Biomacromolecules 2003, 4, 856-858
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Notes Self-Assembly of Poly([R]-3-hydroxybutyric acid)-Block-Poly(ethylene glycol) Diblock Copolymers Franc¸ ois Ravenelle and Robert H. Marchessault* Chemistry Department, McGill University, 3420 University Street, Montre´ al, Qc., Canada H3A 2A7 Received January 16, 2003 Revised Manuscript Received March 10, 2003
Introduction Aliphatic polyesters coupled with monomethoxy poly(ethylene glycol), mPEG, are often used as drug delivery systems, and much of the work done in that field involves polylactides (PLA) coupled to mPEG because of their biodegradability and biocompatibility.1 Zhu et al. first synthesized poly(D,L-lactide)-block-poly(ethylene glycol) for use as a drug carrier.2-4 Diblock5,6 and triblock6 copolymers of chiral poly(L-lactic acid), PLLA, and mPEG have also been reported. The latter systems resemble the one discussed herein as one of our blocks is also a chiral aliphatic polyester. Diblock copolymers of poly([R]-3-hydroxybutyric acid), PHB, and mPEG (Scheme 1) do not generally self-assemble spontaneously in water because of the hydrophobicity and crystallization propensity of the PHB block; that is, the PHB block has no appreciable mobility in water to self-assemble. Once formed by evaporation of a solvent common to both blocks from a water suspension (oil-in-water emulsion), selfassembled structures are not in equilibrium with free chains because the PHB core is in a folded chain lamellae arrangement, as was previously observed from X-ray diffraction.8 They are considered as “dead” or “frozen” organizations.9 This is similar to systems where the core polymer block would be below its glass transition temperature9 or where, after the self-assembly, it would have been crosslinked or a polymerizable function on one of the blocks would be polymerized after the self-assembly.7 This note intends to report interesting morphologies observed with PHB-block-mPEG diblock copolymers synthesized as described previously.8 A casting method was used to generate rods, lamellae clusters, and core-shell particles, as viewed in transmission electron micrographs. These appeared to be similar to those observed by Fujiwara and Kimura for isotactic PLLA and mPEG diblock and triblock copolymers.6 Material and Methods Bacterial PHB was obtained from Imperial Chemicals Ltd., ICI, labeled as BIOPOL, reference number Bx-IRD (Mn * To whom correspondence should be addressed. Phone: 1-514-3986276. Fax: 1-514-398-7249. E-mail:
[email protected].
Scheme 1: Chemical Formula of PHB-Block-mPEG Diblock Copolymer Used in This Study with the Block Number Average Degree of Polymerization
*100% (R) configuration.
300,000; PD 2.0). Monomethoxy poly(ethylene glycol) 5000, mPEG, and bis(2-ethylhexanoate) tin catalyst were purchased from Sigma-Aldrich Canada Ltd. and used as received. PHBblock-mPEG copolymers were synthesized as previously described.8 In a typical experiment at 190 °C, a 25 mL roundbottom flask with magnetic stirrer is loaded with PHB and mPEG. The flask is then plunged into a preheated oil bath at 190 °C under vacuum (water elimination) until the melt viscosity is low enough to be stirred into a homogeneous mixture (10 min wait). Under a flow of Argon, 7 wt % (according to total weight of PHB and mPEG) of liquid catalyst is added through a septum. After 20 min of reaction, the flask is removed from the oil bath and quenched in liquid nitrogen yielding a waxy solid product. The diblock copolymers investigated in this paper have been used as prepared. Reaction times have been set for all mPEG chains to react, according to 1H NMR data and GPC.8 GPC chromatograms showed monomodal molecular weight distribution. Preparation of Aqueous Suspensions. Colloidal suspensions were prepared by solvent evaporation of an oil-in-water emulsion. The “oil” phase is a 0.5% chloroform solution of the diblock copolymer, and 5 mL of that solution was added to 100 mL of distilled water. The mixture was then vigorously and continuously agitated by a 10 mm crosshead magnetic stirrer and/or by a homogenizer (Polytron from Brinkmann Instruments) until chloroform was evaporated and a colloidal suspension was obtained. The rate of chloroform evaporation (heating) and mixing was varied for the different morphologies and is detailed in the text. Transmission Electron Microscopy (TEM). TEM micrographs were recorded using a JEOL JEM-2000 FX electron microscope equipped with a Gatan 792 Bioscan 1k × 1k Wide Angle Multiscan CCD camera. A drop of diluted aqueous suspensions of microparticles or frozen micelles was deposited on Formvar-carbon coated copper grids and airdried prior to observation in the microscope. Results and Discussion In the solid state, both the PHB and mPEG blocks are crystalline8 and melt at very different temperatures. DSC records show that PHB segments in diblocks crystallize around 105 °C, whereas the mPEG moiety in diblocks crystallizes at ca. 5 °C (data not shown). This suggests that a crystalline phase separation of the two blocks must occur
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Notes
Figure 1. TEM micrograph of the PHB17-block-mPEG114 diblock copolymer suspension prepared by slow chloroform evaporation (ca. 2 weeks) under vigorous agitation. The schematic representation shows a complete phase separation of mPEG and PHB.
