Article pubs.acs.org/Biomac
Tuning the Properties of Mucin via Layer-by-Layer Assembly Jiyoung Ahn,† Thomas Crouzier,‡ Katharina Ribbeck,*,‡ Michael F. Rubner,*,§ and Robert E. Cohen*,† †
Department of Chemical Engineering, ‡Department of Biological Engineering, and §Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States S Supporting Information *
ABSTRACT: Multilayer films consisting of bovine submaxillary mucin (BSM) and poly(allylamine hydrochloride) (PAH) were prepared on various substrates using layer-by-layer assembly. The effects of both the assembly pH and ionic strength on multilayer characteristics were investigated by assessing film thicknesses (10−80 nm), surface wetting characteristics, and cell repulsion. Also, the dynamic assembly behavior was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) to further understand the effect of assembly pH on film characteristics. Assembly studies revealed that substantial amounts of BSM adhere to the outermost surface only at low pH conditions. The resulting multilayer films assembled at low pH conditions were found to exhibit hydrophilic and cell repellent behavior. In addition, it was found that batch-to-batch variations of the biopolymer BSM could dramatically alter properties.
1. INTRODUCTION Mucins are high molecular weight (200 kDa−200 MDa) glycoproteins that form the mucus gel on epithelial cell surfaces.1 The mucus gel is a barrier against pathogens and lubricates the underlying cell surface.2 The protein core of mucins is composed of hydrophobic regions separated by serine on threonine rich regions to which glycan moieties are attached.3 These densely glycosylated domains confer to mucins a bottlebrush-like molecular architecture.4 Mucins of different origins are available commercially for relatively low cost depending on the source such as porcine gastric mucin (PGM) and bovine submaxillary mucin (BSM), and share a similar structure in general as described above.5 One way to study and take advantage of the natural properties of mucins is through the formation of mucin surface layers. Adsorption of mucins has been found to lubricate surfaces,6 cause significant changes in contact angle,7 and repel the adhesion of microorganism and mammalian cells.1,8−10 This suggests the potential use of mucin for biomedical applications and practical applicability for industrial purposes.2,8,9 The details in interaction between mucins and various materials are thus of great interest and have been investigated for various polymeric materials.3,7−9,11,12 These studies revealed that the adsorption, as well as the conformation of the resulting mucin layers, depend strongly on the surface properties of the underlying substrates, such as wetting characteristics, types of functional groups present, and surface charge density.4,8,9 For example, mucins form a thick and highly hydrated layer on a gold-coated surface, while a thin and less hydrated layer is formed on silica.14 Also, it was shown that BSM binds very strongly on a hydrophobic and carboxylic acid modified surface but not so strongly on a hydrophilic hydroxyl modified surface.5,13 In addition, it has been reported previously that the © 2014 American Chemical Society
biological function of the resulting mucin layers is also strongly dependent on the type of underlying substrates.5,8−10,14 Manipulating the properties of mucin only by varying the underlying substrate, however, poses a limitation to the applicability of mucin for various applications. Therefore, it would be highly desirable to generate mucin layers with properties that could be controlled independently of the nature of the underlying substrate. For this purpose, the layer-by-layer (LbL) assembly technique was chosen to assemble mucin containing multilayer films. The LbL assembly is a simple and versatile technique to generate conformal films on various substrates where the physicochemical properties of the resulting films can be fine-tuned by simply varying the complementary polymer pair and assembly conditions. Various complementary polymer pairs such as positively charged chitosan,7,15,16 lactoperoxidase,17 and sugar-binding lectin wheat germ agglutinin18 have been used previously to form LbL multilayer films with BSM7,15−17 and PGM.18 However, there lacks a systematic study on the impact of assembly conditions such as pH and ionic strength on the properties of the resulting films. In this work, we focus on BSM, which has a net negative charge in physiological conditions due to its high content of sialic acid. We screened BSM interactions with several polycations and chose poly(allylamine hydrochloride) (PAH) as the complementary interacting polymer due to its interesting interactions with mucins and well-known behavior as a building block in LbL.19,20 The effective charge density of PAH was then varied systematically by changing assembly pH and ionic Received: September 29, 2014 Revised: November 22, 2014 Published: November 23, 2014 228
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Figure 1. (a) Alcian blue absorbance at 626 nm and the amount of adsorbed BSM on various polyelectrolyte multilayers. (b) The number of HeLa cells adhered on various multilayers before and after BSM adsorption. The asterisk is added to emphasize the outermost layers prior to BSM coating. Control experiment data for Alcian blue staining are given in Figure S2a, Supporting Information. with a 2 μm stylus tip, 2 mg stylus force, and a scanning rate of 50 μm/ s. 2.2.2.2. Contact Angle Measurement. The contact angles presented in this article are advancing contact angles, which were measured with the Ramé−Hart Model 590 goniometer. Using the tip of a syringe, a water drop was placed on a sample. The substrate was moved vertically until contact was made between the sample and the water drop. The subsequent addition of a small amount of water to the water drop on the surface produced the steady-state advancing angle in a few seconds (final volume to be about 1 μL). An image of the droplet was taken, and the right and left contact angles of the droplet were then measured by using an enlarged image transmitted to a computer screen. Four separate locations on each sample surface were probed to ensure a representative value of the contact angle. The average value of the measured contact angles was used to represent the wetting characteristics of the sample. 2.2.2.3. Fourier Transform Infrared Spectroscopy (FT-IR). IR spectra of multilayers deposited on ZnSe plates with 0.005 M PAH and 0.5 mg/mL BSM at various pH conditions were taken using a Nicolet 4700 FT-IR spectrometer (Thermo Scientific). Higher concentration for both polymer solutions was applied to intensify the peak signal. To locate the peak of PAH, and BSM at various pHs, solutions of PAH (0.005M) and BSM (0.5 mg/mL) were dropped on ZnSe plates and dried for 24 h in advance of measurement. 2.2.2.4. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). The hydrated mass of the film at various stages of the assembly process was measured by quartz crystal microbalance with dissipation monitoring (QCM-D, E4 system, Q- Sense, Sweden). A gold-covered quartz crystal with a fundamental frequency of 5 MHz (QSX-301, Q-Sense) was used as a substrate for film deposition. As a cleaning procedure, the mixture of 10 mL of deionized water, 2 mL of 25% ammonia, and 2 mL of 30% hydrogen peroxide was used at 75C. The crystals were rinsed with deionized water afterward. Right before the assembly, the crystals were further cleaned by ozone treatment for 15 min. Films were built by manually alternating depositions of mucin and PAH solutions up to 6 bilayers. Mucin was prepared at a concentration of 0.1 mg/mL, and PAH was prepared at 0.001 M in deionized water. Mucin and PAH were left to adsorb for 10 min, followed by 5 min of rinsing with DI water at the pH of assembly. 2.2.2.5. Cell Culture. The epithelial HeLa cell line was maintained at subconfluency in T25 flasks with DMEM media supplemented with 10% fetal bovine serum (Invitrogen) and 1% antibiotics (25 U/mL penicillin and 25 μg/mL streptomycin (Invitrogen). The cells were detached using trypsin-EDTA (Invitrogen) and plated at a density of 45,000 cells/cm2 on top of a selected multilayer film deposited on
strength, and the resulting films were studied in terms of the assembly dynamics and the physicochemical properties.
