pubs.acs.org/Langmuir © 2010 American Chemical Society
Self-Aggregation of Gel Forming PEG-PLA Star Block Copolymers in Water Lucia Calucci,† Claudia Forte,*,† Sytze J. Buwalda,‡ Pieter J. Dijkstra,‡ and Jan Feijen‡ †
‡
Istituto per i Processi Chimico-Fisici del CNR, via G. Moruzzi 1, I-56124 Pisa, Italy, and Department of Polymer Chemistry and Biomaterials, Faculty of Science and Technology, MIRA Institute for Biomedical Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands Received April 22, 2010. Revised Manuscript Received June 28, 2010
The aggregation behavior and dynamics of poly(ethylene glycol) (PEG) and poly(lactide) (PLA) chains in a homologous series of eight-armed PEG-PLA star block copolymers ((PEG65-NHCO-PLAn)8 with n = 11, 13, and 15) in water at different concentrations and temperatures were studied by means of 1H and 13C NMR spectroscopy and 1H longitudinal relaxation time analysis. The state of water in these systems was also investigated through the combined use of 1H and 2H longitudinal relaxation time measurement. On the basis of the NMR experimental findings and of dynamic light scattering measurements, (PEG65-NHCO-PLAn)8 in water can be described as self-aggregated systems with quite rigid hydrophobic domains made of PLA chains and aqueous domains where both PEG chains and water molecules undergo fast dynamics. A smaller number of rigid domains was found for (PEG65-NHCO-PLA11)8 with respect to the homologous copolymers with longer PLA chains. At low concentrations, the PLA domains are mainly formed by chains belonging to the same molecule, thus giving rise to unimolecular micelles. At intermediate concentrations, that is, above the critical association concentration (CAC) but below the critical gel concentration (CGC), nanogels are formed by interconnection of several PLA domains through shared unimers. Above the CGC, the network is extended to the entire system, giving rise to macroscopic gels. In all cases, a fraction of PLA chains remains quite mobile and exposed to water due to topological constraints of the star architecture.
Introduction Block copolymers composed of poly(lactide) (PLA) and poly(ethylene glycol) (PEG) units with an AB or ABA architecture have received broad interest over the last decades for applications in the fields of pharmaceutics and biomedicine. In fact, thanks to both the peculiar properties of PLA and PEG polymers and the capability of the amphiphilic copolymers to self-aggregate in supramolecular structures in water, these materials can be used either as drug carriers in the form of nanoparticles, solutiondispersed micelles, or hydrogels, or as scaffolds for tissue engineering in the hydrogel form.1-7 In particular, PLA is hydrophobic and biodegradable, it adapts well to a biological environment, and it does not have adverse effects on tissues. On the other hand, PEG is hydrophilic and biocompatible. In water, PEG-PLA diblock copolymers typically self-assemble into micellar systems, with the PLA chains packed into a condensed core surrounded by a swollen corona of PEG.8-11 By adjusting specific *To whom correspondence should be addressed. E-mail:
[email protected]. Telephone: þ39-050-3152462. Fax: þ39-050-3152442.
(1) Stolnik, S.; Illum, L.; Davis, S. S. Adv. Drug Delivery Rev. 1995, 16, 195–214. (2) Kumar, N.; Ravikumar, M. N. V.; Domb, A. J. Adv. Drug Delivery Rev. 2001, 53, 23–44. (3) Kissel, T.; Li, Y.; Unger, F. Adv. Drug Delivery Rev. 2002, 54, 99–134. (4) F€oster, S.; Plantenberg, T. Angew. Chem., Int. Ed. 2002, 41, 688–714. (5) Riess, G. Prog. Polym. Sci. 2003, 28, 1107–1170. (6) Gaucher, G.; Dufresne, M.-H.; Sant, V. P.; Kang, N.; Maysinger, D.; Leroux, J.-C. J. Control. Release 2005, 109, 169–188. (7) Torchilin, V. P. Pharm. Res. 2007, 24, 1–16. (8) Hagan, S. A.; Coombes, A. G. A.; Garnett, M. C.; Dunn, S. E.; Davies, M. C.; Harding, S. E.; Purkiss, S.; Gellert, P. R. Langmuir 1996, 12, 2153–2161. (9) Tanodekaew, S.; Pannu, R.; Heatley, F.; Attwood, D.; Booth, C. Macromol. Chem. Phys. 1997, 198, 927–944. (10) Riley, T.; Heald, C. R.; Stolnik, S.; Garnett, M. C.; Illum, L.; Davis, S. S.; King, S. M.; Heenan, R. K.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R.; Washington, C. Langmuir 2003, 19, 8428–8435. (11) Jeong, B.; Lee, D. S.; Shon, J.-In; Bae, J. H.; Kim, S. W. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 751–760.
