Langmuir 1993,9, 945-949
945
Micelles Based on AB Block Copolymers of Poly(ethy1ene oxide) and Poly(&benzyl L-aspartate) G. Kwon,*7tJ M. Naito,tp! M. Yokoyama,t**T. Okano,tJ Y. Sakurai,tJ and K. Kataokatfs International Center for Biomaterials Science, Research Institute for Bioscience, Science University of Tokyo, Yamazaki 2669, Noda-shi, Chiba 278, Japan, Institute of Biomedical Engineering, Tokyo Women’s Medical College, Kawada-cho, Shinjuku-ku, Tokyo 162, Japan, and Department of Materials Science and Technology and Research Institute for Bioscience, Science University of Tokyo, Yamazaki 2641, Noda-shi, Chiba 278, Japan Received July 7,1992. In Final Form: November 23,1992
In aqueous systems, polymeric micelles based on AB block copolymers of poly(ethy1eneoxide) (PEO) and poly(j3-benzyl aspartate) (PBLA)were investigated. First, AB block copolymers were synthesized using amino-terminatedPEO to initiate the polymerization of B-benzyl a aspartate N-carboxy anhydride (BLA-NCA). The composition and molecular weights of the block copolymerswere established using 1H NMR. Micellar solutions of PEO-PBLA block copolymer were characterized by static and dynamic light scattering. Photophysical means were used to study the polymeric micelles. From changes in the fluorescence intensity and shifts in the excitation spectrum of pyrene upon micellization,critical micelle concentrations(cmc)of PEO-PBLA block copolymerswere obtained. T h e vibrationalstructure of pyrene monomer fluorescence was altered in PEO-PBLA micellar solutions consistent with low polarity within the PBLA core. In PEO-PBLA micellar solutions, 1,3-(1,l’-dipyrenyl)propaneintramolecular excimer emission,relative to monomer emission,was very weak, this indicatesvery low mobility of PBLA segments within the micellar core. Further evidence for the limited motion of the PBLA segments in the core was obtained by 1H NMR. This limited motion of the PBLA segments in the micellar core is in contrast to low molecular weight surfactants which commonly show a higher degree of motion within their cores.
Introduction Recently much interest has focused on the study of polymeric micelles formed in aqueous systems.’ While a considerable amount of research has been concentrated on polymericmicellizationin organicmedia,2 fewerstudies have been devoted to polymeric micellar formation in aqueoussystems. Our interest in polymericmicelles stems from their small size, apparent thermodynamic stability, and ability to deliver drugs selectively in vivo with low interactions with biocomponents (e.g., proteins, cells).3In these studies, a hydrophobic drug is covalently coupled to one segment of an AB block copolymer with subsequent micellar formation and retention of high anticancer activity.3 + International Center for Biomaterials Science, Research Institute for Bioscience, Science University of Tokyo. Institute of Biomedical Engineering, Tokyo Women’s Medical College. s Department of Materiale Science and Technology and Research Institute for Bioscience, Science University of Tokyo. (1) (a) Badar, H.; Ringsdorf, H.; Schmidt, B. Angew. Chem. 1984,123/ 124,457. (b) Turro, N. J.; Chung, C. Macromolecules 1984,17,2123. (c) Turro, N. J.; Kuo, P. J. Phys. Chem. 1986,90,4205. (d) Dowling, K. C.; Thomas, J. K. Macromolecules 1990,23,1059. (e) Zhao, C.; Winnik, M. A.; Riess, G.; Croucher, M. D. Langmuir 1990,6,514. (0 Xu, R.; Winnik, M. A.; Hallet, F. R.; Riees, G.; Croucher M. A. Macromolecules 1991,24, 87. (g) Wilhelm, M.; Zhao, C., Wang, Y.; Xu, R.; Winnik, M. A.; Mura, J.; Riess, G.; Croucher M. A. Macromolecules 1991,24,1033. (h) Xu, R.; Winnik, M. A.; Riese, G.; Chu, B.; Croucher