pubs.acs.org/Langmuir © 2009 American Chemical Society
Synthesis of Biocompatible Sterically-Stabilized Poly(2-(methacryloyloxy) ethyl phosphorylcholine) Latexes via Dispersion Polymerization in Alcohol/ Water Mixtures Hasan Ahmad,† Damien Dupin,† Steven P. Armes,*,† and Andrew L. Lewis‡ †
Department of Chemistry, University of Sheffield, Brook Hill, Sheffield, South Yorkshire S3 7HF, U.K., and ‡ Biocompatibles UK Ltd., Chapman House, Farnham Business Park, Weydon Lane, Farnham, Surrey GU9 8QL, U.K. Received May 7, 2009
Poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) is soluble in either 2-propanol or water but becomes insoluble in certain alcohol-rich 2-propanol/water mixtures. We have exploited this unusual cononsolvency behavior in order to prepare new biocompatible sterically stabilized PMPC latexes via nonaqueous dispersion polymerization in 2-propanol/water mixtures. All polymerizations were conducted in the presence of monomethoxy-capped poly(ethylene glycol) methacrylate (PEGMA) as a reactive stabilizer, with some formulations including ethylene glycol dimethacrylate (EGDMA) as a cross-linker. Under optimized conditions, unimodal size distributions could be obtained with a mean latex diameter of approximately 1 μm, as judged by laser diffraction and DLS. The mean latex diameter depended on both the PEGMA and initiator concentration but was almost independent of the cross-linking density. Smaller PMPC latexes were obtained by increasing the alcohol content of the dispersion medium. On dilution with water, these latexes acquired microgel character. The microgel solution viscosity was insensitive to added salt due to the so-called “antipolyelectrolyte” effect, which is characteristic of polyzwitterions. Finally, copolymerization of MPC with a fluorescein-based methacrylic comonomer produced fluorescently labeled PMPC latexes, which may have potential biomedical applications.
Introduction The phosphorylcholine (PC) motif is an essential component of cell membranes. The biomimetic monomer, 2-(methacryloyloxy)ethyl phosphorylcholine (MPC),1,2 has been used to produce a wide range of copolymer coatings that confer excellent blood compatibility as well as resistance to protein adsorption and cell adhesion.3-10 Biomedical applications for MPC-based copolymers include extended-wear soft contact lenses,11,12 biocompatible coatings for guidewires and coronary stents,13 extracorporeal circuits,14 low biofouling coatings on urological devices,15 and *To whom correspondence should be addressed. E-mail: s.p.armes@ sheffield.ac.uk. (1) Umeda, T.; Nakaya, T.; Imoto, M. Macromol. Chem., Rapid Commun. 1982, 3, 457–459. (2) Ishihara, K.; Ueda, T.; Nakabayashi, N. Polym. J. 1990, 22, 355–360. (3) Hayward, J. A.; Chapman, D. Biomaterials 1984, 5, 135–142. (4) Hall, B.; Bird, R. le R.; Kojima, M.; Chapman, D. Biomaterials 1989, 10, 219–224. (5) Nowak, T.; Nishida, K.; Shimoda, S.; Konno, Y.; Ichinose, K.; Sakakida, M.; Shichiri, M.; Nakabayashi, N.; Ishihara, K. J. Artif. Organs 2000, 3, 39–46. (6) Lewis, A. L. Colloids Surf., B 2000, 18, 261–275. (7) Konno, T.; Kurita, K.; Iwasaki, Y.; Nakabayashi, N.; Ishihara, K. Biomaterials 2001, 22, 1883–1889. (8) Murphy, E. F.; Lu, J. R.; Lewis, A. L.; Brewer, J.; Russell, J.; Stratford, P. Macromolecules 2000, 33, 4545–4554. (9) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Langmuir 2005, 21, 5980–5987. (10) Ishihara, K.; Ando, B.; Takai, M. Nanobiotechnology 2007, 3, 83–88. (11) Driver, M. J.; Jackson, D. J. (Biocompatibles Ltd.). U.S. Patent No. 5,741,923, 1998. (12) (a) Browers, R. W. J.; Stratford, P. W.; Jones, S. A. (Biocompatibles Ltd.). World Patent WO 9207885, 1990.(b) Lloyd, A. W.; Faragher, R. G. A.; Wassall, M.; Rhys-Williams, W.; Wong, L.; Hughes, J. E.; Hanlon, G. W. Contact Lens Anterior Eye 2000, 23, 119–123. (c) Andrews, C. S.; Denyer, S. P.; Hall, B.; Hanlon, G. W.; Lloyd, A. W. Biomaterials 2001, 22, 3225–3233. (13) Lewis, A. L.; Stratford, P. W. J. Long-Term Effects Med. Implants 2002, 12, 231–250. (14) De, S. F.; An, B. Y.; Caes, F.; Francois, K.; Arnout, J.; Bossuyt, X.; Taeymans, Y.; Van, N. G. Perfusion 2002, 17, 39–44. (15) Russel, J. C. J. Endocrinol. 2000, 14, 39–42.