2. MATERIALS AND METHODS 2.1. Materials. All polymer solutions were prepared with deionized water, and the pH was adjusted using 1 M HCl and 1 M NaOH. The PAH (poly(allylamine hydrochloride)) used here had a nominal molecular weight of Mw = 70000 g/mol, as quoted by the supplier, PolySciences. PAH solution concentration was 0.001 M (based on a repeat unit molecular weight); 0.05 and 0.1 M of NaCl was added in certain cases to investigate the effect of salt on assembly behavior. Commercially available bovine submaxillary mucin (BSM, SigmaAldrich) was used after being purified to remove some of the serum albumin contanimants.7 For the purification, BSM was dissolved in water at 30 mg/mL and dialyzed in a 100 kDa molecular weight cutoff membrane (Spectrum Laboratories) against water for 7 days. The mucin was then lyophilized for storage. Since commercial BSM solutions are known to form aggregates21,22 (larger than 400 nm depending on the batch) in solution, additional filtration was carried out using bottle top filters with a 0.45 mm PES membrane (VWR, 500 mL) when it is needed. The BSM solution concentration was 0.1 mg/ mL, which was chosen to set the mass ratio with PAH at unity. 2.2. Methods. 2.2.1. Thin Film Assembly. All films were prepared on glass substrates (VWR Scientific) using a Stratosequence VI spin dipper (Nanostrata Inc.) with StratoSmart v6.2 software. Prior to film assembly, the substrates were cleaned by sonication in a 4% (v/v) solution of Micro-90 (Internation Product) for 20 min and then in DI water for 20 min. Oxygen plasma treatment (PDC-32G, Harrick Scientific Products, Inc.) was followed for 2 min at 150 mTorr. The spin dipper was operated with dipping times of 10 min for the polymer solutions without rotation, followed by three rinses of 2, 1, and 1 min in pH adjusted DI water with rotation. The pH of the rinse water was adjusted with 0.1 M HCl or 0.1 M NaOH. The nomenclature for LbL films follows the usual convention (polymer A/polymer B)n, where n is the total number of bilayers deposited. The pH conditions of assembly are specified by providing the pH value after the polymer name, for example, polymerA7.0. The pH conditions of the rinse baths were adjusted to be identical to the pH of the preceding polymer solution. For gold crystal and ZnSe, BSM was used as the first layer to ensure the formation of a stable first layer through hydrophobic interaction between BSM and substrates. 2.2.2. Characterization. 2.2.2.1. Thickness Measurement. Dry film thicknesses were measured using a Tencor P16 surface profilometer 229
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glass. Cells were observed after 24 and 96 h. Images were acquired on an Axio Observer Z1 microscope (Zeiss, Oberkochen, Germany) using a EC-Plan Neofluar 10× 0.3 NA lens (Zeiss) in phase contrast or fluorescence mode. Cells were counted with the image analysis software ImageJ using the cell count plug-in.
3. RESULTS AND DISCUSSION 3.1. BSM Forms Cell-Repulsive, High-Density Coatings on PAH Topped Multilayer Films. Prior to incorporating BSM into multilayers, a preliminary screening procedure was performed to find a promising assembly partner and conditions for BSM. Several well-studied multilayers described in the literature20 composed of a combination of PAH, PAA (poly(acrylic acid)), and SPS (poly(styrenesulfonate)) were assembled with systematic variation in the outermost layer (Figure 1). A single layer of fluorescently labeled BSM (0.1 mg/mL) at pH 7 was deposited on these multilayers, and the amount of BSM adsorbed was quantified by the fluorescence intensity as shown in Figure 1a. Of note, BSM has a net negative charge at this pH since it is above its isoelectric point. Therefore, it was expected that little or no BSM would adsorb onto the films finished with SPS, which has a net negative charge over all pH levels of this study. Adhesion of HeLa cells on these samples was quantified to explore the presence of mucin in the multilayers since BSM coatings can, under certain conditions, prevent the attachment of mammalian cells.23 Moreover, Alcian blue staining was performed to directly quantify the amount of mucins present (Figure 1a and b and Supporting Information, Figures S1−S3).24 Our results show that the outermost polymer species and the pH at which the multilayer was assembled both had a significant impact on the amount of adsorbed BSM. Assemblies with PAH as the outermost layer at pH 9.3 resulted in the strongest reduction of cell adhesion and in the greatest increase in Alcian blue staining after BSM deposition. Of note, there was no clear correlation between the intensity of Alcian blue staining and the reduction of cell adhesion. This indicates that reduction in cell adhesion does not only depend on the amount of BSM present but also on the details of its assembly (nature of pairing polymer, assembly pH) film properties. This suggests that the native properties of mucin can be modified when incorporated in a multilayer. On the basis of these results, we chose PAH as an assembly partner for BSM and investigated the effect of the pH of both BSM and PAH solution and the salt concentration of the PAH solution on the properties of the resulting film. 3.2. Layer-by-Layer Assembly of BSM with PAH. 3.2.1. Layer-by-Layer Assembly of BSM with PAH Is Dependent on pH and Salt Concentration. (PAH/BSM)30 multilayers were successfully assembled on precleaned hydrophilic glass slides with systematic variation in the assembly pH (both PAH and BSM solutions) and salt concentration (PAH solution only) to determine how these parameters influence film growth and average multilayer film thicknesses. The average film thickness of 30 bilayer assemblies as measured by profilometry in the dry state is shown in Figure 2. To indirectly prove that each layer is deposited on the substrate, the thickness of these multilayers was also measured after every 10 bilayer formation (Figure S10, Supporting Information). From Figure 2, it is apparent that both the assembly pH and the salt concentration in PAH solution strongly influence the deposition process. (PAH/BSM)30 built at pH 11 without any additional salt resulted in a noticeably thicker film than that
Figure 2. Average thickness of a 30 bilayer multilayer film of PAH and BSM as a function of assembly pH and salt concentration in the PAH solution.
at any other conditions studied. When salt was present in the PAH solution, the thickness was nearly constant (about 20−30 nm) over a wide range of assembly pH conditions. At low pH conditions, the thickness of the final multilayers was less than 10 nm regardless of the amount of salt. These results are consistent with our studies of interpolymer complex formation (Figures S4 and S9, Supporting Information), where the presence or absence of turbidity of mixtures of the complementary polymer solutions was used as a predictor of whether selected conditions would lead to successful LbL assembly.25,26 Upon mixing salt-free PAH solutions with BSM solutions, the mixture became turbid only at pH 11. When salt was added to the PAH solution prior to mixing, an evident change in solution turbidity was observed from pH 3 to pH 10. Given the strong effect of pH when no salt is present, films assembled without salt were potentially more tunable. Thus, films built in the no-salt condition were further characterized. 3.2.2. Surface Wettability of Multilayers Is Dependent on pH. Adsorption of a single layer of BSM has been shown to make hydrophobic substrates hydrophilic, as manifested by a large decrease in the water contact angle.8 Figure 3 shows the
Figure 3. Advancing water contact angles measured on (PAH/BSM)30 multilayers fabricated with varying pH of assembly.
advancing water contact angles of multilayer films (PAH/ BSM)30 assembled at different pH conditions. As the assembly pH is increased, the contact angle of resulting multilayers is also increased. The height AFM measurements (data not shown) and the average roughness measurement (Table S1, Supporting Information) did not reveal any significant surface morphology changes that can be correlated with this variation in contact angle. Thus, the assembly pH changed both the thickness (Figure 2) and, at least as revealed by surface wettability, the 230
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Figure 4. (a,b) FT-IR spectra of a single layer of BSM, cast from aqueous solutions (0.5 mg/mL) adjusted to various pH conditions. (c,d). FT-IR spectra of (BSM/PAH)30 multilayers on ZnSe substrates assembled at various pH conditions (normalized by the film thickness). Spectra are intentionally overlaid with arbitrary offset for clarity.
Table 1. IR Absorption Ratio of Amide II (v = 1540 cm−1) to Amide I (v = 1652 cm−1) Bands from BSM Cast and BSM/PAH Multilayers (Normalized by Thickness)a vamII/vamI (1540:1652) of BSM cast on ZnSe vamII/vamI(1540:1652)of (BSM/PAH)30 on ZnSe a
pH 2
pH 4
pH 7
pH 9.3
pH 11
0.941 ± 0.064 0.742 ± 0.046
0.810 ± 0.174 0.835 ± 0.128
0.717 ± 0.146 0.667 ± 0.283
0.836 ± 0.055 0.793 ± 0.190
0.830 ± 0.165 0.625 ± 0.332
The ratio is calculated from five independent measurements.
