Lectin Multilayers and Their

Jun 25, 2014 - School of Pharmaceutical Sciences, University of Sao Paulo, USP, Sao Paulo, SP 05508-000, Brazil. ‡. Department of Biological Engineeri...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/Biomac

Sugar-Mediated Disassembly of Mucin/Lectin Multilayers and Their Use as pH-Tolerant, On-Demand Sacrificial Layers Roberta Polak,†,# Thomas Crouzier,‡,# Rosanna M. Lim,§,# Katharina Ribbeck,‡ Marisa M. Beppu,∥ Ronaldo N. M. Pitombo,† Robert E. Cohen,§ and Michael F. Rubner*,⊥ †

School of Pharmaceutical Sciences, University of Sao Paulo, USP, Sao Paulo, SP 05508-000, Brazil Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States § Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ∥ School of Chemical Engineering, University of Campinas, UNICAMP, Campinas, SP 13083-852, Brazil ⊥ Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ‡

S Supporting Information *

ABSTRACT: The layer-by-layer (LbL) assembly of thin films on surfaces has proven to be an extremely useful technology for uses ranging from optics to biomedical applications. Releasing these films from the substrate to generate so-called free-standing multilayer films opens a new set of applications. Current approaches to generating such materials are limited because they can be cytotoxic, difficult to scale up, or have undesirable side reactions on the material. In this work, a new sacrificial thin film system capable of chemically triggered dissolution at physiological pH of 7.4 is described. The film was created through LbL assembly of bovine submaxillary mucin (BSM) and the lectin jacalin (JAC) for a (BSM/JAC) multilayer system, which remains stable over a wide pH range (pH 3−9) and at high ionic strength (up to 5 M NaCl). This stability allows for subsequent LbL assembly of additional films in a variety of conditions, which could be released from the substrate by incubation in the presence of a competitive inhibitor sugar, melibiose, which selectively disassembles the (BSM/ JAC) section of the film. This novel multilayer system was then applied to generate free-standing, 7 μm diameter, circular ultrathin films, which can be attached to a cell surface as a “backpack”. A critical thickness of about 100 nm for the (BSM/JAC) film was required to release the backpacks from the glass substrate, after incubation in melibiose solution at 37 °C for 1 h. Upon their release, backpacks were subsequently attached to murine monocytes without cytotoxicity, thereby demonstrating the compatibility of this mucin-based release system with living cells.

1. INTRODUCTION The generation of thin films that can be disassembled using a specific triggering mechanism has been widely investigated and is now applied to many useful purposes. In addition, the versatility of the layer-by-layer (LbL) assembly methodology has opened a wide range of triggering mechanisms and applications for such sacrificial films. LbL films have been designed to trigger the release of drugs, small molecules, or other thin films, which has proven to be useful for controlled drug delivery and biomimetic materials applications.1−6 In stratified film structures, a bottom sacrificial multilayer film can be selectively dissolved, thereby releasing the more stable parts of the film from the substrate.1,7−17 These so-called freestanding films have found uses in a wide range of applications, from optics to biomedical purposes.18−21 Hydrogen-bonded LbL multilayer systems have been extensively used as sacrificial layers1 since they are typically stable at low pH and dissolve at higher pH. One system, composed of poly(methacrylic) acid and poly(vinylpyrrolidone), relies on hydrogen bonds formed © 2014 American Chemical Society

between carboxylic acids and carbonyls that can be disrupted above pH ∼ 5.6.4,5 An alternative hydrogen bonding system based on poly(methacrylic acid) and poly(N-isopropylacrylamide) dissolves at neutral pH.4 However, mammalian cell cultures and the loading of therapeutic molecules such as proteins will often not sustain the acidic pH required to maintain stability of hydrogen-bonding release layers, thereby limiting their biomedical applicability. Alternative triggering mechanisms that do not depend on pH change have also been reported. Some examples include the use of irradiation (lighttriggered release),22 which can damage DNA; the use of reducing agents on disulfide bonds,23 which can affect the folding of proteins; the use of solvents,24 which, in some instances, can be toxic as well as destructive to cells and proteins; the use of water-soluble polymers,25 which limits their Received: May 13, 2014 Revised: June 18, 2014 Published: June 25, 2014 3093

dx.doi.org/10.1021/bm5006905 | Biomacromolecules 2014, 15, 3093−3098

Biomacromolecules

Article

release when exposed to melibiose. We also demonstrate that backpacks using the (BSM/JAC) release region can be associated with cells and be released in the presence of melibiose, without any sign of cytotoxicity. This new release system thus proved useful in the cell backpack application, broadening the range of polyelectrolyte assemblies possible on top of the sacrificial region and widening the range of drugs and proteins that can be incorporated into the payload regions of the cell backpack.