11442 DOI: 10.1021/la901631a
tympanostomy tube implants.16 The unique properties imparted by MPC copolymers are attributed to their highly hydrophilic character: it has been estimated that there are up to 22 water molecules per MPC repeat unit.17,18 Polymer latexes are widely used for separation technologies, histological studies, immunodiagnostics, and drug delivery.19-25 It is usually important to optimize the latex surface chemistry in order to avoid adverse immunological reactions.26 To date, most MPCbased publications have involved either soluble polymers,27-30 (16) Berry, J. A.; Biedlingmaier, J. F.; Whelan, P. J. Otolaryngol. Head Neck Surg. 2000, 123, 246–251. (17) Ishihara, K.; Nomura, H.; Mihara, T.; Kurita, K.; Iwasake, Y.; Nakabayashi, N. J. Biomed. Mater. Res. 1998, 39, 323–330. (18) Feng, W.; Nieh, M.-P.; Zhu, S.; Harroun, T. A.; Katasaras, J.; Brash, J. L. Biointerphases 2007, 2, 34–43. (19) Boury, F.; Marchais, H.; Benoit, J. P.; Proust, J. E. Biomaterials 1997, 18, 125–136. (20) Bourgeat-Lami, E.; Tissot, I.; Lefebvre, F. Macromolecules 2002, 35, 6185– 6191. (21) Ahmad, H.; Tauer, K. Macromolecules 2003, 36, 648–653. (22) Mahdavian, A. R.; Abdollahi, M. React. Funct. Polym. 2006, 66, 247–254. (23) Alam, M. A.; Miah, M. A. J.; Ahmad, H. Colloid Polym. Sci. 2007, 285, 715–720. (24) Mohanty, P. S.; Dietsch, H.; Rubatat, L.; Stradner, A.; Matsumoto, K.; Matsuoka, H.; Schurtenberger, P. Langmuir 2009, 25, 1940–1948. (25) Duan, L.; Chen, M.; Zhou, S.; Wu, L. Langmuir 2009, 25, 3467–3472. (26) Voronov, I.; Santerre, J. P.; Hinek, A.; Callahan, J. W.; Sandhu, J.; Boynton, E. L. J. Biomed. Mater. Res. 1998, 39, 40–51. (27) Lewis, A.; Tang, Y.; Brocchini, S.; Choi, J.-W.; Godwin, A. Bioconjugate Chem. 2008, 19, 2144–2155. (28) Ma, I. Y.; Lobb, E. J; Billingham, N. C.; Armes, S. P.; Lewis, A. L.; Lloyd, A. W.; Salvage, J. Macromolecules 2002, 35, 9306–9314. (29) Mahon, J.; Zhu, S. Colloid Polym. Sci. 2008, 286, 1443–1454. (30) Matsuda, Y.; Kobayashi, M.; Annaka, M.; Ishihara, K.; Takahara, A. Polym J. 2008, 40, 479–483. (31) Kiritoshi, Y.; Ishihara, K. J. Biomater. Sci., Polym. Ed. 2002, 13, 213–224. (32) Kiritoshi, Y.; Ishihara, K. Sci. Tech. Adv. Mater. 2003, 4, 93–98. (33) Uchiyama, T.; Kiritoshi, Y.; Watanabe, J.; Ishihara, K Biomaterials 2003, 24, 5183–5190. (34) Madsen, J.; Armes, S. P.; Bertal, K.; Lomas, H.; MacNeil, S.; Lewis, A. L. Biomacromolecules 2008, 9, 2265–2275.
Published on Web 07/09/2009
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Article Table 1. Preparation of Cross-linked PEGMA-PMPC Latexes by Dispersion Polymerization in Various 2-Propanol/Water Mixtures at 60°C, 24 h, and 250 rpm run no.
MPC (g)
PEGMA (g)
EGDMA (g)
AIBN (g)
alcohol (g)
water (g)
1 2 10 11
4.4950 4.4950 4.4950 4.4950
0.50 0.50 0.50 0.50
0.005 0.005 0.005 0.005
0.01 0.01 0.01 0.01
42.75 40.50 38.25 36.00
2.25 4.50 6.75 9.00
Table 2. Preparation of Cross-linked PEGMA-PMPC Latex Particles by Dispersion Polymerization with Variable EGDMA Content at 60°C, 24 h, and 250 rpm
Figure 1. Preparation of sterically stabilized cross-linked PEGMA-PMPC latex particles via dispersion polymerization at 60 °C in 2-propanol/water mixtures.