in the solid state when cooled from the melt. Alternately, not all mPEG moieties can crystallize as some may be trapped as a liquid phase between lamellae8 of PHB. One of the first observations resulting from our experiments is that the weight percentage of PHB in the diblock must be less than 50 wt %, otherwise aqueous suspensions are not colloidal; that is, there is a fast precipitation and settling of the diblock copolymers. Under such conditions, mPEG segments fail to sterically stabilize the PHB crystalline lamellae formed during solvent exchange. In the present note, experiments have been carried out using diblock copolymers having a PHB weight percentage of ca. 25% and bearing an mPEG 5000 block (PHB17-block-mPEG114). This is the first report of observed supramolecular assemblies for this new type of diblock copolymers where the PHB moiety is 100% isotactic. Inspired by the morphologies observed by Fujiwara and Kimura,6 who annealed “as-cast” aqueous suspensions of core-shell morphologies of PLLA-PEG and PLLA-PEGPLLA block copolymers, solvent evaporation was used to self-assemble different morphologies. Because chloroform is a solvent for both blocks, initially evaporation forces the PEG segment out of the chloroform droplets. Further evaporation will cause the PHB segments to self-assemble and crystallize. By varying the solvent evaporation rate, the nucleation and crystallization rates of the chiral PHB are altered, leading to conditions similar to annealing. Our first experiment with the “oil-in-water” emulsion (cf. Materials and Methods) was performed by slow chloroform evaporation from 28 × 95 mm (diameter × height) cylindrical glass vials (9.5 drams) with slightly loose screw-caps under vigorous agitation (10 mm crosshead stirrer). Emulsions were stirred for 2 weeks or until the chloroform odor was no longer detectable by smell. Figure 1 shows a coreshell micellar self-assembly obtained from the colloidal suspension. Most micelles have a diameter of ca. 50-100
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Figure 2. TEM micrograph of the PHB17-block-mPEG114 diblock copolymer (scale bar is 200 nm). Rods were prepared by fast evaporation and high shear following a 24 h agitation-evaporation period at room temperature. The low magnification inset shows a random fiber assembly.
nm. Cores of the latter micelles show nonspherical shapes (square or rectangular), supporting the proposed folded chain lamellae texture of the crystalline PHB core.8 In this experiment, phase separation was allowed to develop over a long period. This is supporting evidence for the phase separation model discussed above. One concludes that the darker core of the micelles in Figure 1 comes from the crystalline PHB, whereas the corona is mPEG. The tendency for linear alignment of these particles (cf. Figure 1) could arise from the surface mPEG chains which associate as water evaporates. Thus, mPEG moieties also encourage a higher level of self-assembly during drying. In the next experiment, chloroform in the “oil-in-water” emulsion was allowed to partially evaporate overnight (24 h) from a vial under vigorous agitation with a partially unscrewed cap. The emulsion was then heated (70 °C) and mechanically homogenized until chloroform had evaporated. Subsequent TEM observation of the colloidal suspension showed nanometer scale rods (ca. 20 nm wide, 100-1000 nm long) with irregular surface profiles (Figure 2). Their irregular surface texture suggests a shish-kabob type of arrangement where the PHB forms the longitudinal scaffold (the shish) of the shear-cast structure. The mPEG segments crystallize and/or envelop the shish to form the “kabob” part (cf. Figure 2). Some rods seem to be hollow which may result from shearing of small air bubbles inside the air-saturated particles. The origin of the rod texture most probably derives from the concentration increase in the chloroform droplets during the 24-h evaporation period and the subsequent homogenization and annealing at 70 °C. Using the homogeneizer, concentrated droplets of chloroform are deformed by the shearing effect, giving rise to an elongated, pulp fiberlike, morphology as observed in Figure 2. One of the important
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Figure 3. TEM micrograph of the PHB17-block-mPEG114 diblock copolymer suspension (scale bar is 200 nm). Fast evaporation and high shear were used in the preparation of this suspension. The low magnification inset shows the distribution of lamellae clusters on the grid.