organization of mucin in the film. Bearing in mind that BSM is the top layer, these results suggest changes in the amount of hydrophilic groups at the air−film interface provided by BSM as the assembly pH increases. 3.2.3. Mucin Functional Groups and Protein Structure Are Similar in Cast Films and the Multilayers. The differences in surface wettability could originate from changes in the conformation of the BSM molecules as the assembly pH is varied. FT-IR analysis is rich in information about the nature and conformation of chemical groups present in multilayer films.27,28 We thus used FT-IR analysis on the multilayer system to investigate the origin of its pH-dependent growth behavior. A single-layer deposition of BSM on ZnSe was used as a control to obtain information about the effect of pH on the secondary structure of the protein backbone in BSM. The IR spectra of BSM films (Figure 4a,b) cast on ZnSe substrates are consistent with the structure of mucin as reported by others. Sialic acid in BSM shows two distinct peaks at 1730 and 1570 cm−1, which arise from the carboxyl group and amide groups of sialic acid, respectively.29,30 Also the absorbance peaks at 1653, 1540, 1240 cm−1, and 1039 cm−1 of BSM are attributed to the amide I, II, III bands, and
polysaccharide residues, respectively.30 Between 3000 and 3500 cm−1, very broad absorbance peaks are attributed to the abundant hydroxyl groups in the sugar residues of BSM.31 From 2800 to 3000 cm−1, absorbance peaks from the alkyl groups show up as well.29,32,33 When BSM and PAH are assembled into multilayers, the changes observed by FT-IR mainly reflect the presence of PAH and the relative amount of BSM in the multilayer. To remove the influence of film thickness on the observed peak intensity, the spectra were normalized by thickness prior to further analysis, as shown in Figure 4c,d. The absorbance bands of the multilayers were, for the most part, similar to the spectrum of the pure BSM single layer. Peaks attributed to the amide I, II, and III groups, from the sialic acid and from alkyl groups, are analogous to the peaks identified for cast BSM. An additional peak for PAH at 1310 cm−1 at pH 9.3 and pH 11 appeared for the multilayers assembled at these pHs as shown in the literature.27 The relative peak ratios for the amide groups were found to occur in the same range as the pure cast films of BSM. Put together, these results indicate that there is no major shift in the secondary structure of the protein backbone of BSM during layer-by-layer assembly with PAH, regardless of the 231
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Table 2. IR Absorption for Polysaccharide Bands (v = 1039 cm−1) of BSM Cast versus BSM/PAH Multilayers from Five Independent Measurementsa vpolysaccharides (1039) of BSM cast on ZnSe vpolysaccharides (1039) of (BSM/PAH)30 on ZnSe a
pH 2
pH 4
pH 7
pH 9.3
pH 11
0.013 ± 0.010 0.0021 ± 0.0007
0.013 ± 0.013 0.0039 ± 0.0028
0.014 ± 0.006 0.019 ± 0.006
0.011 ± 0.004 0.015 ± 0.005
0.012 ± 0.013 0.033 ± 0.003
Values were taken prior to normalization with thickness.
Figure 5. Evolution of the hydrated mass for LbL build-up at moderate and high pH of assembly ((a) pH 7, (b) pH 11, (c) pH 2, and (d) pH 4), CPAH = 0.001M, and CBSM = 0.1 mg/mL. B and P indicate the beginning of the BSM or PAH absorption step, respectively. BSM and PAH were left to adsorb for 10 min, followed by 5 min of rinsing with DI water at the pH of assembly.
incorporated mucins. However, the final thickness varied greatly as seen in Figure 2. We thus investigated the dynamics of the adsorption/desorption phenomena that occur during LbL assembly using QCM-D measurements. Real-time adsorption of BSM and PAH onto a gold crystal was observed up to 6 bilayers, and the accumulation of hydrated mass during the multilayer buildup is plotted in Figure 5. In the first bilayer formation, the addition of both BSM and PAH leads to an increase in hydrated mass for all pH conditions investigated. At pHs 7 and 11, a continuous increase in hydrated mass was detected throughout the assembly in a stepwise manner. The final values of hydrated mass after the deposition of 6 bilayers at pHs 7 and 11 were 16.8 mg/m2 and 17.2 mg/m2, respectively. At low pH, a different trend of the multilayer buildup emerged. After the first bilayer, from the second addition of BSM, the total hydrated mass adsorption shows a nonmonotonic cyclical behavior. Exposure to BSM leads to large and stable increases in hydrated mass. However, with the addition of PAH, a sudden and substantial decrease in the total hydrated mass was measured. The hydrated mass loss did not occur during the rinsing step but only when the subsequent PAH is added to the system. Of note, each bilayer resulted in a net positive increment of mass. After 6 bilayers, the total hydrated mass was 13.9 mg/m2 for pH 2 and 20.9 mg/m2 for pH 4, on the same
assembly pH (Table 1). Of note, secondary structures of BSM have been previously confirmed to be dominated by random coil conformation by CD, DLS, and FT-IR measurements.7,34,35 The IR absorption intensity of the polysaccharide residues can be used as an indicator of the amount of BSM embedded in the layers. For the pure BSM layer on ZnSe, the absolute value of the absorption peak arising from the polysaccharide residues (Table 2) remains essentially in the same range regardless of pH. This is expected since the same amount of BSM was used to cast the film at each pH condition. For (BSM/PAH) multilayers on ZnSe, the intensity of the polysaccharide peak (Figure S5, Supporting Information and Table 2) varies by as much as a factor of 10, with a distinct increase in the peak intensity between pH 4 and pH 7. The IR absorbance of a BSM single layer coating at low pH (Figure 4a,b) shows a very strong intensity of these characteristic peaks. Therefore, the absence of strong peaks in the multilayers (Figure S5, Supporting Information) assembled at low pH implies that the amount of BSM in the multilayers assembled at pH 2 and pH 4 is relatively small compared to that in multilayers assembled at higher conditions. 3.3. Adsorption/Desorption Dynamics of BSM and PAH Are pH Dependent. Variation of assembly pH did not seem to significantly impact the conformation of the 232
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Figure 6. (a−e) Phase contrast microscope images of HeLa cells after 4 days in culture on (PAH/BSM)30 multilayers assembled from pH 2 to pH 11 (from a to e) with control growth on bare glass slide (f). The scale bar is 250 μm. (g−h) Numbers of HeLa cells per cm2 after (g) 1 day and (h) 4 days in culture on (PAH/BSM)30 multilayers.
1 and day 4 (Figure 6). To make sure that cell adhesion is not due to film deformation, a stability test was done as shown in Figure S11 (Supporting Information). Multilayers assembled at pH 2 and pH 4 effectively prevent cell adhesion, and this effect persists for more than a month (data not shown, media were changed every 3 days). Considering that the thicknesses of these two multilayers are both less than 10 nm, the effectiveness of cell repulsion and its durability are quite remarkable. Glass substrates coated with a single layer of PAH, BSM, or a (PAH/ BSM) 1 bilayer film at various pH conditions (data not shown) as well as bare glass were densely populated with HeLa cells, meaning the multilayer assembly is necessary for this effect to occur. A noticeable increase in the density of adherent cells on the surface was detected between pH 4 and pH 7. Paired with the contact angle results, we conclude that BSM presents more hydrophilic chemical groups to the surface only when assembled at low pH conditions in LbL multilayers with PAH. 3.5. Batch-to-Batch Variations of BSM. Since BSM is a naturally derived biopolymer, it is inevitable that it will exhibit some batch-to-batch variations. Others have shown significant variations in the amount of aggregates and albumin based on anion exchange chromatography data using two different batches from Sigma (Lot 013 K7028 and Lot 64H7170, released in 2008 and 2009).7 For these two batches, the weightaverage molar masses (Mw), z-average root-mean-square radius (Rrms), hydrodynamic radius (Rh), and the polydispersity index (Mw/Mn) were also measured and shown to vary dramatically depending on the batch.7 On the basis of this information, it was possible to anticipate the batch-to-batch variability on the buildup of (BSM/PAH) films. To investigate this, we repeated a whole set of experiments using two batches obtained from Sigma (batch 1, Lot 039 K7003, and batch 2, Lot SLBC232 V, obtained in 2011 and 2012, respectively). As described in the certificate of analysis from the vendor, batch 1 (Lot 039 K7003) has 17% bound sialic acid with 0.01% free, while batch 2 has 13% bound sialic acid with 0% free. The difference in the amount of sialic acid implies the potential difference in the isoelectric point and FT-IR spectra of these two batches. Of note, all spectra in Figure 4 were measured using batch 1. Unlike the BSM batch 1 used in this study, batch 2 formed
order of scale as the pH 7 and pH 11 cases. By simply decreasing the assembly pH, film growth behavior drastically switched from a typical stepwise growth behavior, in which hydrated mass increases with each polymer deposition step, to a cyclical growth behavior (or zigzag-type growth, i.e., an increase in hydrated mass at one polymer deposition step, followed by some loss in mass during the deposition of the other polymer).36 Similar cyclical growth behavior has been observed previously in various LbL systems where it was attributed to partial redissolution of the polymer complex formed in the prior deposition step by the addition of excess complementary polymer pairs.15,37 In particular, when BSM and chitosan (CHI) were LbL assembled on hydrophobized silica at pH 4.0,15 the addition of BSM and CHI led to the increase and subsequent decrease in hydrated mass for all layers except the first bilayer. Taking into account that the BSM/PAH multilayer assembly in Figure 5 was also performed on a hydrophobic surface (gold crystal) at low pH, the result from Svensson et al.15 is analogous to the result in Figure 5c,d. The first bilayers are insensitive to these phenomena, and all these findings suggest that BSM interacts differently with the already adsorbed PAH depending on the assembly pH. This drastic difference in growth behavior observed by the QCM-D experiment suggests that the resultant film may exhibit significantly different surface characteristics, depending on the assembly pH conditions. Thus, at low pH (pH 2 and pH 4) the assembled multilayers finished with BSM will have more loosely bound BSM adhering to the outermost layer, which is stable toward the typical rinsing condition. This could explain the differences in surface wettability measured and could impact phenomena sensitive to surface wettability such as protein adsorption or cell attachment. 3.4. Cell Adhesion and Proliferation on Multilayers Are pH Dependent. A BSM single layer has been shown to repel mammalian cell adhesion effectively when deposited on hydrophobic polystyrene surfaces.23 We thus investigated the pH dependence of cell adhesion and proliferation on (PAH/ BSM)30 multilayers. The HeLa epithelial cell line was cultured on BSM-topped films, and their densities were recorded at day 233
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Figure 7. Phase contrast microscope images of HeLa cells after 4 days in culture on a BSM single layer built on (PAH4.0/SPS4.0)x multilayers. ((a) Batch 1 BSM (Lot 039 K7003), (b) batch 2 BSM (lot SLBC232 V) without any filtering step, (c) batch 2 BSM (lot SLBC232 V) after filtering with a 450 nm filter.) In each image, the outermost layer prior to BSM coating is SPS (x = 15) on the left and PAH (x = 15.5) on the right. The scale bar is 250 μm.
