Using anisotropic nanoparticles as building blocks for bottomup fabrication of devices and functional structures is a fascinating objective within the field of nanotechnology. Recently,anisotropic micelle of crystalline-coil copolymers have attracted much attention. The crystallization of a crystalline-coil block copolymer as the driving force is a facile method for preparing a variety of anisotropic nano-aggregates [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14]. The crystallization processes not only have important influence on the morphologies of the micelles from a crystalline-coil copolymer,but also are crucial to other properties,such as stability. In our previous works,the amphiphilic crystalline-coil block copolymer based on PPDO can self-assemble into anisotropic micelles with star anise-like morphology in water [13, 14, 15, 16, 17]. A ‘‘crystallization induced hierarchical assembly’’ mechanism was used for explaining the formation of the anisotropic micelles . This kind of anisotropic semicrystalline micelle with special morphology is also a very good candidate for constructing hierarchical nanostructures such as core-shell nanofibers . In many applications,e.g. biomedicine and drug delivery systems,the stability of micelles in water is a key issue; owing to the supramolecular nature of micelles,polymeric micelles and unimers are known to coexist at equilibrium above the critical micelle concentration (CMC) . It is very difficult to keep the polymeric micelles intact and stable when it is diluted within the biological media,where it is below the CMC or interacts with surfactant proteins. The extremely low polymer concentration or interaction with surfactant proteins could accelerate the dissociation of polymeric micelles under physiological conditions .
Recently,much effort has been devoted to improving stability of polymeric micelles. Cross-linking of polymeric micelles is a important strategy to increase particle stability. However,in many cases,crosslinking is performed utilizing covalent bonding within the micelle. The micelle is often achieved with an irreversible stabilization mechanism. Moreover,it is relatively difficult to control the cross-linking process,which does not lend itself to tunable micellar properties. In addition,in some crosslinked micelles,the crosslinks are physically located in the core of the micelle where drugs are loaded,which may interfere with pharmaceutical drug action or drug release from the micelle . It is found that the modification of core-forming blocks of amphiphilic copolymers with hydrophobic alkyl chains or aromatic moieties can enhance stability of polymeric micelles [22, 23]. However,relatively large amounts of the hydrophobic moieties, which may inevitably change the nature of the copolymer,were needed for enhancing the micelle stability. Thus,stability of polymeric micelles used in biological media remains an issue that needs to be settled. In this work,we developed a novel strategy for enhancing the stability of semicrystalline micelles based on amphiphilic diblock copolymers of PPDO by introducing pyrene moieties at the chain end of PPDO blocks. The aggregation of pyrene moieties induced by π-π interactions obviously enhances the micelle stability and crystallization rate of the semicrystalline micelles of the Py-PPDO-b-PEG diblock copolymer because it may serve as a heterogeneous nucleation agent for crystallization of PPDO blocks. 2. Experimental
PDO (99.9%) with a melting point of 25 ℃ was provided by the Pilot Plant of the Center for Degradable and Flame-Retardant Polymeric Materials (Chengdu,China),and dried over CaH2for 48 h and distilled under reduced pressure before use. Poly(ethylene glycol) monomethyl ether (MPEG,degree of polymerization = 12, 16,45,Mn= 0.55 kDa,0.75 kDa,2 kDa) were purchased from Sigma-Aldrich. 1-Pyrenemethanol was purchased from Alfa Aesar. Stannous octoate (SnOct2) (95%) was purchased from Sigma- Aldrich,and was stored in glass ampoules under nitrogen after dilution with anhydrous toluene. Nile Red (analytical grade) was purchased from Sigma-Aldrich. Polyethylene glycol-block-poly (p-dioxanone) diblock copolymers functionalized with (Py-PPDOb-PEG) or without pyrene moieties (PPDO-b-PEG) were synthesized. Other reagents with AR grade were purchased from Aladdin Reagent Company (Shanghai,China) and Bodi Chemical Factory (Tianjin,China) and were used after removing water. 2.1. Preparation of Py-PPDO-b-PEG copolymer The Py-PPDO-b-PEG copolymer was synthesized by a coupling approach reported before [10, 24]. The synthesis process is described briefly as follows. First,the hydroxyl-terminated Py-PPDO precursor was prepared under nitrogen by typical bulk ring-opening polymerization of PDO using 1-pyrenemethanol as an initiator,using Sn(Oct)2 as catalyst. The light yellow powder Py-PPDO-OH precursor was obtained. The MPEG-COOH was prepared by the acylation of the hydroxyl-terminated MPEG with succinic anhydride (SA) using DMAP and TEA as the catalysts. The white powder mPEG end-hydroxyl group (MPEG-COOH) was obtained. The Py-PPDO-b-PEG copolymer was synthesized using DCC as a coupling agent. The synthesis route of Py-PPDO-b-PEG is illustrated in Scheme 1.