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polymer properties, such as the relative block length and the solution conditions, polymeric micelles or micellar-like nanoparticles are formed. This association behavior also applies to PEGPLA-PEG triblock copolymers in water, whereas the PLA-PEGPLA triblock copolymers may, in principle, lead to a variety of self-associated structures, depending on the molecular weight of the copolymer and the relative block lengths.11-14 In fact, flowerlike unimolecular micelles can be formed if the central PEG block in PLA-PEG-PLA takes a loop conformation so that the two end PLA blocks become part of the same micellar core. On the other hand, a branched structure may form with the central PEG block exerting a bridging function between different small clusters of the poorly solvated PLA blocks. In this case, the hydrophobic PLA outer blocks associate with neighboring micelles to form a network of linked micelles or large aggregates, ultimately giving rise to associative gels in certain temperature and concentration ranges,11,13-16 as more generally observed for associative watersoluble polymers.17-19 These physically cross-linked hydrogels are attractive because no chemical cross-linking agent is necessary and gelation can be triggered by temperature. However, these hydrogels are dynamic, their cross-links not being permanent, (12) Drumond, W. S.; Mothe, C. G.; Wang, S. H. Polym. Eng. Sci. 2008, 48, 1939–1946. (13) Tew, G. N.; Sanabria-DeLong, N.; Agrawal, S. K.; Bhatia, S. R. Soft Matter 2005, 1, 253–258. (14) Agrawal, S. K.; Sanabria-DeLong, N.; Coburn, J. M.; Tew, G. N.; Bhatia, S. R. J. Controlled Release 2006, 112, 64–71. (15) Agrawal, S. K.; Sanabria-DeLong, N.; Tew, G. N.; Bhatia, S. R. Macromolecules 2008, 41, 1774–1784. (16) Li, S. M.; Vert, M. Macromolecules 2003, 36, 8008–8014. (17) Yekta, A.; Xu, B.; Duhamel, J.; Adiwidjaja, H.; Winnik, M. A. Macromolecules 1995, 28, 956–966. (18) Furo, I.; Iliopoulos, I.; Stilbs, P. J. Phys. Chem. B 2000, 104, 485–494. (19) Koga, T.; Tanaka, F.; Motokawa, R.; Koizumi, S.; Winnik, F. M. Macromolecules 2008, 41, 9413–9422.
Published on Web 07/12/2010
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Figure 1. Molecular structure of the investigated eight-armed PEG-PLA star block copolymers (PEG65-NHCO-PLAn)8 (n = 11, 13, 15).
and when exposed to an aqueous environment at high dilution, they ultimately dissolve or precipitate and the system loses all mechanical integrity. In order to improve the gel mechanical strength and degradation properties, PEG-PLA star diblock copolymers with three,20,21 four,22-25 or eight arms25-29 have been recently synthesized, with both a hydrophilic central core and hydrophobic arms and vice versa. These copolymers associate into micelles, although differences in the micellization properties with respect to linear diblock copolymers were observed as a result of the topological constraints of the star copolymers.22,23,27 In fact, depending on the block length, the chain architecture, and the number of arms, unimolecular micelles or multimolecular micellar aggregates may form, as pointed out by molecular simulations performed on model amphiphilic block copolymers.30-33 Nevertheless, only a few experimental studies on the characterization of the supramolecular aggregates of PEG-PLA star shaped copolymers with PEG in the central core were reported in the literature.22-24,26 Moreover, in most of these studies, nanoaggregate properties in solution were inferred from measurements in the dry state, since few methods allow a direct analysis of the structure and conformation of such nanoparticles in the solvated state. In the present work, different nuclear magnetic resonance (NMR) spectroscopy and relaxation techniques were applied to investigate the self-aggregation behavior in water of recently synthesized eight-armed PEG-PLA star block copolymers linked by an amide group between the PEG core and the PLA blocks ((PEG65-NHCO-PLAn)8 with n = 11, 13, and 15 in Figure 1). These polymers are water-soluble and form thermosensitive hydrogels with good mechanical properties and, highly important, controlled and slow degradation in vitro.29 Their aggregation behavior, as studied by a dye solubilization method and dynamic light scattering (DLS), is characterized by a critical (20) Park, S. Y.; Han, D. K.; Kim, S. C. Macromolecules 2001, 34, 8821–8824. (21) Park, S. Y.; Han, B. R.; Na, K. M.; Han, D. K.; Kim, S. C. Macromolecules 2003, 36, 4115–4124. (22) Jie, P.; Venkatraman, S. S.; Min, F.; Freddy, B. Y. C.; Huat, G. L. J. Control. Release 2005, 110, 20–33. (23) Salaam, L. E.; Dean, D.; Bray, T. L. Polymer 2006, 47, 310–318. epanek, M.; Uchman, M.; Prochazka, K. Polymer 2009, 50, 3638–3644. (24) St (25) Li, Y.; Kissel, T. Polymer 1998, 39, 4421–4427. (26) Nagahama, K.; Fujiura, K.; Enami, S.; Ouchi, T.; Ohya, Y. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 6317–6332. (27) Hiemstra, C.; Zhong, Z.; Li, L.; Dijkstra, P. J.; Feijen, J. Macromol. Symp. 2005, 224, 119–131. (28) Hiemstra, C.; Zhong, Z.; Li, L.; Dijkstra, P. J.; Feijen, J. Biomacromolecules 2006, 7, 2790–2795. (29) Buwalda, S. J.; Dijkstra, P. J.; Calucci, L.; Forte, C.; Feijen, J. Biomacromolecules 2010, 11, 224–232. (30) Sheng, Y.-J.; Nung, C.-H.; Tsao, H.-K. J. Phys. Chem. B 2006, 110, 21643– 21650. (31) Chou, S.-H.; Tsao, H.-K.; Sheng, Y.-J. J. Chem. Phys. 2006, 125, 194903-1–6. (32) Chou, S.-H.; Tsao, H.-K.; Sheng, Y.-J. J. Chem. Phys. 2008, 129, 224902-1–10. (33) Lin, C.-M.; Chen, Y.-Z.; Sheng, Y.-J.; Tsao, H.-K. React. Funct. Polym. 2009, 69, 539–545.
