Langmuir 1993,9, 1637-1645
1637
Preparation of Polymer Latex Particles with Immobilized Sugar Residues and Their Surface Characterization by X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry M. C. DaVies,*J R. A. p. Lynn,tJ S. S. Davis,t J. Hearn,g J. F. Watts,l J. C. Vickerman,ll and A. J. Paul11
Department of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, U.K., Department of Chemistry and Physics, Nottingham Polytechnic, Clifton Lane, Nottingham N G l l 8NS, U.K., Department of Materials Science and Engineering, University of Surrey, Guildford GU2 5XH, U.K., and Centre for Surface and Materials Analysis, Department of Chemistry, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester M60 lQD, U.K. Received August 20, 1992. In Final Form: March 26, 1993
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS)have been employed for the surfacechemical analpis of a seriesof polymer colloidswith immobilized sugar residues. A thioglycoside, based on galactose (GAL),with an acryloyl group in the aglycon side chain has been prepared, characterized, and copolymerized with styrene in various proportions by surfactantfree emulsion polymerization employing potassium persulfate initiator. The quantitativeelemental and chemical state XPS information suggests the presence of the stericstabilizing water-solublepolymer layer at the colloid surface. These data are complemented by the detection of signals in the ToF-SIMS spectra which are specificallycharacteristic of the sugar derivative, including those attributableto the intact GAL molecule. The changes within the C 1s core level spectra from the XPS analysis and also the area ratios of diagnostic signals in the ToF-SIMS spectra reflected the increase in the surface level of GAL with increasing bulk levels of the galactose derivative. These changes correlate very well with the observed substantial decrease in particle size and electrophoretic mobility of the poly(styrene) (PSI-based colloids as a function of the level of GAL employed, reflecting the presence of a steric stabilizing water-soluble polymer layer at the colloid surface. The data are discussed briefly in terms of the mechanism of colloid formation to provide an explanation of GAL surface enrichment but primarily concentrate on the role of ToF-SIMS and XPS for the surface analysis of these complex polymer colloids. Introduction Many of the biological applications of polymer colloids requirethe presence on the particle surface of active ligands including antibodies, drugs, enzymes, or carbohydrates. It is often advantageous to couplecovalently (alsoreferred to as "grafting") the biomolecule of interest to the particle surface rather than relying on adsorption processes alone, since this eliminates the possibility of desorption or displacement. Several suitable procedures have been developed, and in general, these do not attenuate the activity of the biomolecule in terms of its specificity or affinity.l One disadvantage of this approach is that the stability of the latex may be adverselyaffected during the reactions. Confirmation of the success or otherwiseof the grafting procedure is often achieved via a fluorescent or radioactive marker or from observations of biological activity. In many situations it may be useful to have an indication of the density of the grafted molecule and the nature of the covalent linkage. An alternative to this so-called 'postgrafting" procedure for biomolecule immobilization is the preparation of a polymerizable derivative of the molecule in question. This technique has been used for the preparation of polymeric
* To whom correspondence should be addressed. + University of Nottingham. t Present address: SmithKline Beecham Pharmaceuticals, Great Burgh, Yew Tree Bottom Rd., Epsom, Surrey KT18 SXQ, U.K. 8 Nottingham Polytechnic. 1 University of Surrey. 11 University of Manchester Institute of Science and Technology. (1) Dean, P. D. G., Johnson, W. S., Middle, F. A., Eds. Affinity Chromatography - A Practical Approach; IRL Press: Oxford, 1985.
matrices with pendant drug residues2 and also for the incorporation of peptide3 and glycoside4 residues into water-soluble polymers. In theory, it should also be possible to copolymerize derivatives of peptides, drug molecules, and oligosaccharides with less water-soluble monomers to produce latex particle systems. This approach was exploited by Scholsky and Fitch to prepare latex systems for controlled drug release using acrylate ester derivatives of salicylic acid and chloramphenicol copolymerized with an ethylene diacrylate cross-linker.6 In terms of the incorporation of receptor-specific "targeting" residues, the question then remains as to whether any of these actually reside on the particle surface at the end of the polymerization process. Thiswork is concerned with the surfacecharacterization of polymer latex particles prepared bearing covalentlyattached monosaccharide residues at the particle surface. The significance of saccharides lies in the key role of the carbohydrate determinants of many glycoproteins and glycolipids in biological recognition processes? These determinants are recognized by saccharide-bindingproteins (receptors)on cell surfaces. Thus, Kupffer cells and alveolar macrophages have binding proteins specific for D-mannoselN-acetyl-D-glucosamine while fibroblastshave (2) Ferruti, P.; Tanzi, M. C. CRC Crit. Reu. Ther. Drug Carrier Syst. 1986,Z (2), 175. ( 3 ) Rejmanova,P.;Obereigner,B.;Kopacek, J.Makromol. Chem. 1981,
-182.1899. - -, - - - -.