For comparison,the PPDO-b-PEG copolymer without a pyrene moiety was prepared under nitrogen by typical bulk ring-opening polymerization of PDO using MPEG-OH as an initiator,using Sn(Oct)2 as catalyst. The synthesis route of PPDO-b-PEG is illustrated in Scheme 2.2.2. Characterization The micellar solutions of copolymers with different compositions were prepared by the direct dissolution method. The Py-PPDO-bPEG copolymer or PPDO-b-PEG copolymer was added directly to water at a certain concentration at room temperature. Then thermal treatment was performed at approximate 95 ℃ for 5 min to erase all previous thermal history. Afterwards,the temperature of system was maintained at a certain temperature for 24 h.
The 1H NMR spectra were measured at 25 ℃ using an Avance Bruke-II NMR spectrometer under 400 MHz (Bruker,Germany) using CDCl3 as solvent and tetramethylsilane (TMS) as internal standard.
Gel permeation chromatography (GPC). The molecular weight distribution was measured using a Waters GPC device equipped with a 1515 pump,a Waters model 717 auto sampler,and a 2414 refractive index detector. Chloroform was used as the eluent with flow rate 1.0 mL min-1 at 30 ℃. A calibration curve was obtained using monodisperse polystyrene standards.
The CMCs of the block copolymers in aqueous solution were determined according to reported procedures [25, 26], employing hydrophobic Nile Red as the probe. The CMC experiments are performed as followed: a stock solution of Nile Red (4 × 10 -4 mol L -1 ) in acetone is prepared and stored chilled and protected from light. The Nile Red-loaded micelles samples were diluted with a final Nile Red concentration of 8 × 10 -6 mol L -1 and the micelle concentrations between 2 × 10 -5 and 0.1 mg mL-1 in deionized water. The micelle stability experiments is performed as followed: The same amount of Nile Red-loaded Py-PPDO-b-PEG and PPDO-b-PEG micelles samples diluted with the final micelle concentration of 0.2 mg mL-1 in deionized water. Nile Red-loaded micelles samples incubated in a shaking water bath at 25 ℃ for several months. Fluorescence emission spectra were carried out using a HORIBA JOBINYVON Fluormax-4 spectrofluorometer at room temperature at an excitation wavelength of 310 nm and the emission from 590 to 700 nm. The emission and excitation slit widths were both set to 5 nm.
The copolymer aqueous solution was heated to 95 ℃ and kept for 5 min and then cooled to different temperatures (25 ℃,40 ℃, 50 ℃ and 60 ℃) using a PolyScience temperature controller as the control. The transmittance of copolymers with different composition micelles solutions at the concentration of 1 mg mL-1 were measured with increasing time by using a HITACHI U-1900 spectrophotometer (600 nm) with a temperature-controlled quartz sample cell holder.
The averaged hydrodynamic diameter (Dh) of the polymeric solutions with a concentration of 1 mg mL-1 in water were determined with increasing time by using dynamic light scattering (DLS) on a Brookhaven model BI-200SM spectrometer and a 9000AT correlator using an Innova 304 He-Ne laser (1 W,532 nm) at a fixed scattering angle (u) of 90 ℃.
Differential scanning calorimetry (DSC). Thermal effects of a complete cycle of copolymer with different composition micelles in aqueous solutions were studied with a NANO DSC of TA Instruments-Waters LLC,New Castle,DE at an external pressure of 3.0 atm using deionized water as an external reference. The cell volume was 0.33 mL. The heating rate was 1 ℃ min-1 .
