Mucin Multilayers Assembled through Sugar–Lectin Interactions

Article pubs.acs.org/Biomac

Mucin Multilayers Assembled through Sugar−Lectin Interactions Thomas Crouzier, Colin H. Beckwitt, and Katharina Ribbeck* Department of Biological Engineering, 77 Massachusetts Avenue, Building 56-341c, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States S Supporting Information *

ABSTRACT: Multilayer films of biopolymers are attractive tools to exploit the extraordinary properties of certain biomacromolecules and introduce new functionalities to surfaces. Mucins, the gel-forming constituents of mucus, are versatile glycoproteins that have potential as new building blocks for biomaterial surface coatings. Multilayer films have mostly been assembled through the electrostatic pairing of polyelectrolytes, which results in limited pH and salt stability and screens charges otherwise available for useful payload binding. Here, we aim at assembling mucin multilayer films that differ from conventional paired polyelectrolyte assemblies to obtain highly stable and functional surface modifications. Using the lectin wheat germ agglutinin (WGA) to cross-link mucinbound sugar residues, we show that (Mucin/WGA) films can grow into hydrated films and sustain exceptional resistance to extreme salt conditions and a large range of pH. Furthermore, we show that the addition of soluble N-acetyl-D-glucosamine can induce the controlled release of WGA from (Mucin/WGA) films. Last, we show that (Mucin/WGA) films can repeatedly incorporate and release a positively charged model cargo. The lubricating, hydration, barrier, and antimicrobial properties of mucins open multiple applicative perspectives for these highly stable mucin-based multilayer films.

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biochemical properties, ranging from protons7 to certain pathogenic viruses, bacteria, and neutrophils.8,9 At a functional level, mucins provide a protective barrier toward pathogens and toxins and also serve to hydrate and lubricate the epithelial surfaces.10 Mucins can be isolated from native tissues or from mucus-secreting cell lines, and they can be reconstituted to form hydrogels. Mucins have been used to generate relatively thin monolayer coatings on a variety of surfaces including glass, polystyrene, and implantable materials such as polyurethane.11 Such thin mucin coatings have the capacity to limit bacterial surface adhesion12,13 and appear to trigger low immune responses,14,15 making them potentially biocompatible. Thicker multilayer coatings are also of interest because they have several critical advantages over monolayers: they offer more volume to incorporate molecules of interest such as drugs,16 antibiotics,17 or growth factors,18,19 they provide more structural flexibility to the polymers, thus potentially mimicking more closely their physiological conformation, and they are tunable in thickness, structure, and morphology. In addition, multilayer films generated with mucins could be of specific interest to generate biocompatible surface modifications or to reconstitute native functions of the mucosal barrier on synthetic surfaces. Mucin-based multilayer films can be formed by complexing mucins electrostatically with positively charged molecules such as chitosan,20,21 polyallylamine hydrochloride22 polymers, or

INTRODUCTION Natural hydrogels such as the extracellular matrix or mucus have the capacity to accommodate and regulate a range of biological properties of macromolecules and cells. Accordingly, their constituents have been exploited to create biomimetic materials that recapitulate selected functions. For example, extracellular matrix proteins such as collagen and fibronectin can interact with specific cell surface receptors and accordingly have been used to create coatings or 3D matrices that promote cell adhesion.1 Polysaccharides also find abundant use in biomedical applications. For instance, hyaluronic acid is injected into joints for lubrication in cases of osteoarthritis because its rheological properties are close to those of natural synovial fluids.2 Heparin is an effective anticoagulant, but it can also interact with certain growth factors, and has been used to build scaffolds for the controlled delivery of growth factors.3 Hence, biopolymers have gained increasing attention due to their desirable features for the engineering of biomimetic materials.4,5 One important, yet underexplored, family of biopolymers are mucins, the main gel-forming constituents of mucus. Mucins are glycoproteins that exist in secreted and membrane-bound form and coat all wet surfaces in the human body, including the eyes, and the epithelia of the respiratory, digestive, and female genital tracts. Secreted mucins are densely glycosylated and assume a largely extended conformation, with molecular weights up to several tens of megadaltons.6 Mucins have several exquisite features that make them interesting building blocks for synthetic materials. For example, they can interact with a broad spectrum of particles of different sizes and © 2012 American Chemical Society

