Ind. Eng. Chem. Res. 2005, 44, 8621-8625
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Preparation of Multiphase Polymer Beads Composed of Block Copolymer Amphiphilic Networks Yun Sun and Stephen Rimmer* The Polymer and Biomaterials Chemistry Laboratories, Department of Chemistry, University of Sheffield, Sheffield S3 7HF, United Kingdom
Block copolymer amphiphilic networks with amino functionality have been prepared in bead form. Oligo(butyl methacrylate) with dimethacrylate end groups were prepared as a latex. The oligo(butyl methacylate) was synthesized by ozonolysis of poly(butyl methacrylate-co-butadiene) latexes and reaction of the resultant oligomers with glycidyl methacrylate. This material was then copolymerized with glycidyl methacrylate and ethylene glycol dimethacrylate in aqueous suspension to give spherical beads. Regular, spherical beads in the 100-250 µm size range were produced with low levels of fines. Hydrolysis and amination of the epoxide groups produced beads with hydroxylic and amino functionalities. Scanning electron micrographs revealed that the beads had a phase-separated and porous morphology. Introduction Amphiphilic copolymers have several application in biotechnology, and we have studied these materials, in particular, as potential vehicles for cell culture and as vehicles for transferring cells.1-3 The microstructure of these polymers dictates the material’s morphology, and this, in turn, affects cell adhesion and growth. Also, the diffusion of nutrients is a key feature of the use of these materials as tissue engineering scaffolds.4-6 One advantage of an amphiphilic network is that, if the material is phase-separated, the hydrophilic domains can provide an important route for nutrients and growth factors to diffuse into the cell-material construct. The usual strategy for producing this phase-separated structure is to design block copolymers as either networks or linear polymers, and several strategies are available for the synthesis of these materials. However, the most successful methods involve either sequential feeds of monomers in living polymerizations or chain extension of preformed oligomers. Using the latter strategy, we have shown that polymethacrylate networks composed of hydrophobic poly(butyl methacrylate) (PBMA) and hydrophilic poly(2,3-propandiol-1-methacrylate) domains can be used to culture dermal fibroblasts.3 These materials were produced by preparing an oligo(butyl methacrylate) (OBMA) ditelechelic macromonomer, which was then copolymerized with 2,3-propandiol-1-methacrylate and ethylene glycol dimethacrylate (EGDMA). Telechelic methacrylates can be prepared via controlled radical polymerization7-14 or using dead-end polymerization conditions.15,16 However, we have developed an alternative method that involves the cleavage, using ozonolysis, of poly(methacrylate)s that contain alkenes randomly distributed along the main chain.17-19 In our previous work in this area, we produced flat sheets for use in cell culture.1-3 However, for some clinical applications, it may be of benefit to culture human cells on beads.20,21 Polymer beads with homogeneous and heterogeneous phase morphologies were, * To whom correspondence should be addressed. Tel: +44 114 222 9565. Fax: +44 114 273 8673. E-mail: S.Rimmer@ Sheffield.ac.uk.
of course, pioneered, as aids in organic synthesis, by Sherrington and co-workers22-25 and other researchers26,27 in the 1970s and 1980s; many years before their current popularity. Sherrington’s influence in this area continues to the current time period, and he is one of few authors whose work has spanned the several decades that these systems have been under development.28,29 In this report, we briefly communicate the suspension terpolymerization of PBMA macromonomers with glycidyl methacrylate (GMA) and EGDMA. Beads for use in tissue engineering should also be able to carry biochemical functionality, and, here, we demonstrate that amino functionality can be added via amination of the epoxide groups of the GMA units. The non-aminated GMA units can then be hydrolyzed to provide hydrophilic poly(2,3-propandiol-1-methacrylate) segments. Experimental Section Materials. All materials were purchased from Aldrich. The inhibitor was removed from BMA by washing with two equal volumes of aqueous 2% sodium hydroxide, followed by two equal volumes of deionized water, and then was distilled under reduced pressure, prior to use. GMA was purified by distillation to remove the inhibitor. All other chemicals were used as supplied. Deionized (DI) water was used throughout. Preparation of OBMA with Methacrylate End Groups. The procedure reported in our previous publication was used to prepare OBMAs with methacrylate end groups.3 Thus, starve-fed emulsion polymerization was used to prepare a poly(butyl methacrylate-cobutadiene). The monomer feed in the emulsion polymerization was butyl methacrylate (71 g) and butadiene (10.8 g). The rest of the formulation for the emulsion polymerization was composed of water (400 mL), Dowfax 2A1 (Cytec, 3.2 g), K2S2O8 (1.14 g), and KH2PO4 (0.4 g). The resultant latex was then swollen, ozonized, and worked up with selenium oxide/hydrogen peroxide (35% w/w) to yield a latex of OBMA oligomers with COOH end groups. The carboxylic acid end groups derived from the ozonolysis were then modified by reaction with glycidyl methacrylate (213 g). Preparation of GMA-OBMA-EGDMA Beads. Porous beads were prepared by suspension polymeri-
10.1021/ie050144s CCC: $30.25 © 2005 American Chemical Society Published on Web 05/04/2005
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Ind. Eng. Chem. Res., Vol. 44, No. 23, 2005 Table 1. Recipes for Suspension Polymerization, Percentage Yields, and Degrees of Amination for Materials 1-4 Value
Figure 1. Size-exclusion chromatography (SEC) chromatograms of (a) the poly(butyl methacrylate-co-butadiene, (b) oligo(butyl methacrylate) (OBMA) with COOH end groups, and (c) OBMA with methacrylate end groups.