adhesion, with more hydrophilic and cell-repellent films generated with pH < 7. FT-IR studies showed that the functional groups of BSM in multilayers were not altered during the LbL assembly over the wide range of assembly pH conditions. Also, QCM-D studies showed that a substantial amount of BSM adhered to the outermost surface only at low pH, and this fact can be correlated with distinct differences in the properties of the resulting films. Although batch-to-batch variations limit the direct applicability of these PAH/BSM films, we demonstrate that the properties of mucin films assembled by LbL can be finely tuned by changing assembly pH and ionic strength. In particular, the ability to modulate which particular mucin functional groups are exposed to the surface could be of help in manipulating the virus and bacteria binding capacity of mucins.
cloudy solutions, indicating the presence of insoluble aggregates. Absorbance of transmitted light between 200 and 1200 nm shows a clear difference in aggregate amounts between batches 1 and 2 (Figure S6, Supporting Information). However, the aggregates could be successfully removed by filtering the solution through a membrane with 450 nm pores. The aggregates present in the batch 2 BSM solution affected the (PAH/BSM)30 multilayer growth, yielding multilayers with highly variable thickness (from 10 nm up to 500 nm) when built on glass slides. However, smooth multilayers with thicknesses close to those obtained in this study with batch 1 BSM could be obtained when using filtered batch 2 BSM solutions (Figure S7, Supporting Information). QCM-D measurements show that during the film assembly using filtered batch 2 BSM, a significant amount of BSM and PAH was lost upon rinsing at all pH conditions (Figure S8, Supporting Information). This implies that interaction between PAH and the filtered batch 2 BSM is not as strong as it was with the batch 1 BSM. Finally, we compared the cell-repulsive properties of the two batches by adsorbing a single layer of BSM on (PAH/SPS)15.5 multilayers (Figure 7). The unfiltered BSM from both batch 1 and batch 2 repels cell adhesion effectively when PAH is the outermost layer. However, when batch 2 was filtered prior to adsorption, considerable amounts of cells adhered to the substrate (Figure 7c). Figure S6 (Supporting Information) and Figure 7c imply the possible decrease of BSM concentration after the filtering step. Of note, the multilayers produced from the filtered batch 2 BSM did not exhibit resistance to cell adhesion, regardless of the assembly pH. Therefore, it is necessary to point out that batch-to-batch variation has significant impact on the incorporation and resulting functionalities of BSM multilayers. Although filtering decreases batch-to-batch variations, the interaction between PAH and BSM was adversely affected by the filtering step. This effect limits the access to the novel and advantageous tuning of the properties of BSM that we have demonstrated in the present article. More work will allow a better understanding of the origin of these variations, in particular the role of contaminants such as bovine serum albumin, which has been shown to influence the structure of BSM coatings38 and resolve the inherent issues originating from batch-to-batch variation of BSM.
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ASSOCIATED CONTENT
* Supporting Information S
Alcian blue staining and HeLa cell adhesion on a single layer of BSM on various multilayers; interpolymer complexation studies between PAH and BSM; FT-IR data of multilayers prior to thickness normalization; thickness and dynamic adsorption/ desorption behavior of batch 2 BSM; the attenuation of transmitted light; QCM-D data for batch 2 BSM; the growth behavior of multilayers; the average roughness measurement; and the stability test of multilayers. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(K.R.) E-mail: [email protected]. *(M.F.R.) E-mail: [email protected]. *(R.E.C.) E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Center for Materials Science and Engineering (CMSE), the Institute for Soldier Nanotechnologies (ISN). This work was partially supported by the MRSEC Program of the National Science Foundation under award number DMR0819762. J.A. acknowledges a Samsung Scholarship. We thank Amanda Tsoi and Cassandra Llano for their assistance with this work. T.C. was supported by the Marie Curie International Outgoing Fellowship (project BIOMUC).
4. CONCLUSIONS We have demonstrated that BSM and PAH can successfully be assembled as multilayer films. The properties of the resulting films were strongly dependent on the assembly pH and the salt concentration in the PAH solution. Assembly pH changed film assembly dynamics, final thickness, surface wettability, and cell
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REFERENCES
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(2) McGuckin, M. A.; Lindén, S. K.; Sutton, P.; Florin, T. H. Nature Rev. Microbiol. 2011, 9, 265−278. (3) Amerongen, A. V. N.; Amerongen, A. V. N.; Bolscher, J. G. M.; Bolscher, J. G. M.; Veerman, E. C. I.; Veerman, E. C. I. Glycobiology 1995, 5, 733−740. (4) Bansil, R.; Turner, B. S. Curr. Opin. Colloid Interface Sci. 2006, 11, 164−170. (5) Sheehan, J. K.; Oates, K.; Carlstedt, I. Biochem. J. 1986, 239, 147. (6) Lee, S.; Müller, M.; Rezwan, K.; Spencer, N. D. Langmuir 2005, 21, 8344−8353. (7) Sandberg, T.; Blom, H.; Caldwell, K. D. J. Biomed. Mater. Res., Part A 2009, 91A, 762−772. (8) Shi, L.; Ardehali, R.; Caldwell, K. D.; Valint, P. Colloids Surf., B 2000, 17, 229−239. (9) Bushnak, I. A.; Labeed, F. H.; Sear, R. P.; Keddie, J. L. Biofouling 2010, 26, 387−397. (10) Sandberg, T.; Karlsson Ott, M.; Carlsson, J.; Feiler, A.; Caldwell, K. D. J. Biomed. Mater. Res., Part A 2009, 91A, 773−785. (11) Shi, L.; Caldwell, K. D. J. Colloid Interface Sci. 2000, 224, 372− 381. (12) Yakubov, G. E.; Yakubov, G. E.; McColl, J.; McColl, J.; Bongaerts, J. H. H.; Bongaerts, J. H. H.; Ramsden, J. J.; Ramsden, J. J. Langmuir 2009, 25, 2313−2321. (13) Feldötö, Z.; Pettersson, T.; Dedinaite, A. Langmuir 2008, 24, 3348−3357. (14) Sandberg, T.; Carlsson, J.; Ott, M. K. Microsc. Res. Technol. 2007, 70, 864−868. (15) Svensson, O.; Lindh, L.; Cárdenas, M.; Arnebrant, T. J. Colloid Interface Sci. 2006, 299, 608−616. (16) Dedinaite, A.; Lundin, M.; Macakova, L.; Auletta, T. Langmuir 2005, 21, 9502−9509. (17) Halthur, T. J.; Arnebrant, T.; Macakova, L.; Feiler, A. Langmuir 2010, 26, 4901−4908. (18) Crouzier, T.; Beckwitt, C. H.; Ribbeck, K. Biomacromolecules 2012, 13, 3401−3408. (19) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213− 4219. (20) Lichter, J. A.; Rubner, M. F. Langmuir 2009, 25, 7686−7694. (21) Dedinaite, A.; Bastardo, L. Langmuir 2002, 18, 9383−9392. (22) Bastardo, L.; Claesson, P.; Brown, W. Langmuir 2002, 18, 3848−3853. (23) Crouzier, T.; Jang, H.; Ahn, J.; Stocker, R.; Ribbeck, K. Biomacromolecules 2013, 14, 3010−3016. (24) Steedman, H. F. Q. J. Microsc. Sci. 1950, 3, 477−479. (25) Lee, H.; Mensire, R.; Cohen, R. E.; Rubner, M. F. Macromolecules 2011, 45, 347−355. (26) Kharlampieva, E.; Kozlovskaya, V.; Sukhishvili, S. A. Adv. Mater. 2009, 21, 3053−3065. (27) Choi, J.; Rubner, M. F. Macromolecules 2005, 38, 116−124. (28) Itano, K.; Choi, J.; Rubner, M. F. Macromolecules 2005, 38, 3450−3460. (29) Khajehpour, M.; Dashnau, J. L.; Vanderkooi, J. M. Anal. Biochem. 2006, 348, 40−48. (30) Castillo, E. J.; Koenig, J. L.; Anderson, J. M. Biomaterials 1986, 7, 89−96. (31) Patel, M. M.; Smart, J. D.; Nevell, T. G.; Ewen, R. J.; Eaton, P. J.; Tsibouklis, J. Biomacromolecules 2003, 4, 1184−1190. (32) Skingsley, D. R. J. Molluscan Stud. 2000, 66, 363−372. (33) Bavington, C. D.; Lever, R.; Mulloy, B.; Grundy, M. M.; Page, C. P.; Richardson, N. V.; McKenzie, J. D. Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol. 2004, 139, 607−617. (34) Shanmugam, G.; Polavarapu, P. L. Proteins 2006, 63, 768−776. (35) Lewis, S. P.; Lewis, A. T.; Lewis, P. D. Vib. Spectrosc. 2013, 69, 21−29. (36) Shutava, T.; Prouty, M.; Kommireddy, D.; Lvov, Y. Macromolecules 2005, 38, 2850−2858. (37) Richert, L.; Lavalle, P.; Payan, E.; Shu, X. Z.; Prestwich, G. D.; Stoltz, J.-F.; Schaaf, P.; Voegel, J.-C.; Picart, C. Langmuir 2004, 20, 448−458.
