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Preparation of Nanocomposite Poly(allylamine)-Poly(ethylene glycol) Thin Films Using Michael Addition
Scheme 1. Reaction Scheme for Nanocomposite Poly(allylamine)-Poly(ethylene glycol) Thin Films Prepared on a Mercaptoundecanoic Acid Modified Gold Substrate
Ryan J. Russell, Kaushik Sirkar, and Michael V. Pishko* Department of Chemical Engineering, Texas A&M University, College Station, Texas 77843-3122 Received October 21, 1999. In Final Form: January 26, 2000
Introduction The development of nanoscale polymer films is an emerging field that has potential applications in diverse areas such as biosensing, corrosion control, and cell patterning. The composition and function of the film are obviously of great importance, but other issues include ease, method, and reproducibility of fabrication. Several recently reported approaches have used Michael addition chemistries for preparing thin polymer films using monomethacrylate monomers on silica particles,1,2 glass fibers,3 and gold.4 These approaches focus on nucleophilic addition of primary amines with acrylic monomers, resulting in amide bond formation. Here we report on a similar methodology using acrylate-functionalized macromers for the development of thin, branched, surfacegrafted poly(ethylene glycol) (PEG) films, a polymer whose proven biocompatibility5-8 is desired for many biosensing and biomaterials applications. In this Note we demonstrate the sequential formation of potentially biocompatible ultrathin films on gold. Our synthetic procedure used Michael addition reactions to fabricate nanocomposite thin films consisting of alternating layers of acrylate-functionalized poly(ethylene glycol) (PEG) and poly(allylamine) (PAA). We examined film fabrication in both aqueous and organic solvents for PEGs of differing chain lengths and end group functionalization, for example, diacrylated PEG of molecular weight 575 (PEG 575) and tetraacrylated PEG of molecular weight 18 500 (PEG 18500). Experimental Section A typical thin film growth scheme in deionized water was as follows (see Scheme 1): Sputtered gold surfaces (1000 Å Au, 200 Å Cr on polished Si, Lance Goddard Associates, CA) were acidfunctionalized by immersion for 20 min in 1 mM mercaptoundecanoic acid (MUA) dissolved in ethanol. The initial generation of PAA was grafted by activating the MUA layer carboxylic acids with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and subsequently reacting with a 15 µM PAA solution for 1 h (step 1). Michael addition between PAA pendant amines and acrylated PEG end groups was performed by placing the substrate * To whom correspondence should be addressed. Phone: (409) 847-9395. Fax: (409) 845-6446. E-mail:
[email protected]. (1) Tsubokawa, N.; Ichioka, H.; Satoh, T.; Hayashi, S.; Fujiki, K. React. Funct. Polym. 1998, 37, 75-82. (2) Hayashi, S.; Takeuchi, Y.; Eguchi, M.; Iida, T.; Tsubokawa, N. J. Appl. Polym. Sci. 1999, 71, 1491-1497. (3) Fujiki, K.; Sakamoto, M.; Yoshikawa, S.; Sato, T.; Tsubokawa, N. Compos. Interfaces 1999, 6, 215-226. (4) Zhang, L.; Bo, Z.; Zhao, B.; Wu, Y.; Zhang, X.; Shen, J. Thin Solid Films 1998, 327-329, 221-223. (5) Sawhney, A.; Pathak, C.; Rensburg, J. V.; Dunn, R.; Hubbell, J. J. Biomed. Mater. Res. 1994, 28, 831-838. (6) Sawhney, A.; Hubbell, J. Biomaterials 1992, 13, 863-870. (7) Desai, N.; Hubbell, J. J. Biomed. Mater. Res. 1991, 25, 829-843. (8) Delgado, C.; Francis, G.; Fisher, D. Crit. Rev. Ther. Drug Carrier Syst. 1992, 9, 249-304.