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association concentration (CAC) of 0.48, 0.24, and 0.19 w/v % for n = 11, 13, and 15, respectively. Above the CAC, these star block copolymers form aggregates with diameters showing two distributions around values of 20-40 nm and 100-200 nm.29 Here, 1H and 13C NMR techniques, which have revealed to be of value for the characterization of micelles and gels formed by PEGPLA linear block copolymers34-38 and, in very few cases, also star block copolymers,24,26,29 were used to investigate the structural features and dynamic behavior of the (PEG65-NHCO-PLAn)8 copolymers in water. Preliminary results on a 12 w/v % sample of (PEG65-NHCO-PLA13)8 in the sol and gel phases29 prompted us to study the aggregation and gelation processes and in this respect the role played by the PLA chains. In the present study, the investigation was extended to copolymer concentrations ranging from well below the CAC to above the critical gel concentration (CGC, equal to 12, 10, and 7 w/v % for n = 11, 13, and 15, respectively) and to two other copolymers with different PLA block lengths. In particular, solution state 1H NMR spectra, which show resolved signals only for protons in highly mobile groups, were employed to distinguish mobile and rigid moieties in the polymers and follow their behavior upon changes in concentration and temperature. Solid state 13C cross-polarization (CP) and direct excitation (DE) magic angle spinning (MAS) experiments were used to obtain sitespecific information on polymer dynamics in the gel phase. In fact, whereas CP experiments enhance signals arising from carbons strongly dipolarly coupled to protons, typically 13C nuclei directly bonded to protons and in a rigid environment, DE experiments show all carbon nuclei when performed with a sufficiently long recycle delay, and only the more mobile ones when a short recycle delay is used. Further insight into the dynamics of the polymer chains as a function of concentration and temperature was achieved by measuring proton longitudinal relaxation times (T1). On the other hand, information on the state of water, the other fundamental component of the systems under investigation, was obtained by proton and deuteron T1 analysis on samples of (PEG65-NHCOPLAn)8 in H2O or D2O. Indeed, the use of nuclear longitudinal relaxation for the investigation of polymers in water, either in solution or gel phase, is a well established method to characterize polymer/water interactions as well as the dynamics of water as affected by the presence of polymer aggregates and networks.39 (34) Heald, C. R.; Stolnik, S.; Kujawinski, K. S.; De Matteis, C.; Garnett, M. C.; Illum, L.; Davis, S. S.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R. Langmuir 2002, 18, 3669–3675. (35) Hrkach, J. S.; Peracchia, M. T.; Domb, A.; Lotan, N.; Langer, R. Biomaterials 1997, 18, 27–30. (36) Riley, T.; Stolnik, S.; Heald, C. R.; Xiong, C. D.; Garnett, M. C.; Illum, L.; Davis, S. S.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R. Langmuir 2001, 17, 3168–3174. (37) Yamamoto, Y.; Yasugi, K.; Harada, A.; Nagasaki, Y.; Kataoka, K. J. Control. Release 2002, 82, 359–371. (38) Li, Y.; Qi, X. R.; Maitani, Y.; Nagai, T. Nanotechnology 2009, 20, 055106-1–10. (39) Calucci, L.; Forte, C. Prog. Nucl. Magn. Reson. Spectrosc. 2009, 55, 296–323.
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Experimental Section Materials. The eight-armed poly(ethylene glycol)-poly(L-lactide) star block copolymers (PEG65-NHCO-PLAn)8 were synthesized by ring-opening polymerization of L-lactide in toluene at 110 C as described previously29 using an amine endfunctionalized eight-arm star PEG polymer ((PEG65-NH2)8) and stannous octanoate as initiator and catalyst, respectively. (PEG65NH2)8 was prepared starting from (PEG65-OH)8 (Mn,NMR = 23 700 g/mol)29 following a two-step procedure analogous to that described by Elbert and Hubbell for linear hydroxyl-terminated PEGs.40 For copolymer characterization, see ref 29. DLS Measurements. Dynamic light scattering (DLS) measurements of 0.1, 0.5, and 2.5 w/v % samples of (PEG65-NHCOPLA11)8 in water were performed at 25 C using a Malvern Nano ZS instrument with a laser wavelength of 633 nm and a scattering angle of 173. The particle size was determined by cumulant analysis using the software supplied by the manifacturer. NMR Measurements. NMR experiments were carried out on a Bruker AMX-300 WB spectrometer working at 300.13 MHz for proton and at 75.47 MHz for carbon-13. The temperature was controlled within 0.1 C. 1 H NMR experiments were performed on (PEG65-NHCOPLAn)8 samples in water using a 5 mm probehead for solution state measurements with a 90 pulse of 5.7 μs. The samples, with concentrations ranging from 0.05 to 16 w/v %, were prepared by dissolving the appropriate amount of copolymer in either ultrapure H2O (USF-ELGA Purelab Classic) or D2O (99.98% D, Eurisotop). Repeated heating cycles were necessary to obtain homogeneous samples. For 1D 1H NMR spectra, a recycle delay of 8 s was