Received: August 1, 2012 Revised: August 22, 2012 Published: August 24, 2012 3401

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fluorescent WGA, respectively. The films were assembled in untreated polystyrene 96-well plates (Falcon, 351172) with 0 mM NaCl in 20 mM Hepes buffer (pH 7.4). The fluorescence of each well was measured after the addition of each new layer using a fluorescence plate reader (Spectramax M3, Molecular Devices). Mucin and lectin contents were quantified by calibrating fluorescence in solution with known amounts of mucin and lectin. Dry and Hydrated Mass and Thickness of the (Mucin/WGA) Films. The dry thickness was determined by spectroscopic ellipsometry using a XLS-100 ellipsometer (J.A. Woollam, Lincoln, NE). This surface-sensitive optical technique records changes of light polarization upon reflection on the dried film. The optical thickness and the refractive index of the films were calculated from such measurements performed at an angle of 70° and from wavelength 190 to 993 nm. The films assembled on QCM-D crystals were rinsed with water and dried under nitrogen flow. We performed 70 spectroscopic scans per measurement and 4 measurements per sample. The data were modeled using the WVASE32 software (version 3.768) and assuming a multilayer model composed of a known Si substrate (0.2 mm), a gold layer (75 nm), and a Cauchy layer of unknown thickness and optical properties corresponding to the (Mucin/WGA) film. The mass of the dry film was estimated by assuming a density of 1200 kg/ m3, as previously measured for other similar systems.38 The refractive indices were 1.62 ± 0.06 for films built in 0 mM NaCl, 1.59 ± 0.05 for 200 mM NaCl, 1.59 ± 0.1 for 400 mM NaCl, and 1.59 ± 0.05 for 0 mM NaCl after WGA was released. A refractive index of ∼1.6 is in the upper regime of what has been reported before for biopolymer-based multilayer films.39 The hydrated mass and thickness were measured by quartz crystal microbalance with dissipation monitoring (QCM-D, E4 system, QSense, Sweden). The films were grown on a gold-covered quartz crystal, cleaned with warm 2% SDS and 0.1 M HCl, rinsed with deionized water, and further cleaned by ozone treatment for 15 min. Film growth on polystyrene-coated crystals was similar to that on gold (Figure SI 2 of the Supporting Information). The crystal vibration was followed at its fundamental frequency (∼5 MHz) and the six overtones (15, 25, 35, 45, 55, and 65 MHz). Changes in the resonance frequencies, and in dissipation of the vibration once the excitation is stopped, were followed at the seven frequencies. As suggested by the high dissipation values (Mucin/WGA), films are highly hydrated and possess viscoelastic properties, requiring the measurement data to be modeled. The Voigt-based model40 (i.e., a spring and dashpot in parallel under no slip conditions) was used to calculate the hydrated thickness, assuming a density of 1050 kg/m3 41 and that the film is homogeneous in thickness over the crystal’s surface. The multilayer films were built up to 12 layer pairs. The level of hydration of the film was deduced from the dry and hydrated mass by the relationship:

lactoperoxidase.23,24 In general, multilayer films with oppositely charged polyelectrolytes has enabled the engineering of interesting functional coatings for a wide range of applications, from sensors and solar cells to biomaterial and tissue engineering.25 However, they also have certain shortcomings. First, these films are sensitive to salt and the ionization state of the polymers, making them relatively unstable unless covalently cross-linked. 26,27 Second, the bulk of charges of the polyelectrolyte are complexed by the partner polymer, thus potentially screening charges that could serve to sequester particles or drugs into the multilayer films. Alternative techniques that bypass the need for electrostatic pairing have also been presented and include hydrogen bonding28 and click chemistry,29 but these techniques are best achieved with synthetic polymers of well-controlled chemistry. Other strategies use biological interactions such as biotin/strepativin,30 antibody−antigen pairs,31 and lectin/sugar interactions,32−35 all representing relatively strong interactions that drive the autoassembly of biopolymers and other biomacromolecules in vivo. Lectin/sugar interactions could be wellsuited to generate robust mucin films that are independent of electrostatic pairing of polyelectrolytes, as sugars that are commonly found in mucins; namely, N-acetyl-D-glucosamine (GlcNAc) and sialic acid are ligands to the lectin wheat germ agglutinin (WGA). WGA forms dimers and can bind up to eight sugar residues simultaneously.36 We here describe the use of the WGA lectin to cross-link mucin layers. We study the physicochemical properties of these films such as thickness, level of hydration, morphology, robustness toward ionic strength, and their capacity to bind a model cargo.