variables is the viscosity of the droplets which must play a role in the shear response leading to the observed morphologies. The next set of experiments involved fast evaporation, using a homogenizer as soon as the emulsion was made. The temperature was raised to 70 °C over 30 min until all chloroform was evaporated. TEM micrographs showed starlike clusters of ca. 600 nm diameter made from what resembles PHB single-crystal laths.11 Conclusion These novel morphologies are similar to what was observed by Fujiwara and Kimura6 for PLLA and mPEG diblock copolymers where selected area electron diffraction proved that PLLA was crystalline in the self-assembled organization. This is supporting evidence for our proposed explanation because of the resemblance of the two systems in terms of composition (both crystalline aliphatic polyester block and an mPEG block) and molecular weight. Crystallinity was recorded by using X-ray diffraction for the samples in this study and others.8 Although it is difficult at this point to provide a definite explanation for all observed morphologies, it is clear that
Notes
PHB-block-mPEG diblock copolymers can self-assemble into nanoparticles which could find use as drug carriers, binders, and other specialty applications. Such drug carriers may show a longer lifetime in the bloodstream for they are robust versus dilution; that is, they have no critical micelle concentration because their core is hydrophobic and crystalline and hence will not dissociate because of low concentration. Oil-in-water emulsions and solvent evaporation is a practical and scalable method to generate PHB-block-mPEG particles. An obvious conclusion is that in order to obtain lamellae, rods, or micelles the weight percentage of PHB should be small, e.g. around 25%. Also, by varying the evaporation time and shear conditions, it is possible to observe different morphologies supporting the idea that both the high enthalpy of crystallization and hydrophobicity of PHB are responsible for this self-assembly. Fujiwara and Kimura6 observed a morphological transition by annealing the samples at 60 °C. In the case of the PHB and mPEG diblocks, different morphologies are induced by varying the crystallization onset using the rate of solvent evaporation, annealing, and shear action. Synthesis of PHB-PEG-PHB triblock copolymers by transesterification in the melt8 is easily done by replacing mPEG by PEG and should also prove valuable for broadening the range of possible morphologies. Acknowledgment. F.R. would like to acknowledge Les Fonds de Recherche sur la Nature et les Technologies du Que´bec and the Walter C. Sumner Memorial Foundation for Scholarships. The Natural Sciences and Engineering Council of Canada and Labopharm Inc. supported this work. References and Notes (1) Ignatius, A. A.; Claes, L. E. Biomaterials 1996, 17, 831-839. (2) Zhu, K. J.; Song, B.; Yang, S. J. Polym. Sci., Part C: Polym. Lett. 1986, 24, 331-337. (3) Zhu, K. J.; Song, B.; Yang, S. J. Polym. Sci., Part A: Polym. Chem. 1989, 27, 2151-2159. (4) Zhu, K. J.; Song, B.; Yang, S. J. Appl. Polym. Sci. 1990, 39, 1-9. (5) Kimura, Y.; Matsuzaki, Y.; Yamane, H.; Kitao, T. Polymer 1989, 30, 1342. (6) Fujiwara, T.; Kimura, Y. Macromol. Biosci. 2002, 2, 11-23. (7) Hotz, J.; Meier, W. AdV. Mater. 1998, 10, 1387-1390. (8) Ravenelle, F.; Marchessault, R. H. Biomacromolecules 2002, 3, 1057-1064. (9) Cameron, N. S.; Corbierre, M. K.; Eisenberg, A. Can. J. Chem. 1999, 77, 1311-1326. (10) Sudesh, K.; Abe, H.; Doi, Y. Prog. Polym. Sci. 2000, 25, 15031555. (11) Nobes, G. A. R.; Jurasek, L.; Marchessault, R. H.; Martin, D. P.; Putaux, J.-L.; Chanzy, H. Macromolecules 1996, 29, 8330-8333. (12) Harris, J. M. In Poly(ethylene glycol) Chemistry: Biotechnological and biomedical applications; Plenum Press: New York, 1992; pp 9-14 and references therein.
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