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Microstructure and Physical Properties of a pH-Responsive Gel Based on a Novel Biocompatible ABA-Type Triblock Copolymer Valeria Castelletto* and Ian W. Hamley Department of Chemistry, University of Leeds, Leeds LS2 9JT, United Kingdom Yinghua Ma, Xavier Bories-Azeau, and Steven P. Armes Department of Chemistry, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QJ, United Kingdom Andrew L. Lewis Biocompatibles, Frensham House, Farnham Business Park, Weydon Lane, Farnham, Surrey GU9 8QL, United Kingdom Received January 15, 2004. In Final Form: March 2, 2004
Introduction 2-Methacryloyloxyethyl phosphorylcholine (MPC) is a methacrylic monomer that possesses the phosphorylcholine moiety, the predominant lipid headgroup found on the outside of cell membranes. This biomimetic monomer has been shown to confer clinically proven benefits (enhanced biocompatibility) when incorporated into biomedical polymers.1,2 Moreover, MPC-based statistical copolymers are readily prepared by conventional free radical copolymerization, which makes them attractive from an industrial standpoint. Such copolymers have been exploited for the manufacture of low-irritation soft contact lenses, employed as coatings on implantable devices such as coronary stents and ear grommets, and used for bloodcontacting equipment like guide wires and extracorporeal circuits.3 In 2001, we reported4 that MPC can be polymerized with good control and reasonable living character using atom transfer radical polymerization5 (ATRP). This enables controlled-structure MPC-based block copolymers of relatively low polydispersity to be prepared for the first time.6 One advantage of block copolymers is that they can be designed to undergo spontaneous self-assembly, either in solution or in the solid state.7 For example, MPCtertiary amine methacrylate diblock copolymers form welldefined, stimulus-responsive micelles in aqueous solution.8 Recently, we have also shown that ABA-type triblock copolymer gelators can be synthesized using a commercial bifunctional ATRP initiator.9 Here, the central block B comprises MPC and the outer A blocks are composed of 2-(diisopropylamino)ethyl methacrylate (DPA).9 It was found that DPAm-MPCn-DPAm triblock copolymers (where m and n are the mean degrees of polymerization * To whom correspondence should be addressed. (1) Driver, M. D.; Lewis, A. L. Chem. Br. 1999, 35, 42-45. (2) Nakabayashi, N.; Williams, D. F. Biomaterials 2003, 24, 24312435. (3) Lewis, A. L. Colloids Surf., B: Biointerfaces 2000, 18, 261-275. (4) Lobb, E. J.; Ma, I.; Billingham, N. C.; Armes, S. P.; Lewis, A. L. J. Am. Chem. Soc. 2001, 123, 7913-7914. (5) Patten, T. E.; Matyjaszewski, K. Adv. Mater. 1998, 10, 901-915. (6) Ma, I.; Lobb, E. J.; Billingham, N. C.; Armes, S. P.; Lewis, A. L.; Lloyd, A. W.; Salvage, J. P. Macromolecules 2002, 35, 9306-9314. (7) Hamley, I. W. Angew. Chem., Int. Ed. 2003, 42, 1692-1712. (8) Ma, I.; Tang, Y.; Billingham, N. C.; Armes, S. P.; Lewis, A. L.; Lloyd, A. W.; Salvage, J. P. Macromolecules 2003, 3475-3484. (9) Ma, Y.; Billingham, N. C.; Armes, S. P.; Lewis, A. L. Biomacromolecules 2003, 4, 864-868.