downstream application in aqueous environments; and the use amphoteric copolymer films,17 which selectively dissolves in acidic or in neutral/basic solutions, and narrows their use to a limited pH range. Enzymes are also capable of disassembling some films or structures.26−29 However, the poor diffusion of the enzyme through the multilayer film to reach the substrate−film interface would limit its usefulness and may introduce a protein to other parts of the film.26−29 Thus, unintended enzymatic degradation, in addition to the particular pH, ionic strength, and divalent ions necessary for enzymatic activity, could destabilize the films deposited on top of it. Because of the limitations of other approaches, the development of a sacrificial film system that is biocompatible, stable in a wide pH range, but can be specifically triggered with minimal effects on other aspects of the system is of great importance. We then hypothesized that an alternative to hydrogenbonded sacrificial films could come from the assembly of mucin and lectin proteins. Mucins are a family of densely Oglycosylated glycoproteins that are secreted by higher organisms to both lubricate and protect the epithelial cell surfaces from biological, chemical, and mechanical challenges.30 Since they are negatively charged, they can be paired with positively charged polymers for LbL assembly.31−35 Mucins also present sugars, including N-acetyl-D-glucosamine, N-acetyl-Dgalactosamine (GalNAc), and sialic acid residues, which function as ligands for various carbohydrate-binding proteins called lectins.36−38 Thus, as recently demonstrated by the Ribbeck group, mucin/lectin multilayers can be generated on the basis of such interactions, independent of electrostatic pairing of polyelectrolytes.39 This combination resulted in films that were stable in low and high pH as well as high ionic strength. In the presence of a competitive sugar, a portion of the lectins could be released from the film; however, the film remained intact. To overcome this limitation, we developed a new mucin/lectin film that completely disassembles upon exposure to a competitive inhibitor, while remaining stable in low and high pH and high ionic strengths in its absence. We use bovine submaxillary mucin (BSM) and the lectin jacalin (JAC), a tetrameric protein able to bind the GalNAc moieties40 present in abundance in the BSM sugar chains. We show that they can be successfully assembled into multilayer films using LbL assembly. We also explore the robustness of (BSM/JAC) films in a range of aqueous conditions, including a wide pH range as well as high ionic strength and show that they can be disassembled only in a solution of melibiose sugar. We demonstrate that the films can be employed as sacrificial release layers and determine their biocompatibility in a specific application: the creation of cell backpacks. Cell backpacks are 7−10 μm diameter polymer patches capable of attaching to cells.4 They have the ability to encapsulate and to release drugs while successfully avoiding phagocytic internalization, remaining on the cell surface with minimal reduction in cell viability.6 The fabrication of these cell backpacks involves polymer film deposition onto a photopatterned array to create a stratified LbL multilayer system containing three regions: a release region, a drug payload region, and a cell adhesive region.4 The development of cell backpacks has been hampered by the limitations of the hydrogen-bonded release region, which cannot support the further assembly of drug-loading and cell adhesive regions at neutral and basic pH. We show that the (BSM/JAC) films can support the further assembly of films at high pH and allow their

