In Situ Cross-Linking of Alternating Polyelectrolyte Multilayer Films

Langmuir 2004, 20, 1807-1811

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In Situ Cross-Linking of Alternating Polyelectrolyte Multilayer Films Eric R. Welsh,*,† Caroline L. Schauer,‡ John P. Santos, and Ronald R. Price Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, D.C. 20375 Received September 25, 2003. In Final Form: November 28, 2003 A water-soluble, blocked diisocyanate was proven to support stable growth of bilayers with branched poly(ethylenimine), bPEI, in a layer-by-layer (LBL) technique. A quartz crystal microbalance and ellipsometry were employed to measure bilayer formation and infrared spectroscopy of LBL deposited films of hexamethylene-1,6-di(aminocarboxysulfonate) and bPEI revealed in situ cross-linking between successive layers. Erratic film growth with poly(diallyldimethylammonium chloride) with which covalent linkage was not possible, and film stability in high salt and diethanolamine solutions also supported cross-linking. Silica beads coated with alkaline phosphatase (ALP)-bPEI bilayers were capped with the cross-linking agent, and loss of enzymatic activity and less dissolution of active ALP were observed, relative to uncapped or alternately caped assemblies. Consequently, such a cross-linking agent alone or diluted with other polyanions, like poly(sodium 4-styrenesulfonate), might prove useful in controlling network and dissolution properties of LBL films having a variety of biologic and other functions, such as for controlled release.

Introduction Ultrathin polymer films may be achieved by the versatile and inexpensive method of layer-by-layer (LBL) deposition of oppositely charged polyelectrolytes. This fabrication technique has been adapted to films comprised of a variety of synthetic polymers, biopolymers,1-4 small molecules,5-8 and particulate species9,10 in which favorable intermolecular forces allow for stable adsorption and assembly of layers. This stepwise process produces mutilayer films with well-defined molecular architecture with respect to layer thickness and spatial distribution of molecules or moieties of interest. Recently, the LBL technique has been applied to coat surfaces with controlled biological properties, such as surface bioactivity,11 enzymatic activity,12-16 and controlled release of entrapped species.17,18 For enzymatic activity, the preservation of native structure is achieved through formation of an electrostatic cage,19 with further stabilization gained through cross-linking.1,20,21 Controlled release may be obtained through well-defined decomposition of the carrier or by transport via diffusion from a * To whom correspondence should be addressed. E-mail: welsh@ usna.edu. † Current address: Chemistry Department, U.S. Naval Academy, Annapolis, MD 21402-5026. ‡ Current address: Department of Materials Science and Engineering, Drexel University, Philadelphia, PA 19104. (1) Panchagnula, V.; Kumar, C. V.; Rusling, J. F. J. Am. Chem. Soc. 2002, 124, 12515. (2) Tachaboonyakiat, W.; Serizawa, T.; Endo, T.; Akashi, M. Polym. J. 2000, 32, 481. (3) Serizawa, T.; Goto, H.; Kishida, A.; Endo, T.; Akashi, M. J. Polym. Sci., Polym. Chem. Ed. 1999, 37, 801. (4) Sugama, T.; Cook, M. Prog. Org. Coat. 2000, 38, 79. (5) Shi, X. Y.; Cassagneau, T.; Caruso, F. Langmuir 2002, 18, 904. (6) Ariga, K.; Lvov, Y.; Kunitake, T. J. Am. Chem. Soc. 1997, 119, 2224. (7) Yang, W. J.; Trau, D.; Renneberg, R.; Yu, N. T.; Caruso, F. J. Colloid Interface Sci. 2001, 234, 356. (8) Caruso, F.; Yang, W. J.; Trau, D.; Renneberg, R. Langmuir 2000, 16, 8932. (9) Schmitt, J.; Decher, G.; Dressick, W. J.; Brandow, S. L.; Geer, R. E.; Shashidhar, R.; Calvert, J. M. Adv. Mater. 1997, 9, 61. (10) Caruso, F.; Lichtenfeld, H.; Giersig, M.; Mohwald, H. J. Am. Chem. Soc. 1998, 120, 8523.