used and 8-2000 scans were acquired, depending on the sample concentration; for the less concentrated samples, the spectra were acquired suppressing the intense water signal by presaturation.41 Relative peak intensities within each spectrum were determined by integration of the peaks obtained by spectral deconvolution using the SPORT-NMR software,42 and were then calibrated using 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (DSS) at a known concentration as standard. DSS was also used as reference for the 1H chemical shifts. In the experiments involving Mn2þ, successive aliquots of a 1 mM MnSO4 (MnSO4 3 H2O 99%, Carlo Erba) solution in D2O were added to the samples in the NMR tubes with a micropipet; the highest final Mn2þ concentration reached was 0.1 mM. In the experiments involving lactic acid (85% in H2O, Sigma Aldrich), successive aliquots of a 10 mM solution in D2O were added to the samples in the NMR tubes with a micropipet; the highest final concentration reached was 1 mM. A 2D proton correlation spectroscopy (COSY) spectrum43-45 was recorded on (PEG65-NHCO-PLA13)8 in D2O (1 w/v %) with 256 t1 increments of 8000 data points over a 3.6 kHz spectral width, 80 scans per t1 increment, a recycle delay of 5 s, and with water presaturation. Spin-lattice relaxation times (T1) of water protons were determined in a 12 w/v % sample of (PEG65-NHCO-PLA13)8 in H2O at 25, 40, 55, and 70 C, using the saturation recovery pulse sequence46 with recycle delays ranging from 15 to 40 s, depending on the temperature, and acquiring eight scans. The baseline corrected areas of the water proton peak (I(t)) at different values of the sequence characteristic delay were measured, and T1 values were determined by fitting the saturation recovery curve using the equation: IðtÞ ¼ I0 ð1 - expð - t=T1 ÞÞ
ð1Þ
where I0 is the water signal intensity at time zero. (40) Elbert, D. L.; Hubbell, J. A. Biomacromolecules 2001, 2, 430–441. (41) Hoult, D. I. J. Magn. Reson. 1976, 21, 337–347. (42) Geppi, M.; Forte, C. J. Magn. Reson. 1999, 137, 177–185. (43) Aue, W. P.; Bartholdi, E.; Ernst, R. R. J. Chem. Phys. 1976, 64, 2229–2246. (44) Bax, A.; Freeman, R. J. Magn. Reson. 1981, 44, 542–561. (45) Nagayama, K.; Kumar, A.; W€utrich, K.; Ernst, R. R. J. Magn. Reson. 1980, 40, 321–334. (46) Mao, X.-A.; Ye, C.-H. Concepts Magn. Reson., Part A 1998, 9, 173–187.
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Water deuteron T1 values were determined in neat D2O and in (PEG65-NHCO-PLA13)8 samples in D2O at concentrations of 2, 6, 12, and 16 w/v % at 25, 40, 55, and 70 C using the inversion recovery pulse sequence with a recycle delay of 6 s and acquiring eight scans. The baseline corrected areas of the D2O peak (I(t)) at different values of the sequence characteristic delay were measured, and T1 values were determined by fitting the inversion recovery curve using the equation: IðtÞ ¼ I¥ ð1 - R expð - t=T1 ÞÞ
ð2Þ
where I¥ is the water signal intensity at time ¥ and R is a parameter accounting for noncomplete magnetization inversion.47 For the 12 w/v % (PEG65-NHCO-PLA13)8 samples in both H2O and D2O and for the 2, 6, and 16 w/v % (PEG65-NHCOPLA13)8 samples in D2O, inversion recovery experiments were used to determine T1 values of polymer protons at 25, 40, 55, and 70 C. The peak intensities were obtained from spectral deconvolution by means of the SPORT-NMR software42 and were used to determine T1 values through eq 2. Each saturation recovery and inversion recovery curve was built using at least 15 experimental points and fitted with a nonlinear least-squares procedure in order to determine the T1 values and their standard errors. All T1 measurements were performed on nondegassed samples. 13 C DE-MAS and CP-MAS NMR spectra were recorded under proton decoupling conditions on a sample of (PEG65NHCO-PLA13)8 in the dry state and in the gel state in D2O (16 w/v %) using a 4 mm CP/MAS probehead for solid state measurements. The 90 pulse length was 4.4 μs and the MAS frequency was 6 kHz for the dry sample and 1 kHz for the hydrogel sample. A total of 1200 scans were acquired for all experiments. In the DE-MAS experiments, recycle delays between 1 and 200 s were used in order to select spectral components with different longitudinal relaxation times. In the CP-MAS experiments, a contact time of 1 ms was used for the dry sample and between 1 and 5 ms for the hydrogel sample; the recycle delay was 8 s. Delayed CP spectra were recorded using a 20 or 30 μs delay prior to the contact pulse. All MAS experiments were performed at 25 C. Fourier Transform Infrared (FTIR) Measurements. FTIR spectra were recorded on a Perkin-Elmer Spectrum 100 spectrometer using BaF2 plates, with a resolution of 4 cm-1, and acquiring 32 scans. Samples for FTIR analyses were prepared by dissolving the appropriate amount of (PEG65-NHCO-PLA13)8 copolymer in CHCl3 (1 w/v %), D2O (1 and 12 w/v %), or H2O (1 and 12 w/v %). FTIR spectra were recorded both on solutions and on films prepared by drop casting onto a BaF2 plate and air drying.