2. EXPERIMENTAL SECTION 2.1. Materials. BSM, mucin from bovine submaxillary glands (Sigma or Calbiochem); JAC, unconjugated jacalin (Vector Laboratories); Melibiose, MW = 342.30 g mol−1 (Sigma-Aldrich); PAH, poly(allylamine hydrochloride), MW = 56 000 g mol−1 (SigmaAldrich) or MW = 120−200 g mol−1 (PolySciences, for the magnetic region); SPS, poly(sodium 4-styrenesulfonate), MW = 70 000 g mol−1 (Sigma-Aldrich). MNP, magnetic nanoparticles, EMG 705 from FerroTec, 10 nm in diameter; NeutrAvidin Dylight 633 (Thermo Scientific); NHS-Biotin, (+)-biotin N-hydroxysuccinimide ester (Sigma-Aldrich); antibody, normal mouse IgG biotin (IgG-b) (Santa Cruz Biotechnologies). All materials were used as received. 2.2. LbL Assembly. Each multilayer system is denoted using the following notation: (Poly1X/Poly2Y)n. Poly1 and Poly2 refer to the abbreviation of the polymer or molecule in the assembly process, X and Y refer to the pH of the assembly solution during LbL deposition, and n refers to the number of bilayers that have been deposited. (BSM/JAC)n films were prepared using an automated Zeiss programmable slide stainer. BSM was prepared at 1.0 mg mL−1 and JAC at 0.1 mg mL−1, dissolved in 20 mM HEPES buffer, 300 mM NaCl, pH 7.4. Dipping times: BSM, 10 min followed by two rinses of 1 min each; JAC, 5 min followed by two rinses of 1 min each. After assembly, films were rinsed with DI water and blow dried with compressed nitrogen. The (PAH3/MNP4)10.5 films were prepared as previously described,4 as well as (SPS9.3/PAH9.3)5 films.41 However, in this (SPS9.3/PAH9.3)5 system, 100 mM of NaCl were added to the polymer solutions and 5 mM of NaCl were added to the water rinses to ensure film stability. 2.3. Backpack Fabrication. Films on photopatterned glass slides were prepared using a previous methodology developed by our group.4 (See Supporting Information for more details.) The LbL films were deposited onto the patterned slide in the following order: (a) release region, (BSM7.4/JAC7.4)n; (b) magnetic payload region, (PAH3/ MNP4)10.5; (c) cell attachment region, (SPS9.3/PAH9.3)5. The cell attachment region was only included for the cell compatibility experiments. After deposition, the photoresist was removed by sonication in acetone (20 s) followed by a deionized water rinse. All other chemistry performed on the cell attachment region was done after photoresist removal (see Supporting Information). 2.4. Thickness Determination. Film thicknesses were measured using a surface profilometer (Veeco Dektak 150). 2.5. Mucin/Lectin Film Stability. 2.5.1. Stability Test. Fluorescently labeled (BSM−Alexa488/JAC−FITC)10 films were assembled in a 96-well plate with glass bottom. The films were built with either fluorescently labeled BSM or fluorescently labeled JAC. For each adsorption step, 40 μL of protein was deposited, then the mixture was washed twice with 200 μL of buffer solution each. The films were then washed with ultrapure water adjusted to pH 7 before exposure to the buffered solutions adjusted to various pH using NaOH or HCl (pH 3, 5, 7, 8, 9), to high ionic strength (5 M NaCl, pH 7), to phosphate buffer (PBS), to cell culture media containing 10% serum (DMEM), or to melibiose sugar solution in PBS (50, 100, 200 mM, pH 7) for 1 h at 37 °C. The buffered solutions used to test the stability of the sacrificial layer in different pH environments were the following: 20 mM MES buffer for pH 3 and 5 solutions; 20 mM HEPES buffer for pH 7 and 8 solutions; and 20 mM sodium bicarbonate buffer for pH 9 solutions. After exposure, the films were washed with the buildup solution (300 mM NaCl, 20 mM HEPES, pH 7.4), and the 3094

