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Synthesis of Well-Defined Amphiphilic Block Copolymers Having Phospholipid Polymer Sequences as a Novel Biocompatible Polymer Micelle Reagent Shin-ichi Yusa,*,† Kenichi Fukuda,† Tohei Yamamoto,† Kazuhiko Ishihara,‡ and Yotaro Morishima§ Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan, Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, and Faculty of Engineering, Fukui University of Technology, 6-3-1 Gakuen, Fukui 910-8505, Japan Received August 2, 2004; Revised Manuscript Received November 15, 2004
To realize safer and effective drug administration, novel well-defined and biocompatible amphiphilic block copolymers containing phospholipid polymer sequences were synthesized. At first, the homopolymer of 2-methacryloyloxyethylphosphorylcholine (MPC) was synthesized in water by reversible additionfragmentation chain transfer (RAFT) controlled radical polymerization. The “living” polymerization was confirmed by the fact that the number-average molecular weight increased linearly with monomer conversion while the molecular weight distribution remained narrow independent of the conversion. The poly(MPC) thus prepared is end-capped with a dithioester moiety. Using the dithioester-capped poly(MPC) as a macro chain transfer agent, AB diblock copolymers of MPC and n-butyl methacrylate (BMA) were synthesized. Associative properties of the amphiphilic block copolymer (pMPCm-BMAn) with varying poly(BMA) block lengths were investigated using NMR, fluorescence probe, static light scattering (SLS), and quasi-elastic light scattering (QELS) techniques. Proton NMR data in D2O indicated highly restricted motions of the n-butyl moieties, arising from hydrophobic associations of poly(BMA) blocks. Fluorescence spectra of N-phenyl-1-naphthylamine indicated that the probes were solubilized in the polymer micelles in water. The formation of polymer micelles comprising a core with poly(BMA) blocks and shell with hydrophilic poly(MPC) blocks was suggested by SLS and QELS data. The size and mass of the micelle increased with increasing poly(BMA) block length. With an expectation of a pharmaceutical application of pMPCm-BMAn, solubilization of a poorly water-soluble anticancer agent, paclitaxel (PTX), was investigated. PTX dissolved well in aqueous solutions of pMPCm-BMAn as compared with pure water, implying that PTX is incorporated into the hydrophobic core of the polymer micelle. Since excellent biocompatible poly(MPC) sequences form an outer shell of the micelle, pMPCm-BMAn may find application as a promising reagent to make a good formulation with a hydrophobic drug. Introduction Phospholipids are a component of lipid bilayers comprising cell membranes, which are of interest in biological and biomedical fields as unique substrates.1,2 The lipid bilayer is a molecular assembly formed from phospholipids based on their amphiphilic nature in aqueous media. Numerous studies have been reported on the stabilization of phospholipid assemblies. One of the useful methods to realize this goal is to polymerize phospholipids moieties. 2-Methacryloyloxyethylphosphorylcholine (MPC) is an excellent monomer from the viewpoint of polymerization ability.3,4 MPC can be polymerized with other vinyl compounds, and the properties and functions of MPC polymers can be controlled by changing comonomers. Also, sequence distributions of * To whom correspondence should be addressed. E-mail: yusa@ eng.u-hyogo.ac.jp. † University of Hyogo. ‡ The University of Tokyo. § Fukui University of Technology.
monomer units along the polymer chain is another factor to determine the property and functionality of the polymers. The polymers with water-soluble MPC units showed extremely high biocompatibility and antithrombogenicity. Amphiphilic random copolymers of MPC and n-butyl methacrylate (BMA) were prepared by ordinary radical copolymerization,5 and are of interest as potential materials for drug carriers because aggregates of the copolymers can solubilize hydrophobic compounds into hydrophobic microdomains formed in the polymer aggregates in aqueous solutions.6 The MPC units gave very high water solubility; i.e., even when the content of the BMA unit in the copolymer is 70 mol %, the polymer is soluble in water. In general, the size of hydrophobic microdomains formed from amphiphilic random copolymers is much smaller than hydrophobic cores in polymer micelles formed from amphiphilic block copolymers. Therefore, amphiphilic block copolymer micelles can take up hydrophobic small molecules much more in quantity than random copolymers can. Furthermore, small molecules
10.1021/bm0495553 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/08/2005
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Chart 1. Chemical Structure of pMPCm-BMAn
Yusa et al.