Results and Discussion H NMR Spectra of (PEG65-NHCO-PLAn)8 in Water. In order to study the self-aggregation behavior of (PEG65NHCO-PLAn)8 copolymers in water, 1H NMR spectra of systems with n = 11, 13, and 15 were recorded at room temperature on samples in D2O at different concentrations ranging from 0.05 w/v %, that is well below the CAC, up to 16 w/v %, that is in the gel phase; a selection of exemplifying spectra is presented in Figure 2. All spectra showed a peak ascribable to the PEG methylene protons at 3.698 ppm and several signals ascribable to the PLA methyl protons in the spectral region between 1.3 and 1.6 ppm. Signals arising from the PLA methine protons were observed in the regions 4.2-4.5 and 5.0-5.3 ppm, except in the case of the most diluted samples due to partial overlapping with the signal of HDO (at about 4.8 ppm). The spectra of samples in the gel phase were characterized by broad lines, so that the PLA methyl 1
(47) Gerhards, R.; Diethich, W. J. Magn. Reson. 1976, 23, 21–29.
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Figure 2. Left: 1H NMR spectra of (PEG65-NHCO-PLA13)8 in D2O above (top) and below (bottom) the CGC. Right: 1H NMR methyl
spectral region of (PEG65-NHCO-PLA13)8 in D2O at the indicated concentration values. The spectra were recorded at 25 C. Table 1. 1H NMR Chemical Shifts (ppm) of PLA Methine (δ CH) and Methyl (δ CH3) Groups of (PEG65-NHCO-PLA13)8 in D2O signals
δ CH
δ CH3
assignment
A 4.06 1.314a lactic acid B 4.25 1.361 lactic amide unit C 4.82 1.436 lactyl lactic acid D 4.40 1.441 lactyl lactic acid E 4.47 1.453 terminal lactic unit of PLA-Wb F 5.12 1.487 first lactic unit of PLA-Wb G 5.26 1.581 middle block of PLA-Wb H 5.19 1.54 PLA-Mb a Chemical shift at 0.1 w/v % concentration. b PLA-W = PLA chains dangling in water; PLA-M = mobile PLA chains in hydrophobic aggregates.
and methine signals were both roughly represented by two peaks at about 1.58 and 1.45 ppm, and 5.26 and 5.10 ppm, respectively; on the other hand, the spectra recorded on samples at concentrations below the CGC showed quite narrow PLA signals (Figure 2, left). In fact, besides a broad signal at 1.54 ppm (indicated as H in Table 1), the methyl spectral region was characterized by six quite sharp doublets, with J coupling constants ranging from 6.8 to 7.0 Hz, resonating between 1.3 and 1.5 ppm (signals A-F in Table 1), and by a broad “doublet” at 1.58 ppm (G); minor resonances in the range between 1.44 and 1.48 ppm could also be detected by an accurate spectral deconvolution. The corresponding methine signals were detected in the 4.0-5.3 ppm spectral region and assigned with the aid of a 1H-1H correlation (COSY) experiment (Table 1). All these signals were present, with different relative intensities, in the spectra of the samples with polymer concentrations below the CGC (Figure 2, right) irrespective of the PLA chain length. Variations in chemical shift with concentration were observed only for doublets A, C, and D. The high number of PLA signals in the 1H spectra of (PEG65NHCO-PLAn)8 in water, not observed in nonselective solvents such as dimethyl sulfoxide or chloroform (data not shown), is a clear indication that in water different types of lactic acid chains are present. Signals A-D could be assigned to products of PLA chain hydrolysis, occurring through a backbiting mechanism,48 on the basis of their chemical shift values49 and of specific tests. In (48) de Jong, S. J.; Arias, E. R.; Rijkers, D. T. S.; van Nostrum, C. F.; Kettenesvan den Bosch, J. J.; Hennink, W. E. Polymer 2001, 42, 2795–2802. (49) Espartero, J. L.; Rashkov, I.; Li, S. M.; Manolova, N.; Vert, M. Macromolecules 1996, 29, 3535–3539.
