of R-Amino Acid N-Carboxyanhydrides (NCA). According to Daly,30 ZLL NCA was synthesized from ZLL and 1.5 (24) Napoli, A.; Valentini, M.; Tirelli, N.; Muller, M.; Hubbell, J. A. Nat. Mater. 2004, 3, 183. (25) Koide, A.; Kishimura, A.; Osada, K.; Jang, W. D.; Yamasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2006, 128, 5988. (26) Du, J. Z.; Tang, Y. Q.; Lewis, A. L.; Armes, S. P. J. Am. Chem. Soc. 2005, 127, 17982. (27) Che´cot, F.; Bruˆlet, A.; Oberdisse, J.; Gnanou, Y.; Mondain-Monval, O.; Lecommandoux Langmuir, S. 2005, 21, 4308. (28) Holowka, U. P.; S, V. Z.; Kamei, D. T.; Deming, T. J. Nat. Mater. 2007, 6, 52. (29) Santella, R. M.; Li, H. J. Biochemistry 1975, 14, 3604. (30) Daly, W. H.; Poche´, D. Tetrahedron Lett. 1988, 29, 5859.
Langmuir, Vol. 23, No. 16, 2007 8309 equiv triphosgene in THF at 50 °C. After 1.5 h, the mixture was precipitated by an excess of petroleum ether and then purified by three times of recrystallization with ethyl acetate and petroleum ether (yield: 79%). The preparation of L-phenylalanine NCA was similar to that of ZLL NCA, except the reaction temperature of 40 °C and the reaction time of 2.5 h (yield: 69%). 2.1.3. Synthesis of Poly(-benzyloxycarbonyl L-lysine)-block-poly(L-phenylalanine) (PZLL-b-PPA). The PZLL-b-PPA was synthesized in two steps: (1) The synthesis of PZLL, ring-opening polymerization (ROP) of ZLL NCA carried out in DMF solution (5 wt %) with proportional n-hexylamine as initiator. The reaction mixture was stirred for 3 days at 30 °C and then precipitated with an excess of diethyl ether under vigorous stirring. Then, the viscous polymer was dissolved in chloroform again and precipitated with diethyl ether to give a white solid. The product PZLL-NH2 was dried under vacuum at room temperature for 48 h. (2) The synthesis of PZLL-b-PPA, ROP of L-phenylalanine NCA at 40 °C in DMF solution (2 wt %) for 24 h in the presence of PZLL-NH2 as macroinitiator. The product PZLL-b-PPA was collected and purified as in step (1). 2.1.4. Deprotection of PZLL-b-PPA. The benzyloxycarbonyl (Z) groups on PZLL-b-PPA were removed by reacting with 4 equiv of HBr (in HAc, C ) 33%) with respect to Z-lysine repeat units in CF3COOH (0.04 g/mL) at 0 °C for 1.5 h. The product PLL-b-PPA was precipitated with an excess of diethyl ether to get a white solid and was dried in vacuum at room temperature for 48 h. 2.1.5. Measurements of the Block Copolymers. 1H NMR spectra were measured in D2O and in DMSO-d6 at room temperature (20 ( 1 °C) by an AV-400 NMR spectrometer from Bruker. Molecular weights and their distributions were determined by size exclusion chromatography (SEC) coupled with multiangle laser light scattering (MALLS). The system included a Styragel HMW6E column, a 515 HPLC pump, an IR OPTILAB DSP detector, and a DAWN EOS multiangle laser light scattering detector (Wyatt Technology). The MALLS operated at 18 angles with a vertically polarized He-Ne laser (laser wavelength of 690 nm). The absolute molecular weight was determined in 10 mmol/L LiBr in DMF at 30 °C at a flow rate of 1 mL/min. DSC was recorded on DSC Q100 instruments (TA) with N2 at a rate of 50 mL/min. The samples were scanned from 0 to 180 °C at a rate of 10 °C/min. 2.1.6. Preparation of Polymer Solutions. All copolymers were directly dissolved in water (pH