dx.doi.org/10.1021/bm5006905 | Biomacromolecules 2014, 15, 3093−3098

Biomacromolecules

Article

fluorescence was measured using a fluorescence plate reader (SpectraMax). 2.5.2. Stability of Backpacks for the Cell Work. During deposition of the (SPS/PAH)5 top layers, we found that the backpacks would tend to dissolve in the pH 9.3 rinsewater, and the addition of 5 mM of NaCl to the pH 9.3 rinse solutions ensured film stability. Thus, a small amount of salt may be necessary to promote JAC binding to BSM at high pH. This is supported by a preliminary experiment showing the salt-dependency of JAC binding to BSM (Supporting Information Figure S6). 2.6. Release of Backpacks. The BSM/JAC film was used as a sacrificial multilayer for the release of cell backpacks. The backpacks used for the release experiments consisted of the BSM/JAC release region topped with the magnetic nanoparticle payload region. Glass slides with backpacks were incubated with 100 mM of melibiose in PBS for 1 h at 37 °C. After incubation, the slides were rinsed with deionized water to remove backpacks that had been released from the substrate. Multiple phase contrast microscope images were taken at various points on the substrate to determine the number of backpacks remaining on the slide. The percentage of release for each sample was calculated by averaging the fraction of released backpacks for all images. 2.7. Viability Assay. The WEHI 265.1 mouse monocyte cell line (American Type Culture Collection) was used for the cell experiments, since monocytes circulate in the bloodstream and can be used for targeted drug delivery via cell backpacks. The cells were rinsed twice in HBSS buffer (Hank’s Balanced Salt Solution, Gibco) and resuspended into HBSS buffer at 3 million cells mL−1 before introduction to the multilayer patches. Cells settled onto the surface of backpacks for 30 min at 37 °C and 5% CO2 to form a cell monolayer. The glass slide was then rinsed twice in HBSS buffer to remove non-adherent cells. The release solution was then added to the surface to cover the top of the glass slide (the control release solution consisted of PBS without melibiose). In addition to 100 mM melibiose, the release solution also contained 4 μM calcein AM and 8 μM ethidium homodimer for the live/dead assay (Molecular Probes). The cells were incubated with the backpacks in the release solution for 1 h at 37 °C and 5% CO2. An incubation of 1 h was chosen because it is the amount of time necessary for cell attachment. The cells were immediately imaged after incubation with the release solution. Confocal laser scanning microscope images for the live/dead assay were obtained on an inverted Zeiss LSM 510 at 10×, so that more cells can be sampled in each field of view. Excitation wavelengths of 488 and 543 nm were used to image cells stained with calcein and ethidium homodimer, respectively. The live and dead cells in each image were counted using CellProfiler software. The percent viability, defined as the number of live cells over the number of total cells, was calculated for each image and averaged over 10 images to get the final values for incubation of cells with and without melibiose. Fluorescent images of the cell backpacks were obtained using an excitation wavelength of 633 nm, to excite the Dylight 633 conjugated NeutrAvidin.

Figure 1. Growth curve of (BSM/JAC)n films on glass microscope slides. Thicknesses were measured by profilometry (n = 6).

and 60 bilayer (BL) films and 12 ± 3 nm for 80 BL, as measured by profilometry. The linear growth behavior and range of roughness values measured for the (BSM/JAC) film are characteristics similar to another mucin/lectin multilayer system composed of pig gastric mucin and wheat germ agglutinin, which was first explored by Crouzier et al.39 3.2. (BSM/JAC) Films Withstand pH and Ionic Strength Variations and Are Destabilized by Melibiose. Since the (BSM/JAC) film is based on specific and multivalent interactions between the lectin and sugar residues,36−38 we hypothesized that this architecture would be stable enough to allow the deposition of additional layers from solutions with a wide range of pH and ionic strengths. To demonstrate these features, we studied the stability of (BSM/JAC)10 films under a variety of pH and ionic strengths by monitoring the amount of fluorescently labeled BSM−Alexa488 and JAC−FITC before and after treatment (Figure 2). Since most applications of sacrificial films require rapid dissolution of the films, we limited the treatment time to 1 h.

3. RESULTS AND DISCUSSION 3.1. (BSM/JAC)n Films Grow Linearly. To characterize the growth behavior of the system, (BSM/JAC)n films were assembled onto nonpatterned glass slides, where the subscript n refers to the number of bilayers deposited. Figure 1 depicts the growth curve for (BSM/JAC)n films through 80 bilayers as assembled from 1.0 mg mL−1 of BSM and 0.1 mg mL−1 of jacalin in 300 mM of NaCl, in 20 mM HEPES buffer at pH 7.4. A preliminary investigation of the assembly parameters and film growth that lead to the adoption of these conditions can be found in Supporting Information (Table S1). The growth curve has a linear trend, owing to the strong mucin/lectin interactions that suppress diffusion of the components in the alreadyassembled regions of the growing film. The films were optically clear and smooth with a roughness of 4−6 nm for the 20, 40,

Figure 2. Stability of releasable films in different aqueous environments. Fluorescently labeled (BSM−Alexa488/JAC−FITC)10 films assembled in a 96-well glass plate were monitored for film stability after incubation for 1 h in different buffered solutions (n = 3).