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MATERIAL AND METHODS

Materials and Reagents. Pig gastric mucin was purified from pig stomachs and lyophilized as reported in ref 37, omitting the cesium chloride density gradient. This preparation is enriched in the secreted mucin MUC5AC. Lyophilized mucin samples were dissolved overnight at 4 °C in deionized water at 2 mg/mL. Purified pig gastric mucin was labeled with the fluorescence dye Alexa488-carboxylic acid succinimidyl ester (Invitrogen). The dye was dissolved and stored at 10 mg/mL (15.5 mM) in DMSO. For the labeling reaction, 4 mg of mucin was dissolved in 0.1 M bicarbonate buffer (pH 9) and combined with 0.1 mg of the dye. The mixture was incubated for 1 h at room temperature; then, the pH was lowered to 7, and the unbound dye was removed by centrifuge filtration (Pall, MWCO 50 kDa). WGA lectin, poly L-lysine (PLL), fluorescein-labeled PLL (PLL-FITC), and N-acetyl-D-glucosamine were purchased from Sigma (St. Louis, MO). (Mucin/WGA) Film Buildup. Films were built by alternating depositions of mucin and the lectin WGA from dilute solutions. Mucin was prepared at a concentration of 0.2 mg/mL and lectin was prepared at 0.1 mg/mL in buffer containing 20 mM Hepes (pH 7.4) and 0, 200, or 400 mM NaCl. Mucin was left to adsorb for 15 min, and lectin was left for 5 min. Between the layering steps, two washes of 5 min were performed with the same buffer as used for buildup. For 96-well plates, 50 μL of mucin and WGA solution and 150 μL of washing solution were used. For QCM-D experiments, 300 μL of WGA and mucin solution and 1 mL of washing solution were used. The resulting films are termed (Mucin/WGA)n, with n being the number of layer pairs. The (Mucin/WGA)12 films were assembled on both the gold surface used for AFM measurement and the polystyrene surface for all other analyses. No differences in thickness or growth curve were measured on these two surfaces by QCM-D (Figure SI 2 of the Supporting Information). (Mucin/WGA) Film Composition. To determine the composition of the (Mucin/WGA) multilayer films, we spiked the mucin and WGA solutions with 40% (w/w) fluorescent mucin and 10% (w/w)

hydration =

hydrated mass − dry mass hydrated mass

(Mucin/WGA) Film Imaging. For fluorescence microscopy, the films were built using the fluorescent mucin-Alexa488 conjugate in 96well plates with thin untreated polystyrene bottoms, optimized for optical observation (Costar 3615 Corning, Corning, NY). The films were scratched with a pipet tip and imaged using an Observer Z1 inverted fluorescent microscope (Zeiss, Oberkochen, Germany) and a 10× 0.3 NA or 100× 1.4 NA lens (Zeiss, Germany). For AFM imaging, the films were built on QCM-D gold-covered crystals, kept in 20 mM Hepes buffer (pH 7.4), and observed in tapping mode using pyramidal cantilevers (NP-S10, Veeco, Santa Barbara, CA) and an Asylum MFP-3D-BIO AFM (Asylum Research, Santa Barbara, CA). WGA Release and Film Resistance to pH, Salt, and Media. WGA was released from the mucin-capped films by adding 200 μL of a 100 mM N-acetyl-D-glucosamine solution to each well. Resistance tests to various chemicals were done on films built in 20 mM Hepes buffer without NaCl. For low-pH treatment, 200 μL of acetate buffer (0.1 M, pH 3) was used. The high-pH treatment consisted of 200 μL of carbonate buffer (0.1 M sodium carbonate and 0.1 M sodium 3402