copolymer hydrogels,31-36 or surface coatings for either planar37-40 or colloidal substrates.41-44 Only a few reports have described MPCbased latexes. For example, Sugiyama and Aoki prepared a series of copolymer microspheres of MPC with various comonomers such as methyl-, ethyl-, n-butyl-, hexyl methacrylates, and styrene by emulsifier-free emulsion copolymerization.45 Ishihara et al. prepared poly (L-lactic acid) or polystyrene nanoparticles by solvent evaporation in the presence of a highly polydisperse statistical terpolymer comprising MPC, n-butyl methacrylate, and p-nitrophenyloxycarbonyl poly (ethylene glycol) methacrylate.46-48 These MPC nanoparticles were evaluated in the context of biomedical diagnostics, drug delivery, and for protein separation. (35) Schexnailder, P.; Schmidt, G. Colloid Polym. Sci. 2009, 287, 1–11. (36) Liu, W.; Deng, C.; McLaughlin, C. R.; Fagerholm, P.; Lagali, N. S.; Heyne, B.; Scaiano, J. C.; Watsky, M. A.; Kato, Y.; Munger, R.; Shinozaki, N.; Li, F.; Griffith, M. Biomaterials 2009, 30, 1551–1559. (37) Kihara, T.; Yoshida, N.; Mieda, S.; Fukazawa, K.; Nakamura, C.; Ishihara, K.; Miyake, J. Nanobiotechnology 2007, 3, 127–134. (38) Ishihara, K.; Iwasaki, Y.; Ebihara, S.; Shindo, Y.; Nakabayashi, N. Colloids Surf., B: Biointerfaces 2000, 18, 325–335. (39) Huang, X.-J.; Xu, Z.-K.; Wan, L.-S.; Wang, Z.-G.; Wang, J.-L. Langmuir 2005, 21, 2941–2947. (40) Jang, K.; Sato, K.; Mawatari, K.; Konno, T.; Ishihara, K.; Kitamori, T. Biomaterials 2009, 30, 1413–1420. (41) Konno, T.; Watanabe, J.; Ishihara, K. Biomacromolecules 2004, 5, 342–347. (42) Chen, X. Y.; Armes, S. P.; Greaves, S. J.; Watts, J. F. Langmuir 2004, 20, 587–595. (43) Vo, C.-D.; Schmid, A.; Armes, S. P.; Sakai, K.; Biggs, S. Langmuir 2007, 23, 408–413. (44) Yuan, J.-J.; Schmid, A.; Armes, S. P.; Lewis, A. L. Langmuir 2006, 22, 11022–11027. (45) Sugiyama, K.; Aoki, H. Polym. J. 1994, 26, 561–569. (46) Watanabe, J.; Ishihara, K. Biomacromolecules 2006, 7, 171–175. (47) Park, J.; Kurosawa, S.; Watanabe, J.; Ishihara, K. Anal. Chem. 2004, 76, 2649–2655. (48) Goto, Y.; Matsuno, R.; Konno, T.; Takai, M.; Ishihara, K. Biomacromolecules 2008, 9, 828–833.
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run no.
MPC (g)
3 2 4 5 6 7 8 9 12
4.4975 4.4950 4.4937 4.4925 4.4884 4.4750 4.4500 4.4000 4.2500
PEGMA EGDMA (g) (g) 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
0.0025 0.005 0.0063 0.0075 0.0116 0.0250 0.050 0.100 0.250
AIBN (g)
alcohol (g)
water (g)
0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
40.50 40.50 40.50 40.50 40.50 40.50 40.50 40.50 40.50
4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50
Recently, Ishihara and co-workers have demonstrated that PMPC homopolymer exhibits an unusual property known as “cononsolvency”.30-32 Although soluble in both water and 2-propanol, PMPC is actually insoluble in certain 2-propanol/ water mixtures. We have recently confirmed that this phenomenon is also true for PMPC brushes.49 In the present work, we have exploited this cononsolvency behavior in order to prepare sterically stabilized latexes via dispersion polymerization of MPC. Thus the PMPC chains form the latex cores, while monomethoxycapped poly(ethylene glycol) methacrylate (PEGMA) acts as a reactive steric stabilizer, see Figure 1. Ethylene glycol dimethacrylate (EGDMA) was optionally used as a cross-linker and the effect of independently varying the solvent composition and the EGDMA, PEGMA, and initiator concentrations on the final particle diameter was studied. The resulting PEGMA-PMPC latexes were characterized using dynamic light scattering (DLS), laser diffraction, scanning electron microscopy (SEM), optical microscopy, confocal laser scanning microscopy, gel permeation chromatography (GPC), and 1H and 13C NMR spectroscopy. The effect of adding salt on the solution viscosity of PMPC latexes prepared with different cross-linked EGDMA densities was also measured.
Experimental Section Materials. MPC (>99%) was donated by Biocompatibles (UK) and used as received. EGDMA (98%) was purchased from Aldrich, and its inhibitor was removed by column chromatography using activated basic alumina. PEGMA (mean degree of polymerization=45; Mw/Mn=1.10) was donated by Cognis Performance Chemicals (UK) and was used as received. 2,20 -Azobis (isobutyronitrile) (AIBN) was recrystallized from methanol and stored in the refrigerator prior to use. Fluorescein O-methacrylate (FMA) obtained from Sigma-Aldrich was used as a fluorescent comonomer in selected latex syntheses without any purification. 2-Propanol (HPLC grade) was purchased from Fisher Scientific (UK) and used without purification. Both D2O and 2-propanol-d8 were purchased from Aldrich. All other chemicals used were of reagent grade. Deionized water was used in all measurements.