A small fraction of lectin and mucin is released when the films are exposed to solutions different from the assembly solution, probably due to the disruption of electrostatic interactions between some components of the film. The (BSM/JAC)10 film was stable in PBS, serum-containing DMEM, a broad pH range (from pH 3 to pH 9), and high ionic strength (up to 5 M NaCl, pH 7). While the high density 3095

dx.doi.org/10.1021/bm5006905 | Biomacromolecules 2014, 15, 3093−3098

Biomacromolecules

Article

of multivalent specific interactions between the lectin and mucin sugar residues offers good stability to the films when challenged with a variety of aqueous environments, exposure to solutions of melibiose causes significant dissolution of the multilayer film (Figure 2). This sugar functions as a competitive inhibitor for the jacalin lectin, destabilizing the intermolecular interactions between BSM and JAC. The remaining fluorescence measured after melibiose treatment is likely due to residual BSM or JAC that has adsorbed to the substrate and could not be removed by buffer washes of the well. The combination of high pH and ionic strength stability and the ability to trigger its dissolution by exposure to melibiose make (BSM/JAC) films ideal candidates as sacrificial films. As such, (BSM/JAC) would have the potential to unlock a wide diversity of potential applications previously inaccessible with other releasable films. 3.3. (BSM/JAC)60 Can Release (PAH/MNP)10.5 Backpacks. Having established the extended range stability in various aqueous environments and triggerable dissolution behavior of (BSM/JAC) films, we then explored their use as a sacrificial multilayer for the fabrication of cell backpacks. Cell backpack conjugates are biohybrid materials consisting of a polymer patch, hundreds of nanometers thick and microns wide, that can attach to a cell surface.4−6 Adapting a previous methodology developed by our group4 (see Experimental Section for details), we built a photopatterned array of patches of (BSM/JAC)n topped with a magnetic multilayer stack of (poly(allylamine hydrochloride)/magnetic nanoparticles)10.5, or (PAH/MNP)10.5, as shown in Figure 3a. (PAH/MNP)10.5 multilayers are used in the cell backpack system to provide magnetic manipulation and to create mechanically robust patches5 that do not fold once suspended in solution (Figure S1). The final backpacks had a mean diameter of 7−10 μm with a 15 μm edge-to-edge spacing on the glass substrate. The measured thicknesses of the backpacks topped with (PAH/ MNP)10.5 are reported in Figure 3b, showing that the backpack film thickness reaches a saturation point at about 60 BL. The same saturation point is also observed when (BSM/JAC)n films are topped with (PAH/MNP)10.5 layers on a nonpatterned glass slide (Figure S4). As will be discussed later, diffusion of PAH into the (BSM/JAC) region is likely possible and could alter the growth regime of the thin films, limiting backpack film growth beyond 60 bilayers. This diffusion-related saturation in growth has an effect on the melibiose-induced release of the cell backpacks. As shown in Figure 3c, a critical number of bilayers is necessary for maximal yield of released backpacks. Upon incubation in 100 mM melibiose, the percentage of backpacks released increases as the number of bilayers increases and levels off after 60 BL, consistent with the trend in backpack film growth. In fact, the releasable system is already robust using 40 BL of (BSM/JAC), since more than 90% of the backpacks release from the substrate. Figure 3d shows representative images of backpacks remaining on the glass slide after the release step. Of note, the backpacks did not release when exposed to solutions free of melibiose at various pH and ionic strength conditions (Figure S2). Two other assembly conditions for (BSM/JAC)n films were tested by varying the mucin and/or salt concentrations (0.4 mg/mL of BSM, 300 mM NaCl, 120 BL; and 0.2 mg/mL BSM, 150 mM NaCl, 180 BL), as seen in Table S2 and Figure S3. The growth rate was slower for these assembly conditions, but release was observed when increasing the number of bilayers to

Figure 3. (a) Scheme of the cell backpack composition for the release tests. (b) Growth curve of final backpack patches topped with (PAH/ MNP)10.5. (c) Percentage of released backpacks and (d) corresponding phase contrast microscope images of slides after incubation in melibiose 100 mM for 1 h at 37 °C and subsequent removal of detached backpacks. Backpack composition: (BSM/JAC)n and (PAH/ MNP)10.5, where n = 20, 40, 60, or 80 BL. All scale bars are 200 μm. Backpacks were 7−10 μm diameter and 15 μm edge-to-edge spacing on the substrate.