of each block) could be molecularly dissolved in mildly acidic solution (pH < 4) without requiring any cosolvents. On adjusting to neutral pH, the DPA blocks became deprotonated and, hence, hydrophobic, leading to attractive interchain interactions and consequently to the formation of “flower” micelles.9 If the central MPC block is sufficiently long to bridge between adjacent micelles, macroscopic gelation occurs at high copolymer concentrations. In particular, it was found that free-standing gels were formed at pH 8 at triblock copolymer concentrations higher than 10 wt %, while a free-flowing liquid was observed in dilute solution (∼ 0.1 wt %).9 Herein we report the microstructural characteristics of the aqueous solutions and gels formed by one such ABA triblock, namely, DPA50-MPC250-DPA50. A series of samples has been characterized by NMR spectroscopy, small-angle neutron scattering (SANS), and rheology measurements. NMR data indicate that gelation is due to hydrophobic interactions between the deprotonated DPA blocks. SANS provides information on the micellar shape and correlation length, and rheometry was used to examine the frequency dependence of the dynamic shear moduli. Materials and Methods The DPA50-MPC250-DPA50 triblock copolymer [Mr ) 130 000 and Mr/Mn ) 1.63, as judged by aqueous GPC using poly(ethylene oxide) calibration standards] was identical to that used previously.9 Although this triblock has a relatively broad molecular weight distribution, the polydispersity of the central MPC block was relatively low at around 1.20. Moreover, there is reasonable evidence that the polydispersity of the triblock is actually an over-estimate.10 The copolymer was initially molecularly dissolved in a dilute DCl/D2O solution (pH 2), and then the solution pH was adjusted to 9 by addition of NaOD. Aqueous solutions or gels with copolymer concentrations of 0.7, 1, 2, 4, 6, and 9 wt % DPA50-MPC250-DPA50 were studied at pH 9. Copolymer concentrations in the range 0.7-4.0 wt % DPA50-MPC250-DPA50 remained as free-flowing fluids at pH 9 while the more concentrated solutions became free-standing gels, in general agreement with preliminary data.9
Results and Discussion Tube inversion experiments were performed to estimate macroscopic variations in the flow behavior of the samples as a function of temperature. The measurements were carried out from 20 to 70 °C for all the samples mentioned previously. Samples were prepared in small tubes and observed while slowly heating in a water bath. No change in the macroscopic flow behavior was observed at elevated temperatures; thus, all measurements were carried out at 25 °C. Proton NMR spectra were recorded at 25 °C using a 300-MHz Bruker Avance DPX300 spectrometer. The first spectrum was recorded from a 9 wt % DPA50-MPC250DPA50 copolymer solution in DCl/D2O (approximately pH 2). This spectrum was referenced using the HOD signal at δ ) 4.70, and 16 scans were recorded. Then, the solution pH was adjusted in situ by the addition of a drop of 40 wt % NaOD to ensure that there was no significant increase in solution volume. This pH adjustment caused immediate macroscopic gelation, and pH paper was used to estimate a pH of 9 for the aqueous gel. The NMR tube was then agitated until all the entrapped air bubbles were removed (10) Liu, S.; Ma, I.; Armes, S. P.; Perruchot, C.; Watts, J. F. Langmuir 2002, 18, 7780-7784.
10.1021/la049859a CCC: $27.50 © 2004 American Chemical Society Published on Web 04/03/2004
Notes
Figure 1. 1H NMR spectra obtained for the DPA50-MPC250DPA50 triblock copolymer (a) as a free-flowing aqueous solution at pH 2 in DCl/D2O and (b) as a macroscopic physical gel at pH 9 after addition of NaOH. Note that the signals assigned to the protonated DPA residues in spectrum (a) disappear completely from spectrum (b) because the deprotonated DPA blocks become hydrophobic and, hence, much less solvated in the gel state.