pMPCm-BMAn and its solubilizing ability for poorly soluble compounds were investigated. Experimental Section
incorporated into block copolymer micelles can be much better protected from the bulk phase than those taken up by random copolymer aggregates. Therefore, block copolymers with well-defined poly(MPC) and poly(BMA) block lengths should be of interest, in comparison with random copolymers, from a practical point of view, because hydrophobic cores may be confined deep in an assembly of hydrophilic phosphorylcholine groups, hence yielding polymer micelles with excellent blood compatibility. In recent years, much effort has been focused on the development of “living” free radical polymerization methods which are useful for the syntheses of homopolymers and block copolymers with controlled molecular weight and narrow molecular weight distribution. Nitroxide-mediated polymerization (NMP),7,8 metal-catalyzed atom transfer radical polymerization (ATRP),9,10 and reversible additionfragmentation chain transfer (RAFT) radical polymerization11 are among these methods. In particular, ATRP and RAFT radical polymerization methods can be easily applied to various monomers in various solvents. Armes et al.12-14 have reported that MPC can be polymerized by ATRP in water and in methanol, and that well-defined homopolymers and block copolymers of MPC can be obtained. However, metal catalysts must be removed with silica gel from the obtained MPC-based polymers for biomedical applications, and the complete removal of the ATRP metal catalyst from the MPC polymers should be very difficult. RAFT-controlled radical polymerization is a metal-free method with the use of a dithioester chain transfer agent (CTA) added to an ordinary free radical polymerization system. As reported by McCormick et al.,15 methacrylic-based sulfobetaine monomers can be polymerized in aqueous solution by the RAFT process, and the obtained homopolymers with a dithioester chain end functionality can be used as a macro-CTA for the preparation of AB block copolymers. Recently, Stenzel et al.16 reported the preparation of amphiphilic block copolymers composed of phosphorylcholine acrylate and butyl acrylate by RAFT polymerization. From a practical point of view, methacrylate polymers should be more desirable than acrylate polymers because the former is much more stable than the latter toward spontaneous hydrolysis in aqueous media. In this paper, we report on the synthesis and some associative properties of amphiphilic block copolymers composed of poly(MPC) and poly(BMA) sequences (pMPCmBMAn) (Chart 1) in aqueous media. We first synthesized poly(MPC) by RAFT radical polymerization in water and subsequently used the polymer as a macro-CTA for the block copolymerization with BMA. The association behavior of
Materials. MPC was synthesized as previously reported and recrystallized from acetonitrile.3 n-Butyl methacrylate (BMA) was dried over 4 Å molecular sieves and purified by distillation under reduced pressure. Random copolymers composed of MPC and BMA (poly(MPC-r-BMA)) were synthesized by a conventional radical polymerization and purified by the method reported previously.5 In this study, poly(MPC-r-BMA)s with MPC mole fractions 0.80 and 0.50 were used. 4-Cyanopentanoic acid dithiobenzoate was synthesized according to the method reported by McCormick and co-workers.17 N-Phenyl-1-naphthylamine (PNA) was purified by recrystallization from methanol. Paclitaxel (PTX) was obtained from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Water was purified with a Millipore Milli-Q system. Other reagents were used as received. Homopolymerization of MPC by RAFT. MPC (47.1 g, 159 mmol) was dissolved in 275 mL of water, and then 4-cyanopentanoic acid dithiobenzoate (650 mg, 2.16 mmol) and 4,4′-azobis(4-cyanopentanoic acid) (75.0 mg, 0.268 mmol) were added to the solution. The mixture was degassed by purging with Ar gas for 30 min. Polymerization was carried out at 70 °C for 2 h. The reaction mixture was dialyzed against pure water for 1 week; the pure water was changed twice a day. The polymer was recovered