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Tuning the Properties of Mucin via Layer-by-Layer Assembly Jiyoung Ahn,† Thomas Crouzier,‡ Katharina Ribbeck,*,‡ Michael F. Rubner,*,§ and Robert E. Cohen*,† †
Department of Chemical Engineering, ‡Department of Biological Engineering, and §Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States S Supporting Information *
ABSTRACT: Multilayer films consisting of bovine submaxillary mucin (BSM) and poly(allylamine hydrochloride) (PAH) were prepared on various substrates using layer-by-layer assembly. The effects of both the assembly pH and ionic strength on multilayer characteristics were investigated by assessing film thicknesses (10−80 nm), surface wetting characteristics, and cell repulsion. Also, the dynamic assembly behavior was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) to further understand the effect of assembly pH on film characteristics. Assembly studies revealed that substantial amounts of BSM adhere to the outermost surface only at low pH conditions. The resulting multilayer films assembled at low pH conditions were found to exhibit hydrophilic and cell repellent behavior. In addition, it was found that batch-to-batch variations of the biopolymer BSM could dramatically alter properties.
1. INTRODUCTION Mucins are high molecular weight (200 kDa−200 MDa) glycoproteins that form the mucus gel on epithelial cell surfaces.1 The mucus gel is a barrier against pathogens and lubricates the underlying cell surface.2 The protein core of mucins is composed of hydrophobic regions separated by serine on threonine rich regions to which glycan moieties are attached.3 These densely glycosylated domains confer to mucins a bottlebrush-like molecular architecture.4 Mucins of different origins are available commercially for relatively low cost depending on the source such as porcine gastric mucin (PGM) and bovine submaxillary mucin (BSM), and share a similar structure in general as described above.5 One way to study and take advantage of the natural properties of mucins is through the formation of mucin surface layers. Adsorption of mucins has been found to lubricate surfaces,6 cause significant changes in contact angle,7 and repel the adhesion of microorganism and mammalian cells.1,8−10 This suggests the potential use of mucin for biomedical applications and practical applicability for industrial purposes.2,8,9 The details in interaction between mucins and various materials are thus of great interest and have been investigated for various polymeric materials.3,7−9,11,12 These studies revealed that the adsorption, as well as the conformation of the resulting mucin layers, depend strongly on the surface properties of the underlying substrates, such as wetting characteristics, types of functional groups present, and surface charge density.4,8,9 For example, mucins form a thick and highly hydrated layer on a gold-coated surface, while a thin and less hydrated layer is formed on silica.14 Also, it was shown that BSM binds very strongly on a hydrophobic and carboxylic acid modified surface but not so strongly on a hydrophilic hydroxyl modified surface.5,13 In addition, it has been reported previously that the © 2014 American Chemical Society
biological function of the resulting mucin layers is also strongly dependent on the type of underlying substrates.5,8−10,14 Manipulating the properties of mucin only by varying the underlying substrate, however, poses a limitation to the applicability of mucin for various applications. Therefore, it would be highly desirable to generate mucin layers with properties that could be controlled independently of the nature of the underlying substrate. For this purpose, the layer-by-layer (LbL) assembly technique was chosen to assemble mucin containing multilayer films. The LbL assembly is a simple and versatile technique to generate conformal films on various substrates where the physicochemical properties of the resulting films can be fine-tuned by simply varying the complementary polymer pair and assembly conditions. Various complementary polymer pairs such as positively charged chitosan,7,15,16 lactoperoxidase,17 and sugar-binding lectin wheat germ agglutinin18 have been used previously to form LbL multilayer films with BSM7,15−17 and PGM.18 However, there lacks a systematic study on the impact of assembly conditions such as pH and ionic strength on the properties of the resulting films. In this work, we focus on BSM, which has a net negative charge in physiological conditions due to its high content of sialic acid. We screened BSM interactions with several polycations and chose poly(allylamine hydrochloride) (PAH) as the complementary interacting polymer due to its interesting interactions with mucins and well-known behavior as a building block in LbL.19,20 The effective charge density of PAH was then varied systematically by changing assembly pH and ionic Received: September 29, 2014 Revised: November 22, 2014 Published: November 23, 2014 228
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Figure 1. (a) Alcian blue absorbance at 626 nm and the amount of adsorbed BSM on various polyelectrolyte multilayers. (b) The number of HeLa cells adhered on various multilayers before and after BSM adsorption. The asterisk is added to emphasize the outermost layers prior to BSM coating. Control experiment data for Alcian blue staining are given in Figure S2a, Supporting Information. with a 2 μm stylus tip, 2 mg stylus force, and a scanning rate of 50 μm/ s. 2.2.2.2. Contact Angle Measurement. The contact angles presented in this article are advancing contact angles, which were measured with the Ramé−Hart Model 590 goniometer. Using the tip of a syringe, a water drop was placed on a sample. The substrate was moved vertically until contact was made between the sample and the water drop. The subsequent addition of a small amount of water to the water drop on the surface produced the steady-state advancing angle in a few seconds (final volume to be about 1 μL). An image of the droplet was taken, and the right and left contact angles of the droplet were then measured by using an enlarged image transmitted to a computer screen. Four separate locations on each sample surface were probed to ensure a representative value of the contact angle. The average value of the measured contact angles was used to represent the wetting characteristics of the sample. 2.2.2.3. Fourier Transform Infrared Spectroscopy (FT-IR). IR spectra of multilayers deposited on ZnSe plates with 0.005 M PAH and 0.5 mg/mL BSM at various pH conditions were taken using a Nicolet 4700 FT-IR spectrometer (Thermo Scientific). Higher concentration for both polymer solutions was applied to intensify the peak signal. To locate the peak of PAH, and BSM at various pHs, solutions of PAH (0.005M) and BSM (0.5 mg/mL) were dropped on ZnSe plates and dried for 24 h in advance of measurement. 2.2.2.4. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). The hydrated mass of the film at various stages of the assembly process was measured by quartz crystal microbalance with dissipation monitoring (QCM-D, E4 system, Q- Sense, Sweden). A gold-covered quartz crystal with a fundamental frequency of 5 MHz (QSX-301, Q-Sense) was used as a substrate for film deposition. As a cleaning procedure, the mixture of 10 mL of deionized water, 2 mL of 25% ammonia, and 2 mL of 30% hydrogen peroxide was used at 75C. The crystals were rinsed with deionized water afterward. Right before the assembly, the crystals were further cleaned by ozone treatment for 15 min. Films were built by manually alternating depositions of mucin and PAH solutions up to 6 bilayers. Mucin was prepared at a concentration of 0.1 mg/mL, and PAH was prepared at 0.001 M in deionized water. Mucin and PAH were left to adsorb for 10 min, followed by 5 min of rinsing with DI water at the pH of assembly. 2.2.2.5. Cell Culture. The epithelial HeLa cell line was maintained at subconfluency in T25 flasks with DMEM media supplemented with 10% fetal bovine serum (Invitrogen) and 1% antibiotics (25 U/mL penicillin and 25 μg/mL streptomycin (Invitrogen). The cells were detached using trypsin-EDTA (Invitrogen) and plated at a density of 45,000 cells/cm2 on top of a selected multilayer film deposited on
strength, and the resulting films were studied in terms of the assembly dynamics and the physicochemical properties.