from the gel, and the second spectrum was recorded using the same acquisition parameters. SANS experiments were performed at the ISIS pulsed neutron source of the Rutherford Appleton Laboratory, Didcot, U.K., on station LOQ. LOQ is a time-of-flight instrument for which the neutrons have a range of wavelengths. The scattering vector, q, covered the range 0.007 e q e 0.29 Å-1 (|q| ) 4π(sin θ)/λ, where the scattering angle is 2θ and λ is the neutron wavelength). The samples were mounted in standard 1-mm quartz cells. The cell temperature (25 °C) was controlled using a water bath. The SANS data were corrected to allow for sample transmission and background scattering (using a D2O sample as the reference). The data were collected using a two-dimensional area detector and reduced to a onedimensional form by radial averaging. The resulting intensity curves are denoted I(q). Shear rheometry experiments were performed using a Rheometric Scientific SR5 controlled stress rheometer equipped with a cone-and-plate geometry (radius 2.0 cm, gap 0.038 mm) and a solvent trap that prevents the evaporation of solvent during longer experiments. Measurements of the dynamic shear moduli were conducted for frequencies in the range ω ) 0.1-100 rad s-1. All measurements were carried out at frequencies and strains that led to a linear response. The polymer solution was gently loaded onto the plate and given about 30 min to allow the stresses to relax and to attain thermal equilibrium before starting measurements. Figure 1a shows the 1H NMR spectrum obtained for the DPA50-MPC250-DPA50 triblock copolymer, as a freeflowing aqueous solution at pH 2 in DCl/D2O. The 1H NMR spectrum for a free-standing physical gel at pH 9, obtained after addition of NaOH, is shown in Figure 1b. As expected, the signals assigned to the protonated DPA residues in spectrum (a) disappeared completely in spectrum b. This is because the deprotonated DPA blocks become hydrophobic and, hence, much less mobile and solvated in the gel state. Figure 2 shows the SANS intensity profiles obtained for DPA50-MPC250-DPA50 triblock copolymer concentrations between 1 and 9 wt % at pH 9 and for the 9 wt % copolymer solution at pH 3. All the SANS curves at pH 9 showed a continuous decay in intensity with increasing q. Figure 2e confirms that the micellar structural order
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Figure 2. SANS curves obtained at 25 °C for (a) 1, (b) 2, (c) 4, (d) 6, and (e) 9 wt % DPA50-MPC250-DPA50 triblock copolymers at pH 9 and at (e) pH 3. The full line corresponds to the fit obtained using eq 1 for 1, 2, and 4 wt % and using eq 2 for 6 and 9 wt %.
observed at pH 9 was lost upon addition of acid because the scattering curve at pH 3 corresponds to that of a uniform solution. Several attempts were made to fit the SANS curves for concentrations between 1 and 4 wt % using a model of polydisperse spheres.11 Unfortunately, this model did not provide satisfactory fits to the experimental data. The SANS data for the liquid samples could be modeled assuming that the solution contains flowerlike micelles which behave (at least in regard to small-angle scattering) like star polymers with a large number of arms. Bearing this in mind, the micelle model introduced by Grest et al.,12 which accounts for the overall size of the particles and the monomer-monomer correlation function in the corona, was used:
(
I(q) ∝ I0 exp -
)
q2RG2 sin[µ tan-1(qξ)] +β 3 qξ(1 + q2ξ2)µ/2
(1)
where RG is the radius of gyration of the micelles as a whole [the micelle radius, RM ) (5/3)1/2RG], β is a prefactor depending on the number of arms, ξ is the average blob size in the corona, and µ ) 1/ν - 1 (ν ) 3/5 or 1/2 for a polymer in a good solvent or in a theta solvent, respectively). It has been suggested that the microstructure of DPA50MPC250-DPA50 gels probably consists of interconnected micelles, with some of the DPA blocks self-assembling into two micelles (rather than just one micelle) and the central MPC block acting as an intermicelle bridge.9 Therefore, SANS data from DPA50-MPC250-DPA50 gels were modeled according to the following expression, which corresponds to a micellar gel with a mesh structure:13,14
I(q) ∝
() 2 π
1/2
〈c〉2ζ2
(1 + q2ζ2)
(
+ 〈∆c2〉∆R2 exp -
)
∆R2q2 2
(2)
where ζ is the polymer-polymer correlation length in the first term which is a Lorentzian describing liquidlike or (11) Kotlarchyk, M.; Chen, S.-H. J. Chem. Phys. 1983, 79, 24612469. (12) Grest, G. S.; Fetters, L. J.; Huang, J. S. Adv. Chem. Phys. 1996, 94, 67-163.