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polymer scaffold, as dictated by network characteristics. As cross-linking provides a method for preserving enzymatic activity and polymer stability, both in terms of decomposition and network parameters, the use of a charged, bifunctional cross-linking agent with controlled reactivity, or stability, in aqueous solutions would be wellsuited to multilayer assembly and consequent control of properties. To this end, the current investigation focuses on the use of a diisocyante as a cross-linking agent that was recently employed with chitosan films and hydrogels.22-24 Due to the reactivity with and insolubility of most commercially available diisocyanates in water, the isocyanate is reacted with a bisulfite to protect, or block, reactivity and to impart water solubility (Figure 1).25-27 While stable in acidic aqueous solutions, with increased pH or temperature the adduct readily reacts with amines, (11) Serizawa, T.; Yamaguchi, M.; Matsuyama, T.; Akashi, M. Biomacromolecules 2000, 1, 306. (12) Santos, J. P.; Welsh, E. R.; Gaber, B. P.; Singh, A. Langmuir 2001, 17, 5361. (13) Caruso, F.; Trau, D.; Mohwald, H.; Renneberg, R. Langmuir 2000, 16, 1485. (14) Caruso, F.; Schuler, C. Langmuir 2000, 16, 9595. (15) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Biotechnol. Bioeng. 1996, 51, 163. (16) Lee, Y.; Stanish, I.; Rastogi, V.; Cheng, T.-c.; Singh, A. Langmuir 2003, 19, 1330. (17) Vazquez, E.; Dewitt, D. M.; Hammond, P. T.; Lynn, D. M. J. Am. Chem. Soc. 2002, 124, 13992. (18) Farhat, T. R.; Schlenoff, J. B. J. Am. Chem. Soc. 2003, 125, 4627. (19) Schwinte, P.; Ball, V.; Szalontai, B.; Haikel, Y.; Voegel, J. C.; Schaaf, P. Biomacromolecules 2002, 3, 1135. (20) Kohli, P.; Blanchard, G. J. Langmuir 2000, 16, 8518. (21) Kohli, P.; Rini, M. C.; Major, J. S.; Blanchard, G. J. J. Mater. Chem. 2001, 11, 2996. (22) Welsh, E. R.; Schauer, C. L.; Qadri, S. B.; Price, R. R. Biomacromolecules 2002, 3, 1370. (23) Lin-Gibson, S.; Walls, H. J.; Kennedy, S. B.; Welsh, E. R. Carbohydr. Polym. 2003, 54, 193. (24) Welsh, E. R.; Price, R. R. Biomacromolecules 2003, 4, 1357. (25) Guise, G. B.; Jackson, M. B.; Maclaren, J. A. Aust. J. Chem. 1972, 25, 2583. (26) Dalton, J. R.; Kirkpatrick, A.; Maclaren, J. A. Aust. J. Chem. 1976, 29, 2201. (27) Hoagland, P. D.; Buechler, P. R. J. Am. Leather Chem. Assoc. 1983, 78, 223.

This article not subject to U.S. Copyright. Published 2004 by the American Chemical Society Published on Web 01/14/2004

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Figure 1. Structure of hexamethylene-1,6-di(aminocarboxysulfonate) (HDACS).