in a 10% (v/v) solution of PEG diacrylate for 2 h at room temperature (step 2). Please note that Scheme 1 depicts additions only to primary amines. However, other additions are possible, including additions to amides. Additional PAA and PEG layers were applied by repeating the PAA (excluding EDAC activation of the MUA layer) and PEG addition steps (step 3). Between each immersion, the coated substrate was washed with ethanol and dried under N2. Both aqueous and alcohol reaction media were examined, although films fabricated in ethanol were allowed to react overnight (∼12 h) for steps 2 and 3. For nanocomposite films of PEG 575, three PEG generations were deposited. Additional films were fabricated by replacing the PEG 575 solution with a dilute solution of tetraacrylated PEG 18500. The acrylate-functionalized PEG macromer was prepared from tetrahydroxy-PEG 18500 (Polysciences, Warrington, PA), a linear polymer formed from the linking of two PEG chains (MW 9000) by a bisphenol A bisepoxide spacer, using a published procedure.9 Acrylation results in two terminal acrylate groups and two acrylate groups in the middle of the polymer at the bisphenol moiety. Attenuated total reflectance FTIR (FTIR-ATR) of the polymer dissolved in ethanol was used to confirm acrylation of the PEG hydroxy groups. Due to macromer length, only one layer of PEG 18500 grafted onto PAA was examined. Increases in film thickness were quantified using ellipsometry and the introduction of various functional groups (amine, amide, carboxyl, carbon-carbon stretching) was verified with Fourier transform infrared external reflectance spectroscopy (FTIR-ERS). Prior to spectral acquisition, films prepared in aqueous media were first dehydrated overnight under vacuum to remove excess water from the hygroscopic PEG layers. FTIR-ERS measurements were made using a Bio-Rad FTS-40 spectrometer outfitted with a Harrick Scientific Seagull reflection accessory (Harrick Instruments, Inc., Ossining, NY) and a liquid N2 cooled MCT detector. Ellipsometry was performed using a Gaertner model L2W26D ellipsometer (Gaertner Scientific Corp., Chicago, IL) using a 633 nm HeNe laser. Refractive indexes and film thicknesses were determined by assuming a homogeneous film model using Gaertner software.
Results and Discussion Nanoscale thin films of poly(ethylene glycol) and poly(allylamine) macromers were fabricated using Michael addition between PEG acrylated end groups and PAA pendant amines. Previous surface modification techniques using Michael addition have primarily used methacrylates monomers to grow thin films.1-4 While film thickness was not reported, use of monomers likely resulted in extremely small growth in film thickness over multiple generations. (9) Quinn, C.; Pathak, C.; Heller, A.; Hubbell, J. Biomaterials 1995, 16, 389-396.
10.1021/la991388i CCC: $19.00 © 2000 American Chemical Society Published on Web 03/17/2000
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Figure 1. Ellipsometric thicknesses (n ) 6) of three generations of diacrylate-terminated PEG (MW 575) polymer films constructed from Michael-type addition with pendant primary amines on poly(allylamine) (MW 65000) in deionized water. Error bars reflect standard deviation.
Between generations these films underwent an amination step before the end groups were available for subsequent Michael addition reactions. While more complex, this approach prevented both ends of an R,ω-functionalized monomer from reacting with the substrate and inhibiting further film growth due to lack of residual functionality. Our approach was to use macromers as Michael-type substrates to prepare nanoscale thin films. We anticipated macromer length and polymer conformation would minimize the probability of reacting both terminal acrylates. Multifunctional macromer chains eliminated need for an amination step between Michael additions, minimized film growth termination due to reactions occurring to both the R- and ω-acrylates, and produced thicker films with each grafted layer. Measured layer thicknesses are shown in Figure 1 for PAA and PEG 575 thin films made using aqueous solutions as the reaction medium. Three generations of PEG 575 were grafted with PAA by Michael addition of acrylated end groups on PEG 575 and pendent amines on PAA. Ellipsometric measurements revealed a linear increase in film thickness through three generations, excluding the first grafted PEG layer, which consistently yielded high values. Film thickness increases during the PEG additions averaged 18.5 Å, while PAA increases were about 10 Å. For comparison, we calculated a random coil length for PEG 575 to be 17 Å using Cerius2 v3.5 molecular modeling software (Molecular Simulations, Inc., San Diego, CA).10 Thickness changes for PAA layers tended to be smaller than the PEG 575 chains, a reflection of the horizontal conformation one would expect from the PAA, due to multiple pendant amide linkages to the underlying layer. The large thickness of the first PEG layer is likely due to the different extent of reactions between Michael addition of acrylated PEG with PAA pendant amines and the initial reaction between PAA and activated MUA. We believe the initial EDAC-activated MUA covalently coupled to a large percentage of the available pendant amines on the first PAA layer, resulting in few amines for the initial PEG addition. As a result, the first PEG layer was less dense than subsequent PEG layers and possibly underwent greater compression when the second PAA layer was grafted. A control experiment to rule out electrostatic complexing between the MUA and PAA pendant amines, conducted by removing EDAC from the reaction scheme, resulted in negligible thickness changes. FTIR-ERS and ATR spectra distinguished the alternating layers through the presence of characteristic carbon-carbon stretching, amine, and amide peaks. The IR spectra (Figure 2) revealed the evolution of characteristic structures one would predict after each film (10) Cerius2 simulations were conducted using the Molecular Dynamics module with quenching after every 500 steps of molecular dynamic simulation. A total of 300 000 steps were performed.