by freezedrying (41.5 g, 88.1% conversion). The obtained MPC homopolymer could be used as a macro-CTA to prepare block copolymers. To investigate the relationship between polymerization time and conversion, monomer conversion was determined by 1H NMR spectroscopy. Predetermined amounts of MPC, 4-cyanopentanoic acid dithiobenzoate, and initiator were dissolved in D2O. This solution was transferred to several NMR tubes and deoxygenated by purging with Ar gas for 30 min. After deoxygenation, the cap was sealed and the solutions were heated at 70 °C in a preheated oil bath for varying lengths of time. The polymerization was terminated by rapid cooling with an ice bath. The monomer conversion was monitored as a function of polymerization time. Gelpermeation chromatography (GPC) for the reaction mixture was measured to estimate the number-average molecular weight (Mn), weight-average molecular weight (Mw), and molecular weight distribution (Mw/Mn). Block Copolymerization of MPC with BMA. A typical procedure for block copolymerization is as follows. MPC macro-CTA (2.30 g, Mw ) 2.84 × 104; Mw/Mn ) 1.27), BMA (1.33 g, 9.35 mmol), and 4,4′-azobis(4-cyanopentanoic acid) (5.91 mg, 0.0210 mmol) were dissolved in 24.4 mL of methanol. The solution was placed in a glass ampule and outgassed on a vacuum line by six freeze-pump-thaw cycles, and then the ampule was vacuum-sealed. Polymerization was carried out at 70 °C for 24 h. The reaction mixture was poured into a large excess of diethyl ether to precipitate the resulting polymer. The polymer was purified by reprecipitating from methanol into a large excess of
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diethyl ether (3.18 g, 66.2% conversion). The method to determine the relationship between polymerization time and monomer conversion was similar to that described above. Measurements. Gel-permeation chromatography (GPC) was performed with a JASCO GPC-900 equipped with a refractive index (RI) detector and two sets of Tosoh TSKgel R-M columns working at 40 °C under a flow rate of 1.0 mL/min. A mixed solvent of a 0.1 M NaNO3 aqueous solution and acetonitrile (8/2 v/v) was used as eluent. Molecular weights of the sample polymers were calibrated with standard sodium poly(styrenesulfonate) samples of 11 different molecular weights ranging from 1.37 × 103 to 2.61 × 106. 1 H and 13C NMR spectra were obtained with a Bruker DRX-500 spectrometer. The sample solutions of the block copolymers at a polymer concentration (Cp) of 5.0 g/L for 1 H NMR measurements were prepared in D2O containing 0.1 M NaCl and methanol-d4. Fluorescence spectra were recorded on a Hitachi F-2500 fluorescence spectrophotometer. Sample solutions were prepared by mixing aliquots of stock solutions of the polymer and PNA (1.0 × 10-6 M) in 0.1 M NaCl aqueous solutions. The sample solutions were excited at 340 nm. Excitation and emission slit widths on the spectrometer were maintained at 20 and 5.0 nm, respectively. Static light scattering (SLS) and quasi-elastic light scattering (QELS) measurements were performed at 25 °C with an Otsuka Electronics Photal DLS-7000DL light scattering spectrometer equipped with an ALV-5000E multi-τ digital time correlator. Sample solutions for SLS and QELS measurements were filtrated with a 0.45-µm pore size membrane filter. For SLS measurements, a He-Ne laser (10 mW at 632.8 nm) was used as a light source. The weightaverage molecular weight (Mw), z-average radius of gyration (Rg), and second virial coefficient (A2) values were estimated from the relation18 KCp 1 1 ) 1 + 〈Rg2〉q2 + 2A2Cp Rθ Mw 3
(
)
(1)
where Rθ is the Rayleigh ratio; K ) 4π2n2(dn/dCp)2/NAλ4, with n the refractive index of the solvent, dn/dCp the refractive index increment against Cp, NA Avogadro’s number, and λ the wavelength ()632.8 nm); q ) (4πn/λ) sin(θ/2), with θ the scattering angle. By measuring Rθ for a set of Cp and θ, values of Mw, Rg, and A2 were estimated from Zimm plots. Toluene was used for the calibration of the instrument. Values of dn/dCp were determined with an Otsuka Electronics Photal DRM-1020 differential refractometer at a wavelength of 632.8 nm. For QELS measurements, an Ar+ laser (30.0 mW at 488 nm) was used as a light source. To obtain the relaxation time distribution, τA(τ), the inverse Laplace transform (ILT) analysis was performed using the algorithm REPES.19,20 g(1)(t) )