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bicarbonate, adjusted to pH 9) or KCl/NaOH buffer (0.1 M, pH 12). Of note, the labeled mucin retained its fluorescence intensity after exposure to pH 12 solutions, suggesting that the fluorophore and its linkage to mucin are stable under these conditions (Figure SI 5 of the Supporting Information). To test the resistance to salts, the films were subjected to 200 μL of a 5 M NaCl, 75 mM MgCl2, or 75 mM CaCl2 solution buffered with 20 mM Hepes to pH 7.4. Resistance to cell culture media was followed over 14 days by subjecting the films to 200 μL of DMEM supplemented with 10% fetal bovine serum (Invitrogen), 25 U/mL penicillin, and 25 μg/mL streptomycin (Invitrogen). The medium was exchanged after each measurement. Films were washed three times with 150 μL of 20 mM Hepes buffer (pH 7.4) after treatment. Compositional changes in (Mucin/WGA))12 films were measured by comparing the total fluorescence before and after treatment, relative to the fluorescence of nontreated wells. PLL Incorporation and Release. To investigate the capacity of (Mucin/WGA) films to incorporate positively charged molecules, 40 μL of a 0.5 mg/mL solution of PLL-FITC was deposited on (Mucin/ WGA)12 or (Mucin/WGA)11.5 films built in 0 mM NaCl buffered with 20 mM Hepes at pH 7.4. The films were either untreated or treated with GlcNAc to release WGA. After 30 min, the PLL-FITC solution was removed, and the wells were washed five times with 150 μL of 20 mM Hepes solution (pH 7.4) before fluorescence was quantified. Fluorescence was converted to mass of PLL by calibration in solution. The PLL incorporation relative to the mucin content in the film was calculated based on the mucin mass obtained by fluorescence measurements in separate experiments. (See the (Mucin/WGA) Film Composition section.) PLL was released from the film by adding 150 μL of 20 mM Hepes (pH 7.4) supplemented with either or 0.15 or 5 M NaCl. At each time point, the film’s fluorescence was measured in 150 μL of a 20 mM Hepes solution at pH 7.4. For repeated loading/ unloading, the PLL was released for 30 min using a 5 M NaCl solution. (Mucin/WGA) Film Cytotoxicity. HeLa cells were grown to 70% confluency in T25 flasks with DMEM media supplemented with 10% fetal bovine serum (Invitrogen), 25 U/mL penicillin, and 25 μg/mL streptomycin (Invitrogen). The cells were detached using trypsinEDTA (Invitrogen). The detached cells were washed to remove the trypsin before being plated at a density of 27 000 cells/cm2 on the films constructed in the wells of 96-well plates. The cells were incubated at 37 °C, 5% CO2 under a humidified atmosphere for 1, 3, or 7 days before being stained with the live-dead stain (2 μM calcein and 2 μM Ethidium homodimer-1 in DMED media, Invitrogen) for 20 min. 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. Live (green) and dead (red) cells were counted with the image analysis software ImageJ using the cell count plug-in.

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Figure 1. (Mucin/WGA) film growth. Hydrated thickness obtained by QCM-D measurements, as a function of layer number for (Mucin/ WGA) films built in the presence of 0, 200, or 400 mM NaCl (A). The growth of the films was also followed using fluorescently labeled mucin (B) and WGA (B′). The corresponding masses were calculated based on calibration with mucin and WGA in solution.

Figure 2. (Mucin/WGA) film hydration. The hydrated and dry thicknesses (scale on the left) and the corresponding hydration (scale on the right) for (Mucin/WGA)12 were determined for films built in buffer containing 0, 200, or 400 mM NaCl.