Figure 2. FTIR-ERS spectra of multiple alternating poly(allylamine)/poly(ethylene glycol) layers grafted in water. The bottom spectrum is that of MUA-EDAC-PAA. Each spectrum above that represents additions of PEG 575 and PAA in alternating sequence.
assembly step such as CdC bonds spectra at 1636 cm-1 after a PEG addition or amine and amide peaks at 1652 and 1558 cm-1 after PAA additions. The initial PAA layer resulted in primary amine absorbance at 1652 cm-1 and a smaller secondary amide peak at 1558 cm-1 (from covalent binding with EDAC-activated MUA). PEG addition was evidenced in the FTIR spectra by alkane stretching peaks at 1351 and 1456 cm-1. Terminal carboncarbon double bond peaks at 1408 cm-1 provided evidence that both acrylate termini did not react during PEG addition steps. Absorption at 1639 cm-1, characteristic of terminal carbon-carbon double bonds, was present in some spectra but frequently obscured by overlapping amine and amide peaks. An ester stretching peak at 1729 cm-1, associated with acrylate moieties, was readily apparent. Michael addition of PEG resulted in decreased primary amine absorption at 1652 cm-1 and increased secondary amine peak intensity at 1558 cm-1, due to Michael addition of acrylates with primary amines. Continued grafting via Michael addition showed increases in primary amine peaks upon PAA steps and increases in amides, esters, and alkane stretching peaks when PEG was grafted to the surface. Magnitude of the carboncarbon double bond peak at 1408 cm-1 decreased during PAA grafting steps, while increasing during PEG grafting steps. Similar to earlier generations, the final spectra for the third generation of Michael addition PEG grafting indicates the continued existence of both primary amines and terminal carbon-carbon double bonds. The remaining functionalized terminal and pendant groups are available for subsequent applications, such as enzyme immobilization for a biosensor and ligand attachment for affinity assays. Additional experiments were conducted using tetraacrylated PEG 18500 as a higher molecular weight, tetrafunctional analogue for diacrylated PEG 575. We anticipated the longer macromer would generate a thicker film and provide additional carbon-carbon double bonds accessible for further grafting. Unconstrained PEGs of MW 18500 in non-theta solvents have unswollen characteristic radii of 261 Å,11 and molecular modeling of constrained PEG 18500 chains had extended lengths approaching 1000 Å.12 Ellipsometry measurements for the PEG 18500 layer revealed a bimodal thickness distribution with averages at 440 and 980 Å. On the basis of molecular (11) Merrill, E. W.; Dennison, K. A.; Sung, C. Biomaterials 1993, 14, 1117-1126.
