Langmuir 2007, 23, 2339-2345
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Synthesis and Supramolecular Organization of Amphiphilic Diblock Copolymers Combining Poly(N,N-dimethylamino-2-ethyl methacrylate) and Poly(E-caprolactone) Franc¸ ois Bougard,†,‡ Me´lanie Jeusette,† Laetitia Mespouille,‡ Philippe Dubois,‡ and Roberto Lazzaroni*,† Laboratory for Chemistry of NoVel Materials and Laboratory of Polymeric and Composite Materials, UniVersity of Mons-Hainaut, Place du Parc 20, B-7000 Mons, Belgium ReceiVed July 17, 2006. In Final Form: NoVember 23, 2006 Well-defined poly(-caprolactone) (PCL)/poly(N,N-dimethylamino-2-ethyl methacrylate (PDMAEMA) diblock copolymers were synthesized, and their self-assembly was investigated as micelles both in aqueous solutions and in thin solid deposits. The synthetic approach combines controlled ring opening polymerization (ROP) of -caprolactone (CL) and atom transfer radical polymerization (ATRP) of N,N-dimethylamino-2-ethyl methacrylate (DMAEMA). Diblock copolymers were prepared by ROP of CL initiated by (Al(OiPr)3), followed by quantitative reaction of the PCL hydroxy end-groups with bromoisobutyryl bromide. The R-isopropyloxy ω-2-bromoisobutyrate poly(-caprolactone) (PCL-Br) obtained was used as a macroinitiator for the ATRP of DMAEMA. The molecular characterization of those diblock copolymers was performed by 1H NMR spectroscopy and gel permeation chromatography (GPC) analysis. The self-assembly of the copolymers into micellar aggregates in aqueous media was followed with dynamic light scattering (DLS), as a function of concentration and the pH. In parallel, the morphology of the solid deposits of those micelles was examined with atomic force microscopy (AFM).
Introduction One particularly interesting aspect of polymer compounds is the self-organization of copolymer chains into polymeric micelles in a selective solvent. The first reports of polymeric micelles appeared in the late 1950s, when stable turbidity was observed in solutions of amphiphilic copolymers.1,2 Micelle formation is due to the different affinities of the two polymer segments for the solvent. The polymer segments with the highest affinities are oriented to the exterior, forming a solvated corona that stabilizes a core made of the low-affinity segments. Various micellar structures are possible, the most common one being the multimolecular spherical micelle.3 These spherical assemblies have a diameter of tens to hundreds of nanometers and show promise in numerous applications such as emulsifiers, foaming agents, and controlled drug delivery. Polymeric micelles4 indeed show many advantages for biomedical applications: they are highly stable in aqueous solution as their low critical micelle concentration (cmc) (∼10-3 mol/L) prevents micelle dissociation upon dilution in the bloodstream after intravenous injection.5,6 Furthermore, the nanometric size of polymeric micelles can facilitate their extravasation at tumor sites while avoiding renal clearance and nonspecific reticuloendothelial uptake.7 For biomedical applications, the choice of polymers is obviously restricted to those that are biocompatible; for temporary applications, biodegradable amphiphilic copolymers are ideal. Systems such as poly(ethylene oxide)-poly(propylene oxide) * To whom correspondence should be addressed. Telephone: ++32 (0)65 37 38 60. Fax: ++32 (0)65 37 38 61. E-mail:
[email protected]. † Laboratory for Chemistry of Novel Materials. ‡ Laboratory of Polymeric and Composite Materials. (1) Merret, F. M. J. Polym. Sci. 1957, 24, 467. (2) Schlicks, S.; Levy, M. J. Phys. Chem. 1960, 64, 883. (3) Jo¨nsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactant and Polymer in Aqueous Solution; Wiley: Chichester, U.K., 1998. (4) Torchilin, V. P. J. Controlled Release 2001, 73, 137. (5) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliVery ReV. 2001, 47, 113. (6) Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai, Y. J. Controlled Release 1993, 24, 119.