∫τA(τ) exp(-t/τ) d ln τ
(2)
Here, τ is the relaxation time and g(1)(t) is the normalized
autocorrelation function. The relaxation time distributions are given as a τA(τ) versus log τ profile with an equal area. The relaxation rate, Γ ()τ-1), is a function of Cp and θ.21 The diffusion coefficient D is calculated from D ) (Γ/q2)qf0. The translational diffusion coefficient at finite dilution (D0) is calculated from D ) D0(1 + kdCp)
(3)
where kd is the hydrodynamic virial coefficient. The hydrodynamic radius (Rh) is calculated using the Einstein-Stokes relation Rh ) kBT/6πηD0, where kB is Boltzmann’s constant, T is the absolute temperature, and η is the solvent viscosity.22-24 Determination of PTX Solubility in Various MPC Copolymer Aqueous Solutions. A given amount of PTX was dissolved in 100 µL of ethanol, and the PTX solution was added to 900 µL of an aqueous solution containing various amounts of MPC polymer (PTX concentration 1.0 and 3.0 g/L), followed by vortexing of the mixture solution. The ethanol was removed under reduced pressure. When the PTX crystal appeared, the suspension was heated at 70 °C for 10 min. Then the suspension or solution was allowed to stand for 3 h at 25 °C. Then the solubility was evaluated by the transparency of the solution. Results and Discussion RAFT Radical Polymerization of MPC in Water. We performed homopolymerization of MPC by the RAFT process employing 4,4′-azobis(4-cyanopentanoic acid) as a water-soluble initiator and 4-cyanopentanoic acid dithiobenzoate as a water-soluble chain transfer agent (CTA). Figure 1a shows the time-conversion relationship for the homopolymerization of MPC as well as the pseudo-firstorder kinetic plot in D2O. The monomer consumption was monitored by 1H NMR spectroscopy as a function of time. There was an induction period of ca. 10 min, which may be due to a slow rate of formation of the 4-cyanopentanoic acid radical fragment.25 The RAFT polymerization of MPC in aqueous solution was rapid at 70 °C; monomer conversions of 90 and 99.4% were reached within 60 and 240 min, respectively. The pseudo-first-order kinetic plot shown in Figure 1a is consistent with that for a controlled polymerization, which indicates that the concentration of active species remains constant during the polymerization. Figure 1b shows GPC elution curves for the homopolymerization of MPC. The GPC elution curves clearly show a peak shift to higher molecular weights with increasing polymerization time. The GPC elution curves are all unimodal with no sign of coexisting low or high molecular weight species that may be yielded from uncontrolled polymerization. In Figure 1c, values of Mn and Mw/Mn for poly(MPC) in the RAFT radical polymerization are plotted as a function of the conversion. The increase in Mn with conversion is linear, and the resulting polydispersity is somewhat narrow (Mw/Mn ) 1.26). A notable observation is the marked deviation of the GPC molecular weight from
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Figure 2. Time-conversion (O) and pseudo-first-order kinetic plots (4) for polymerization of BMA in the presence of MPC macro-CTA (Mw(SLS) ) 2.84 × 104; Mw/Mn(GPC) ) 1.27) in methanol-d4 at 70 °C. [M] and [M]0 are concentrations of the monomer at polymerization time ) 0 and corresponding time, respectively.
Figure 1. (a) Time-conversion (O) and pseudo-first-order kinetic plots (4) for polymerization of MPC in the presence of 4-cyanopentanoic acid dithiobenzoate in D2O at 70 °C. [M] and [M]0 are concentrations of the monomer at polymerization time ) 0 and at corresponding time, respectively. (b) Evolution of GPC elusion curves during synthesis of MPC homopolymer with polymerization time. (c) Dependence of Mn (O) and Mw/Mn (4) on monomer conversion in the polymerization of MPC in D2O at 70 °C. The broken line represents the theoretical line.