2. MATERIALS AND METHODS 2.1. Materials. All polymer solutions were prepared with deionized water, and the pH was adjusted using 1 M HCl and 1 M NaOH. The PAH (poly(allylamine hydrochloride)) used here had a nominal molecular weight of Mw = 70000 g/mol, as quoted by the supplier, PolySciences. PAH solution concentration was 0.001 M (based on a repeat unit molecular weight); 0.05 and 0.1 M of NaCl was added in certain cases to investigate the effect of salt on assembly behavior. Commercially available bovine submaxillary mucin (BSM, SigmaAldrich) was used after being purified to remove some of the serum albumin contanimants.7 For the purification, BSM was dissolved in water at 30 mg/mL and dialyzed in a 100 kDa molecular weight cutoff membrane (Spectrum Laboratories) against water for 7 days. The mucin was then lyophilized for storage. Since commercial BSM solutions are known to form aggregates21,22 (larger than 400 nm depending on the batch) in solution, additional filtration was carried out using bottle top filters with a 0.45 mm PES membrane (VWR, 500 mL) when it is needed. The BSM solution concentration was 0.1 mg/ mL, which was chosen to set the mass ratio with PAH at unity. 2.2. Methods. 2.2.1. Thin Film Assembly. All films were prepared on glass substrates (VWR Scientific) using a Stratosequence VI spin dipper (Nanostrata Inc.) with StratoSmart v6.2 software. Prior to film assembly, the substrates were cleaned by sonication in a 4% (v/v) solution of Micro-90 (Internation Product) for 20 min and then in DI water for 20 min. Oxygen plasma treatment (PDC-32G, Harrick Scientific Products, Inc.) was followed for 2 min at 150 mTorr. The spin dipper was operated with dipping times of 10 min for the polymer solutions without rotation, followed by three rinses of 2, 1, and 1 min in pH adjusted DI water with rotation. The pH of the rinse water was adjusted with 0.1 M HCl or 0.1 M NaOH. The nomenclature for LbL films follows the usual convention (polymer A/polymer B)n, where n is the total number of bilayers deposited. The pH conditions of assembly are specified by providing the pH value after the polymer name, for example, polymerA7.0. The pH conditions of the rinse baths were adjusted to be identical to the pH of the preceding polymer solution. For gold crystal and ZnSe, BSM was used as the first layer to ensure the formation of a stable first layer through hydrophobic interaction between BSM and substrates. 2.2.2. Characterization. 2.2.2.1. Thickness Measurement. Dry film thicknesses were measured using a Tencor P16 surface profilometer 229
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glass. Cells were observed after 24 and 96 h. Images were acquired on an Axio Observer Z1 microscope (Zeiss, Oberkochen, Germany) using a EC-Plan Neofluar 10× 0.3 NA lens (Zeiss) in phase contrast or fluorescence mode. Cells were counted with the image analysis software ImageJ using the cell count plug-in.
3. RESULTS AND DISCUSSION 3.1. BSM Forms Cell-Repulsive, High-Density Coatings on PAH Topped Multilayer Films. Prior to incorporating BSM into multilayers, a preliminary screening procedure was performed to find a promising assembly partner and conditions for BSM. Several well-studied multilayers described in the literature20 composed of a combination of PAH, PAA (poly(acrylic acid)), and SPS (poly(styrenesulfonate)) were assembled with systematic variation in the outermost layer (Figure 1). A single layer of fluorescently labeled BSM (0.1 mg/mL) at pH 7 was deposited on these multilayers, and the amount of BSM adsorbed was quantified by the fluorescence intensity as shown in Figure 1a. Of note, BSM has a net negative charge at this pH since it is above its isoelectric point. Therefore, it was expected that little or no BSM would adsorb onto the films finished with SPS, which has a net negative charge over all pH levels of this study. Adhesion of HeLa cells on these samples was quantified to explore the presence of mucin in the multilayers since BSM coatings can, under certain conditions, prevent the attachment of mammalian cells.23 Moreover, Alcian blue staining was performed to directly quantify the amount of mucins present (Figure 1a and b and Supporting Information, Figures S1−S3).24 Our results show that the outermost polymer species and the pH at which the multilayer was assembled both had a significant impact on the amount of adsorbed BSM. Assemblies with PAH as the outermost layer at pH 9.3 resulted in the strongest reduction of cell adhesion and in the greatest increase in Alcian blue staining after BSM deposition. Of note, there was no clear correlation between the intensity of Alcian blue staining and the reduction of cell adhesion. This indicates that reduction in cell adhesion does not only depend on the amount of BSM present but also on the details of its assembly (nature of pairing polymer, assembly pH) film properties. This suggests that the native properties of mucin can be modified when incorporated in a multilayer. On the basis of these results, we chose PAH as an assembly partner for BSM and investigated the effect of the pH of both BSM and PAH solution and the salt concentration of the PAH solution on the properties of the resulting film. 3.2. Layer-by-Layer Assembly of BSM with PAH. 3.2.1. Layer-by-Layer Assembly of BSM with PAH Is Dependent on pH and Salt Concentration. (PAH/BSM)30 multilayers were successfully assembled on precleaned hydrophilic glass slides with systematic variation in the assembly pH (both PAH and BSM solutions) and salt concentration (PAH solution only) to determine how these parameters influence film growth and average multilayer film thicknesses. The average film thickness of 30 bilayer assemblies as measured by profilometry in the dry state is shown in Figure 2. To indirectly prove that each layer is deposited on the substrate, the thickness of these multilayers was also measured after every 10 bilayer formation (Figure S10, Supporting Information). From Figure 2, it is apparent that both the assembly pH and the salt concentration in PAH solution strongly influence the deposition process. (PAH/BSM)30 built at pH 11 without any additional salt resulted in a noticeably thicker film than that
Figure 2. Average thickness of a 30 bilayer multilayer film of PAH and BSM as a function of assembly pH and salt concentration in the PAH solution.
at any other conditions studied. When salt was present in the PAH solution, the thickness was nearly constant (about 20−30 nm) over a wide range of assembly pH conditions. At low pH conditions, the thickness of the final multilayers was less than 10 nm regardless of the amount of salt. These results are consistent with our studies of interpolymer complex formation (Figures S4 and S9, Supporting Information), where the presence or absence of turbidity of mixtures of the complementary polymer solutions was used as a predictor of whether selected conditions would lead to successful LbL assembly.25,26 Upon mixing salt-free PAH solutions with BSM solutions, the mixture became turbid only at pH 11. When salt was added to the PAH solution prior to mixing, an evident change in solution turbidity was observed from pH 3 to pH 10. Given the strong effect of pH when no salt is present, films assembled without salt were potentially more tunable. Thus, films built in the no-salt condition were further characterized. 3.2.2. Surface Wettability of Multilayers Is Dependent on pH. Adsorption of a single layer of BSM has been shown to make hydrophobic substrates hydrophilic, as manifested by a large decrease in the water contact angle.8 Figure 3 shows the
Figure 3. Advancing water contact angles measured on (PAH/BSM)30 multilayers fabricated with varying pH of assembly.
advancing water contact angles of multilayer films (PAH/ BSM)30 assembled at different pH conditions. As the assembly pH is increased, the contact angle of resulting multilayers is also increased. The height AFM measurements (data not shown) and the average roughness measurement (Table S1, Supporting Information) did not reveal any significant surface morphology changes that can be correlated with this variation in contact angle. Thus, the assembly pH changed both the thickness (Figure 2) and, at least as revealed by surface wettability, the 230
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Figure 4. (a,b) FT-IR spectra of a single layer of BSM, cast from aqueous solutions (0.5 mg/mL) adjusted to various pH conditions. (c,d). FT-IR spectra of (BSM/PAH)30 multilayers on ZnSe substrates assembled at various pH conditions (normalized by the film thickness). Spectra are intentionally overlaid with arbitrary offset for clarity.