(PEO-b-PPO-b-PEO),8-10 multiblock poly(ethylene oxide)poly(lactic acid) (PEO-b-PLA),11 and poly(ethylene oxide)poly(-caprolactone) (PEO-b-PCL)12,13 have been extensively studied for drug delivery applications. In recent years, gene delivery, especially using nonviral synthetic vectors based on PEO-modified cationic polymers such as PEO-b-PDMAEMA14 and PEO-b-polyethyleneimine (PEI),15 has also attracted considerable scientific interest. Polymeric pH-sensitive micelles have also been extensively studied for drug delivery systems with pH targeting. A remarkable example of pH-sensitive micelles, developed by Muller and co-workers,16 is based on poly(Nisopropylacrylamide)-b-poly(acrylic acid). The behavior of these micelles can be studied either directly in solution, in particular with dynamic light scattering (DLS), or in the solid state with atomic force microscopy (AFM) or transmission electron microscopy (TEM). Eisenberg and coworkers have used TEM to image thin films of amphiphilic copolymers deposited by the Langmuir-Blodgett technique.17-19 They have also studied micelles on glass with AFM.20 Similarly, the structure of poly(ethylene)-b-poly(styrenesulfonic acid) (7) Batrakova, E. V.; Li, S.; Li, Y.; Alakhov, V. Y.; Elmquist, W. F.; Kabanov, A. V. J. Controlled Release 2004, 100, 389. (8) Rapoport, N.; Pitt, W. G.; Sun, H. J. Controlled Release 2003, 91, 85. (9) Bromberg, L.; Temchenko, M.; Hatton, T. A. Langmuir 2003, 19, 8675. (10) Kabanov, A.; Batrakova, E. V.; Alakhov, V. Y. AdV. Drug DeliVery ReV. 2002, 54, 99. (11) Xiong, X. Y.; Tam, K. C.; Gan, L. H. Polymer 2005, 46, 1841. (12) Kissel, T.; Li, Y. X.; Unger, F. AdV. Drug DeliVery ReV. 2002, 54, 99. (13) Soo, P. L.; Luo, L. B.; Maysinger, D. Langmuir 2002, 18, 9996. (14) Steenis, J. H.; Maarseveen, E. M.; Hennink, W. E. J. Controlled Release 2003, 87, 167. (15) Tang, G. P.; Zeng, J. M.; Gao, S. J.; Too, H. P.; Wang, S. Biomaterials 2003, 24, 2351. (16) Schilli, C. M.; Zhang, M.; Rizzardo, E.; Thang, S. H.; Chong, Y. K.; Edwards, K.; Karlsson, G.; Muller, A. H. E. Macromolecules 2004, 37, 7861. (17) Zhu, J.; Eisenberg, A.; Lennox, R. B. J. Am. Chem. Soc. 1991, 113, 5583. (18) Zhu, J.; Lennox, R. B.; Eisenberg, A. J. Phys. Chem. 1992, 96, 6. (19) Li, S.; Hanley, S.; Khan, I.; Varshney, S. K.; Eisenberg, A.; Lennox, R. B. Langmuir 1993, 9, 2243. (20) Zhu, J.; Hanley, S.; Eisenberg, A.; Lennox, R. B. Makromol. Chem., Macromol. Symp. 1992, 53, 211.
10.1021/la0620657 CCC: $37.00 © 2007 American Chemical Society Published on Web 01/10/2007
2340 Langmuir, Vol. 23, No. 5, 2007
Bougard et al. Scheme 1
micelles adsorbed on solid substrates from aqueous solution has been investigated by AFM of dried samples.21 The influence of the substrate hydrophobicity was examined by comparing deposits on graphite and on mica modified by a poly(ethyleneimine) coating. Various structures were observed, from simple micelles to network-like structures, by varying the ionic strength. The micelles deposited by simple casting on strongly charged substrates, such as mica, can adsorb on the surface and form novel structures by fusion:22 cylindrical micelles, wormlike micelles, reverse micelles, and other complex mesophases such as porous polymer films. Those structures can then be used for the template growth of a variety of materials, according to welldefined nanoscale patterns.23-28 The advantages of this approach are the simplicity of the sample preparation procedure and the control of the morphologies obtained by modifying the solution characteristics (pH, temperature, presence of electrolytes, and organic cosolvent). In this work, we investigate a novel series of biocompatible amphiphilic diblock copolymers combining poly(-caprolactone) (PCL) and poly(N,N-dimethylamino-2-ethyl methacrylate) (PDMAEMA). PCL is a