M. Macromolecules 1992,25, 644. (i) Prochazka, K.; Kiserow, D.; Ramirreddy, C.; Tuzar, 2.; Munk, P.; Webber, S. E. Macromolecules 1992,25,454. (2) Tuuu, 2.; Kratochvil, P. Adu. Colloid Interface Sci. 1976,6,201. (3) (a) Yokoyama, M.; Inoue, S.; Kataoka, K.; Yui, N.; Okano, T.; Sakurai, Y. Makromol. Chem. 1989, 190, 2041. (b) Yokoyama, M.; Miyauchi, M.; Yamada, N.; Okano, T.; Sakurai, Y.; Kataoka, K.; Inoue, S . J. Controlled Release 1990,11,269. (c) Yokoyama, M.; Miyauchi, M.; Yamnda, N.; Okano, T.; Sakurai, Y.; Kataoka, K.; Inoue, S. Cancer Res. 1990,50,1693. (d) Yokoyama, M.; Okano, T.; Sakurai, Y.; Ekimoto, H.; Shibazaki, C.; Kataoka, K. Cancer Res. 1991,51, 3229. (e) Yokoyama, M.; Kwon, G. S.;Okano,T.; Sakurai, Y .;Seto, T.;Kataoka, K. Bioconjugate Chem., in press. (0Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai, Y. Submitted for publication in J. Controlled Release.
*
Drugs may be physically incorporated within polymeric micelles. Indeed, a host of hydrophobic solutes have been solubilized in the interior of polymeric micelles.4 To this end, polymeric micelles based on AB block copolymers of PEO and PBLA were prepared. PEO is a well-known biomedical polymer which expresses low toxicity5 and, when present at surfaces and interfaces, the ability to suppress cellular and protein adsorption.5~6The hydrophobic poly(amino acid) block would provide a biodegradable componentas well as the required amphiphilicity for micellization. In principle, a systematic alteration in the structure of PEO-poly(amino acid) block copolymers through polymerizationof varying amino acids or through side chain modifications is attainable. There are few examples of block copolymers which are soluble in water and form micelles. In such cases, the hydrophobic blocks are usually based on poly(styrene) (PSI or poly(propy1ene oxide) (PPO). lH NMR studies on micellar solutions of AB block copolymers based on PEO and PS suggest that the PS core is in a glassy state.7 This premise was further substantiated by recent experimental results using fluorescently-labeled AB block copolymers of poly(methacry1ic acid) and PS.” On the other hand, micellar solutions of AB block copolymers based on PEO and PPO exhibited lH NMR signals from the PPO block indicating the core of the micelle was in a liquidlike statea7a Reported viscosities for the interiors of PEO-PPO micelles, as determined by fluorescentprobes, are consistent with liquidlike cores.lb Of interest is the physical nature of the PEO-PBLA micellar core; knowledge about the micellar core is expectedto facilitate studies (4) Nagarajan, R.; Barry,M.; Ruckenstein, E. Langmuir 1986,2,210. (5) Merril, E. W.; Salzman E. W. ASAIO J. 1983, 6,60. (6) Jeon, S. I.; Lee J. H.; Andrade, J. D.; de Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (7) (a) Nakamura, K.; Endo, R.; Takeda, M.J. Polym. Sci., Polym. Phys. Ed. 1977,15,2095. (b) Bahadur,P.; Sastry, N. V.;Rao, Y. KO;R i w , G. Colloids Surf. 1988,29, 343.
0143-1463/93/2409-0945$04.00/00 1993 American Chemical Society
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946 Langmuir, Vol. 9, No.4, 1993
on drug solubilization by polymeric micelles and subsequent drug release characteristics. The PEO-PBLA block copolymers were synthesized using an amino-terminated PEO to initiate the polymerization of BLA-NCA. Micellar solutionsof the AB block copolymers were studied by light scattering, 'H NMR, and photophysical means. The studies demonstratePEOPBLA block copolymer micelle formation and further evidence that polymer chains within the cores of polymeric micelles may exhibit very low mobility.