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modeling, we believe that two types of PEG addition occurred on the gold surface: (1) PEG 18500 molecules attached by one end group, resulting in a linear chain attached to the substrate, and (2) PEG 18500 molecules attached by at least one of the central carbon-carbon double bonds associated with the central bisphenol linkage, resulting in decreased film thickness. Another potential explanation is that PEG 18500 was cleaved during acrylation and the resulting PEG diacrylate chains of MW 9000 grafted to the film. However, this is unlikely as PEG 18500 chain scission during tetraacrylation does not occur as demonstrated by a published gel permeation chromatography (GPC) analysis of a similar polymer reported by Hubbell and co-workers.13 FTIR-ERS spectra for PEG 18500 were similar to those reported earlier for PEG 575. Upon the Michael addition of PEG 18500 the initial PAA primary amine peak was reduced, with a subsequent increase in the secondary amine peak. Aqueous solutions have frequently been reported to be a less efficient solvent than alcohols for a Michael-type addition,14 although environmental concerns have recently led to more focus upon water as a solvent. Aqueous-based reactions are desirable when working with biomolecules. These reactions have been reported for a wide range of temperature and time conditions, with some reactions requiring as long as 8 days.15 Michael addition of amines to chalcone,16 nitroalkanes with methylvinyl ketone under neutral conditions,17 mercapto groups with 3-nitrophenyl acrylate,18 and nitroalkanes with electrophilic alkenes in a weak solution of sodium hydroxide19 have all recently been reported. Michael addition of methyl acrylate to amine surfaces was previously reported to proceed more effectively under basic conditions, although similar monomers such as methacrolein and acrolein underwent either Michael addition or Schiff base formation under both acidic and basic conditions.20 Loh and co-workers reported yields for Michael addition reactions between amines and R,βethylenic compounds in distilled water between 20 and 62%; higher yields were obtainable using an indium(III) (12) Cerius2 simulations were conducted using the Polymer Builder and Molecular Dynamics modules with quenching after every 500 steps of molecular dynamic simulation. A total of 100 000 steps were performed. (13) Cruise, G. M.; Scharp, D. S.; Hubbell, J. A. Biomaterials 1998, 19, 1287-1294. (14) March, J. Advanced Organic Chemistry: Reactions, Mechanisms, and Structure; Wiley: New York, 1992. (15) Caldwell, G.; Neuse, E. W. S. Afr. J. Chem. 1992, 45, 93-102. (16) Toda, F.; Takumi, H.; Nagami, M.; Tanaka, K. Heterocycles 1998, 47, 469-479. (17) Lubineau, A.; Auge, J. Tetrahedron Lett. 1992, 33, 8073-8074. (18) Ichinose, N.; Shimo, N.; Masuhara, H. Chem. Lett. 1995, 3, 237238. (19) Ballini, R.; Bosica, G. Eur. J. Org. Chem. 1998, 2, 355-357. (20) Nitzan, B.; Margel, S. J. Polym. Sci., Part A: Polym. Chem. 1997, 35, 171-181.
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trichloride catalyst.21 While deionized water was primarily used as the reaction medium in this study, the high concentration of poly(allylamine) pendant amines resulted in a very basic microenvironment on the substrate surface which likely made the Michael-type addition possible. Although we have yet to optimize the reaction conditions, the most consistent thickness increases were obtained for those films whose IR spectra and film thickness measurements indicted a dense initial layer of PAA covalently linked by EDAC to the MUA monolayer. Dense PAA surface layers implies a high concentration of primary amines and thus a more basic microenvironment pH. Equivalent Michael addition results to those reported above were also obtained using ethanol instead of deionized water. However, the Michael-type addition between amines and carbon-carbon double bonds required additional time in ethanol. Similar monomer-based Michael additions reported by others have required between 2 and 24 h in methanol for completion, influenced in part by the reaction temperature.1,2,4 Possibly as a result of the lengthy reaction time in ethanol, IR spectra of our thin films constructed in water exhibited CdC absorption peaks at 1636 cm-1 more defined than those reacted in ethanol. This is indicative of more free carbon-carbon double bonds than those reacted in ethanol, where the amide spectra were predominant. Conclusion We fabricated nanoscale thin PEG-PAA films by repeated Michael addition reactions between PEG acrylate groups and primary pendant amines on PAA. While gold was used in the present study due to ease of monolayer fabrication with thiols, these films could similarly be formed on other functionalized surfaces such as plastics, in a manner similar to recent results reported by Crooks and co-workers.22 The presence of terminal vinyl and pendant amine groups within the polymer layers, aqueous enzyme-friendly reaction medium, and biocompatible macromers suggest these films have potential use for a wide variety of biomaterial applications. Acknowledgment. Support of this work in part by the National Science Foundation (CTS 9875372) and the Whitaker Foundation is gratefully acknowledged. We thank Professor Richard M. Crooks and his research group at Texas A&M University for numerous enlightening discussions and use of their ellipsometer and FTIR-ERS instruments. We also thank the Center for Asphalt Research at Texas A&M University for use of their FTIRATR instrument. M.V.P. thanks the Alfred P. Sloan Foundation for its support through a research fellowship. LA991388I (21) Loh, T.-P.; Wei, L.-L. Synlett 1998, 9, 975-979. (22) Ghosh, P.; Crooks, R. M. J. Am. Chem. Soc., in press.