forming a urea linkage, at a rate much greater than that of competing reactions with alcohols or water.28,29 Furthermore, stability in the presence of amines and consequent long-term storage may be increased by the choice of an aliphatic diisocyanate, rather than a more reactive aromatic one.25 That hexamethylene-1,6-di(aminocarboxysulfonate) (HDACS) would be amenable to LBL assembly is supported by the work of Ariga et al.,6 where lowmolecular-weight dyes were successfully employed in a large variety of LBL assemblies. Experimental Section Chemicals and Materials. Solutions of branched poly(ethylenimine) (bPEI), (Sigma, Mw 60 000, 1.0 mg/mL, pH 8.0), poly(sodium 4-styrenesulfonate) (PSS) (Aldrich, Mw 70 000, 1.0 mg/mL, pH 6.0), poly(diallyldimethylammonium chloride) (PDDA) (Aldrich, 1.0 mg/mL, pH 8), and alkaline phosphatase (ALP) (EC 3.1.3.1) (Sigma, bovine intestinal mucosa, 1.0 mg/mL, 10 mM Tris, pH 8.0) were prepared with Milli-Q water and filtered through a 0.2 µm filter. All other materials were used without further purification from the manufacturer. HDACS was synthesized as previously described by Welsh et al.22 and was prepared in buffered solution immediately prior to use (10 mg/ mL, pH 6.0). Glass beads (Polysciences, Inc., 30-50 µm) were cleaned according to the method of Santos et al.12 Gold-coated silica slides were prepared by electrolytically depositing a 5 nm chromium layer on a piranha-cleaned silica substrate and then depositing a 55 nm gold layer. Alternate Adsorption. Substrates were submerged in polyelectrolyte solution for a given time, rinsed with reverse osmosis water, and dried in a stream of dry nitrogen, prior to quartz crystal microbalance (QCM), ellipsometry, or IR measurements. Glass beads were coated with a similar procedure without intermediate drying and with centrifugation (5 min, 100g) between successive adsorption and wash steps. Final drying was achieved in a vacuum at ambient temperatures. For the QCM and glass bead deposition, a precursor of (bPEI-PSS)3-bPEI was used to condition the substrates. Quartz Crystal Microbalance (QCM). Quartz plates with gold electrodes (0.196 cm2) were used as resonators (10 MHz) to monitor the change in frequency, after equilibration to (5 Hz under ambient conditions. The deposited thickness (d, Å) was calculated from frequency change (∆F, Hz) according to a modified Sauerbrey equation.30,31 Results are reported as the average value of n measurements, as indicated in the relevant data tables, with the corresponding error representing 1 standard deviation. Ellipsometry. The ellipsometry measurements were taken on a J. A. Woollam Co, Inc., multiwavelength ellipsometer (Lincoln, NE) with an incidence angle of 70°. The indices of the gold and chromium-coated silica substrates were first measured and entered as the blank. Films were dried with a stream of N2 before each measurement. Reflectance IR. Reflectance FT-IR spectra were taken of films that were dipped onto gold-coated silica substrates, using a Nicolet Magna IR 750 spectrophotometer, series II (Madison, WI). Bulk powder consisted of dried precipitate collected from mixing solutions of PEI and HDACS (1:1, w/w) that was then pressed into an IR pellet using an Imperial press 2000 (Hartland, WI) and the FT-IR spectrum was taken on a Nicolet Impact 400D (Madison, WI). (28) Wicks, D. A.; Wicks, Z. W. Prog. Org. Coat. 1999, 36, 148. (29) Wicks, D. A.; Wicks, Z. W. Prog. Org. Coat. 2001, 41, 1. (30) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (31) Sauerbrey, G. Z. Phys. 1959, 155, 206.

Figure 2. Average QCM frequency decrease (-∆F) for alternating electrolyte layer assembly with bPEI (filled symbols) adsorption for 20 min, HDACS for 5 min (O, A), and PSS for 5 min (O, B) and 10 min (3, C), respectively. Enzymatic Activity. Beads modified with adsorbed ALP layers (ca. 25 mg) were suspended in 1.0 mL of DEAM buffer (1.0 M diethanolamine, 0.5 mM MgCl2, pH 9.8) and 0.1 mL of 0.109 mM p-nitrophenyl phosphate (pNPP). The absorbance at 405 nm was monitored after mixing and allowing the beads to settle to the bottom of a cuvette. Unit activity is reported as the micromoles of pNPP hydrolyzed per minute per milligram of enzyme or coated beads, as determined from initial reaction velocity, and is the average of three measurements.

Results and Discussion QCM. The relationship between the frequency change ∆F (Hz) and mass gain M (g) on a QCM electrode with a resonant frequency of 10 MHz and with an area of A (cm2) is given by

∆F ) -2.262 × 108 (cm2‚Hz/g) M/A

(1)

Consequently, a frequency decrease of 1 Hz corresponded to a mass increase of 0.87 ng and, assuming a density of 1.2 g/cm3 for the adsorbed species, a film thickness (d, Å) on both sides of the electrode estimated by

d ) -0.1842∆F

(2)

which, due to variations in both film density and area, is reliable to (10%.30 For each adsorption cycle, QCM data were collected with three separate crystals in separate chambers containing freshly prepared electrolyte solutions. A plot of decreasing frequency (increasing adsorbed mass) versus layer number revealed a mass increase for both the adsorption of bPEI and HDACS (Figure 2a), indicating stable film growth with a corresponding bilayer thickness of 20 ( 3 Å (Table 1). When PSS was substituted for HDACS with the same adsorption times, film growth was