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Table 1. DPA50-MPC250-DPA50 Solution and Gel Structural Parameters Extracted from Fits to the SANS Data Using Equations 1 and 2 c (wt %)
RGa (Å)
RMa (Å)
ξa (Å)
νa
1 2 4 6 9
86.6 86.6 91.8
111.7 111.7 118.4
20.1 20.1 20.1
0.56 0.56 0.53
ζb (Å)
∆Rb (Å)
90.3 130
73.5 80
a Parameters extracted assuming micellar liquids containing flowerlike micelles. b Parameters extracted assuming a micellar gel with a mesh structure.
short-range order. The second term allows for spatial heterogeneities in concentration. It is assumed that the concentration fluctuations, 〈∆c2〉, are due to elastic constraints randomly located in the system that can be described by a Gaussian distribution with a characteristic length ∆R. Therefore, ∆R is governed by long-range permanent elastic constraints, while ζ is dominated by liquidlike short-range fluctuations. Equations 1 and 2, convoluted with the resolution function of the LOQ instrument, provided a satisfactory fit to our SANS data (Figure 2): the parameters extracted from these fitted curves are listed in Table 1. The presence of micelles in dilute solution at around neutral pH had already been confirmed by our earlier dynamic light scattering (DLS) experiments.9 For example, DLS studies indicated an intensity-average radius, RDLS, of 340 Å for “flower micelles” produced from a 0.1 wt % aqueous solution of the DPA50-MPC250-DPA50 copolymer at pH 8.9 It was also observed that, if the central MPC block was sufficiently long (to bridge between adjacent micelles), gelation occurred at higher copolymer concentrations.9 The present more detailed study indicates that the minimum concentration required for gelation of the DPA50-MPC250-DPA50 copolymer is actually only around 6 wt %, which is somewhat lower than the 10 wt % concentration estimated earlier.9 The results shown in Table 1 for the liquid samples indicate that the micellar structural parameters depend very weakly on the copolymer concentration. The values of ν listed in Table 1 suggest that the copolymer chains in the corona adopt a Gaussian conformation. The micelle radii RM (Table 1) are substantially lower than the DLS micelle radius mentioned previously. It is well-known that, compared to SANS, DLS is much more sensitive to the presence of larger aggregates in solution.14 We, therefore, ascribe the large radius from DLS to the presence of a proportion of aggregates that are significantly larger than single micelles. At this stage, it should be pointed out that the models described by eqs 1 and 2 consider only one level of density contrast between the polymer and the solvent. The scattering length density is Ftriblock ) 1.29 × 1010 cm-2, assuming a polymer density of 1.0 g cm-3. Considering the individual contributions, the main contribution to the excess scattering length density (with respect to the solvent) comes from the corona. However, we have shown that the SANS data can be fitted without considering the density of the core/corona structure. However, differences in contrast cannot explain the discrepancy between SANS and DLS. As already discussed, the parameter ζ is the correlation length of the short-range thermodynamic concentration (13) Mallam, S.; Hecht, A. M.; Geissler, E.; Pruvost, P. J. Chem. Phys. 1989, 91, 6447-6454. (14) Berne, B. J.; Pecora, R. Dynamic Light Scattering; WileyInterscience: New York, 1976.