reach the same thickness as the 60 BL film shown in Figure 3. These results emphasize the role of film thickness as the critical parameter for optimal release of the backpacks. Critical thicknesses for sacrificial layers have also been previously reported in the literature.1,4 Here, we hypothesize that some of the PAH is diffusing into or through the (BSM/JAC) layer and stabilizing it via electrostatic interactions. BSM is negatively charged at most pH’s, and JAC is slightly negatively charged at neutral pH. Thus, the positively charged PAH polymer diffusing into the release region cross-links the BSM and JAC. Thicker (BSM/JAC) layers provide a region at the base of the film that is unmodified by diffusing PAH, leaving it susceptible to melibiose attack and release. Such interdiffusion could explain the saturation point in the backpack growth curves and the mismatch between thicknesses of individual (BSM/JAC) films (69 nm for 20 BL), (PAH/MNP) films (110 nm for 10.5 BL), and the final backpack system (109 nm for 20 BL (BSM/JAC) region) (Figure 3b). In support of this hypothesis, backpack release did not occur when using a lower molecular weight PAH polymer (56 kDa as opposed to the 120−200 kDa PAH used in the main set of experiments), (data not shown). The lack of backpack release may originate from the increased diffusion of PAH through the film, since low molecular weight polymers tend to diffuse more quickly 3096

dx.doi.org/10.1021/bm5006905 | Biomacromolecules 2014, 15, 3093−3098

Biomacromolecules

Article

Figure 4. (a) Scheme showing the final backpack composition used for cell attachment experiments. (b) Cell viability of murine monocytes after 1 h of incubation in PBS buffer (control) or a 100 mM melibiose solution in PBS. The data is reported as the average (n = 10) ± SD. (c) Monocytes (dyed with a fluorescently green live cell stain) docked onto backpacks (pink) after 1 h of incubation in PBS. (d) Monocytes (green) attached to released backpacks (pink) floating in solution after 1 h of incubation in 100 mM melibiose. Scale bars are 10 μm.

through multilayers than higher molecular weight polymers.42,43 Reproducibility of the releasable backpacks was tested with films assembled using BSM from another provider (Calbiochem), to account for variations between mucin sources. A solution of 100 mM melibiose in PBS triggered release for an 80 BL film from this BSM provider, demonstrating that this methodology is applicable to various mucin sources (Figure S5). 3.4. The (BSM/JAC) Release Region Is Compatible with Monocyte Attachment to Backpacks. We next assembled a cell attachment region on the top of the magnetic multilayer region (Figure 4a). This region could not have been built with typical pH sensitive hydrogen-bonded sacrificial films, as it is composed of poly(styrenesulfonate)/poly(allylamine hydrochloride) (SPS/PAH) deposited at pH 9.3, or (SPS 9.3/ PAH 9.3)5. First, this allowed us to test if the (BSM/JAC) release region would sustain the addition of a film assembled at high pH. Second, by attaching cells to this region and exposing the cells/backpacks to the melibiose release solution, we also determined the biocompatibility of the (BSM/JAC) sacrificial release system. IgG antibodies were grafted to the backpack through a biotin/NeutrAvidin bridge utilizing the reactive free amine groups of the SPS/PAH system.41 The IgG antibodies in the backpack are able to bind to Fc receptors on cell surfaces, thus providing the system with cell-attachment capability. The use of antibodies for cell attachment adds versatility to the backpack, since the different antibodies can be used to specifically attach to different types of cells. IgG antibodies work well for the WEHI 265.1 mouse monocytes used in this study. The complete backpack system (Figure 4a) assembled well, although it should be noted that a low concentrations of salt were added to the assembly rinse solutions in this case to promote better stability at pH 9.3 (see experimental section and Figure S6). Once assembled, the backpacks were incubated with murine monocytes in the melibiose release solution, and cell viability was determined by a cell membrane integrity viability assay. For the cell attachment experiments, the monocytes were incubated with the backpacks in a pH 7.4 HBSS buffer, since this buffer is devoid of competing serum proteins that could interfere with backpack-cell attachment. To induce cellbackpack release, melibiose sugar dissolved in PBS buffer was

added to the glass slide. PBS buffer alone was used as a control for the release trigger solution. As shown in Figure 4b, the viability of murine monocytes exposed to melibiose dissolved in PBS at pH 7.4 (87.9 ± 6.8%) is comparable to that of cells incubated in PBS pH 7.4 alone (89.1 ± 5.3%). This shows that the high sugar concentration does not adversely affect cell viability, at least after 1 h of incubation. Cell viability and function over the course of a few days after exposure to melibiose is currently under investigation. Backpacks attached to cells remained well aligned in their original lattice when exposed to PBS at pH 7.4 (Figure 4c). The green fluorescence of the cells indicates that the cells maintained their membrane integrity while they are interacting with the backpacks. About 90% of the surface-anchored backpacks had cells docked onto them (Figure S7). More optimization would be required to achieve a perfect lattice of cells docked on backpacks, since many cells resided in the interstitial regions between the backpacks. During incubation with melibiose, the backpacks attached to cells were successfully released from the substrate, showing that the (BSM/JAC) release region remained functional after the addition of the cell-attachment region (Figure 4d). Again, the green fluorescence of the cells indicates that that the integrity of the membrane is preserved after conjugation to the backpack and release from the substrate.