(4) Chytry, V.;Kopacek, J.; Leibnitz, E.; OHare, K.; Scarlett, L.; Duncan, R. New Polym. Mater. 1987, 1 (l),21. (5) Scholsky, K. M.; Fitch, R. M. J. Controlled Release 1986, 3, 87. (6) Neufeld, E. F.; Ashwell, G. In The Biochemistry of Glycoproteins and Proteoglycans; Lennarz, W. J., Ed.;Plenum Press: New York, 1880, p 241.
0743-7463/93/2409-1637$04.00/00 1993 American Chemical Society
Davies et al.
1638 Langmuir, Vol. 9, No. 7, 1993 a receptor for ~-mannwe-6-phosphateand hepatocytes possess receptors recognizing D-gdaCtoSe and N-acetyl~-galactosamine.~The potential of sugar residues as targeting groups in drug delivery has been investigated using liposomes8and water-soluble polymers?JOand has been reviewed by Shen7 The phagocytic activityof retinal cells has been investigated using carboxylated PS latex particles with immobilized monosaccharide residues." In the present study, a major requirement was to prepare a polymerizable derivative of galactose. Several methods have been reported in the literature for synthesizing such materials. These include coupling of the lactone derivatives of mono- or oligosaccharides to @-vinylbenzy1)amine to produce styrene-type macromonomers.l2JSThe major advantagesof this synthetic route are that hydroxyl group protection on the sugar is not required and that it is applicable to a range of carbohydrates including both mono- and oligosaccharides. A series of alternative synthetic routes was described by Lee et al. involving the synthesis of glycosides having acryloyl groups at the terminus of the aglyconportion of the derivative.lk17These were then copolymerized with acrylamide and methylene bhcrylamide to form gels bearing immobilizedglycosides, and used to investigate cellular adhesion phenomena.la21 In order to establish a structure-activity relationship between the surface sugar derivative and the colloidal performance in vitro and in uiuo,a detailed understanding of the interfacial chemicalstructure of the colloidalsurfaces is required. In this paper, we explore the potential of time-of-flight secondary ion mass spectrometry (ToFSIMS)and X-ray photoelectron spectroscopy (XPS) for the surface chemical analysis of a range of polymer latex systems prepared by the polymerization of a galactose derivative (GAL,) with styrene in the absence of emulsifier. The qualitative molecular information from ToF-SIMS coupled with the quantitative elementaland chemicalstate data from X P S has been shown to provide detailed insight intothe surfacechemistryof polymers,~23 biomaterials,24p26 and, more recently and to a limited extent, polymer colloids.529 In this work, the proportion of saccharide (7) Shen, T. Y. Biological Approaches to the Controlled Delivery of Drugs. In Annab ofthe New York Academy ofScience8;Juliano, R. L., Ed.;New York Academy of Sciences: New York, 1987; Vol. 607, p 272. (8) Dae,P. K.; Murray, G. J.; Zirzow, G. C.; Brady, R. 0.; Barranger, J. A. Biochem. Med. 1986,33, 124. (9) Seymour, L. W.; Duncan, R.;Kopeckova,P.; Kopacek,J. J. Bioact. Compat. Polym. 1987,2, 97. (10) Bridges, J. F.; Woodley, J. F.; Duncan, R.; Kopacek, J. Znt. J. Pharm. 1988,44, 213. (11) Seyfried-Williams,R.; McLaughlin,B. J. VisionRes. 1983,23 (51, 485. (12) Kobayaehi,K.; Sumitomo,H.J. Macromol. Sci., Chem. 1988,A25 (5-7), 655. (13) Kobayaehi, K.; Sumitomo,H.; Ina, Y. Polym. J. 1983,lS (9), 667. (14) Chipowsky, 5.;Lee, Y. C. Carbohydr. Res. 1973,31,339. (15)Lee, R. T.; Cascio, 5.;Lee, Y. C. A d . Biochem. 1979,96, 260. (16) Lee, R. T.; Lee, Y. C. Methods Enzymol. 1982,83,299. (17) Stowell, C. P.; Lee, Y. C. Method8 Enzymol. 1982,83,278. (18) Kuhle"idt,M. S.;S h e l l , E.;Lee,R.T.;Lee, Y. C.;Roaeman, S . J. Biol. Chem. 1979,254 (21), 10830. (19) Weigel, P. H.; Schmell,E.; Lee, Y. C.; Roseman, S. J. Biol. Chem. 1978,253 (2), 330. (20) Schnaar, R. L.; Weigel, P. H.;Kuhlenechmidt, M. S.; Lee, Y. C.; Roeeman, S. J. Biol. Chem. 1978,253 (211, 7940. (21) Guamaccia, S. P.; Kuhlenschmidt, M. S.; Slife, C. W.; Schnaar, R. L.J. Biol. Chem. 1982,257 (23), 14293. (22) Brigga, D. Br. Polym. J. 1989,21, 3. (23) Andrade, J. D. In Surface ondZnterfacia1 Aspects of Biomedical Polymers, Volume 1, Surface Chemistry and Physics; Andrade, J. D., Ed.; Plenum Press: New York, 1985; p 105. (24) Davies, M. C.; Lynn, R. A. P. Crit. Reu. Biocompat. 1990,5 (4) 297. (25) Ratner, B. D.; In Polymers in Medicine ZZZ; Migliaresi, C., et al., Eds.; Elsevier: Amsterdam, 1988; p 87. (26) Pijpers, A. P.; Donners,W. A. B. J. Polym. Sci.: Polym. Chem. Ed. 1985,23,453.