zation of GMA, OBMA, and EGDMA in an aqueous dispersion. The aqueous phase was comprised of DI water (300 mL) and poly(vinyl alcohol) (PVA, 1.2 g, 8789% hydrolyzed, average molecular weight of 85 000124 000). The organic phase was comprised of the monomers (GMA, OBMA, and EGDMA in varying ratios), toluene, and azobisisobutyronitrile (AIBN) (0.125 g). Polymerization was performed in a 500-mL parallelsided, fluted reaction vessel that was equipped with a mechanical stirrer with three blades, a nitrogen inlet, and a reflux condenser. The polymerizations were conducted at 60 °C in a nitrogen atmosphere for 24 h. A premixing stage, prior to initiation, was conducted at 800 rpm to mix the aqueous and monomer phases. The stirring was reduced to 400 rpm after addition of the AIBN. After completion of the reaction, the copolymer beads were washed with DI water (3 × 300 mL) and methanol (3 × 200 mL) and then dried in a vacuum oven at room temperature. The beads were examined
Figure 2. Distributions of particle diameters derived from sieving.
property
1
2
3
4
GMA amount (g) OBMA amount (g) EGDMA amount (g) toluene (mL) yield (wt %) amine density (mmol/g) conversion of epoxide (mol %)
8.4 0 4.2 25 90.1 1.30 28
4.2 4.2 4.2 19 87.8 0.85 35
4.2 4.2 4.2 25 85.3 0.96 39
5.6 2.8 4.2 19 85.5 1.13 35
by scanning electron microscopy (SEM), particle sizing by sieving, and solid-state nuclear magnetic resonance (NMR). Preparation of Ammonia-Functionalized Particulate Polymer. The cleaned and dried beads were fractionated according to their sizes, using a series of sieves. A fraction of beads 106-250 µm in diameter was selected for further reaction of epoxide groups. The epoxide groups of the beads were reacted with 30% ammonia solution at 85 °C with stirring for 6.5 h. The amino-functionalized particles were washed at room temperature with DI water (3 × 100 mL) and then dried in a vacuum oven at 50 °C. Characterization. All the samples were analyzed by 1H NMR (JEOL model GSX 400 spectrometer), using CDCl3 as the solvent. Average molecular weights and polydispersities were measured via size-exclusion chromatography (SEC), using Styragel 5-mm mixed-gel columns (Polymer Laboratories) and a RI detector system. The calibration was performed with poly(methyl methacrylate) (PMMA) standards. Tetrahydrofuran (THF) was used as the solvent, at a flow rate of 1.0 cm3/ min. Sample concentrations were ∼2 g dm-3. Vapor
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Figure 3. Scanning electron microscopy (SEM) microphotographs of the beads.
pressure osmometry (VPO) was performed on a Osmomat 070-SA (Gontotec) system, using THF (SEC-grade) as the solvent and benzile as the calibrant. SEM was performed using a Camscan MKII (Camscan) system. Results and Discussion The emulsion copolymerization gave a product in the form of a latex with a solids content of 19.1 wt % (equivalent to ∼100% conversion of monomer). The ozonolysis then gave an OBMA with a number-average
molecular weight of Mn ) 2345 g/mol and a ratio of weight-average molecular weight to number-average molecular weight of Mw/Mn ) 2.5, as measured by SEC. The methacrylation reaction gave a product with values of Mn ) 3194 g/mol and Mw/Mn ) 2.1, as measured by SEC. The increase in Mn and the decrease in Mw/Mn was due to fractionation following the methacrylation; that is, at the end of the methacrylation step, the latex is no longer stable and it forms two distinct phases. The oil phase contains the OBMA with methacrylate end groups, and the aqueous phase has a lower molecular
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Ind. Eng. Chem. Res., Vol. 44, No. 23, 2005
Figure 4. Solid-state NMR spectra (a) before amination and (b) after amination.
weight and water-soluble fraction of OBMA. VPO gave an absolute Mn value of 5730 g/mol, whereas the Mn value obtained from 1H NMR, calculated by assuming that all of the end groups are methacrylate, was 6510 g/mol. The alkene resonances of the methacrylate end groups were observed as broad resonances at 5.60 and 6.12 ppm in the 1H NMR spectra. Combining these two measurements, we can conclude that 88 mol % of the end groups have the desired functionality. Figure 1 shows SEC chromatograms that illustrate the decrease in molecular weight following the ozonolysis process and the small increase in molecular weight following methacrylation. Suspension polymerizations were conducted using the formulations shown in Table 1 (denoted as materials 1-4). All of the polymerizations produced high yields of regular spherical beads, with polydisperse particlesize distributions. Beads produced in the presence of OBMA were more buoyant than those prepared without OBMA. This feature affected the settling time during the washing procedure. Also, slightly more material was produced as fines, and this was lost in the washing process. However, in all of the polymerizations that we attempted, the yields of usable beads were high and the level of production of fines was negligible. The particlesize distributions were determined by sieving and are shown in Figure 2. As shown in Figure 2, the majority of the beads were produced with diameters of ∼100-250 µm, with the main distribution of sizes in the 106-250 µm range. An important aspect of the application of the particles as cell-carrier vehicles is the fraction of material with diameters of