the theoretical number-average molecular weight (Mn(theor)), which can be calculated from Mn(theor) )
[MPC]0 x M + MCTA [CTA]0 m m
(4)
where [MPC]0 is the initial monomer concentration, [CTA]0 is the initial CTA concentration, xm is the conversion of monomer, Mm is the molecular weight of monomer, and MCTA is the molecular weight of CTA. To verify the true molecular weight of the poly(MPC), SLS measurements were performed. An Mw of 2.84 × 104 determined by SLS in fair agreement with a theoretical Mn value of 2.18 × 104, assuming Mw/Mn ∼ 1. It should be mentioned that Mn values estimated by GPC are only apparent values probably because sodium poly(styrenesulfonate), a polymer with no bulky side chain compared to poly(MPC) with a bulky phosphorylcholine side chain, was used as a standard for molecular weight calibration. Preparation of Amphiphilic pMPCm-BMAn by RAFT. To obtain amphiphilic pMPCm-BMAn, we performed polym-
erization of BMA by the RAFT process in methanol in the presence of MPC macro-CTA. In Figure 2, a timeconversion relationship is depicted along with the pseudofirst-order kinetic plot for the polymerization of BMA in the presence of MPC macro-CTA (Mw(SLS) ) 2.84 × 104; Mw/ Mn(GPC) ) 1.27) in methanol-d4 under an Ar atmosphere. The monomer consumption was monitored by 1H NMR spectroscopy as a function of time. A monomer conversion of 61.4% was reached after 21 h. The pseudo-first-order kinetic plot for the RAFT polymerization deviated significantly from the first-order kinetics. The downward curvature was observed, which may indicate a decrease in the concentration of propagating radicals. Similar deviation from the first-order kinetics has been reported for the RAFT polymerization of N-acryloylmorpholine,26,27 N,N-dimethylacrylamide,25,28 and sodium 6-acrylamidohexanoate.29 We prepared a series of block copolymers of different poly(BMA) sequence lengths using the same MPC macroCTA (Mw(SLS) ) 2.84 × 104; Mw/Mn(GPC) ) 1.27). These block copolymers are abbreviated as pMPCm-BMAn where m and n are the number-average degrees of polymerization (DP) of poly(MPC) and poly(BMA) blocks calculated from SLS (assuming Mw/Mn ) 1) and 1H NMR, respectively. Some molecular characteristics of pMPCm-BMAn are listed in Table 1. Characterization of pMPCm-BMAn. Figure 3 shows 1H NMR spectra for pMPC96-BMA76 in D2O at 27 °C and in methanol-d4 at 50 °C. In D2O containing 0.1 M NaCl, the 1 H NMR spectrum for pMPC96-BMA76 is about the same as that of poly(MPC). The resonance bands observed at 3.34.5 ppm are attributed to the phosphorylcholine moieties, those observed at 1.8-2.4 ppm are attributed to the methylene protons of the main chain, and those observed at 0.81.4 ppm are attributed to the R-methyl protons which are split due to tacticity. The resonance bands due to the poly(BMA) blocks were not observed, suggesting the association of the hydrophobic poly(BMA) blocks. Similar disappearance of 1H NMR signals for BMA units has been reported by Ishihara et al.5 for poly(MPC-r-BMA). In methanol-d4 at 50 °C, however, the resonance bands related to the poly(BMA) unit are observed. The resonance peaks observed at 1.0, 1.4, 1.7, and 3.9 ppm were assigned to the methyl and methylene protons of the n-butyl moieties in the poly(BMA) blocks. The molar ratio of the MPC and the BMA units was
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Novel Biocompatible Polymeric Micelle Reagent Table 1. Characteristics of pMPCm-BMAn
d
sample code
DP of MPCa
DP of BMAb
Mn (NMR)b 10-4
Mw (SLS)c 10-6
A2 × 105 c (mol mL g-2)
Rgc (nm)
Rhd (nm)
µ2/Γ2 d
Rg/Rh
Nagg
pMPC96-BMA22 pMPC96-BMA76
96 96
22 76
3.15 3.93
1.24 8.79
2.43 0.40
13.0 35.6
14.2 29.1
0.053 0.080
0.915 1.22
39.4 224
a Degree of polymerization (DP) determined by SLS. b Estimated by 1H NMR in methanol-d . c Estimated by SLS in 0.1 M NaCl aqueous solutions. 4 Estimated by QELS at Cp ) 5.0 g/L in 0.1 M NaCl aqueous solutions.
Figure 5. Emission maximum in PNA (1.0 × 10-6 M) fluorescence spectra as a function of Cp for pMPC96-BMA22 (4) and pMPC96-BMA76 (O) in 0.1 M NaCl aqueous solutions.
Figure 3. 1H NMR spectra for pMPC96-BMA76 at Cp ) 5.0 g/L in D2O containing 0.1 M NaCl at 27 °C (a) and in methanol-d4 at 50 °C (b).