(49) Edmondson, S.; Nguyen, N. T.; Armes, S. P. Langmuir, in preparation.
DOI: 10.1021/la901631a
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Synthesis of Cross-linked PEGMA-PMPC Latexes. Dispersion copolymerizations were conducted in two-necked roundbottomed glass reactors immersed in a thermostatted oil bath with a total comonomer (i.e., MPC, EGDMA, and PEGMA) content fixed at 10 wt %. The composition of the 2-propanol/water solvent mixture was varied from 80 to 95 wt % 2-propanol to examine its effect on latex formation and the kinetics of copolymerization. Each polymerization was carried out under a nitrogen atmosphere at 60 °C for 24 h, and reaction mixtures were magnetically stirred at 250 rpm. The particular formulation used for the preparation of each PEGMA-PMPC latex is summarized in Table 1. Additional polymerizations were conducted with varying EGDMA content (0-5 wt %) using a 90/10 2-propanol/water mixture, see Table 2. In some cases, the concentrations of AIBN initiator and PEGMA stabilizer were also varied. All latexes were purified by repeated centrifugation followed by serum replacement using the appropriate 2-propanol/water mixture used for the original latex synthesis. This protocol ensured that any nongrafted PEGMA or unreacted MPC was removed prior to further analyses, Synthesis of Fluorescent PEGMA-PMPC Latex. To allow confocal laser scanning microscopy studies, a fluorescent PMPC latex was synthesized using 1.0 wt % FMA relative to total comonomer (MPC (3.70 g), EGDMA (0.25 g), PEGMA (1.00 g), and FMA (0.05 g)) in a 90/10 2-propanol/water mixture. Polymerization was conducted under a nitrogen atmosphere at 60 °C for 24 h using magnetic stirring at 250 rpm. Overall Monomer Conversion. Small aliquots were withdrawn from the reaction solution at desired times, quenched by the addition of one drop of a 1% hydroquinone solution and immediately cooled using an ice bath. The overall monomer conversion was calculated by comparison of the vinyl signals with the polymer backbone signals in each 1H NMR spectrum. Dynamic Light Scattering (DLS). Hydrodynamic diameters were measured at 25 °C using a Malvern Zetasizer ZEN 3600 instrument equipped with a 4 mW He-Ne laser operating at 633 nm. Backscattered light was detected at 173°, and the mean particle diameter was calculated from the quadratic fitting of the correlation function over 30 runs of 10 s duration. All measurements were performed in triplicate on roughly 0.01 w/v % dispersions after dilution using a 90/10 2-propanol/water mixture at 25 °C. The solution viscosity of this 90/10 2-propanol/water mixture was taken to be 2.3259 cP according to the literature.50 Laser Diffraction. Measurements were conducted using a Malvern Mastersizer Hydro2000SM instrument equipped with a Hydro2000SM sample dispersion unit. The volume-average particle size distribution, D4/3, of each PEGMA-PMPC latex was calculated by assuming a spherical morphology. Particle size distributions were recorded for five successive measurements, and the final results were averaged. The uniformity gives an indication of the breadth of the particle size distribution. Scanning Electron Microscopy (SEM). For SEM observation, two sample preparation methods were examined. The first involved placing a few drops of diluted latex on a silicon wafer (Compart Technology, Peterborough, UK, with a silicon dioxide layer of 2 nm) and immediately drying by spin-coating (30005000 rpm) at room temperature. The silicon wafer was then affixed to a aluminum stub via adhesive carbon tape. The second protocol involved drying a highly dilute (∼0.1% solids) latex dispersion directly onto an aluminum stub in an oven set at 70 °C, followed by further drying at 50 °C overnight. Both samples were then sputter-coated with an ultrathin overlayer of gold prior to observation using a FEI Inspect F FEG instrument operating at 20 kV. Confocal Laser Scanning Microscopy (CLSM). Fluorescent PEGMA-PMPC latex particles were observed by CLSM (Zeiss LSM 510 Meta laser accessory mounted on an Axiovert 200 M
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Figure 2. Time-conversion curves for the preparation of crosslinked PEGMA-PMPC latex particles (runs 2 and 11 in Table 1) by dispersion polymerization at 60 °C in various 2-propanol/water mixtures.