S
STEP I Acetobromoa-D-Galactose
0
II
II
Michael Addition (methylene-bis-acrylamide)
STEP 3
0
II
CH,=CH-C-NH-CH,-NH-C-CH=CH2
j
: z e NaOMe tylation
0 0 ~ ~ ~ ~ - ~ c H , ~ c - NI1H - c H ~ - N H - c - c H = c H ~
II
OH D-Galactose Derivative with Acryloyl Group in Aglycone Side.Chain
Figure 1. Syntheticroute to S-glycosides with acryloyl groups in the aglycon side chain.
monomer was varied in an attempt to produce particles with differentsurface coveragesof galactcme. In particular, our aim was to establish whether ToF-SIMS and X P S can provide valuable information on the molecular structure and surfacepresence of the GAL derivativeto compliment other colloid characterization techniques such as particle size and electrophoretic mobility measuremente. Where appropriate, the data are discussed briefly in terms of the particle formationmechanismbut in the main concentratea on the role of Tof-SIMSand X P S for the surface chemical analysis of these complex colloids.
Preparative and Characterization Method8 Synthesis and Characterization of the Galactore Derivative. T h e synthetic prduresummanzed belowis baaed upon the methods described by Lee et al.lC1'with minor modifications introducedto improve the yield and removeunreadad materialg more effectively. This route is shown in Figure 1 and involves reaction of a mixture of 2,3,4,6-tetra-O-acetyl-a-~galactopyranosy1 bromide (Sigma,Dorset, U.K.) and fiiely-groundthiourea (BDH, Doreet, U.K.) to form (2,3,4,Btetra-O-acstyl-yl-gDgalactopyranosy1)-1-isothioureahydrobromide(step1). Thisproduct is then converted to 2,3,4,6-tetra-O-acetyl-l-thio-gD-galacto ranoside by reduction (step 2), followed by an addition reaction with NJ-methylenebisacrylamide (Aldrich, Gillingham, U.K.) to form 1-[ [2-[[(N-acrylamidomethyl)aminolcarbonyllethyl]thio1-2,3,4,6-tetra-O-acetyl-gDgalactopyranoede (step3) which fiially undergoes deacetylation to yield an S-glycoside with an acryloyl group in the aglycon side chain, l-E[2-[[(N-acrylamid o m e t h y l ~ a m i n o 3 ~ ~ n y l l ~ y l l e t h y l l t h i o 3 - B(step Dga 4). T h e producta of steps 3 and 4 (Le., the acetylated and deacetylated versions of the product) were analyzed by proton NMR using a 90-MHzVarian EM390 spectrometer (Varian Associates). The product of step 3 was dissolved in deuterated (27) Zhao,C.-L.;Holl, Y.; Pith, T.;Lambla, M. Br. Polym. J. 1989,21, 155. (28) Zhao, C.-L.; Dobler, F.; Pith, T.; Holl,Y.; Lambla, M. J. Colloid Znteface Sci. 1989, 128 (2), 437. (29) K&, F.; Muller, R. H.;Davis,5.5.;Daviea, M.C. J. Controlled Release 1989, 9, 149.