Figure 4. 13C NMR spectrum for pMPC96-BMA76 at Cp ) 100 g/L in methanol-d4 at 30 °C.
calculated to be 96/76 from the integral intensity ratio of the resonance bands at 3.7 and 1.7 ppm for the methylene protons neighboring the quaternary ammonium group in the phosphorylcholine moiety and in the n-butyl moiety, respectively. DP of the poly(BMA) sequences was calculated from 1 H NMR, as summarized in Table 1. 13 C NMR spectra were measured with gated decoupled and can therefore be accurately integrated. Figure 4 shows a 13C NMR spectrum for pMPC96-BMA76 in methanol-d4. The resonance peaks of the pendent n-butyl group for the poly(BMA) sequences were observed at 14.8, 20.9, 32.1, and 66.5 ppm. The molar ratio of the MPC and the BMA units was calculated to be 96/76 from an integral intensity ratio of the resonance bands associated with the methylene carbon of the phosphorylcholine moiety at 61.1 ppm and the methylene carbon of the n-butyl moieties at 32.1 ppm. The molar ratio of the MPC and BMA units estimated from 13C NMR agreed well with that estimated from 1H NMR data. Aggregation of pMPCm-BMAn in Aqueous Medium. The formation of micellar aggregates of pMPCm-BMAn in
water was confirmed by a fluorescence technique using N-phenyl-1-naphthylamine (PNA)30-32 as a fluorescence probe. It is well established that a decrease in the polarity around PNA leads to a blue shift of its fluorescence emission maximum. Fluorescence spectra of PNA probes dissolved in 0.1 M NaCl aqueous solutions in the presence of pMPCmBMAn were measured at varying Cp values. Emission maxima are plotted as a function of Cp in Figure 5. The emission maxima are practically constant at 464 nm in a low Cp region, indicative of the absence of the hydrophobic association of PNA with the block copolymers. The emission maxima exhibit a substantial decrease with increasing Cp, suggesting an onset of the incorporation of PNA molecules into the core of the polymer micelle at a certain Cp level. The emission maxima for pMPC96-BMA76 begin to decrease with increasing Cp around 0.001 g/L Cp, reaching a blueshifted wavelength of 394 nm at Cp > 0.2 g/L. In the case of pMPC96-BMA22, on the other hand, the blue shift of the emission maximum is much less than that for pMPC96BMA76, with the blue shift saturating at 408 nm at Cp > 0.5 g/L. The polymer concentration at which the emission maximum starts to blue shift may correspond to a critical micelle concentration (cmc). If that is the case, the cmc seems to decrease by more than 1 order of magnitude when the poly(BMA) block length is increased from 22 to 76. Kim et al.33 reported that the cmc values for poly(2-ethyl-2-oxazoline)-block-poly(-caprolactone) with different block lengths are in the range 0.0018-0.0039 g/L. As the length of hydrophobic poly(-caprolactone) block increases, the cmc value decreases. This tendency is similar to the case of pMPCm-BMAn. As suggested by 1H NMR and fluorescence data, the block copolymers exist as aggregates in aqueous solutions. Zimm plots for the block copolymers obtained by SLS measurements are shown in Figure 6. Considering the chemical structure of the block copolymer, the polymer aggregates are expected to be a core-shell type micelle with hydrophobic poly(BMA) blocks forming cores and hydrophilic
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Figure 7. Typical examples of QELS relaxation time distributions for pMPC96-BMA22 (4) and pMPC96-BMA76 (O) at Cp ) 5.0 g/L in 0.1 M NaCl aqueous solutions at θ ) 90°.
Figure 6. Zimm plots for pMPC96-BMA22 (a) and pMPC96-BMA76 (b) in 0.1 M NaCl aqueous solutions at 25 °C at angles from 30° to 130° with a 20° increment. The Cp was varied from 1.0 to 8.0 g/L.