microscope). This instrument was equipped with both argon ion and HeNe gas lasers; wavelengths were set at λex=488 nm and λem >514 nm for excitation and emission of fluorescein, respectively. Prior to observation, a few drops of diluted latex was placed on a glass slide and covered with a coverslip to prevent evaporation of the 2-propanol/water continuous phase. 1 H and 13C NMR Spectroscopy. Selected linear (i.e., noncross-linked) PEGMA-PMPC latexes were dried and then dissolved in either D2O or 95/5 2-propanol-d8/D2O. 1H NMR spectra were recorded using a Bruker Avance 400 MHz NMR spectrometer. For 13C NMR spectra, D2O containing a few drops of DMSO was used as a solvent. X-ray Photoelectron Spectroscopy (XPS). The surface composition of PEGMA-PMPC latex particles prepared using 2 wt % EGDMA and 20 wt % PEGMA based on total monomer was evaluated using a Kratos Axis Ultra DLD X-ray photoelectron spectrometer equipped with a monochromatic Al X-ray source operating at 6.0 mA and 15 kV at a typical base pressure of 10-8 torr. The step size was 1.0 eV for the survey spectra (pass energy 160 eV) and 0.1 eV for the high resolution spectra (pass energy 80 eV). Spectra were typically acquired from two separate sample areas of dried latex. The latex dispersion (purified by serum replacement after repeated centrifugation) was dried onto silicon wafer prior to analysis. Rheology Studies. The effect of adding salt to swollen aqueous PEGMA-PMPC microgels was investigated by measuring the solution viscosity. PEGMA-PMPC latexes prepared with varying degrees of cross-linking were diluted in distilled deionized water to obtain 1.0 wt % aqueous dispersions. Correspondingly, the same PEGMA-PMPC latex was also diluted using either 0.2 or 0.5 M aqueous NaCl solution. The zero-shear rate viscosity (η0) of these PMPC microgels was measured at 25 °C using an AR-G2 rheometer equipped with a concentric cylinder (which is routinely used for low viscosity fluids). Gel Permeation Chromatography (GPC). GPC was used to assess the molecular weight distribution of a linear PEGMA-PMPC latex prepared using 20 wt % PEGMA. The GPC setup comprised a Polymer Laboratories PLgel 5 μm Mixed “C” column operating at 40 °C in combination with a refractive index detector. The eluent was a 3:1 chloroform:methanol mixture at a flow rate of 1.0 mL min-1, and calibration was carried out using five near-monodisperse poly (methyl methacrylate) calibration standards. The data were processed using Cirrus GPC offline GPC/SEC software.
Results and Discussion (50) Flick, E. W. Industrial Solvents Handbook, 5th ed.; Noyes Data Corporation: Westwood, NJ, 1998; Chapter 6 .
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A series of cross-linked PEGMA-PMPC latex particles were prepared in various 2-propanol/water mixtures under the Langmuir 2009, 25(19), 11442–11449
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Figure 3. Optical micrographs of cross-linked PEGMA-PMPC latex particles prepared in various 2-propanol/water mixtures: (a) 95/5, (b) 90/10, (c) 85/15, and (d) 80/20.
conditions summarized in Table 1. In all cases, colloidally stable PEGMA-PMPC latexes were obtained with little or no coagulum. However, at stabilizer concentrations of 5.0 wt % PEGMA (data not shown), the latex particles proved to be colloidally unstable. Figure 2 shows the typical conversion vs time curves obtained for such formulations. Clearly the composition of the continuous phase influences the polymerization kinetics. Higher water contents lead to greater solvency for the growing oligomeric PMPC chains, which inevitably prolongs the prenucleation period.51,52 Polymerization initially proceeds homogeneously in the continuous phase and only becomes heterogeneous after particle nucleation. Water is a good solvent for both the MPC monomer and PEGMA stabilizer, so these two reactants will remain soluble as the 2-propanol/water composition is varied. Thus delayed nucleation most likely accounts for the observed slower rate of polymerization at higher water contents. The nature of the continuous phase also affected the final mean diameter of the latex particles. Larger PEGMA-PMPC latexes were obtained as the water content of the continuous phase was increased. This is evident from the optical micrographs shown in Figure 3. DLS studies also show similar results: the intensityaverage particle diameter increased from 1.6 to 4.8 μm as the water content of the continuous phase was increased from 5 to 20 wt %. However, particle size analysis by laser diffraction using a Mastersizer (data not shown) often indicated bimodal size distributions for the same latexes. This apparent discrepancy can be rationalized because the resolution of optical microscopy is relatively poor, while DLS has a strong bias toward larger particles because the scattered light intensity increases with the sixth power of the particle radius; these two instrumental limitations mean that submicrometer-sized latex populations are not detectable. Scanning electron microscopy of these PEGMA(51) Barrett, K. E. J. Dispersion Polymerization in Organic MediaLJohn Wiley: New York, 1975; p135. (52) Tauer, K.; K€uhn, I. Macromolecules 1995, 28, 2236–2239.
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Table 3. Characterization of Cross-linked PEGMA-PMPC Latex Particles Prepared with Variable EGDMA Content DLS run no.