Langmuir, Vol. 9, No. 7, 1993 1639
Preparation of Polymer Latex Particles Table I. Polymerization Recipes for Poly(8tyrene)-Bared Colloids Prepared in the Presence of Galactose Derivative (Total Monomer Composition 2% w/v) volume added (mL) 1 2 3 4 styrene 1.105 0.995 0.885 0.775 galactuse derivative - (0) 3.05 (10) 6.05 (20) 9.09 (30) (3.3% W/Vin H2O)b potassiumpereulfate 1.5 1.5 1.5 1.5 solution (0.1% w/v) double-dmtilled water 47.4 44.5 41.6 38.6 totalvolume 50.0 50.0 50.0 50.0 0 Based on density of 0.905g mL-1. b Values in parenthesesindicate (w/w) percent galactose derivative in the monomer mixture. ~~
chloroform (CDCld (Aldrich, Gillingham,U.K.) while the product of step 4 was dissolved in deuterated dimethyl sulfoxide (DMSOde) (Aldrich, Gilliigham, U.K.) in the presence of a few drops of D10 to aid solubility. In each case, tetramethylsilane (TMS) (Aldrich, Gdingham, U.K.) was added as a reference. Analysis of the final product by positive ion fast atom bombardment mass spectrometry (FAB-MS) was performed using a modified AEI -902 instnunent (AEUKratos, Mancheeter, UK), employing a krypton atom source. The sample was prepared by dispersing a few mg of the product in a glycerol matrix. Preparation of Polymer Latex Particles. Monomer Purification a n d Colloid Production. Styrene monomer (ST) (Aldrich, Gillingham, U.K.) contained 10-15 ppm p-tert-butylcatechol as inhibitor and was purified before use by distillation a t 40-60 OC at 5 mmHg of pressure under nitrogen. Purified monomer was stored under nitrogen at 4 OC protected from light until required. Having confirmed the identity of the galactose derivative from NMR and fast atom bombardment mass spectrometry (FABMS), it was used in polymer formation without further purification. Potassium persulfate initiator (BDH, Poole, U.K.) was recrystallized twice from double-distilled water and dried in a desiccator. This process was repeated after two weeks of storage. A series of latices based on PS was prepared by surfactantfree emulsion polymerization with various proportions of the galactose derivative (Table I). The colloids were prepared on a small scale with a typical fiial volume of 50 mL and a total monomer concentration of 2% w/v. The required quantity of double-distilledwater was heated to 70 OC (A1 "C) under nitrogen. The required volume of a 3.3% w/v solution of the galactose derivative in water was then added to the flask. AB soon as the temperature had returned to 70 "C, the appropriate volume of styrene was added, followed immediately by 1.5 mL of a 0.1% w/v potassium persulfate solution (equivalent to a final concentration of 11.1X 1odmol dm-9). The reaction solutions were stirred continuously for 24 h. Prior to cleaning, aggregates were removed from the colloids by fiitration through Whatman No. 1filter paper (Whatman, UK). Cleaningand Storage of Latex Systems. The colloids were cleaned by exhaustively dialyzing 10-mLsamples against doubledistilled water using Spectropor membrane tubing (MW cutoff, OOO,Spectrum Medical Industries, Los Angeles, CA), 12 -14 which was rigorously cleaned before use. The dialysate was changed every 24 h for a period for 14 days, by which time ita conductivity was equal to that of water. The removal of residual monomer was confirmed by centrifuging 5-mL samples of the cleaned colloids at 10 000 rpm on a Beckman JS-21 Centrifuge (Beckman Instruments Inc., Palo Alto, CA) and performing a UV scan on the supernatant. Purified latex samples were stored in chromic acid-cleaned glass tubes at 4 OC prior to analysis. In each m e , the yield of polymer as latex was greater than 80 % . Characterization of Polymer Colloids. Size a n d Electrophoretic Mobility Determination. Measurements on the particle size of the latex were carried out by photon correlation spectroscopy (PCS)Bo*B1 (Malverm Instruments Ltd., U.K.). A (30)Cummine, H. Z., Pike, E. R., Ede. Photon Correlation and tight Beating Speectroacopy; NATO AS1 Series B (No. 3);PlenumPreea: New York, 1974.