water-insoluble aliphatic polyester, which is known for its enzymatic biodegradability, its permeability, and the biocompatibility of its metabolites.29 PDMAEMA is a pH- and temperature-sensitive polymer. PDMAEMA has a pKa of 7.4 and a lower critical solution temperature (LCST) of 50 °C.30 It is nontoxic and water-soluble in its protonated form; it can be absorbed by endocytosis and can be used as a nonviral DNA vector.31 When dispersed in aqueous media of appropriate pH, PCL-b-PDMAEMA copolymers are thus expected to (21) Regenbrecht, M.; Akari, S.; Forster, S.; Mo¨hwald, H. J. Phys. Chem. B 1999, 103, 6669. (22) Hou, X.; Sun, L.; Zou, B.; Wu, L. Mater. Lett. 2004, 58, 369. (23) Bormashenko, E.; Pogreb, R.; Stanevsky, O.; Bormashenko, Y.; Stein, O.; Gengelman, T. Langmuir 2005, 21, 9604. (24) Yu, K.; Hurd, A. J.; Eisenberg, A.; Brinker, C. J. Langmuir 2001, 17, 7963. (25) Gleiche, M.; Chi, L. F.; Fychs, H. Nature 2000, 403, 173. (26) Lu, N.; Gleiche, M.; Zhang, J. W.; Lenhert, S.; Xu, B.; Chi, L. F.; Fuchs, H. AdV. Mater. 2002, 14, 1812. (27) Gunther, J.; Stupp, S. I. Langmuir 2001, 17, 6530. (28) Duffy, D. C.; Jackman, R. J.; Vaeth, K. M.; Jensen, K. F.; Whitesides, G. M. AdV. Mater. 1999, 11, 546. (29) Zeng, F.; Shen, Y.; Zhu, S. Macromol. Rapid. Commun. 2002, 23, 1113. (30) Jeong, H. J.; Seok, H. K.; Yang, S. R.; Kim, J.-D. Polymer 2003, 44, 583. (31) Matyjaszewski, K. Chem.sEur. J. 1999, 5, 3095.
associate into micelles, which could lay the basis for new drug delivery systems. Zheng and Sto¨ver32 have recently studied PCL-b-PDMAEMA diblock copolymers grafted on poly(DVB80-co-HEMA) microspheres (where DVB and HEMA are divinylbenzene and hydroxyethylmethacrylate, respectively). Here, our objective is to study the self-assembly properties of the PCL/PDMAEMA block copolymers in aqueous media. In a recent paper, we have demonstrated that the corresponding graft copolymers show an amphiphilic behavior in water.33 PCL-b-PDMAEMA copolymers have been recently studied by Xu et al. in cisplatin-releasing pH-responsive nanoparticles.34 Since the micelle size and shape are strongly dependent on the copolymer molecular structure (block length, block ratio, and molecular weight distribution), the synthesis was carried out following a strategy that allows efficient control of those parameters: PDMAEMA was produced via atom transfer radical polymerization (ATRP),33,35 while PCL was synthesized by ring opening polymerization (ROP).36 Throughout the paper, the diblock copolymers will be referred to as Bxy, where xy is the PDMAEMA weight percentage (for example, B70 contains 70 wt % PDMAEMA and 30 wt % PCL). After complete characterization of the various synthesized copolymers, micelles were generated in water using the procedure first proposed by Eisenberg:37 the copolymers were dissolved in a minute amount of a good solvent for both constituents, to which an aqueous solution was gradually added. These micelles were then investigated both in the liquid medium, with DLS, and as thin deposits on a solid substrate, with AFM. The influence of various parameters (polymer composition, concentration, and pH) on the micellar aggregation was examined. Experimental Section Copolymer Synthesis. On one hand, aluminum alkoxides are very effective initiators for the ROP of lactones and lactides.36 This (32) Zheng, G.; Sto¨ver, D. H. Macromolecules 2003, 36, 7439. (33) Mespouille, L.; Dege´e, Ph.; Dubois, Ph. Eur. Polym. J. 2005, 41, 1187. (34) Xu, P.; Van Kirk, E. A.; Murdoch, W. J.; Zhan, Y.; Isaak, D. D.; Radosz, M.; Shen, Y. Biomacromolecules 2006, 7, 829. (35) Matyjaszewski, K. Macromolecules 1998, 31, 4710. (36) Baran, J.; Duda, A.; Kowalski, A.; Szymanski, R.; Penczek, S. Macromol. Symp. 1997, 123, 93. (37) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728.