Mastersizer
EGDMA % (w/w)
dμm
PDI
d[3,4]μm
uniformity
3
0.05
3.24
0.58
2.58
2
0.100
3.15
0.75
4
0.126
3.16
1.00
5
0.150
3.16
0.82
6
0.232
3.04
1.00
7
0.5
3.12
0.93
8
1.0
2.06
0.81
9
2.0
2.15
0.79
12
5.0
2.73
1.00
peak 1: 0.157 peak 2: 1.525 peak 1: 0.146 peak 2: 1.645 peak 1: 0.149 peak 2: 1.565 peak 1: 0.149 peak 2: 1.564 peak 1: 0.143 peak 2: 1.680 peak 1: 0.143 peak 2: 1.604 peak 1: 0.157 peak 2: 1.525 peak 1: 0.143 peak 2: 1.645 peak 1: 0.157 peak 2: 1.487
2.34 2.35 2.38 2.65 2.58 2.26 2.61 2.25
PMPC latexes was also somewhat problematic because the 2propanol/water composition tends to change during drying, leading to plasticization and coalescence of the particles. To improve this situation, it was decided to introduce a bifunctional crosslinker during the dispersion polymerization of the MPC. A series of PEGMA-PMPC latexes were prepared with EGDMA contents ranging from 0.05 to 5.0 wt % relative to total monomer using a fixed 90/10 2-propanol/water composition (see Table 2). In all cases, stable colloidal dispersions were obtained with no coagulum. Optical micrographs (not shown) did not suggest any significant differences in particle size, and DLS measurements were consistent with these observations (see Table 3). Bimodal size distributions were again typically obtained using the Mastersizer, with a significant population of submicrometer-sized latex particles being observed. SEM analysis DOI: 10.1021/la901631a
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Figure 4. SEM images of cross-linked PEGMA-PMPC latexes prepared using the following EGDMA and PEGMA contents: (a) 2.0 wt % EGDMA þ 10 wt % PEGMA (SEM sample prepared by spin-coating); (b) 5.0 wt % EGDMA þ 10 wt % PEGMA; (c) 2.0 wt % EGDMA þ 10 wt % PEGMA (SEM sample prepared by oven-drying); (d) 2.0 wt % EGDMA þ 20 wt % PEGMA.
Figure 5. Particle size distribution of cross-linked PEGMAPMPC latex particles prepared with variable PEGMA contents measured by Malvern Mastersizer.
Figure 6. Variation of mean particle diameter of cross-linked PEGMA-PMPC latex particles against AIBN initiator concentration. Latex particles were prepared with identical MPC (3.90 g), EGDMA (0.10 g), and PEGMA (1.0 g) formulation in 90/10 (w/w) 2-propanol/water mixtures.
proved somewhat problematic because these PMPC particles were relatively soft and rather prone to film formation. To try to address this problem, samples were prepared by spin-coating dilute dispersions of the more heavily cross-linked latexes directly onto the SEM stubs. This approach ensured both rapid drying (,1 s) and isolated latex particles. A typical SEM image of such a PEGMA-PMPC latex prepared using 2 wt % EGDMA is shown in Figure 4a. The particles are spherical and reasonably monodisperse, with diameters ranging from around 300 to 400 nm; somewhat larger mean diameters are obtained at higher EGDMA contents (see Figure 4b). However, these SEM diameters are typically intermediate between the two latex diameters observed by laser diffraction (see Figure 5). This suggests that some degree
of spreading/flattening of the smaller latex particles occurs during spin-coating. SEM sample preparation by oven-drying allowed both the small and the large latex populations indicated by laser diffraction to be observed by SEM (see the two red arrows in Figure 4c). Compared to our standard formulation conditions (see run 9 in Table 2), increasing the PEGMA concentration from 10 to 20 wt % reduced both the mean particle diameter and the polydispersity index, as judged by laser diffraction (see Figure 5). Moreover, the latter technique indicates that the bimodal size distribution obtained using 10 wt % PEGMA stabilizer becomes a relatively narrow, unimodal size distribution at 20 wt % PEGMA. Similar stabilizer concentration effects have been previously reported for sterically stabilized latexes prepared by
11446 DOI: 10.1021/la901631a
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Figure 8.
13
C NMR spectra of PEGMA, PMPC homopolymer and cross-linked PEGMA-PMPC latex particles prepared with 2 wt % EGDMA and 20 wt % PEGMA in a 90/10 2-propanol/ water mixture.
Figure 7. 1H NMR spectra of (A) cross-linked PEGMA-PMPC latex particles prepared with 2 wt % EGDMA and 20 wt % PEGMA, and (B) fluorescent cross-linked PEGMA-PMPC latex particles prepared with 5 wt % EGDMA and 20 wt % PEGMA.