In Situ Cross-Linked Polyelectrolytes

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Table 1. QCM Characteristics of Multilayer Assemblies electrolytesa

-∆F polycation (Hz)b

-∆F anion (Hz)b

bilayer thickness, d (Å)c

bPEI20′-HDACS5′ bPEI20′-PSS5′ bPEI20′-PSS10′ PDDA20′-HDACS5′

15 ( 20 1.6 ( 10 -7.1 ( 30 -25 ( 80

91 ( 20 65 ( 9 128 ( 30 81 ( 30

20 ( 3 12 ( 1 23 ( 3 8.1 ( 2

a Subscript refers to adsorption time. b n ) 27 for bPEI-HDACS, n ) 15 for bPEI-PSS5′, bPEI-PSS10′, and PDDA20′-HDACS5′. c Determined from slopes in Figure 2 and d ) -0.1842∆F.

Figure 4. Reflectance FT-IR spectrum of two, three, and five bilayer films on gold-coated silica. Increasing urea peak appears at 1648 cm-1 as the bilayer number increases.

Figure 3. Ellipsometry measurements of bPEI-HDACS films. Each bar represents the mean ( the standard deviation for three independent experiments.

less significant and more erratic with a smaller PSS mass increase and often a greater mass loss for PEI adsorption (Figure 2b). When the PSS adsorption time was doubled to 10 min (Figure 2c), mass gain also doubled with similar trends in polyion sorption noted for shorter times. The mass loss observed for PEI adsorption was attributed to the disruption of underlying polyelectrolyte layers due to ion exchange (i.e., displacement) or charge neutralization32 and represents a common phenomenon for the LBL process.12,16 In contrast to the erratic results observed by Ariga et al.6 with bPEI and ionic dyes with the same charged moieties of HDACS (i.e., sulfite), the data presented in Figure 2a indicated an optimal assembly process in which desorption of the anion was minimized, perhaps due to cross-linking. To test this hypothesis, PDDA was substituted for bPEI, which, due to the quaternary amine, was incapable of reacting with HDACS to form a covalent linkage, yet, based on the work of Ariga et al., was expected to be more favorable in minimizing desorption of the anionic species. However, the data presented in Table 1 revealed similar mass gain for HDACS layers to those observed when alternated with bPEI, although the construct was not stable with a highly variable and often negative mass gain for PDDA. While no significant efforts were made to optimize this latter LBL process, the fact that, under the same conditions, film growth was not sustained in the same manner implicated cross-linking between HDACS and bPEI. Ellipsometry. The LBL process on gold-coated, silica substrates was also monitored via ellipsometry using an index of refraction of 1.45 for both bPEI and HDACS (Figure 3). Film growth was proportional to the number of bilayers with an average thickness of 9.5 ( 5 Å, which is half of that estimate by QCM. This disparity was attributed to the lack of preconditioning layers, such as (32) Schlenoff, J. B.; Ly, H.; Li, M. J. Am. Chem. Soc. 1998, 120, 7626.