Synthesis and Assembly of PCL/PDMAEMA Copolymers procedure relies on a coordination-insertion controlled mechanism, which allows for the synthesis of polyester chains with predictable molecular weight. On the other hand, the ATRP mechanism is intensely utilized for the controlled polymerization of methacrylates and many other vinyl monomers. The use of a CuBr catalytic complex, in the presence of a halogenated alkyl initiator, transforms the radical active species into a halogenated dormant species and thus maintains the presence of radical species to a concentration as low as 10-8 mol/L. As a result, the molecular weight distribution of the copolymer is more homogeneous than for conventional free radical polymerization. In the present study, these two approaches have been combined. The poly(-caprolactone)-b-poly(N,N-dimethylamino-2ethyl methacrylate) (PCL-b-PDMAEMA) diblock copolymers were synthesized by the (i) ROP of -caprolactone (CL), (ii) esterification reaction of PCL hydroxyl end-groups with bromoisobutyryl bromide, and (iii) ATRP polymerization of DMAEMA onto this macroinitiator. Materials. -Caprolactone (CL, from Acros, 99%) was dried over calcium hydride (CaH2) for 48 h at room temperature and then distilled under reduced pressure before use. Aluminum triisopropyloxide (Al(OiPr)3, from Acros, 98%) was distilled under vacuum, quenched in liquid nitrogen, rapidly dissolved in dry toluene, and then stored under a nitrogen atmosphere. The Al(OiPr)3 concentration was determined by back complexometric titration of Al3+ using ethylenediaminetetraacetic (EDTA) acid disodium salt and ZnSO4 at pH 4.8. Ethyl 2-bromoisobutyrate (EBiB, from Aldrich, 98%), 2-bromoisobutyryl bromide (Br-iBuBr, Aldrich), 1,1,4,7,10,10-hexamethylenetetramine (HMTETA, from Aldrich, 97%), and copper bromide (CuBr, from Fluka, 98%) were used without further purification. N,N-Dimethylamino-2-ethyl methacrylate (DMAEMA, from Aldrich, 98%) was passed through a column of basic alumina to remove the stabilizing agents and then stored under a nitrogen atmosphere at -20 °C. Toluene (Labscan, 99%) was dried by refluxing over CaH2 and then distilled before utilization. Tetrahydrofuran (THF) was passed through a column of basic alumina. Synthesis of PCL-b-PDMAEMA Diblock Copolymers. Diblock copolymers were obtained with a three-step technique (Scheme 1). The first step was the ROP of CL by the coordination-insertion mechanism initiated by Al(OiPr)3 to selectively form R-isopropyloxy ω-hydroxy poly(-caprolactone) chains (PCL-OH). The second step was the quantitative conversion of PCL-OH into the R-isopropyloxy ω-bromoisobutyrate poly(-caprolactone) macroinitiator (PCL-Br). The last step was the ATRP polymerization of DMAEMA initiated by the PCL-Br. The equipment was first flame-dried, resulting in a clean airtight system. Particular attention was given to the bottom flask equipped with a three-way stopcock and a rubber septum. The flask was cleaned, dried, and then purged with nitrogen before use as the polymerization vessel. -Caprolactone (30 mL, 0.27 mol) was added to 200 mL of freshly dried toluene. The solution was cooled to 0 °C, and then 7.5 mL of the initiator solution (Al(OiPr)3, 0.83 M in toluene) was added. After 30 min, a few drops of an aqueous HCl solution (1 M) was added to stop the polymerization. The polymer was then selectively precipitated in a large volume of cold heptane, filtrated, and dried under reduced pressure until constant weight. Al residues were extracted by liquid/liquid extraction by washing the polyester solution in chloroform once with an aqueous EDTA acid solution (0.1 mol/ L) buffered at pH 4.8 and then twice with water. The organic layer was finally poured into cold heptane to recover the R-isopropoxy ω-hydroxy poly(-caprolactone) by precipitation. PCL-OH (30 g) was dissolved in 100 mL of THF with 3.8 mL of triethylamine. Bromoisobutyryl bromide (3.4 mL) was then added dropwise to the solution. Triethylamine was used to trap the hydrobromic acid produced as an insoluble ammonium salt and thus completely displace the equilibrium. The reaction was carried out in a round-bottom flask under atmospheric pressure for 72 h at room temperature. Afterward, the PCL-Br in THF solution was filtered to remove the ammonium salt, precipitated in cold methanol, and then dried at 40 °C. The PCL/PDMAEMA diblock copolymers were synthesized by controlled ATRP of DMAEMA using the brominated macroinitiator (PCL-Br). The polymerization was performed in THF at 60 °C in