Experimental Section The synthesis of PEO-PBLA has been detailed eleewhere.l Briefly, BLA-NCA was polymerized using u-methyl-w-aminoPEO (NOF Co., Japan) as an initiator to form the AB block copolymer. The PEO had molecular weights ranging from 2000 to 12 OOOas determined by vapor preeeure osmometry. The PEOPBLA block copolymer was precipitated in diethyl ether, collected, and dried under vacuum. 1H NMR spectra of PEO-PBLA were obtained in CDCh or D20 using a NMR instrument (Gemini 5005,Varian, USA) at 500 MHz. The concentration of the polymer in CDCh or D2O was 3.0 and 0.5% (w/v), respectively. The solubility of the block copolymers based on PEO-PBLA in water was determined. Polymericsolutions,ranging from 0.010 to 0.10% (w/v), were prepared and their solubility observed. Samples that were not soluble at 25 "C were heated to 60 "C to facilitate solubilization. All block copolymersexamined had PEO contents above 40 % by weight. The hydrodynamicradii of polymericmicelles were determined by dynamic light scattering measurements. A light scattering spectrophotometer (DLS-700,Photal, OtsukaElectronk~, Japan) equiped with a He-Ne laser was used at a wavelength of 633 nm. A scattering angle of 90" was used. The concentrations of the PEO-PBLA studied were 1.0 mg/mL for sample designated 5-10, andO.lOmg/mLforsamplesdesignated5-2Oand 12-20. Diffusion coefficients, D, were obtained from the average decay constants of the autocorrelation functions. The diameter, d, of the micelles was calculated using the Stokes-Einstein equation d = kT/3uvD (1) The Boltzmann constant, temperature, and viscosity of medium are denoted by k, T,and 7 , respectively. Static light scattering studies were carried out on PEO-PBLA in doubly distilled water. The concentrations of the PEO-PBLA ranged from 0.10 to 1.0 mg/mL. The scattering angles ranged from 40" to 130". Weight average molecular weights were established by a Zimm plot. The refractive index increments of the polymer were obtained at 25 OC using a double beam differential refractometer (DRM-1020, Otsuka Electronics, Japan). All light scattering experiments were done at 25 "C. Fluorescence measurements were carried out using pyrene (Wako Chemicals, Japan) and 1,3-dipyrenylpropane (Dojindo, Japan) as probes to analyze PEO-PBLA micelles in doubly distilled water. Emission and excitation spectra were measured at varying PEO-PBLA concentrations on a fluorometer (770F, JASCO, Japan). Probe dissolution was accompliihed by a protocol developed for the PEO-PS block copolymer.'# A pyrene concentration of 6.0 X 1V7M was used. Experiments with pyrene were done with excitation and emission wavelengths of 339 and 390 nm, respectively. Excitation and emission bandwidths were 3.0 and 1.5 nm, respectively. For 1,3-(l,l'-dipyrenyl)propane, experiments were done using a concentration of 2.0 x lV7 M. Excitation and emkion bandwidths were 5.0 and 5.0 nm, respectively. Excitation was carried out at 333 nm. The polymer solutions were deoxygenated with Nz prior to measurements, and experiments were done at 25 "C unless otherwise noted.
Results and Discussion The PEO-PBLA copolymer synthesis was achieved using a-methyl-o-amino-PEOas an initiator (Figure 1). The polymerization was assumed to proceed by the primary amine mechanism whereby the initiator undergoes nu-
b
b
Figure 1. Synthesis of PEO-PBLA block copolymers.
Table I. Properties of PEO-PBLA Block Copolymers and Micelles diameter cmc PEO sample (wt %)" MnO npm np(Bu)' (nm)b CmglW ZJL 5-10 73.0 7000 110 9.0 18,67 10 0.048 5-20 55.8 9100 110 19 17,108 5.0 0.033 12-20 75.0 18ooo 270 20 21,93 10 0.041 a Estimated bylH NMR. Determined by dynamiclight scattering, weight average.