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particular, changes observed in 1H spectra upon addition of a water solution of Mn2þ, a water-soluble relaxing agent, or of lactic acid, containing both monomeric and oligomeric species,50 allowed A signals to be assigned to lactic acid and signals C and D to lactyl lactic acid. These species, amounting at most to 3% of the PLA units, are present in higher relative concentration in samples below the CAC than in those above the CAC, suggesting that the formation of aggregates disfavors hydrolysis, as already observed in PLA-PEG-PLA triblock copolymers.51 On the basis of the chemical shift value and its reduced intensity, which corresponds to much less than 1% of the PLA units for all the investigated samples, the B signals could be ascribed to the residual lactic amide unit after complete hydrolysis of a PLA chain. The assignment of signals A-D was confirmed by comparison with the 1H NMR spectrum in D2O of a sample of almost fully degraded (PEG65-NHCO-PLA15)8 prepared by dissolving the copolymer in 2-(cyclohexylamino)ethanesulfonic acid (CHES) buffer at pH 10.4 and stirring for 18 h at room temperature. The remaining PLA signals belong to copolymer chains in different environments. The E-G signals could be ascribed, on the basis of relative intensities and chemical shift values,49,52 to relatively long PLA chains in water (indicated in the following as PLA-W), with E and F signals corresponding to the terminal and the first lactic acid units, respectively, and the G signals to the central units; shorter chains give rise to the minor methyl peaks in the 1.44-1.48 spectral region. On the other hand, on the basis of the mean chemical shift values and of the line shape, signals H could be ascribed to relatively mobile chains in hydrophobic domains, named from now on PLA-M. Aggregation Behavior of (PEG65-NHCO-PLAn)8 in Water. The presence of PLA chains in different environments, highlighted by the above-described 1H NMR spectral assignment, represents a piece of evidence of the aggregation behavior of (PEG65-NHCO-PLAn)8 star copolymers in water. However, an accurate analysis of the spectral signal intensities is required for a thorough description of the structure and dynamics of PEG and (50) Kroschwitz, J. I. In Encyclopedia of Chemical Technology, 4th ed.; Kroschwitz, J. I., Ed.; Wiley: New York, 1995; Vol. 13, pp 1042-1054. (51) Rashkov, I.; Manolova, N.; Li, S. M.; Espartero, J. L.; Vert, M. Macromolecules 1996, 29, 57–62. (52) Rashkov, I.; Manolova, N.; Li, S. M.; Espartero, J. L.; Vert, M. Macromolecules 1996, 29, 50–56.
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Figure 3. Relative populations of PLA units in aqueous (PLA-W) and hydrophobic environments (PLA-R and PLA-M with very low and relatively high mobility, respectively) determined for (PEG65-NHCO-PLAn)8 copolymers (n = 11, 13, and 15 from left to right) in D2O at different concentrations.
PLA blocks in water. To this end, the signal intensities of the PEG methylene and PLA methyl protons in the 1H NMR spectra were determined by deconvolution techniques and compared with those of a standard. It was found that all the PEG protons were detected for all copolymers at all concentrations, whereas a different proportion of PLA methyl protons was observed depending on the PLA chain length and copolymer concentration. In particular, for copolymer concentrations up to 0.1 w/v %, the relative intensities of PEG and PLA signals reflected the stoichiometric ratios as in nonselective solvents. On the contrary, at concentrations above 0.1 w/v %, the PLA signals, comprising both PLA-W and PLA-M units, were significantly less intense than expected, accounting for 30-60%, 25-55%, and 20-40% of the total PLA units for (PEG65-NHCO-PLA11)8, (PEG65NHCO-PLA13)8, and (PEG65-NHCO-PLA15)8, respectively, with their amount sensibly decreasing with increasing copolymer concentration (see Figure 3). Analogous findings have been reported for linear and star PEG-PLA block copolymers by other authors,24,26,34-38 and by us for (PEG65-NHCO-PLA13)8 at a concentration of 12 w/v % and for the analogous (PEG65-OCOPLA13)8 copolymer at a concentration of 22 w/v %.29 This behavior can be ascribed to the formation of PLA aggregates in which most of the chains have reduced mobility, differently from the PEG chains which are in all cases quite mobile. The observable PLA protons correspond to mobile chains, either in an aqueous (PLA-W) or in a hydrophobic (PLA-M) environment, whereas the fraction of PLA units which is not detected in the 1H NMR solution state spectra, in the following indicated as PLA-R, belongs to relatively rigid chains in the hydrophobic aggregates. It must be pointed out that NMR signals similar to those of PLAW (peaks E-G in Table 1) have been reported also for PLA-PEG diblock copolymers34 and PLA-PEG-PLA triblock copolymers12 with relatively short PLA chains with respect to PEG ones, and ascribed to mobile PLA units at the interface between PLA cores and PEG shells. In our systems, where the star shape of PEG, together with the arm length and the PEG/PLA block length ratio, constitutes constraints for the formation of hydrophobic PLA domains, it seems more probable that the PLA-W units belong to pendant chains in water. Below the CGC, the intensity of PLA-W signals corresponds to 5-10% of the total lactic units for (PEG65-NHCO-PLA11)8 and to 4-7% for the other two copolymers, while that of PLA-M accounts for 20-40% of the PLA units in the case of (PEG65-NHCO-PLA11)8 and 10-30% in the other cases. At concentrations above the CGC, where the 12894 DOI: 10.1021/la101613b
Figure 4. 1H NMR spectra of a 2 w/v % sample of (PEG65NHCO-PLA13)8 in D2O at the indicated temperatures.
spectral resolution is reduced because of the lower overall mobility (Figure 2, left), the overall intensity in the methyl region, arising from PLA-W and PLA-M units, corresponded to 2530% of the PLA units. In order to better characterize the nature of the PLA aggregates, the influence of the temperature on the 1H NMR spectra was investigated on representative samples of (PEG65-NHCOPLAn)8 at different concentrations below and above the CGC. By increasing the temperature from 25 up to 70 C, thus reaching the sol phase, the main effect observed was the progressive growth and narrowing of the broad unresolved methyl signal at 1.54 ppm (see Figure 4) and of the corresponding methine peak at 5.19 ppm. At the highest temperature investigated, the intensity of the detected PLA methyl signals represented 85-90% of the total methyl protons; on the other hand, all PEG methylene protons were detected at all the investigated temperatures. This behavior, which was independent of copolymer concentration, indicates that the PLA-M and PLA-R units have the same average Langmuir 2010, 26(15), 12890–12896
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Figure 6. Schematic structures of (a) unimolecular micelles and (b) nanoaggregates of (PEG65-NHCO-PLAn)8 in water.