Then, for the 12 subsequent mucin−WGA layer pairs, the film grew almost linearly, suggesting that mucins and WGA can indeed autoassemble into multilayer films. It appears that the lectins are able to cross-link soluble mucins onto already existing mucin layers. To analyze the robustness of assembly of (Mucin/WGA) films in further detail, we performed the assembly reaction at three different ionic strengths: 0, 200, and 400 mM NaCl. Figure 1A shows that the final hydrated thickness of the (Mucin/WGA) films was comparable under the three conditions. For comparison, the growth of films composed of mucins and the positively charged PLL polymer was sensitive to salt (Figure SI 3 of the Supporting Information), resulting in about a two-fold difference in the final thickness with no salt and 200 mM NaCl. High ionic strength can influence the growth of polyelectrolyte-based assemblies as it affects charge shielding and changes the number of charged groups available for ionic paring.44 The relative insensitivity of (Mucin/WGA) film growth toward ambient ionic strength when measured by QCM-D suggests that specific lectin/sugar interactions are driving the film’s assembly by mechanisms that differ from the salt-sensitive ionic pairing occurring in (Mucin/PLL) films. Also of note is that the growth of the (Mucin/WGA) films was

RESULTS AND DISCUSSION

Mucin Can Be Cross-Linked via Lectins to Form (Mucin/WGA) Films. When a dilute solution of mucins is subjected to the substrate of a quartz crystal microbalance with dissipation monitoring (QCM-D), a 40−60 nm thick and hydrated mucin coating forms.11 It is not trivial to increase the coating thickness by depositing further mucin layers, possibly because adsorbed mucins prevent further binding of mucins from solution by steric and electrostatic effects (Figure SI 1 of the Supporting Information).42,43 We tested if this limitation could be overcome by cross-linking individual mucin layers with lectins. In this experiment, the deposition of mucins was alternated with the deposition of the lectin WGA. The QCM-D measurements were modeled to obtain the hydrated thickness of the films as the mucin and lectin layers were deposited. Figure 1A shows that the initial mucin layer decreased in thickness on deposition of the first lectin layer, possibly due to a collapse of the glycan chains protruding from the mucins. 3403

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Figure 3. (Mucin/WGA) film morphology. (Mucin-Alexa488/WGA)12 films observed by epifluorescence microscopy at different magnifications: 10× objective; scale bar is 100 μm (A) and 100× objective; scale bar is 20 μm (B); and by AFM, the image is 10 × 10 μm (C). All observations were made with films in the hydrated state.

properties such as thickness, porosity, diffusion, and functionalities such as lubrication. The hydration of the (Mucin/WGA) films was calculated based on the hydrated mass obtained by QCM-D and the dry mass obtained by ellipsometry. (See the Material and Methods.) Figure 2 shows that ionic strength had no significant effect on the hydrated thickness of the film. However, the dry thickness increased from 26 ± 3 (0 mM NaCl) to 46 ± 11 nm (200 mM NaCl) (Figure 2). This increase may be attributed to the more effective incorporation of WGA at higher ionic strength (Figure 1B′). Combining the changes in hydrated and dry mass implies that the level of hydration decreased from 84 ± 0.6 (0 mM NaCl) to 78 ± 0.6% (400 mM NaCl), suggesting that the films are mildly sensitive to ionic strength. In the three buildup conditions tested here, (Mucin/WGA) films consisted of ∼80% water, which is comparable to previous measurements on mucin- and other biopolymer-based multilayer films.20,45 Although this level of hydration is lower than would be provided by a single mucin coating (estimated at ∼90% water24,47), it appears that the mucin molecules maintain their water binding capacity to a certain degree when arranged as multilayer films. Morphology of (Mucin/WGA) Films. The morphology of the films was characterized both by fluorescence microscopy using fluorescently labeled mucins and by atomic force microscopy (AFM). For this experiment, films were assembled in Hepes buffer without NaCl. At low (10×) magnification, the films appeared relatively homogeneous (Figure 3A). However, at 100× magnification, aggregates in the micrometer range were visualized (Figure 3B). AFM measurements on hydrated films revealed these ∼1 μm aggregates in more detail (Figure 3C). The film’s root mean-square-average roughness was 6.5 (±3.7) nm, which is comparable to the roughness in cross-linked (PLL/hyaluronic acid) multilayer films.48,49