dispersion polymerization.53,54 This observation is confirmed by SEM (see Figure 4d), which also confirms that partial particle coalescence occurs on drying at 70 °C. Figure 6 shows the effect of varying the AIBN initiator concentration on the mean DLS particle diameter of a series of PEGMA-PMPC latexes each prepared using 2 wt % EGDMA cross-linker and 20 wt % PEGMA stabilizer. Smaller latexes are produced at higher initiator concentrations, as expected. Because the number of free radicals (and hence the PMPC oligomer radicals) is increased, the nucleation stage becomes more extensive, which leads to a larger number of smaller final latex particles being formed. Despite the higher latex surface area, the PEGMA stabilizer remained effective at this relatively high concentration: polydispersities were almost constant and no coagulum was observed. The chemical composition of selected cross-linked PEGMAPMPC latexes was analyzed by 1H and 13C NMR spectroscopy and XPS. Figure 7A shows the 1H NMR spectra recorded for a PEGMA-PMPC latex prepared using 2 wt % EGDMA and 20 wt % PEGMA prepared in a 90/10 2-propanol/water mixture. The spectrum recorded in D2O shows all the characteristic peaks of PMPC chains, indicating that substantial latex swelling (i.e., microgel character) occurs under these conditions. Unfortunately, the expected oxyethylene signal due to the PEGMA stabilizer cannot be observed due to the large overlapping (53) Baines, F. L.; Dionisio, S.; Billingham, N. C.; Armes, S. P. Macromolecules 1996, 29, 3096–3102. (54) Baines, F. L. Ph.D. Thesis. University of Sussex: Brighton, U.K., 1995.
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azamethylene signal due to PMPC at 3.6 ppm. The effect of cononsolvency can also be observed for the spectrum recorded in 95/5 2-propanol-d8/D2O. As expected, no PMPC signals are observed in this mixture because it is a poor solvent for these chains. The presence of PEGMA stabilizer in the PMPC latex was qualitatively confirmed by 13C NMR spectroscopy. Figure 8 shows the 13C NMR spectra recorded in D2O (using DMSO as a reference) for PEGMA stabilizer, PMPC homopolymer, and a 2 wt % cross-linked PEGMA-PMPC latex, respectively. A weak signal at 70.24 ppm indicated that the PEGMA macromonomer is incorporated in the PMPC latex, as expected.36 XPS was used to examine the surface composition of the same PEGMA-PMPC latex. Figure 9 shows the XPS spectra recorded for the PEGMA stabilizer, PMPC homopolymer and the PEGMA-PMPC latex. O1s and C1s core-line spectra of PEGMAPMPC latex (not shown) do not differ significantly from the corresponding spectra obtained for PEGMA and PMPC homopolymer reference materials. However, the P2p signal intensity corresponds to ∼4.28 atom % in the PMPC-PEGMA latex and ∼5.0 atom % in PMPC homopolymer. Similarly, the N1s signal intensity was also reduced from 3.14 atom % in PMPC homopolymer to around 2.7 atom % in the PEGMA-PMPC latex. In both cases, this reduction is attributed to the presence of the PEGMA stabilizer, which contains neither P nor N. From these data, we estimate that the surface coverage of chemically grafted PEGMA chains is around 14%. Figure 10 shows how variation of the solvent composition affects the mean PEGMA-PMPC latex diameter for several crosslinking densities at fixed concentrations of 20 wt % PEGMA stabilizer and 0.20 wt % AIBN initiator. Smaller latexes were obtained on increasing the 2-propanol content of the dispersion medium. As reported by Ishihara and co-workers, PMPC is fully soluble in either water or 2-propanol, but becomes insoluble in certain 2-propanol/water mixtures (typically those containing 80-95% 2-propanol) due to an unusual phenomenon known as cononsolvency.30-32 It is likely that this behavior is due to subtle changes in hydrogen bonding between the PMPC chains and the two cosolvents.55 The molecular weight distribution of a linear PEGMA-PMPC latex prepared using 20% PEGMA stabilizer was assessed by gel permeation chromatography. This analysis indicated a number-average molecular weight of 80200 (expressed (55) Franks, F.; Desnoyers, J. E. In Alcohol-Water Mixtures Revisited; Franks, F., Ed.; Cambridge University Press: London, 1985, p 171-232.
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Figure 9. XPS spectra of PEGMA, PMPC homopolymer, and cross-linked PEGMA-PMPC latex particles prepared with 2 wt % EGDMA and 20 wt % PEGMA in a 90/10 2-propanol/water mixture.
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Figure 11. Effect of NaCl concentration on the variation of
solution viscosity at 25 °C for 1.0% aqueous solutions of crosslinked PEGMA-PMPC microgel particles prepared with varying EGDMA cross-link densities.
Figure 10. Variation of mean particle size at 25 °C for cross-linked PEGMA-PMPC latex particles prepared with different EGDMA cross-linker contents in 90/10 2-propanol/water.