Figure 5. Aggregate size distributions of 0.1, 0.5, and 2.5 w/v % samples of (PEG65-NHCO-PLA11)8 in water at 25 C determined by DLS measurements.
chemical shift and therefore experience environments with the same hydrophilic/hydrophobic properties. The two types of units strongly differ in their degree of mobility at room temperature, with such a difference decreasing with raising the temperature thanks to a progressive increase in the motional rates of PLA-R units. On the basis of the above observations, the PLA-M and PLAR units could be ascribed either to chains with different mobility within the same type of aggregate or to chains belonging to different types of aggregates. Indeed, two distinct aggregate size distributions, which could be associated to PLA-M and PLA-R, were found for 0.5 w/v % (PEG65-NHCO-PLAn)8 in water by DLS measurements,29 giving evidence in favor of the second hypothesis. Further confirmation was obtained by DLS experiments performed on (PEG65-NHCO-PLA11)8 samples at different concentrations. In fact, as shown in Figure 5, the proportion of aggregates with average diameter on the order of few hundreds nanometers increased, by increasing the concentration, at the expense of those with average diameters of 20-40 nm. In addition, at concentrations quite higher than the CAC, aggregates with even larger dimensions formed, giving rise to a macroscopic network above the CGC. Considering the copolymer arm lengths and their number, the smallest aggregates are compatible with unimolecular micelles with a single intramolecular PLA domain (Figure 6a), although the formation of flowerlike micelles by the aggregation of few molecules, as suggested for PEG-PLA 4-arm star block copolymers24 and PLA-PEG-PLA triblock copolymers,14,15 cannot be excluded. The other type of aggregates is nanoaggregates containing several intermolecular PLA domains (Figure 6b). It must be noted that at high concentration a bimodal distribution of particle size may be erroneously derived from DLS measurements, the bimodal character being instead due to collective and self-diffusion.53 However, in the present case, we are (53) Pusey, P. N.; Fijnaut, H. M.; Vrij, A. J. Chem. Phys. 1982, 77, 4270–4281.
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confident that the observed DLS data reflect a bimodal aggregate size distribution, since the same two average dimensions are found also at low concentration. Polymer and Water Dynamics. The formation of PLA solidlike domains by chain aggregation was confirmed by 13C solid state NMR experiments on samples in the gel phase, where the copolymer concentration is sufficiently high to give good spectra in relatively short experimental times. A selection of CPand DE-MAS NMR spectra is shown for a 16 w/v % sample of (PEG65-NHCO-PLA13)8 in D2O in Figure 7, where spectra recorded in the dry state are also reported for comparison. As it can be seen, in the CP and DE (with a long recycle delay of 60 s) spectra of the dry sample, all the expected signals for the copolymer were detected, that is a narrow peak at 70.3 ppm and a broad one at 71.5 ppm ascribable to methylene carbons in amorphous and crystalline PEG, respectively,54 and peaks at 16.3 and 171.0 ppm, ascribable to methyl and carbonyl PLA carbons, respectively, with the methine carbons, resonating at about 69 ppm, giving rise to a shoulder of the PEG signal.55 On the other hand, for the sample in the gel phase, only the signal relative to the PEG methylene carbons was observed in the DE spectra, even using a recycle delay as long as 200 s, whereas the CP spectrum showed a signal for the PEG methylene carbons and one for the PLA methyl carbons. It must be pointed out that in the gel the peak relative to the PEG methylene carbons was observed at lower chemical shift (by approximately 0.6 ppm) as expected in an aqueous environment.56 Moreover, in the CP spectrum, the peak of PLA methyl carbons was three times more intense compared to the dry state. Since cross-polarization is more efficient for carbon nuclei in rigid environments, the observation of similar spectra under CP and DE conditions for the dry sample indicates that the mobility of the PEG and PLA chains is not significantly different. On the contrary, in the gel phase, the dynamic behavior of PEG and PLA moieties was quite different, with PLA remaining solidlike and PEG showing an increased mobility ascribable to the plasticizing effect of water; only a small fraction of PEG was sufficiently rigid to give rise to cross-polarization. Moreover, in variable contact time CP experiments (data not shown), the PEG CH2 signal progressively increased in intensity with increasing contact time, whereas the PLA CH3 signal showed maximum intensity at short contact time (1 ms), therefore indicating that PLA chains are more rigid than the most rigid among PEG chains. Delayed CP experiments, performed with a preacquisition delay during which relatively rigid protons, having sufficiently short spin-spin relaxation time, dephase causing a depleted CP effect, gave further evidence of the high rigidity of PLA chains. In fact, a reduction in the PLA methyl signal with respect to the PEG (54) Dechter, J. J. J. Polym. Sci., Polym. Lett. 1985, 23, 261–266. (55) Howe, C.; Vasanthan, N.; MacClamrock, C.; Sankar, S.; Shin, I. D.; Simonsen, I. K.; Tonelli, A. E. Macromolecules 1994, 27, 7433–7436. (56) Bjorling, M.; Karlstriim, G.; Linse, P. J. Phys. Chem. 1991, 95, 6706–6709.