Transmission electron microscopy (TEM). The morphology of the Py-PPDO-b-PEG micelle was observed by Transmission Electron Microscope (TEM,Tecnai G2 F20 S-TWIN electron microscope (FEI Co.) operated at an acceleration voltage of 200 kV. In the TEM study,the micelles were collected onto a copper grid for about 10 min at room temperature,and then recorded after staining by 2% OsO4solution for 4 min. 3. Results and discussion 3.1. Synthesis of Py-PPDO-b-PEG and PPDO-b-PEG The synthesis of a hydroxyl-terminated Py-PPDO precursor was prepared by typical bulk ring-opening polymerization of PDO using pyrenemethanol as an initiator and Sn(Oct)2 as catalyst. 1H NMR (400 MHz,CDCl3): δ 8.16-8.29 (m,1H,CHCHC or CHCHCH), 5.93 (s,2H,CHCH2C),4.34 (m,2H,CH2CH2OC(≡0O)),4.16 (s,2H, OC(≡0O)CH2O),4.10 (s,2H,CHCH2OC(≡0O)CH2O),3.78 (s,2H, CH2CH2OC(≡0O)),3.65-3.75 (m,2H,OCH2CH2OH),3.65-3.75 (m,2H,OCH2CH2OH) (Fig. 1).
|Fig. 1. 1 H NMR spectra of methoxyl-PEG-COOH (MPEG-COOH) (A),Py-PPDO-OH (B), and Py-PPDO-b-PEG (C) in CDCl3,respectively.|
The degree of polymerization (Dp) of Py-PPDO-OH was calculated by 1H NMR using following equation:
The Mn,NMRof Py-PPDO-OH was calculated by 1H NMR using following equation:
MPEG-COOH was prepared by the acylation of the mPEG endhydroxyl group with succinic anhydride using DMAP and TEA as catalysts according to the procedure widely reported previously. MPEG-COOH precursor: 1H NMR (400 MHz,CDCl3): δ 4.25 (t,2H, CH2C(≡0)OCH2CH2),3.65 (s,4H,OCH2CH2O),3.38 (s,3H, CH2CH2OCOCH3),2.65 (m,4H,C(≡0)CH2CH2COOH). The carboxyl group of mPEG and hydroxyl group of PPDO with an OH/COOH mole ratio of 1:1 was connected by a moderate esterification reaction in the presence of DCC as dehydrant. Py-PPDO-b-PEG copolymer: 1H NMR (400 MHz,CDCl3): δ 8.16-8.29 (m,1H,CHCHC or CHCHCH), 5.93 (s,2H,CHCH2C),4.33 (t,2H,CH2CH2OC(≡0)CH2O),4.30 (t,2H, OCH2CH2OC(≡0)CH2CH2),4.24 (t,2H,CH2C(≡0)CH2CH2),4.20 (s,2H,OC(≡0)CH2O),4.10 (s,2H,CHCH2OC(≡0)CH2O),3.78 (s,2H, CH2CH2OC(≡0)),3.64 (s,4H,OCH2CH2O),3.37 (s,3H,CH2CH2OCH3), 2.65 (s,4H,C(≡0) CH2CH2COOCH2). The Mn,NMR of Py-PPDO-b-PEG copolymer was calculated by 1H NMR as follows:
The PPDO-b-PEG copolymer was prepared by typical bulk ringopening polymerization of PDO using MPEG as an initiator and Sn(Oct)2 as catalyst. 1H NMR (CDCl3,400 MHz): δ 3.40 (s,CH3OCH2CH2O-),3.66 (m,-OCH2CH2O-),3.76 (t,COCH2OCH2-CH2OH),3.82 (t,-COCH2OCH2CH2O-),4.20 (s,-COCH2OCH2CH2O-), 4.32 (t,-CH2CH2OCO-),4.37 (t,-COCH2OCH2CH2O-) (Fig. 2).
The degree of polymerization (DP) of PPDO block was then calculated as follows:
The copolymers were successfully synthesized and characterized by 1H NMR spectroscopy and GPC experiment (Fig. 3),the results were listed in Table 1. The copolymers were recorded as Py-PPDOxb-PEGy,where x and y represent the degree of polymerization of PPDO and PEG blocks,respectively. Meanwhile,in comparison to Py-PPDO6.5-b-PEG16,the control sample of the PPDO-b-PEG copolymer without a pyrene group has similar molecular weight to that of Py-PPDO6.5-b-PEG16,and was recorded as PPDO7-b-PEG16.