poly(MPC) blocks forming shells. SLS data for the block copolymers determined from Zimm plots are presented in Table 1. The dn/dCp values at 632.8 nm for pMPC96-BMA22 and pMPC96-BMA76 were 0.141 and 0.142 mL/g, respectively. Values of Mw were estimated by extrapolation of Cp and θ to zero, and values of Rg and A2 were estimated from the slope of the angular and concentration dependence in the Zimm plots, respectively. A value of the aggregation number (Nagg) for the polymer micelle can be calculated from the ratio of an apparent molecular mass of the micelle and Mw for a single polymer chain (i.e., unimer). The numberaverage molecular weight (Mn(NMR)) of the unimer was determined by 1H NMR in methanol-d4 (Table 1). Values of Nagg thus estimated for pMPC96-BMA22 and pMPC96BMA76 are 39.4 and 224, respectively (Table 1). Values of Mw, Nagg, and Rg for the polymer micelles increase with increasing poly(BMA) block length. The A2 values for pMPC96-BMA22 and pMPC96-BMA76 were estimated to be 2.43 × 10-5 and 0.40 × 10-5 mol mL g-2, respectively. The small A2 values for the block copolymers are consistent with the formation of polymer micelles because the polymer micelles indicate small A2 values owing to an electrostatic shielding effect by counterion condensation on the aggregates.34,35 The interpolymer micellization of the block copolymers was supported by QELS data. As compared in Figure 7, QELS relaxation time distributions for pMPC96-BMA22 and pMPC96-BMA76 in 0.1 M NaCl are unimodal with different relaxation times at the peak top. As the poly(BMA) block length is increased, the peak for the distributions shifts toward longer relaxation times. The relaxation rates (Γ) estimated from QELS at different measuring angles are plotted as a function of the square of the scattering vector (q2) in Figure 8. A roughly linear relation passing through the origin indicates that all the relaxation modes are virtually due to diffusive process. These findings suggest that the polymer micelles are a spherical shape.36 Approximate values of the hydrodynamic radii (Rh) were calculated from the Stokes-Einstein relation using the values
Figure 8. Relationship between relaxation rate (Γ) and square of the scattering vector (q2) for pMPC96-BMA22 (4) and pMPC96-BMA76 (O) at Cp ) 5.0 g/L in 0.1 M NaCl aqueous solutions at 25 °C.
Figure 9. Plots of diffusion coefficient (D) for pMPC96-BMA22 (4) and pMPC96-BMA76 (O) as a function of Cp in 0.1 M NaCl aqueous solutions.
of diffusion coefficients (D) estimated from the slopes of the Γ-q2 plots, as listed in Table 1. In Figure 9, D values estimated from the Γ-q2 plots (Figure 8) are plotted against Cp. The D values for the block copolymer micelles are practically constant in the Cp regime 1.0 e Cp e 10 g/L. The diffusion coefficients are independent of Cp, and the hydrodynamic virial coefficient (kd) in eq 3 is almost zero. Hence, these observations suggest that the hydrodynamic size of the polymer micelles and Nagg are constant independent of Cp. The ratio of Rg/Rh is often useful to characterize the shape of a molecular aggregate. The theoretical value of Rg/Rh for a homogeneous hard sphere is 0.778, and it increases substantially for a less dense structure and a polydisperse mixture; e.g., Rg/Rh ) 1.5-1.7 for flexible linear chains in a good solvent while Rg/Rh g 2 for a rigid rod.37-39 The Rg/Rh values for pMPC96-BMA22 and pMPC96-BMA76 are 0.915 and 1.22, respectively (Table 1). These values suggest that the polymer micelles are close to a spherical shape and
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Novel Biocompatible Polymeric Micelle Reagent Table 2. Solubilization of PTX in MPC Polymer Aqueous Solutions polymer none poly(MPC-r-BMA) poly(MPC-r-BMA) poly(MPC-r-BMA) poly(MPC-r-BMA) poly(MPC-r-BMA) poly(MPC-r-BMA) pMPC96-BMA22 pMPC96-BMA22 pMPC96-BMA22 pMPC96-BMA76 pMPC96-BMA76 pMPC96-BMA76
exploitation of pMPCm-BMAn for pharmaceutical applications is now under way.
solubility of PTX a
MPC unit mole fraction
Cp (g/L)
1.0 g/L
3.0 g/L
0.80 0.80 0.80 0.50 0.50 0.50 0.81 0.81 0.81 0.56 0.56 0.56
0 0.01 0.1 1.0 0.01 0.1 1.0 0.01 0.1 1.0 0.01 0.1 1.0
+/+/+ +/+ ++ + ++ ++
+/+ +/++ ++
a Solubility of PTX was indicated as -, insoluble; +/-, partially soluble; +, soluble by heating; and + +, stably soluble.
are somewhat polydisperse in size as may be implied by the relaxation distribution data shown in Figure 7. The polydispersity index (µ2/Γ2) by a cumulant analysis is listed in Table 1. The value of the index is not sufficiently small for a monodisperse sample.40 Although Mw/Mn of the block copolymer is close to unity, the micelle size distribution is not narrow; Nagg values for the polymer micelles are somewhat polydisperse. Solubilization of PTX by pMPCm-BMAn in Aqueous Media. PTX is one of the most effective and commonly used drugs for the treatment of various cancers, especially ovarian and breast cancers. Due to the poor solubility of PTX in water (