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Figure 7. (a) 13C DE- and (b) CP-MAS NMR spectra of dry (PEG65-NHCO-PLA13)8; (c) 13C DE- and (d) CP-MAS NMR spectra of 16 w/v % (PEG65-NHCO-PLA13)8 in D2O.
methylene one was observed; in particular, the CH3/CH2 intensity ratio was reduced to half when a delay of 20 μs was used, whereas no CH3 signal was detected with a delay of 30 μs. The dynamic behavior of (PEG65-NHCO-PLAn)8 copolymers in water was investigated more in detail by analysis of the longitudinal relaxation times (T1) of polymer protons and FTIR spectra, both below and above the CGC (see the Supporting Information). Relaxation coupling between water and polymer protons was not present, although it could be expected for PEG given the observed strong hydrogen bonds with water molecules.56-58 In fact, only minor differences were found between the polymer T1 values determined in D2O and H2O, and, moreover, the polymer and water proton T1’s at the same temperature were significantly different (see the Supporting Information). Therefore, the polymer T1’s were analyzed in terms of chain dynamics. The findings indicated that PEG chains undergo fast dynamics, as observed for pure PEG59,60 and PEG-PLA linear copolymers34 in water, which is thus not affected by the constraints exerted by the autoaggregation of PLA chains and by the molecular architecture. Considering the high mobility of the PEG chains and the absence of groups which could strongly interact via dipolar coupling or proton exchange, relaxation coupling between PEG and PLA protons is not expected. The PLA T1 data suggested that the dynamics of the chains dangling in water (PLA-W) is in the fast motion regime, differently from that of the PLA units in the hydrophobic domains (PLA-M þ PLA-R) for which, however, a detailed characterization is hampered by the fact that the relaxation times result from qualitatively and quantitatively different PLA moieties at the different temperatures investigated. Water dynamics was also studied by 1H and 2H longitudinal relaxation time analysis in samples at concentrations below and above the CGC (see the Supporting Information); a bulklike dynamic behavior was inferred at all concentrations, with stronger hindrance in the gel phase due to the polymer network.
Conclusions The self-assembly of (PEG65-NHCO-PLAn)8 star copolymers in water at various concentrations and temperatures was investigated by means of 1H and 13C NMR techniques. The applied experiments allowed a characterization of the distribution of PEG and PLA chains in domains with different hydrophilic/hydrophobic properties and degree of mobility to be made. The PEG chains resulted to undergo rapid motions at all concentrations, both (57) Benko, B.; Buljan, V.; Vuk-Pavlovic, S. J. Phys. Chem. 1980, 84, 913–916. (58) L€usse, S.; Arnold, K. Macromolecules 1996, 29, 4251–4257. (59) Liu, K.-J.; Ullman, R. J. Chem. Phys. 1968, 48, 1158–1168. (60) Hey, M. J.; Ilett, S. M.; Davidson, G. J. Chem. Soc., Faraday Trans. 1995, 91, 3897–3900.
12896 DOI: 10.1021/la101613b
in the sol and gel phases, even at room temperature, which is a clear indication that the PEG blocks are always well swollen in water, as expected for hydrophilic polymers, albeit the eight-arm star architecture. Water molecules did not show strong interactions with the polymer, and their dynamics was practically as fast as that in pure water for concentrations up to the CGC and slightly more restricted only in the gel phase. On the other hand, PLA chains were distributed in at least three different environments, with their relative proportions changing with copolymer concentration and composition. The majority of PLA units belonged to chains forming two types of hydrophobic aggregates characterized by a considerably different degree of mobility at room temperature. On the basis of the NMR and DLS data, the more mobile units (PLA-M) could be reasonably associated to unimolecular micelles with a single intramolecular PLA domain and average diameter of 20-40 nm. On the other hand, the more rigid units (PLA-R) were ascribed to nanoaggregates containing several intermolecular PLA domains having average diameter on the order of a few hundreds of nanometers; thanks to the good solvation of PEG blocks, these supramolecular aggregates can be considered nanogels. Above the CGC, the PEG network is sufficiently extended to give rise to macroscopic gels. At all concentrations, a minor proportion of PLA units (PLA-W) belonging to chains dangling in water was detected. Although energetically penalized due to the hydrophobic character of PLA, the presence of these chains is entropically favored since it avoids the formation of constrained PEG loops.61 It is interesting to observe that, whereas no significant differences were observed in the aggregation behavior between (PEG65-NHCO-PLA13)8 and (PEG65-NHCOPLA15)8, the homologue with shorter PLA chains showed a higher fraction of unimolecular micelles and of dangling chains at all concentrations. This behavior can be explained on the basis of the smaller hydrophobicity of (PEG65-NHCO-PLA11)8 due to the shorter PLA blocks, which is also responsible for the higher critical association and gel concentrations.11 Acknowledgment. Dr. Emilia Bramanti is kindly acknowledged for having performed the FTIR measurements. Supporting Information Available: Polymer 1H and water H and 2H longitudinal relaxation rates measured on (PEG65-NHCO-PLA13)8 samples in D2O and H2O at different concentrations and temperatures; FTIR spectra of (PEG65-NHCO-PLA13)8 in the dry state and in water. This material is available free of charge via the Internet at http:// pubs.acs.org. 1
(61) Zhou, Z.; Chu, B.; Peiffer, D. G. Langmuir 1995, 11, 1956–1965.
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