Figure 3. (9) Storage modulus G′ and (0) loss modulus G′′ measured for (a) 0.7 and (b) 9.0 wt % DPA50-MPC250-DPA50 with pH 9 (the shear stress was fixed at σ ) 0.1 and 1 Pa for the 0.7 and 9 wt % samples, respectively). The full lines show the fits to the dynamic data according to G′ ∝ ω1.5 and G′′ ∝ ω1.1.
fluctuations, while the parameter ∆R is associated with random mechanical constraints in the system that generate permanent spatial concentration fluctuations. In our case, the mechanical constraints presumably result from the bridging of copolymer chains between micelles. Table 1 shows that ∆R and ζ increase with increasing concentration. It is evident that ∆R and ζ are close to the values obtained for the micellar radius in the liquid. It is therefore clear that the SANS data are consistent with the formation of a meshlike gel structure with the formation of bridges between pairs of adjacent micelles. Figure 3 compares the effects of increasing copolymer concentration on the viscoelastic properties of the DPA50MPC250-DPA50 triblock copolymer. Figure 3a shows the dependence of the elastic shear moduli on frequency for the 0.7 wt % copolymer. The solution is in the liquid regime (G′′ > G′). The exponents in the power-law scalings with frequency in the terminal zone were 1.5 and 1.1 for G′ and G′′, respectively, which are close to the expected values of 2.0 and 1.0, respectively, for a Maxwellian fluid. A relaxation time of τ ) 0.83 s could be determined from the crossover of G′ and G′′ at high frequencies (Figure 3a). As already discussed, an increase in copolymer concentration (at a fixed pH 9) results in a free-standing gel, as indicated by the viscoelastic data shown in Figure 3b for the 9 wt % DPA50-MPC250-DPA50 copolymer. In contrast to Figure 3a, the elastic moduli do not exhibit a crossover point in Figure 3b; G′ is greater than G′′ over the whole frequency range, and G′ is almost frequency-independent. Values of G′ and G′′ in Figure 3b are rather low, corresponding to a “soft gel” in the terminology adopted for Pluronic copolymers.15,16 This suggests that the extent of bridging (i.e., content of elastically active chains) is rather low. Strictly, comparison should be made to the dynamic mechanical behavior of “reverse Pluronics” with the same ABA architecture with hydrophobic poly(propylene oxide) A blocks and hydrophilic poly(ethylene oxide) B blocks. Here, the polymer self-assembles in disordered micellar networks with excess water, for determined conditions of concentration and temperature.17,18 The storage modulus G′ of this phase is ∼1000 (15) Hvidt, S.; Jørgensen, E. B.; Schille´n, K.; Brown, W. J. Phys. Chem. 1994, 98, 12320-12328. (16) Hamley, I. W. Philos. Trans. R. Soc. London 2001, 359, 10171044. (17) Mortensen, K. Macromolecules 1997, 30, 503-507.
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Pa, which is somewhat higher than the storage modulus in Figure 3b, suggesting that the extent of bridging is higher than in the DPA50-MPC250-DPA50 gels. Conclusions In summary, our combined NMR, SANS, and rheology data confirms that the DPA50-MPC250-DPA50 triblock copolymer self-assembles to form micelles on deprotonating the DPA blocks. Liquidlike behavior is obtained in dilute solution, while free-standing micellar gels are observed at higher copolymer concentrations (6 wt % or higher). The SANS data from the mobile solutions obtained at low copolymer concentrations are consistent with the (18) Mortensen, K.; Brown, W.; Jørgensen, E. Macromolecules 1994, 27, 5654-5666.
formation of flowerlike micelles, with a micellar radius of 111.7-118.4 Å. The MPC-based chains in the corona are not fully stretched. The SANS data from the free-standing gels could be satisfactorily modeled according to a “mesh” of micelles with DPA cores, where individual micelles are interconnected through bridges made by the solvated MPC blocks. Acknowledgment. The authors are grateful to the Engineering and Physical Sciences Research Council for financial support (Platform Grant GR/S25845 to S.P.A. and Grant GR/N08353 to I.W.H.). V.C. acknowledges Richard Heenan for providing the analytical expression for the incident neutron beam profile, used in the SANS data smearing correction. LA049859A