Figure 4. (Mucin/WGA) film resistance to degradation. (MucinAlexa488/WGA)12 and (Mucin/WGA-Alexa488)12 films were tested for their resistance to a range of pH values and ionic strengths. The reported values are the percentage of remaining mucin and WGA inside the films after the indicated treatments. The films remained largely intact under these tested conditions, except at pH 12.

roughly linear. This profile is indicative for relatively strong interactions that generate tightly stacked polymers, resulting in a linear increase in thickness at each deposition step.45,46 To measure the interaction of WGA and mucin with the film independently, the components were labeled with Alexa488, and their incorporation was followed by fluorescence imaging. (See the Materials and Methods.) WGA adsorbed to the film more efficiently at high salt concentrations, whereas no difference in incorporation was measured for mucin at low or high ionic strength (Figure 1B,B′). This experiment also revealed that 10−20% of WGA was released from the film on each mucin deposition step, and conversely, 5−10% of mucins were released on WGA deposition. This may suggest that a certain fraction of newly formed Mucin−WGA complexes dissociated from the surface of the film in each deposition step. (Mucin/WGA) Films Are Highly Hydrated. Hydration corresponds to the fraction of the film’s mass that is composed of water and is an important parameter that relates to

Figure 5. GlcNAc-induced WGA release. (Mucin/WGA)12 films were built in 96-well plates and subjected to three consecutive treatments with 100 mM N-acetyl-D-glucosamine (GlcNAc). The percentage of mucin and WGA remaining in the film in response to each release treatment is plotted (A). The GlcNAc-mediated selective release of WGA results in an enrichment of the film in mucin (B). 3404

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Figure 6. (Mucin/WGA) film loading with PLL. (Mucin/WGA)12 films with a top layer of mucin or WGA were built in wells of 96-well plates. PLL incorporation is depicted as the mass ratio of PLL to mucin in the film. GlcNAc-induced WGA release increased the relative content of PLL loaded in the film (A). The release profile of PLL from GlcNAc-treated films when placed in a solution containing NaCl was affected by the salt concentration (B). PLL could be released from a GlcNAc-treated film with 5 M NaCl and reloaded over several cycles (C).

mM MgCl2 or CaCl2. Indeed, already established films remained largely intact even under extreme conditions, since neither the mucin nor WGA content decreased by more than 20% when exposed to pH 3 and 9 or 5 M NaCl. The films were also resistant toward divalent salts at a supraphysiological concentration of 75 mM. Only a pH of 12 dismantled the structure of the film (Figure 4). By fluorescence microscopy, no changes could be observed after the permissive treatments (Figure SI 4 of the Supporting Information). These results suggest that (Mucin/WGA) films are more robust than mucin multilayer films that are based on electrostatic interactions. For example (Mucin/Chitosan) tend to dismantle when exposed to a different pH or ionic strength than used for the buildup.20 Several other polyelectrolyte multilayer systems and, in particular, synthetic polymer systems such as poly(allylamine hydrochloride)/poly(acrylic acid) films, can resist pH changes within the range tested here.50,51 However, resistance to 5 M NaCl is unusual. This effect may be brought about by the relatively high concentrations of WGA and WGA-binding sites within the film or the specific nature of hydrogen bonding that largely contributes to the WGA− carbohydrate interactions.52 Controlled Release of WGA from (Mucin/WGA) Films by N-Acetyl-D-Glucosamine. Lectins have successfully been used to modulate the delivery and targeting of certain drugs.53,54 Hence, if it is possible to release the mucin-bound lectins from the films, for example, by introducing small molecules that specifically compete for lectin binding, then this system may provide an interesting carrier for lectin-coupled drugs. To test this possibility, we exposed (Mucin/WGA)12 films to N-acetyl-D-glucosamine, a known ligand for WGA. The films were treated three consecutive times with a 100 mM GlcNAc solution at pH 7.4 for 30 min. Figure 5A shows that the WGA content of the film decreased by ∼80% after GlcNAc treatment. For comparison, the amount of mucin dropped by only 20% under the same conditions, suggesting that mucin remained largely associated with the films even after a

Figure 7. (Mucin/WGA) film cytotoxicity. The release in the culture media of both mucin and WGA from a (Mucin/WGA)12 film not treated with GlcNAc (A). The viability of HeLa cells cultured on (Mucin/WGA)12 films not treated with GlcNAc, as tested by LiveDead stain (B). Phase images (10×) of HeLa cells on the films after 1, 3, or 7 days (C, C′, and C″) and on plastic after 1 day (C‴).