Table 1. IR Absorption Ratio of Amide II (v = 1540 cm−1) to Amide I (v = 1652 cm−1) Bands from BSM Cast and BSM/PAH Multilayers (Normalized by Thickness)a vamII/vamI (1540:1652) of BSM cast on ZnSe vamII/vamI(1540:1652)of (BSM/PAH)30 on ZnSe a
pH 2
pH 4
pH 7
pH 9.3
pH 11
0.941 ± 0.064 0.742 ± 0.046
0.810 ± 0.174 0.835 ± 0.128
0.717 ± 0.146 0.667 ± 0.283
0.836 ± 0.055 0.793 ± 0.190
0.830 ± 0.165 0.625 ± 0.332
The ratio is calculated from five independent measurements.
organization of mucin in the film. Bearing in mind that BSM is the top layer, these results suggest changes in the amount of hydrophilic groups at the air−film interface provided by BSM as the assembly pH increases. 3.2.3. Mucin Functional Groups and Protein Structure Are Similar in Cast Films and the Multilayers. The differences in surface wettability could originate from changes in the conformation of the BSM molecules as the assembly pH is varied. FT-IR analysis is rich in information about the nature and conformation of chemical groups present in multilayer films.27,28 We thus used FT-IR analysis on the multilayer system to investigate the origin of its pH-dependent growth behavior. A single-layer deposition of BSM on ZnSe was used as a control to obtain information about the effect of pH on the secondary structure of the protein backbone in BSM. The IR spectra of BSM films (Figure 4a,b) cast on ZnSe substrates are consistent with the structure of mucin as reported by others. Sialic acid in BSM shows two distinct peaks at 1730 and 1570 cm−1, which arise from the carboxyl group and amide groups of sialic acid, respectively.29,30 Also the absorbance peaks at 1653, 1540, 1240 cm−1, and 1039 cm−1 of BSM are attributed to the amide I, II, III bands, and
polysaccharide residues, respectively.30 Between 3000 and 3500 cm−1, very broad absorbance peaks are attributed to the abundant hydroxyl groups in the sugar residues of BSM.31 From 2800 to 3000 cm−1, absorbance peaks from the alkyl groups show up as well.29,32,33 When BSM and PAH are assembled into multilayers, the changes observed by FT-IR mainly reflect the presence of PAH and the relative amount of BSM in the multilayer. To remove the influence of film thickness on the observed peak intensity, the spectra were normalized by thickness prior to further analysis, as shown in Figure 4c,d. The absorbance bands of the multilayers were, for the most part, similar to the spectrum of the pure BSM single layer. Peaks attributed to the amide I, II, and III groups, from the sialic acid and from alkyl groups, are analogous to the peaks identified for cast BSM. An additional peak for PAH at 1310 cm−1 at pH 9.3 and pH 11 appeared for the multilayers assembled at these pHs as shown in the literature.27 The relative peak ratios for the amide groups were found to occur in the same range as the pure cast films of BSM. Put together, these results indicate that there is no major shift in the secondary structure of the protein backbone of BSM during layer-by-layer assembly with PAH, regardless of the 231
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Table 2. IR Absorption for Polysaccharide Bands (v = 1039 cm−1) of BSM Cast versus BSM/PAH Multilayers from Five Independent Measurementsa vpolysaccharides (1039) of BSM cast on ZnSe vpolysaccharides (1039) of (BSM/PAH)30 on ZnSe a
pH 2
pH 4
pH 7
pH 9.3
pH 11
0.013 ± 0.010 0.0021 ± 0.0007
0.013 ± 0.013 0.0039 ± 0.0028
0.014 ± 0.006 0.019 ± 0.006
0.011 ± 0.004 0.015 ± 0.005
0.012 ± 0.013 0.033 ± 0.003
Values were taken prior to normalization with thickness.
Figure 5. Evolution of the hydrated mass for LbL build-up at moderate and high pH of assembly ((a) pH 7, (b) pH 11, (c) pH 2, and (d) pH 4), CPAH = 0.001M, and CBSM = 0.1 mg/mL. B and P indicate the beginning of the BSM or PAH absorption step, respectively. BSM and PAH were left to adsorb for 10 min, followed by 5 min of rinsing with DI water at the pH of assembly.
incorporated mucins. However, the final thickness varied greatly as seen in Figure 2. We thus investigated the dynamics of the adsorption/desorption phenomena that occur during LbL assembly using QCM-D measurements. Real-time adsorption of BSM and PAH onto a gold crystal was observed up to 6 bilayers, and the accumulation of hydrated mass during the multilayer buildup is plotted in Figure 5. In the first bilayer formation, the addition of both BSM and PAH leads to an increase in hydrated mass for all pH conditions investigated. At pHs 7 and 11, a continuous increase in hydrated mass was detected throughout the assembly in a stepwise manner. The final values of hydrated mass after the deposition of 6 bilayers at pHs 7 and 11 were 16.8 mg/m2 and 17.2 mg/m2, respectively. At low pH, a different trend of the multilayer buildup emerged. After the first bilayer, from the second addition of BSM, the total hydrated mass adsorption shows a nonmonotonic cyclical behavior. Exposure to BSM leads to large and stable increases in hydrated mass. However, with the addition of PAH, a sudden and substantial decrease in the total hydrated mass was measured. The hydrated mass loss did not occur during the rinsing step but only when the subsequent PAH is added to the system. Of note, each bilayer resulted in a net positive increment of mass. After 6 bilayers, the total hydrated mass was 13.9 mg/m2 for pH 2 and 20.9 mg/m2 for pH 4, on the same
assembly pH (Table 1). Of note, secondary structures of BSM have been previously confirmed to be dominated by random coil conformation by CD, DLS, and FT-IR measurements.7,34,35 The IR absorption intensity of the polysaccharide residues can be used as an indicator of the amount of BSM embedded in the layers. For the pure BSM layer on ZnSe, the absolute value of the absorption peak arising from the polysaccharide residues (Table 2) remains essentially in the same range regardless of pH. This is expected since the same amount of BSM was used to cast the film at each pH condition. For (BSM/PAH) multilayers on ZnSe, the intensity of the polysaccharide peak (Figure S5, Supporting Information and Table 2) varies by as much as a factor of 10, with a distinct increase in the peak intensity between pH 4 and pH 7. The IR absorbance of a BSM single layer coating at low pH (Figure 4a,b) shows a very strong intensity of these characteristic peaks. Therefore, the absence of strong peaks in the multilayers (Figure S5, Supporting Information) assembled at low pH implies that the amount of BSM in the multilayers assembled at pH 2 and pH 4 is relatively small compared to that in multilayers assembled at higher conditions. 3.3. Adsorption/Desorption Dynamics of BSM and PAH Are pH Dependent. Variation of assembly pH did not seem to significantly impact the conformation of the 232
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Figure 6. (a−e) Phase contrast microscope images of HeLa cells after 4 days in culture on (PAH/BSM)30 multilayers assembled from pH 2 to pH 11 (from a to e) with control growth on bare glass slide (f). The scale bar is 250 μm. (g−h) Numbers of HeLa cells per cm2 after (g) 1 day and (h) 4 days in culture on (PAH/BSM)30 multilayers.