*
cleophilic addition of the C5 carboxyl group of the NCA.* Earlier studies indicated no contamination of PEO homopolymer?" The composition of the PEO-PBLA copolymers was determined by 'H NMR spectroscopy in CDCb. The molar ratio of PEO and PBLA was calculated from the intensities of the characteristic peaks (Le., PEO, OCH2CH2, u = 3.6 ppm; PBLA, COOCH2C&, u = 5.0 ppm). Samples with low PBLA block lengths (ca. 120 BLA unite) and long PEO block (ca. 15OOO g/mol) were found to be soluble. Only sample designated 5-10showed appreciable water solubility (ca. 40 mg/mL). PEO-PBLA solutions exhibited two subpopulations of particles by dynamic light scattering (Table I). Experimente were carried out at least 1order of magnitude above the cmc of the polymers. The smaller particles were approximately 20 nm and arc attributed to individual PEO-PBLA micelles. The larger particles are thought to be micelles that further associated, but the poseibility of suspended impurities cannot be excluded. This distribution of particle sizes was noted for low molecular weight PEO-containing amphiphilese and later for polymeric micelles.lfJO Static light scattering studies were carried out for PEO-PBLA sample designated 5-10 in doubly distilled water. The micellar molecular weight was eetimated to be 2.8 X lo6 g/mol, which was a much elevated value compared to free polymer chain (ca. 7000 g/mol). This is consistent with the closed association of single polymer chains into micelles.2 Assuming a preponderance of polymer chains are in the micellar form, an association number of 400 was calculated for the polymeric micelle. The determinedmicellar molecular weight and association number will reflect contributions from individual micelles as well as secondary aggregates. In Figures 2 and 3 the emission and excitation spectrum of pyrene are shown in the presence of varying concentrations of PEO-PBLA, respectively. Pyrene will preferentially partition into hydrophobic microdomains (e.g., interior of micelles) with a concurrent change in the molecule's photophysical properties.ldJ*J1 An increase in the total fluorescent intensity and a change in the vibrational structure of monomer fluorescence were observed with increasing concentrations of PEO-PBLA ~
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(8) Goodman, M.;Hutchinson, J. J. Am. Chem. Soc. 1966,88,3627. (9) Tanford, C. The Hydrophobic Effect: Formation of Micclka and
Biological Membrane, 2nd ed.;John Wiley and Sons: New York, lW, Chapter 8.
(10) Khan,T.N.;Mobbe,R.H.;Price,C.;Quintana,J.R.;Stubberfield, R. B. Eur. Polym. J. 1987,23,191.
(11) Kalyanaeundaram, K.;Thomae,J. K. J. Am. Chem. SOC.1977,99, 2039.
Langmuir, Vol. 9, No. 4, 1993 947
Polymeric Micelles with Poly(amino acid) Core
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(Figure 2). A red shift was observed in the excitation spectrum with increasing concentrations of PEO-PBLA (Figure3). This shift in the excitation spectrum of pyrene was observed in the study of PEO-PS block copolymers in water and the spectral shift was used to determine cmc values of PEO-PS micelle systems.le Specifically,the (0,O) band for pyrene, which is at 334 nm in water, has been shown to shift to 339 nm upon addition of PEO-PS block copolymer. Total fluorescence intensity increases of a fluorescent probe upon micellization have been utilized to determine cmc for a host of surfactants12and for PEOPS.le Figure 4 illustrates plots of total fluorescence intensity and red shift in the excitation spectrum (i.e., 339 nm/334 nm ratio) as a function of logarithm PEO-PBLA concentration of different compositions. A t low concen~
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Figure 4. (a) Total fluorescence intensity of pyrene monomer emission versus logarithm of PEO-PBLA concentration. (b) Intensity ratio (339/334) of pyrene from excitation spectrum versus logarithm of PEO-PBLA concentration. The compositions of the PEO-PBLA block copolymers are given in Table I.