bPEI-PSS or PDDA-PSS, applied to the same gold substrates used for reflective IR studies (see below) to avoid additional moieties. Preconditioning layers were used for QCM and, in general, provide a more uniformly charged base surface for subsequent electrolyte attraction and adsorption of successive layers.6,12,16,17,30,33,34 To further investigate the effect of preconditioning layers, fresh goldcoated silica substrates were coated with the same precursor layers as the QCM crystals, (bPEI-PSS)3-bPEI, and film thickness was monitored via ellipsometry. The average bilayer thickness of bPEI-HDACS on two distinct substrates was determined to be 27 ( 17 Å (data not shown),35 which is in better agreement with QCM results and indicates that preconditioning layers provided an increased and sustained charge base for multilayer assembly. Cross-Linking. The chemical composition of the bilayer films was investigated using reflectance FT-IR and compared to FT-IR of bulk powder. Multilayer assemblies for IR analysis were created similar to those used for QCM by alternately dipping of the substrate into bPEI and HDACS solutions for 20 and 5 min, respectively, with intermediate washing, Scheme 1. The carbonyl region of the IR spectrum of two, three, and five bilayers (Figure 4) revealed increasing resonance consistent with crosslinking. Specifically, the isocyanate reaction (i.e., urea, carbamate, allophanate, biuret) was evident in the presence of a carbonyl peak at 1690 cm-1 for the HDACS isocyanate and an increasing urea carbonyl peak (1648 cm-1) with increasing number of bilayers. These data indicate that, during the LBL process, the ionic intermolecular forces that favored layer assembly were converted into covalent bonds (cross-links), the extent of which increased upon raising the pH during deposition of successive layers. That such reactions occur in situ was supported by the IR spectra of a bulk sample created from combining solutions of bPEI and HDACS, which quickly react to form a white, insoluble precipitate. IR spectroscopy of the isolated powder showed a shoulder peak for CdO at 1680 cm-1 and a shoulder peak for SdO at 1205 cm-1 indicating unreacted HDACS (Figure 5), which is consistent with partial reaction, or grafting of HDACS onto bPEI, or both.36 (33) Chluba, J.; Voegel, J. C.; Decher, G.; Erbacher, P.; Schaaf, P.; Ogier, J. Biomacromolecules 2001, 2, 800. (34) Chen, K. M.; Jiang, X. P.; Kimerling, L. C.; Hammond, P. T. Langmuir 2000, 16, 7825. (35) Reported value is an average of six measurements: three bilayers on two distinct substrates.

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Scheme 1. In Situ Cross-Linking of Alternating Layers of HDACS and bPEI

Figure 6. Activity of ALP on glass beads layered with (ALP15′bPEI15′)8 without capping (b) or with PSS7′-bPEI20′ (1), MES7′bPEI20′ (O), or HDACS5′-bPEI20′ (3). Figure 5. Bulk powder FT-IR of HDACS (A), bPEI (B), and bPEI-HDACS (C).

Table 2. Effect of Capping on ALP Multilayer Activity unit activity (mmol/(min/mg))a

Film stability was examined by dipping into either 0.6 M NaCl or DEAM buffer (pH 9.8) for 20 min, conditions known to deconstruct electrostatic multilayers.37,38 The film that was dipped into DEAM swelled from 164 to 219 Å, while the other, which was dipped into NaCl lost the outermost bilayer (189-163 Å), indicating that the majority of bilayers were not solubilized, due to crosslinking, and that the extent of cross-linking in the outermost layer increased at the higher pH of DEAM. The cross-linked film swelled in DEAM due to changing the pH from 6 to 9.8, which altered the charges on the electrolytes39,40 and due to charge shielding from the buffer.41 Enzyme Activity. Alkaline phosphatase (ALP) was adsorbed on silica spheres after the method of Santos et al.12 with the intention to further stabilize enzyme activity via cross-linking or capping the LBL assembly.16 Specifically, eight bilayers of ALP and bPEI were applied for 15 and 20 min each layer, respectively. A batch of beads containing this base film, given by (ALP15′-bPEI20′)8, was divided into four groups that were capped as follows: (1) no capping; (2) a (MES-bPEI) cap that represents a pH control for which 10 mM MES (pH 6.0) buffer was substituted for the polyanion; (3) a (PSS-bPEI) cap with an outermost bilayer consisting of layers without enzymatic activity; (4) a (HDACS-bPEI) cap that is capable (36) Petersen, H.; Kunath, K.; Martin, A. L.; Stolnik, S.; Roberts, C. J.; Davies, M. C.; Kissel, T. Biomacromolecules 2002, 3, 926. (37) Dubas, S. T.; Schlenoff, J. B. Macromolecules 2001, 34, 3736. (38) Dubas, S. T.; Farhat, T. R.; Schlenoff, J. B. J. Am. Chem. Soc. 2001, 123, 5368. (39) Yoo, D.; Shiratori, S. S.; Rubner, M. F. Macromolecules 1998, 31, 4309. (40) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213. (41) Dubas, S. T.; Schlenoff, J. B. Langmuir 2001, 17, 7725.