1 and day 4 (Figure 6). To make sure that cell adhesion is not due to film deformation, a stability test was done as shown in Figure S11 (Supporting Information). Multilayers assembled at pH 2 and pH 4 effectively prevent cell adhesion, and this effect persists for more than a month (data not shown, media were changed every 3 days). Considering that the thicknesses of these two multilayers are both less than 10 nm, the effectiveness of cell repulsion and its durability are quite remarkable. Glass substrates coated with a single layer of PAH, BSM, or a (PAH/ BSM) 1 bilayer film at various pH conditions (data not shown) as well as bare glass were densely populated with HeLa cells, meaning the multilayer assembly is necessary for this effect to occur. A noticeable increase in the density of adherent cells on the surface was detected between pH 4 and pH 7. Paired with the contact angle results, we conclude that BSM presents more hydrophilic chemical groups to the surface only when assembled at low pH conditions in LbL multilayers with PAH. 3.5. Batch-to-Batch Variations of BSM. Since BSM is a naturally derived biopolymer, it is inevitable that it will exhibit some batch-to-batch variations. Others have shown significant variations in the amount of aggregates and albumin based on anion exchange chromatography data using two different batches from Sigma (Lot 013 K7028 and Lot 64H7170, released in 2008 and 2009).7 For these two batches, the weightaverage molar masses (Mw), z-average root-mean-square radius (Rrms), hydrodynamic radius (Rh), and the polydispersity index (Mw/Mn) were also measured and shown to vary dramatically depending on the batch.7 On the basis of this information, it was possible to anticipate the batch-to-batch variability on the buildup of (BSM/PAH) films. To investigate this, we repeated a whole set of experiments using two batches obtained from Sigma (batch 1, Lot 039 K7003, and batch 2, Lot SLBC232 V, obtained in 2011 and 2012, respectively). As described in the certificate of analysis from the vendor, batch 1 (Lot 039 K7003) has 17% bound sialic acid with 0.01% free, while batch 2 has 13% bound sialic acid with 0% free. The difference in the amount of sialic acid implies the potential difference in the isoelectric point and FT-IR spectra of these two batches. Of note, all spectra in Figure 4 were measured using batch 1. Unlike the BSM batch 1 used in this study, batch 2 formed
order of scale as the pH 7 and pH 11 cases. By simply decreasing the assembly pH, film growth behavior drastically switched from a typical stepwise growth behavior, in which hydrated mass increases with each polymer deposition step, to a cyclical growth behavior (or zigzag-type growth, i.e., an increase in hydrated mass at one polymer deposition step, followed by some loss in mass during the deposition of the other polymer).36 Similar cyclical growth behavior has been observed previously in various LbL systems where it was attributed to partial redissolution of the polymer complex formed in the prior deposition step by the addition of excess complementary polymer pairs.15,37 In particular, when BSM and chitosan (CHI) were LbL assembled on hydrophobized silica at pH 4.0,15 the addition of BSM and CHI led to the increase and subsequent decrease in hydrated mass for all layers except the first bilayer. Taking into account that the BSM/PAH multilayer assembly in Figure 5 was also performed on a hydrophobic surface (gold crystal) at low pH, the result from Svensson et al.15 is analogous to the result in Figure 5c,d. The first bilayers are insensitive to these phenomena, and all these findings suggest that BSM interacts differently with the already adsorbed PAH depending on the assembly pH. This drastic difference in growth behavior observed by the QCM-D experiment suggests that the resultant film may exhibit significantly different surface characteristics, depending on the assembly pH conditions. Thus, at low pH (pH 2 and pH 4) the assembled multilayers finished with BSM will have more loosely bound BSM adhering to the outermost layer, which is stable toward the typical rinsing condition. This could explain the differences in surface wettability measured and could impact phenomena sensitive to surface wettability such as protein adsorption or cell attachment. 3.4. Cell Adhesion and Proliferation on Multilayers Are pH Dependent. A BSM single layer has been shown to repel mammalian cell adhesion effectively when deposited on hydrophobic polystyrene surfaces.23 We thus investigated the pH dependence of cell adhesion and proliferation on (PAH/ BSM)30 multilayers. The HeLa epithelial cell line was cultured on BSM-topped films, and their densities were recorded at day 233
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Figure 7. Phase contrast microscope images of HeLa cells after 4 days in culture on a BSM single layer built on (PAH4.0/SPS4.0)x multilayers. ((a) Batch 1 BSM (Lot 039 K7003), (b) batch 2 BSM (lot SLBC232 V) without any filtering step, (c) batch 2 BSM (lot SLBC232 V) after filtering with a 450 nm filter.) In each image, the outermost layer prior to BSM coating is SPS (x = 15) on the left and PAH (x = 15.5) on the right. The scale bar is 250 μm.
adhesion, with more hydrophilic and cell-repellent films generated with pH < 7. FT-IR studies showed that the functional groups of BSM in multilayers were not altered during the LbL assembly over the wide range of assembly pH conditions. Also, QCM-D studies showed that a substantial amount of BSM adhered to the outermost surface only at low pH, and this fact can be correlated with distinct differences in the properties of the resulting films. Although batch-to-batch variations limit the direct applicability of these PAH/BSM films, we demonstrate that the properties of mucin films assembled by LbL can be finely tuned by changing assembly pH and ionic strength. In particular, the ability to modulate which particular mucin functional groups are exposed to the surface could be of help in manipulating the virus and bacteria binding capacity of mucins.
cloudy solutions, indicating the presence of insoluble aggregates. Absorbance of transmitted light between 200 and 1200 nm shows a clear difference in aggregate amounts between batches 1 and 2 (Figure S6, Supporting Information). However, the aggregates could be successfully removed by filtering the solution through a membrane with 450 nm pores. The aggregates present in the batch 2 BSM solution affected the (PAH/BSM)30 multilayer growth, yielding multilayers with highly variable thickness (from 10 nm up to 500 nm) when built on glass slides. However, smooth multilayers with thicknesses close to those obtained in this study with batch 1 BSM could be obtained when using filtered batch 2 BSM solutions (Figure S7, Supporting Information). QCM-D measurements show that during the film assembly using filtered batch 2 BSM, a significant amount of BSM and PAH was lost upon rinsing at all pH conditions (Figure S8, Supporting Information). This implies that interaction between PAH and the filtered batch 2 BSM is not as strong as it was with the batch 1 BSM. Finally, we compared the cell-repulsive properties of the two batches by adsorbing a single layer of BSM on (PAH/SPS)15.5 multilayers (Figure 7). The unfiltered BSM from both batch 1 and batch 2 repels cell adhesion effectively when PAH is the outermost layer. However, when batch 2 was filtered prior to adsorption, considerable amounts of cells adhered to the substrate (Figure 7c). Figure S6 (Supporting Information) and Figure 7c imply the possible decrease of BSM concentration after the filtering step. Of note, the multilayers produced from the filtered batch 2 BSM did not exhibit resistance to cell adhesion, regardless of the assembly pH. Therefore, it is necessary to point out that batch-to-batch variation has significant impact on the incorporation and resulting functionalities of BSM multilayers. Although filtering decreases batch-to-batch variations, the interaction between PAH and BSM was adversely affected by the filtering step. This effect limits the access to the novel and advantageous tuning of the properties of BSM that we have demonstrated in the present article. More work will allow a better understanding of the origin of these variations, in particular the role of contaminants such as bovine serum albumin, which has been shown to influence the structure of BSM coatings38 and resolve the inherent issues originating from batch-to-batch variation of BSM.
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ASSOCIATED CONTENT
* Supporting Information S
Alcian blue staining and HeLa cell adhesion on a single layer of BSM on various multilayers; interpolymer complexation studies between PAH and BSM; FT-IR data of multilayers prior to thickness normalization; thickness and dynamic adsorption/ desorption behavior of batch 2 BSM; the attenuation of transmitted light; QCM-D data for batch 2 BSM; the growth behavior of multilayers; the average roughness measurement; and the stability test of multilayers. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(K.R.) E-mail: [email protected]. *(M.F.R.) E-mail: [email protected]. *(R.E.C.) E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Center for Materials Science and Engineering (CMSE), the Institute for Soldier Nanotechnologies (ISN). This work was partially supported by the MRSEC Program of the National Science Foundation under award number DMR0819762. J.A. acknowledges a Samsung Scholarship. We thank Amanda Tsoi and Cassandra Llano for their assistance with this work. T.C. was supported by the Marie Curie International Outgoing Fellowship (project BIOMUC).
4. CONCLUSIONS We have demonstrated that BSM and PAH can successfully be assembled as multilayer films. The properties of the resulting films were strongly dependent on the assembly pH and the salt concentration in the PAH solution. Assembly pH changed film assembly dynamics, final thickness, surface wettability, and cell
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