trations of PEO-PBLA, negligible changes in the total fluorescenceintensity and shifts in the excitationspectrum were observed. As the concentration of PEO-PBLA was increased, at a certain polymer concentration (i.e., cmc), the total fluorescence intensity and red shift in the excitation spectrum increased dramatically in a sigmoidal manner. A clear correlation between the two plots is seen; low cmc values for the PEO-PBLA polymers were determined (Table I). The PEO-PBLA block copolymer with a higher weight content of PBLA, 5-20, had a slightly lower cmc (ca. 5.0 mg/L) than other compositions. The low cmc values of PEO-PBLA block copolymers are consistentwith known low cmc values for polymeric micelle systems which cannot usually be determined by standard methods for cmc determination (e.g., surface tension, light scattering). As was noted earlier, there was a change in the vibrational structure of pyrene monomer emission upon micellization of PEO-PBLA (Figure 2). The vibrational structure of pyrene monomer emission is known to be dependent on local polarity (i.e., the Ham effect).1°J3 Specificallyfor pyrene, the ratio of the (0,O)band, I, to the intensity of the (0,2) band, 111,exhibits sensitivity toward local polarity. The preferential partitioning of pyrene into hydrophobic domains may be used to determine m i c r o p larities of molecular assemblies. In Figure 5, the dependence of the I/III band ratios upon logarithm PEO-PBLA (13) (a) Nakajima, A. Bull. Chem. SOC.Jpn. 1971,44,3272. (b) Dong, D. C.; Winnik, M. A. Can J . Chem. 1984,62,2560.
Kwon et al.
948 Langmuir, Vol. 9,No.4, 1993 1.9 1
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a 7 6 5 A 3 2 (ppml Figure 7. (a) lH NMR of PEO-PBLA in D20. (b) lH NMR of PEO-PBLA in CDCla. Sample is 5-10.
(14) (a) Fchariasse, K. A.; Vaz, W.; Sotomayor, C.; Kunhnle, W. Eiochrm. Brophys. Acta 1982,688, 323. (b) Dangreau, H.; Joniau, M.; Cuyper, M.; Hanssens, I. Biochemistry 1982,21,3594.
group of PBLA segment, respectively. The small, broad signalsindicate restricted motions of theee protons within the micellar core? On the other hand, these characteristics peaks of the PBLA segment are readily apparent by lH NMR in CDC13 where micelle formation is not expected (Figure 7). This suggests that the core of the polymeric micelle is a very rigid structure. This behavior of PEOPBLA micelles is in contrast with low molecular weight amphiphiles and PEO-PPO block copolymers which typically exhibit liquidlike cores and relatively higher mobility. In summary, evidence was presented which suggeststhat PEO-PBLA copolymers may associate in water to form micelles. First, the AB block copolymer based on PEOPBLA was synthesized and characterized by ‘HNMR.
~
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Polymeric Micelles with Poly(amino acid) Core
Light scattering studies of the PEO-PBLA suggest a bimodal particle size distribution; this was presumed to be due to individual micelles and secondary aggregates of micelles. Through the use of fluorescent probes, the cmc values of PEO-PBLA block copolymerswere determined. The polarity felt by pyrene in PEO-PBLA solutions waa found to be decreased relative to water consistent with micelle formation. Intramolecular excimer formation, relative to monomer emission, of 1,341,l'-dipyreny1)propane in PEO-PBLA micellar solutions indicates high viscosity within the micellar core consistent with low mobility of PBLA segments. This premise was further substantiated by lH NMR. The solubilization of hydro-
Langmuir, Vol. 9, No. 4, 1993 949
phobic molecules into low molecular weight, amphiphile micelles is a clear indication of the liquidlike nature of their cores, and it is of interest to examinethe solubilization of molecules into PEO-PBLA polymeric micelles. In this study, PEO-PBLA was used as a paradigm for watersoluble, amphiphilic AB block copolymers based on poly(amino acids); future studies will extend the number of examples of this class of amphiphiles and interesting structureJpropertyrelationships may be obtained.
Aoknowledgment. G.K.acknowledges support from the Japanese Society for the Promotion of Science postdoctoral fellowship program.