(ALP15′-bPEI15′)8-(X-Y) aloned

ALP no capping PSS7′-bPEI20′ MES7′-bPEI20′ HDACS5′-bPEI20′

initial

overnightb

supernatantc

23 ( 4 16 ( 1 9.2 ( 0.3 6.4 ( 1 1.1 ( 0.2

0.14 ( 0.05 0.83 ( 0.2 0.41 ( 0.09 0.46 ( 0.08 0.047 ( 0.05

8.1 ( 0.4 3.9 ( 0.2 1.6 ( 0.2 0.09 ( 0.05

a For all samples, except ALP, ×10-6. b 16 h in DEAM at room temperature. c Layered beads extracted for 30 min in DEAM under constant agitation. d Determined at 0.1 mg/mL DEAM, supplier activity given as 20 units/mg.

of forming a covalent film or network. For the MES and PSS capping treatments, an adsorption time of 7 min was used to ensure similar time exposure at low pH and equal mass adsorption of PSS and HDACS, based on the adsorption rates reported in Table 1. ALP unit activity, as determined from the initial velocity of hydrolysis curves (Figure 6) and reported as micromoles of pNPP hydrolyzed per minute per milligram, is presented in Table 2. The activity of the uncapped beads was the greatest, in agreement with that reported by Santos et al. (i.e., 1.7 ( 0.5 A405/(min/mg)), and, based on the activity of free ALP, indicated that the fraction of active enzyme adsorbed was indeed small. Activity decreased with the addition of PSS-bPEI, which is most likely due to a loss in activity expected with the lowered pH during PSS adsorption, rather than forming a physical barrier.12 This effect of pH was also seen with the MES-bPEI cap, yet was more significant than that for PSS. The greatest loss in activity was observed for the HDACS-bPEI cap, indicating that HDACS either cross-linked the entire structure, including the enzyme, formed a barrier (physical or chemical) to diffusion, and/or facilitated denaturing ALP. The decreased activity from exposure to either MES

In Situ Cross-Linked Polyelectrolytes

or HDACS further indicated that these small molecules, relative to PSS, were capable of diffusion into the multilayers and sulfonate ion exchange that may have disrupted complementary ionic interactions and corresponding enzyme conformation, resulting in decreased activity.18,32,42,43 These data indicate that this deleterious effect was further enhanced by ion exchange with HDACS that would allow for extensive cross-linking, upon raising the pH to 8.0 for bPEI adsorption and then higher to pH 9.8 for assay conditions. For all samples, ALP activity was found in the supernatant, when exposed to the assay buffer for 30 min with agitation, and was found to decrease by greater than 90% after overnight exposure (Table 2). In contrast to observations by Santos et al., where 10 mM Tris buffer (pH 8) was used and no activity was observed in the supernatant, DEAM buffer (1.0 M diethanolamine, 0.5 mM MgCl2, pH 9.8) was more disruptive to the layer structure with 51, 42, 25, and 8% of the enzyme activity found in the supernatant, after just 30 min for the uncapped, PSSbPEI capped, MES-bPEI capped, and HDACS-bPEI capped samples, respectively. While HDACS denatured or otherwise reduced ALP activity and no long-term activity was achieved for the applied experimental condi(42) Dubas, S. T.; Schlenoff, J. B. Macromolecules 1999, 32, 8153. (43) Schlenoff, J. B.; Dubas, S. T. Macromolecules 2001, 34, 592.

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tions, HDACS prevented the dissolution of ALP via the formation of a barrier, chemical or physical, as suggested above. Taken together these data indicate that HDACS was able to support stable growth of bilayers with bPEI, with which covalent linkage was possible, as was supported by erratic film growth with PDDA, IR spectroscopy, film stability in high salt and DEAM solutions, and loss of enzymatic activity in an LBL construct with less dissolution of active ALP. Consequently, such a cross-linking agent alone or diluted with other polyanions, like PSS, might prove useful in controlling network and dissolution properties of LBL films having a variety of biological and other functions, such as controlled release. Acknowledgment. This work was supported by the Naval Research Laboratory 6.2 development program and was preformed while C. L. Schauer held a National Research Council Research Associateship Award at the same facility. Supporting Information Available: Complete description of all infrared spectra. This material is available free of charge via the Internet at http://pubs.acs.org. LA035798P