total of 10 measurements were recorded for each sample, and mean size (dz)and polydispersity (Q)values were calculated. The value of Q is determined from Koppel's method of cumulanta. Using this approach, monodisperse latices have a value of Q = 0.03, but correlative data from electron microscopy suggest that values less than 0.1 reflect particle sizes that are narrowly distributed. The determination of the colloid electrophoretic mobility (EPM) and 5 potential (ZP) was performed using the technique of laser Doppler anemometry.Measurements were carried out using a Malvern Zetasizer I1 (Malvern Instruments, Malvern, U.K.). EPM measurements were made over the pH range of 3.0-8.0 using phosphate-citrate buffers of constant ionic strength (0.01 M). Samples were prepared by the addition of 200-300 pL of latex to 5 mL of the appropriate buffer solution. This level of dilution provided a suitable scattered light intensity without compromisingbuffering capacity. Five measurements were made for each latex sample at each pH. X-ray Photoelectron Spectroscopy (XPS). XPS spectra were obtained using a VG Scientific ESCALAB Mk I1 electron spectrometer (VG Scientific Ltd., East Grinstead, Sussex, U.K.) employing Mg Ka X-rays (photon energy, hv = 1253.6 eV), and an electron takeoff angle of 45O. The base pressure of the spectrometer was typically 108 mbar. The X-ray gun was operated at 10 kV and 20 mA, corresponding to a power of 200 W. A wide scan (0-1000 eV) was recorded for each sample (single scan) followed by the C Is, N Is, 0 Is, and S 2p regions where appropriate (five scans). The analyzer was operated in fixed analyzer transmission (FAT) mode with a pass energy of 50 eV (wide scan) and 20 eV (C Is, 0 Is, N Is, and S 2p regions). Data analysis was performed on a VGS 5000 data system based on a DEC PDP 11/73computer. The methodology employed for peak fitting of the C 1s envelopes has been described in detail elsewhere.98 Typically, 1.51.6-eV line widths and Gaussian/ Lorentzian ratios of 30% were employed for the components of the C 1s envelope. Atomic percentage values and elemental ratios were calculated from the peak areas using sensitivity factore and background subtraction. Spectra were corrected for sample charging by referencing photoelectron peaks to C-C/C-H at 285 eV." Time-of-Flight SIMS. ToF-SIMS studies were performed using a VG Ionex 1x235 instrument based on a Poschenrieder design.u The primary ions were Ga+of 30-keV energy generated from a liquid metal ion source. The Ga+ ions were focused into a sample area of 0.6 mm X 0.6 mm in pulses of 20-ne duration, with an incident angle of 38' normal to the surface. The total ion dose during setup and spectral acquisition was about 1X 10" cm-2 per sample which lies well within the "static" SIMS regime.Both positive and negative secondary ion mass spectra were acquired over the m/z range of 0-1600. The secondary ions were accelerated to 5 keV before entry into the analyzer by applying a bias of the appropriate polarity to the sample. A DEC PDP 11 computer system was for spectral acquisition, storage, and data processing. ToF-SIMS and XPS analyses were also carried out on a f i b deposited onto aluminum foil prepared from a dilute aqueous solutionof the galactose derivative alone. This provided a means of identifying spectral features unique to this monomer, thereby facilitating its identification on the polymer latex surfaces.
Results and Discussion Characterization of the Galactose Derivative. In the positive ion FABMS spectrum for the final product (31) Cu"ina,H.Z.,Pike,E.R.,Eds.Photon Correlotion Spectroscopy
and Velocimetry; NATO AS1 Series B (No. 23); Plenum Press: New
York, 1977. (92) Earnshaw, J. C., Steer, M. W., Eds. The Application of Laser tight Scattering to the Study of Biological Motion; Plenum Press: New York, 1983. (33) Sherwood,P. M. A. In Practical Surface Analysis (by Auger and X-ray Photoelectron Spectroscopy);Briggs, D., Seah, M. P., E&; John Wilay: Chichester, 1983; p 445. (34) Eccla, A. J.; Vickerman, J. C.J. Vac. Sci. Technol. 1989, A7 (21, 234. (35)Briggs, D.;Heam, M.J. Vacuum 1986,36,1005. (36) H e m , M.J.; Briggs, D. Surf. Interface Anal. 1986, 9,411.
1640 Langmuir, Vol. 9, No.7, 1993
Davies et al.
also observed considerablereductions in the particle size of emulsifier-freelatices prepared with increasingfractions of AMin the initial monomer charge.s7 This was attributed monomer commition to the presence of a water-soluble (i.e., AM-rich) polymer layer on the particle surfacea which was capableof sterically (5% W/W) EPMb ZPb ST GAL dza(nm) Q ( r r m ~ - ~ c m V - 9(mV) stabilizing the colloid at a smaller particle size than that achieved by the electrostatic barrier on PS particles. Thie -66.3 0.052 5.08 100 234 hypothesis was corroborated by a series of critical coag-39.0 0.034 2.99 90 10 165 2.21 -29.6 0.070 80 20 156 ulation concentrationexperimentsin which they observed -26.2 30 153 0.045 2.04 70 that homocoagulation of PS/AM colloids did not occur even at high electrolyte In contrast,rapid Mean of lomeasurements,coefficients of variation,