Langmuir, Vol. 23, No. 5, 2007 2341 the presence of a CuBr/HMTETA catalyst system and under a nitrogen atmosphere. The system must be oxygen-free to prevent (poly)peroxide formation and transfer reactions. The catalytic system was placed in the system purged of oxygen by three repeated vacuum/nitrogen cycles. In a second round-bottom flask, THF and the PCL-Br initiator were placed, with nitrogen bubbling. The DMAEMA monomer was injected into the first flask. The contents of the first flask were then transferred to the second flask. Polymerization was carried out at 60 °C for an appropriate time. The polymer solution was finally diluted in an excess of THF followed by the addition of cold heptane, resulting in precipitation. The copper catalyst system was extracted by passing the copolymer in the THF solution through a column of basic alumina. The purified copolymer solution was reprecipitated in cold heptane, filtered, and then dried under reduced pressure at 40 °C. Adjusting the initial amount of monomer in the reaction medium allowed the modification of the length of the PDMAEMA segment. 1H NMR Spectroscopy. The chemical structures of the diblock (and the intermediate species, PCL-OH and PCL-Br) were confirmed by 1H NMR spectroscopy, which was also used to determine the composition of the copolymers. Those 1H NMR spectra were recorded on BRUKER AMX-300 MHz or 500 MHz spectrometers in CDCl3 (with 0.03% tetramethylsilane (TMS)). Gel Permeation Chromatography (GPC) Characterization. GPC was employed to determine the molecular weight and the molecular weight distribution of the copolymers. GPC analysis was carried out using a Plgel 10 µm (50 mm × 7.5 mm) precolumn and two Plgel 10 µm mixed-B (300 mm × 7.5 mm) gradient columns with THF as the eluent (1 mL/min) and polystyrene standards for column calibration. Twenty microliter samples were injected. The eluent was analyzed with a differential refractive index (RI) detector. Thermal Analysis. Thermal analysis was carried out by differential scanning calorimetry (DSC) under a nitrogen flow with a DSC quest apparatus from T.A. Instruments (heating rate: 10 °C/min). Preparation of the Aqueous Micellar Solutions. Polymeric micelles formed by those copolymers in water are expected to be composed of PCL as the hydrophobic core and PDMAEMA as the surrounding hydrophilic corona. They were prepared by the dissolution of the copolymers (5 mg) in acetone (1 mL), a good solvent for both constituents, followed by the progressive addition (1 drop/10 s) of 100 mL of Millipore water at pH 6.5 and 25 °C to the acetone solution under vigorous stirring. Various final concentrations were considered: 0.5, 0.05, and 0.01 mg/mL. Modification of the pH was carried out after completion of the water addition at pH 6.5, by adding a few drops of NaOH (0.1 M) or HCl (0.1 M) solution under the control of a pH meter. The residual acetone was eliminated by evaporation under ambient temperature and pressure for 24 h. Particle Size Measurements. The mean hydrodynamic diameter and the size distribution of the PCL/PDMAEMA polymeric micelles were measured at a 90° angle with a BI-160 dynamic light scattering apparatus (Brookhaven Instruments Corporation, USA) equipped with a He-Ne laser, which produces a vertically polarized incident beam at 633 nm. After being filtered through a 1.2 µm Acrodisk filter, the samples were measured at 25 °C. The errors in the measurements of micellar sizes from the DLS diagrams are within 5% around the mean value over 10 measurements for each sample corresponding to a cumulative time of 1 min. The particle sizes and size distribution were calculated using CONTIN algorithms. Atomic Force Microscopy. Thin deposits of micelles were prepared by applying 10 µL of the corresponding aqueous solution onto freshly cleaved mica. Mica is a hydrophilic substrate (freshly cleaved mica has a water contact angle of 60% in PDMAEMA) are consistent with the controlled living coordination-insertion mechanism for the PCL segment and the controlled ATRP mechanism for the diblock copolymers. The initiation reaction of the ATRP from PCL-Br is probably slower than the propagation of the monomer and leads to slightly larger polydispersities for diblock copolymers with low content in PDMAEMA. The graft copolymers also have a low dispersity ( 7), the deprotonation of the PDMAEMA segment induces coagulation of the spherical micelles into larger aggregates, such as cylindrical micelles, with a constant width of ∼30 nm and a compact arrangement. Acknowledgment. This work was partially supported by the “Re´ gion Wallonne” and the European Commission in the frame of the “Phasing-Out Hainaut: Materia NoVa” program, by the Belgian Federal Government Office of Science Policy (SSTCPAI 5/3), and by FNRS-FRFC. F.B. is grateful to “F.R.I.A.” for his Ph.D. grant. Supporting Information Available: 1H NMR spectra, GPC chromatograms, and AFM images of the macroinitiator and the diblock copolymers. This material is available free of charge via the Internet at http://pubs.acs.org. LA0620657