(Mucin/WGA) Films Are Exceptionally Resistant to High Salt Concentrations. Because the hydrated thickness of mucin multilayer films is independent of the ionic strength used for assembly (Figure 2), it is also possible that established films remain resistant to high ionic strength and possibly extreme pH conditions. This feature would be desirable because it may significantly prolong the lifetime of the film if put in contact with harsh biological fluids, such as the gastric acid of the stomach. (Mucin/WGA)12 films were built in 0 mM NaCl at pH 7.4 and then subjected to pH 3, 9, or 12 or 5 M NaCl, 75 3405

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Figure 8. (Mucin/WGA) films as a multilfuntional delivery vehicle. Mucins (protein core in black and attached glycans in blue) interact with lectins (orange cross) to form nanothick mutlilayer films. The loading and release of molecules of interest inside the (Mucin/WGA) films is possible through multiple strategies. The cargo can electrostatically interact with the mucin molecules and be released with salt. This does not alter the film’s structure, and hence loading−unloading cycles can be repeated. In addition, the specific release of lectins from the films can be induced by soluble sugars. If drugs are conjugated to the lectins, then the films could act as a sugar-triggered drug delivery system.

intact even at 5 M NaCl, the same structure can be discharged and recharged with PLL at least four times with no loss of PLL binding capacity (Figure 6C). While we studied the loading of mucin multilayer films with the model cargo PLL, we note that the mucus barrier accommodates a range of bioactive molecules such as growth factors and antimicrobial peptides in vivo.56 Hence, one might envision mucin-based coatings with complexed bioactive molecules for biomedical applications such as drug delivery, wound healing, and antimicrobial surfaces. In addition, sialic acid is recognized as a ligand with high biological significance57 because it can interact with a range of different viruses and bacteria. We foresee that mucin-multilayers may be used to detect or inactivate such biological functional entities. (Mucin/WGA) Films Are Noncytotoxic. In the context of biomedical applications or as a model substrate to study cell− mucin interactions, mucin multilayer films would be placed in contact with cells and must thus prove to be noncytotoxic. Previous studies report a certain toxicity of lectins toward cancer cell lines, for example.58,59 Components of multilayers can potentially be released during cell culture procedures and influence cell behavior.60 We found that over 14 days, (Mucin/ WGA)12 films released ∼20% of the lectin and less than ∼5% of mucin (Figure 7A). Next, we directly tested if the (Mucin/ WGA)12 films and the released film components were cytotoxic toward epithelial HeLa cells. Our data revealed that HeLa cells grown on (Mucin/WGA)12 films for 1, 3, and 7 days remained almost as viable as those on standard polystyrene surfaces as judged by a fluorescence-based live-dead assay (Figure 7B). While the cells adhered to the films as depicted in Figure 7C,C′,C″, they were less spread than on standard tissue culture surface (Figure 7C‴). Lectins represent an integral part of our diet, and one may thus argue that these molecules should be, to some degree, biocompatible. Nevertheless, few studies directly test the toxicity of recombinant or purified lectins in vivo. Our system consisting of HeLa cells clearly is a simplification and cannot be extrapolated to live organisms; however, it may provide an approximation to mucin-associated lectin behavior in context of living cells. WGA appears to be cytotoxic when uptaken by the cell,58 hence its relatively stable sequestration to the mucins may limit toxicity by reducing its ability to penetrate into cells.