in poly(methyl methacrylate) equivalents) and a polydispersity of 2.0, as expected for free-radical polymerization under dispersion conditions.56,57 Thus this new dispersion polymerization protocol may provide a convenient “low viscosity” route to high molecular weight linear PMPC homopolymer (if the relatively low PEGMA stabilizer content is ignored). In this context, we note that PMPC homopolymers and certain statistical copolymers are already used in various cosmetics formulations, particularly in Japan. Figure 11 shows the effect of added salt on the solution viscosity of a series of PEGMA-PMPC latexes prepared using 10 wt % PEGMA and 0.20 wt % AIBN initiator with varying amounts of EGDMA cross-linker. The solution viscosities remained essentially unchanged irrespective of the salt concentration. This is due to the so-called “antipolyelectrolyte effect”, which characterizes the aqueous solution properties of polyzwitterions such as PMPC.29,58 In contrast, conventional polyelectrolytes such as ionized poly(methacrylic acid) or protonated poly (2-vinylpyridine) are highly swollen at zero or low salt concentration but collapse on the addition of salt due to charge screening reducing the electrostatic repulsions between the anionic (56) Bourgeat-Lami, E.; Guyot, A. Polym. Bull. 1995, 35, 691–696. (57) Cho, M. S.; Yoon, K. J.; Song, B. K. J. Appl. Polym. Sci. 2002, 83, 1397– 1405. (58) Matsuda, Y.; Kobayahsi, M.; Annaka, M.; Ishihara, K.; Takahara, A. Chem. Lett. 2006, 35, 1310–1311.
11448 DOI: 10.1021/la901631a
Figure 12. Confocal laser scanning microscopy image of fluorescent cross-linked PEGMA-PMPC latex particles prepared with 5 wt % EGDMA and 20 wt % PEGMA in a 90/10 2-propanol/ water mixture.
(or cationic) chains.59-61 Figure 11 also suggests a weak maximum in solution viscosity at approximately 0.23 wt % EGDMA. Below this optimal degree of cross-linking, it is likely that there is a significant soluble fraction of noncross-linked PMPC chains, whereas overcross-linking (and hence reduced swelling) occurs at higher EGDMA contents.62 In this investigation, fluorescent PEGMA-PMPC latex was also prepared using fluorescent comonomer, FMA. The incorporation of FMA was confirmed by both visible absorption spectroscopy (data not shown) and 1H NMR spectroscopy; see Figure 7B. Additional aromatic protons appeared at 6.46-8.33 ppm, and integration of this relatively weak signal suggests that at least 40% of the FMA comonomer was incorporated into the PMPC latex. Figure 12 shows the CLSM image obtained for this (59) Eliassaf, J.; Silberberg, A. J. Polym. Sci., Polym. Chem. Ed. 2003, 41, 33–51. (60) Guo, L.; Tam, K. C.; Jenkins, R. D. Macromol. Chem. Phys. 1998, 199, 1175–1184. (61) Dupin, D. Ph.D. Thesis. University of Sheffield: Sheffield, U.K., 2007. (62) Tan, B. H.; Tam, K. C.; Lam, Y. C.; Tan, C. B. Adv. Colloid Interface Sci. 2005, 120, 111–120.
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fluorescently labeled PEGMA-PMPC latex. The particles are spherical and fairly monodisperse, with an approximate numberaverage particle diameter of 750 to 900 nm. Taking into account the effect of polydispersity, this is consistent with the Mastersizer diameter of 940 nm diameter obtained for this latex. Given the highly biocompatible nature of the PMPC and PEGMA chains, such fluorescently labeled latexes may have potential biomedical applications, e.g., as model particles for studies of phagocytosis.
Conclusions PMPC latex particles were prepared by dispersion polymerization using PEGMA macromonomer as a reactive stabilizer in various 2-propanol/water mixtures. The resulting sterically stabilized latexes were spherical and colloidally stable. Both DLS measurements and optical microscopy studies suggest that larger latexes are obtained as the solvency of the PMPC chains in the continuous phase is increased. Laser diffraction studies indicated bimodal size distributions for PMPC latexes prepared in the presence of 10 wt % PEGMA, with a significant population of submicrometer-sized particles. SEM studies confirmed these bimodal size distributions, and it was also shown that unimodal size distributions could be achieved by conducting PMPC latex syntheses in the presence of 20 wt % PEGMA stabilizer. The incorporation of PEGMA stabilizer into the latex particles was
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confirmed by 13C NMR spectroscopy. The mean PEGMAPMPC latex diameter also varied according to the 2-propanol content of the continuous phase as well as the initiator concentration. Gel permeation chromatography analysis of the linear latexes confirmed that relatively high molecular weight PMPC chains were obtained (Mw ∼ 160 000 vs poly(methyl methacrylate) calibration standards). Cross-linking could be achieved by introducing ethylene glycol dimethacrylate comonomer with relatively little effect on the mean latex diameter. The cross-linked PMPC latexes swelled on dilution with water as expected and, irrespective of their cross-linking density, the solution viscosities of the resulting PMPC microgels were relatively insensitive to salt concentration due to the so-called “antipolyelectrolyte” effect exhibited by polyzwitterions. Cross-linked fluorescent PEGMAPMPC latexes were prepared by the same polymerization protocol using a commercially fluorescent methacrylic comonomer. Acknowledgment. The authors are grateful to the Commonwealth Commission, U.K. for financial support of HA during his visit to the University of Sheffield, UK. Biocompatibles Ltd. are thanked for providing the MPC monomer. SPA is the recipient of a five-year Royal Society-Wolfson Research Merit Award. DD thanks the University of Sheffield for financial support of his postdoctoral studies.
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