The successful coupling of the Py-PPDO precursor and MPEGCOOH was verified by the disappearance of the resonance of Py-PPDO-CH2OH (3.65-3.75 ppm) and an increase of molecular weight in the GPC experiment. Moreover,it can be found that the number average molecular weight of the Py-PPDO precursor and the copolymer obtained from 1H NMR both showed good accordance with the value estimated by GPC.
|Fig. 3.The GPC traces of (A) Py-PPDO6.5-OH,(B) PPDO7-b-PEG16,(C) Py-PPDO6.5-bPEG12,(D) Py-PPDO6.5-b-PEG16,(E) Py-PPDO6.5-b-PEG45in CHCl3.|
|Fig. 4.The nano DSC cooling curves (A) and subsequent heating curves (B) of Py-PPDO6.5-b-PEG16,PPDO7-b-PEG16copolymers in water after erasing the thermal history at 95 ℃|
The micellization kinetics of PPDO-b-PEG copolymers with or without pyrene moieties under different temperature were monitored by a UV-Vis spectrometer. After heating treatment at 95 ℃,all PPDO crystalline were melted and the copolymers were homogeneously dispersed in water. When cooled to certain temperatures, the transmittance gradually decreased. The micellization of copolymers and the crystallization of PPDO blocks in micelles,especially the latter,are responsible for the decrease in transmittance. As showed in Fig. 5,the temperature plays a key role in determining of crystallization and micellization of the copolymer because supercooling is needed for homogeneous nucleation. Compared to the PPDO7-b-PEG16copolymer without pyrene moieties,the Py-PPDO6.5-b-PEG16showed much faster decreasing rate of transmittance under same temperature. Meanwhile,the equilibrium transmittance of Py-PPDO6.5-b-PEG16after a long time of deposition at a certain temperature (e.g. 25 ℃ and 40 ℃) are also much lower than those of PPDO7-b-PEG16. These phenomena suggest that the copolymer functionized with pyrene moieties may have stronger micellization ability even at high temperatures and faster crystallization rate of PPDO blocks in micelles than those of copolymer without pyrene moieties. These results also proved that the pyrene moieties could promote the crystallization of the PPDO blocks in micelles.
|Fig. 5.The variation of transmittance versus time from 1 mg mL-1 (A) Py-PPDO6.5-b-PEG16; (B) PPDO7-b-PEG16 aqueous dispersions at different temperatures after erasing the thermal history at 95 ℃.|
Fig. 6 shows the variation of micelle diameter at different temperatures after heating treatment at 95 ℃. As showed in Fig. 6, the Py-PPDO-b-PEG copolymer can self-assemble into micelles in water in very short time. When micellization occurs at 60 ℃, micelles with relatively small size (about 100 nm) are formed. As shown in the TEM image of Py-PPDO6.5-b-PEG16micelles (Fig. 7A), the copolymer formed isotropic spherical micelles suggested that the PPDO blocks did not crystallize at this temperature. Moreover, some black dots were observed in the micelles which may be attributed to the aggregation of pyrene moieties. In comparison,no micelle was observed for PPDO7-b-PEG16copolymer during DLS and TEM tests,indicating that the interaction between pyrene moieties may also promote the micellization of Py-PPDO-b-PEG copolymer at high temperature (60 ℃). The micellization at 50 ℃ led to a slightly smaller Dh of Py-PPDO6.5-b-PEG16micelles than at 60 ℃,while some anisotropic flake-like morphology of these micelles was observed in TEM image (Fig. 7B),suggesting that the PPDO blocks had already crystallized at this temperature (50 ℃). This promotion of crystallization of PPDO blocks at relatively high temperature could be attributed to the aggregation of pyrene moieties induced by π-π interactions,which may serve as the heterogeneous nucleation agent for PPDO blocks. For micellization at low temperature (25 ℃ and 40 ℃),the Dhof Py-PPDO-b-PEG micelles gradually increased and reached equilibrium after several hours,and the obtained micelles had relatively large size and star anise-like morphology (Fig. 7C and D). This phenomenon could be explained by a crystallization induced hierarchical assembly mechanism .