substantial fraction of WGA had been released. One direct consequence of WGA dissociation from the film is a relative enrichment of the film in mucin, increasing the Mucin/WGA ratio from 2 to nearly 6 after three GlcNAc treatments (Figure 5B). A competitor-induced release of lectins has been demonstrated on several occasions in 3D hydrogels and multilayer films, mostly in the context of autoregulated insulin delivery systems.55 The related characteristics of (Mucin/WGA) films bear potential for the design of novel sugar-specific inducible delivery systems, in particular if combined with the film’s resilience toward a wide range of ionic strengths and pH values. (Mucin/WGA) Films Can Repeatedly Incorporate and Release the Positively Charged Polymer PLL. The potential to release mucin-bound WGA is of interest in the context of drug delivery, where WGA-tethered molecules could be specifically dissociated in the presence of sugars. However, secreted mucins are acidic proteins that contain abundant negative charge at neutral pH. Accordingly, they may interact with positively charged molecules such as polycationic polymers, growth factors, antimicrobial peptides, and certain small drugs. To test if (Mucin/WGA) films can be loaded with positively charged molecules, we analyzed their ability to incorporate PLL. Figure 6A depicts that mucin multilayers can indeed incorporate PLL. However, the ratio of mucin to PLL was lower than that on simple mucin coatings, possibly due to the occupation of binding sites on the mucins by WGA and the presence of repulsive positive charges introduced by WGA (Ip = 9). The release of WGA should uncover charged groups like sialic acid, which could serve as further binding sites for positively charged molecules. Therefore, the controlled release of WGA from the mucins could potentially be used to adjust the binding capacity for positively charged molecules within the film. Indeed, the PLL-to-mucin ratio increased slightly after partial release of WGA (Figure 6A). If PLL adsorbs to mucins based on electrostatic interactions, then it should be possible to release it with salt. Figure 6B shows the release of PLL over time at relatively low (0.15 M NaCl) or high ionic strength (5 M NaCl). At 0.15 M NaCl, a smaller fraction of the loaded PLL was released than at 5 M NaCl, suggesting that the extent, and to some degree the rate, of the release can be tuned by varying the salt concentration. One further feature of the mucin-multilayer films that can be exploited for drug delivery applications is their resistance to high ionic strength: because the mucin multilayer films remains

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CONCLUSIONS We here report a new type of biopolymer-based multilayer film made from mucins and the lectin WGA. These films are 3406

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resistant to extreme NaCl and divalent ion concentrations as well as a broad range of pH values. In addition, they can undergo multiple rounds of loading and release of a model substrate, PLL, without disintegrating (Figure 8). Because of its unique properties, this system may be used to generate mucinbased materials for a range of biomedical applications. For example, the high stability could enable the generation of permanent drug-eluting coatings for materials that are otherwise poorly biocompatible and which should remain coated after delivery of the drug cargo. The uptake of certain drugs by the body can be improved by coupling them to lectins.53,54 Hence, mucin multilayers in which lectins can be released in a controlled way may be suitable vectors for such lectin-bound drugs (Figure 8). Last, the creation of thin films with mucins could be a starting point for exploiting the native ability of the biomolecules to trap specific pathogens, such as certain viruses. Mucin multilayers may be used to deplete such viruses from, for example, blood, and potentially be the starting point for diagnostic tools.

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ASSOCIATED CONTENT

S Supporting Information *

QCM-D data showing five experiments: (1) that mucins cannot spontaneously form multilayer films in the conditions used; (2) that the (Mucin/WGA) films grow on both gold and polystyrene covered surfaces, (3) the growth curves of the electrostatically paired (Mucin/PLL) films, fluorescence microscopy images of (Mucin/WGA) films after treatment with salts or sugar (4); we also show a control experiment verifying the stability of the Alea488 dye and its linkage to mucin when exposed to pH 12 solutions (5). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +617-715-4575. Fax: 617-3247554. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Johnson & Johnson Corporate Office of Science and Technology for the postdoctoral fellowship provided to T.C, and MIT startup funds to K.R. This work made use of the MRSEC Shared Experiment Facilities supported by the National Science Foundation under award number DMR − 819762. We are also grateful to Ronn Friedlander for his assistance with the AFM experiments.

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REFERENCES

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