|Fig. 6.The variation of average hydrodynamic diameter of 1 mg mL-1 Py-PPDO6.5-b-PEG16micelle solutions at different temperatures as a function of time.|
|Fig. 7.TEM images of Py-PPDO-b-PEG micelles obtained at different temperatures: (A) 60 ℃,(B) 50 ℃,(C) 40 ℃,and (D) 25 ℃.|
|Fig. 8.(A) Flurescence emission spectra of Nile Red (λex= 310 nm) in aqueous solutions of Py-PPDO6.5-b-PEG16at different concentrations. (B) Plot of intensity at 621 nm as a function of concentration of Py-PPDO6.5-b-PEG16.|
In addition,the stability of Py-PPDO-b-PEG micelle solution was also evaluated by monitoring the variation of fluorescent intensity of Nile Red probe in micelle solutions. Nile Red is nearly insoluble in water and becomes nonfluorescent within 20-25 min. In the presence of micelles,Nile Red can transfer into the hydrophobic phase and remain very stable . Fig. 9 shows the fluorescence intensity at 621 nm of fluorescent emission spectra of copolymers micelle solutions as a function of time. It has been clearly seen that the fluorescent intensity increased quickly at the initial stage of the measurement,suggesting that the Nile Red probe gradually transferred from a water environment into the hydrophobic core of micelles. After achieving equilibrium,the fluorescence intensity of Nile Red in Py-PPDO-b-PEG micellar solution almost remained unchanged even after four months,indicating that the Py-PPDOb-PEG micelles can keep stable for a long time. In comparison,the fluorescence intensity of Nile Red in PPDO-b-PEG micelle decreased with time. The improved micelle stability of Py-PPDO-b-PEG copolymer could be attributed to the aggregation of pyrene moieties induced by intermolecular interactions. Pyrene is one of the few condensed aromatic hydrocarbons with strong hydrophobicity and it tends to aggregate in water medium even when the temperature was higher than the melting temperature of PPDO blocks. These aggregated pyrene moieties may also serve as heterogeneous nucleation agent and promote the crystallization of PPDO blocks with low supercooling degree. Therefore,the Py-PPDO-b-PEG micelles showed much higher stability than those of PPDO-b-PEG copolymer without pyrene moieties.
|Fig. 9.Flurescence emission spectra of Nile Red (λex= 310 nm) in aqueous solutions of Py-PPDO6.5-b-PEG16,PPDO7-b-PEG16 at different time.|
|||W.N. He, J.T. Xu, Crystallization assisted self-assembly of semicrystalline block copolymers, Prog. Polym. Sci. 37 (2012) 1350-1400.|
|||P.A. Rupar, L. Chabanne, M.A. Winnik, I. Manners, Non-centrosymmetric cylindrical micelles by unidirectional growth, Science 337 (2012) 559-562.|
|||H. Qiu, G. Cambridge, M.A. Winnik, I. Manners, Multi-armed micelles and block co-micelles via crystallization-driven self-assembly with homopolymer nanocrystals as initiators, J. Am. Chem. Soc. 135 (2013) 12180-12183.|
|||N. Petzetakis, A.P. Dove, R.K. O'Reilly, Cylindrical micelles from the living crystallization-driven self-assembly of poly(lactide)-containing block copolymers, Chem. Sci. 2 (2011) 955-960.|
|||N. Petzetakis, D. Walker, A.P. Dove, R.K. O'Reilly, Crystallization-driven sphere-to-rod transition of poly(lactide)-b-poly(acrylic acid) diblock copolymers: mechanism and kinetics, Soft Matter 8 (2012) 7408-7414.|
|||J. Schmelz, M. Karg, T. Hellweg, H. Schmalz, General pathway toward crystallinecore micelles with tunable morphology and corona segregation, ACS Nano 5 (2011) 9523-9534.|
|||Y. Zhao, X. Shi, H. Gao, et al., Thermo-and pH-sensitive polyethylene-based diblock and triblock copolymers: synthesis and self-assembly in aqueous solution, J. Mater. Chem. 22 (2012) 5737-5745.|
|||Z.Y. Li, R. Liu, B.Y. Mai, et al., Temperature-induced and crystallization-driven selfassembly of polyethylene-b-poly(ethylene oxide) in solution, Polymer 54 (2013) 1663-1670.|
|||D.D. Yao, Y.J. Guo, S.G. Chen, J.N. Tang, Y.M. Chen, Shaped core/shell polymer nanoobjects with high antibacterial activities via block copolymer microphase separation, Polymer 54 (2013) 3485-3491.|
|||A.M. Mihut, J.J. Crassous, J.F. Dechézelles, et al., Towards smart self-assembly of colloidal silica particles through diblock copolymer crystallization, Polymer 54 (2013) 3874-3881.|
|||W.N. He, B. Zhou, J.T. Xu, B.Y. Du, Z.Q. Fan, Two growth modes of semicrystalline cylindrical poly(e-caprolactone)-b-poly(ethylene oxide) micelles, Macromolecules 45 (2012) 9768-9778.|
|||L.G. Yin, M.A. Hillmyer, Disklike micelles in water from polyethylene-containing diblock copolymers, Macromolecules 44 (2011) 3021-3028.|
|||S.C. Chen, G. Wu, J. Shi, Y.Z. Wang, Novel "star anise"-like nano aggregate prepared by self-assembling of preformed microcrystals from branched crys-talline-coil alternating multi-block copolymer, Chem. Commun. 47 (2011) 4198-4200.|
|||H. Wang, C.L. Liu, G. Wu, et al., Temperature dependent morphological evolution and formation mechanism of anisotropic nano aggregates from crystalline-coil block copolymer of poly(p-dioxanone) and poly (ethylene glycol), Soft Matter 9 (2013) 8712-8722.|
|||G. Wu, S.C. Chen, X.L. Wang, et al., Dynamic origin and thermally induced evolution of new self-assembled aggregates from an amphiphilic comb-like graft copolymer: a multiscale and multimorphological procedure, Chem. Eur. J. 18 (2012) 12237-12241.|
|||F. Song, W.T. Shi, X.T. Dong, et al., Fennel-like nanoaggregates based on polysaccharide derivatives and their application in drug delivery, Colloids Surf. B 113 (2014) 501-504.|
|||S.C. Chen, L.L. Li, H. Wang, et al., Synthesis and micellization of amphiphilic multibranched poly(p-dioxanone)-block-poly(ethylene glycol), Polym. Chem. 3 (2012) 1231-1238.|
|||F.Y. Zhai, W. Huang, G. Wu, et al., Nanofibers with very fine core-shell morphology from anisotropic micelle of amphiphilic crystalline-coil block copolymer, ACS nano 7 (2013) 4892-4901.|
|||G. Riess, Micellization of block copolymers, Prog. Polym. Sci. 28 (2003) 1107-1170.|
|||T.C. Lai, H. Cho, G.S. Kwon, Reversibly core cross-linked polymeric micelles with pH-and reduction-sensitivities: effects of cross-linking degree on particle stability, drug release kinetics, and anti-tumor efficacy, Polym. Chem. 5 (2014) 1650-1661.|
|||J. RiosDoria, A. Carie, T. Costich, et al., A versatile polymer micelle drug delivery system for encapsulation and in vivo stabilization of hydrophobic anticancer drugs, J. Drug Deliv. 2012 (2012) (Article ID 951741).|
|||M. Harada, I. Bobe, H. Saito, et al., Improved anti-tumor activity of stabilized anthracy-cline polymeric micelle formulation, NC-6300, Cancer Sci. 102 (2011) 192-199.|
|||P. Opanasopit, M. Yokoyama, M. Watanabe, et al., Block copolymer design for camptothecin incorporation into polymeric micelles for passive tumor targeting, Pharm. Res. 21 (2004) 2001-2008.|
|||X.L. Wang, Y.R. Mou, S.C. Chen, et al., A water-soluble PPDO/PEG alternating multiblock copolymer: synthesis, characterization, and its gel-sol transition behavior, Eur. Polym. J. 45 (2009) 1190-1197.|
|||L. Yan, W. Wu, W. Zhao, et al., Reduction-sensitive core-cross-linked mPEG-poly (ester-carbonate) micelles for glutathione-triggered intracellular drug release, Polym. Chem. 3 (2012) 2403-2412.|
|||C.J. Chen, Q. Jin, G.Y. Liu, et al., Reversibly light-responsive micelles constructed via a simple modification of hyperbranched polymers with chromophores, Polymer 53 (2012) 3695-3703.|
|||M. Krishna, Excited-state kinetics of the hydrophobic probe Nile Red in membranes and micelles, J. Phys. Chem. A 103 (1999) 3589-3595.|