4. CONCLUSIONS In conclusion, we developed a new mucin/lectin film that can be used as a robust and versatile sacrificial multilayer system for triggered release of polymer thin films. We show that (BSM/ JAC) films assembled by lectin−sugar interactions can be selectively disassembled in the presence of the competitive inhibitor sugar, melibiose. This feature allows for additional LbL assembly at a wide range of pH (pH 3−9) and ionic strength (up to 5 M NaCl). Because of these unique properties, these films widen the range of drugs and proteins that can be integrated into releasable polymer multilayer systems such as cell backpacks. The biocompatibility of the release trigger enables work with sensitive cells, which will open new biomedical applications for releasable multilayer films. Such films could also find applications in other areas that require sacrificial films such as the generation of free-standing cell sheets. 3097

dx.doi.org/10.1021/bm5006905 | Biomacromolecules 2014, 15, 3093−3098

Biomacromolecules

■

Article

(19) Jiang, C. Y.; Markutsya, S.; Tsukruk, V. V. Adv. Mater. 2004, 16, 157−+. (20) Larkin, A. L.; Davis, R. M.; Rajagopalan, P. Biomacromolecules 2010, 11, 2788−2796. (21) Lynn, D. M. Adv. Mater. 2007, 19, 4118−4130. (22) Pennakalathil, J.; Hong, J. D. ACS Nano 2011, 5, 9232−9237. (23) Chassepot, A.; Gao, L. C.; Nguyen, I.; Dochter, A.; Fioretti, F.; Menu, P.; Kerdjoudj, H.; Baehr, C.; Schaaf, P.; Voegel, J. C.; Boulmedais, F.; Frisch, B.; Ogiert, J. Chem. Mater. 2012, 24, 930−937. (24) Greco, F.; Zucca, A.; Taccola, S.; Menciassi, A.; Dario, P.; Mattoli, V. Sacrificial Layer and Supporting Layer Techniques for the Fabrication of Ultra-Thin Free-Standing PEDOT:PSS Nanosheets. In Multifunctional Polymer-Based Materials; Lendlein, A., Behl, M., Feng, Y., Guan, Z., Xie, T., Eds.; Cambridge University Press: Cambridge, NY, 2012; Vol. 1403, pp 253−258. (25) Riva, E. R.; Desii, A.; Sartini, S.; La Motta, C.; Mazzolai, B.; Mattoli, V. Langmuir 2013, 29, 13190−13197. (26) Becker, A. L.; Johnston, A. P. R.; Caruso, F. Macromol. Biosci. 2010, 10, 488−495. (27) Picart, C.; Schneider, A.; Etienne, O.; Mutterer, J.; Schaaf, P.; Egles, C.; Jessel, N.; Voegel, J. C. Adv. Funct. Mater. 2005, 15, 1771− 1780. (28) Ren, K. F.; Ji, J.; Shen, J. C. Biomaterials 2006, 27, 1152−1159. (29) Barthes, J.; Mertz, D.; Bach, C.; Metz-Boutigue, M.-H.; Senger, B.; Voegel, J.-C.; Schaaf, P.; Lavalle, P. Langmuir 2012, 28, 13550− 13554. (30) Andrianifahanana, M.; Moniaux, N.; Batra, S. K. Biochim. Biophys. Acta, Rev. Cancer 2006, 1765, 189−222. (31) Dedinaite, A.; Lundin, M.; Macakova, L.; Auletta, T. Langmuir 2005, 21, 9502−9509. (32) Svensson, O.; Lindh, L.; Cardenas, M.; Arnebrant, T. J. Colloid Interface Sci. 2006, 299, 608−616. (33) Lindh, L.; Svendsen, I. E.; Svensson, O.; Cardenas, M.; Arnebrant, T. J. Colloid Interface Sci. 2007, 310, 74−82. (34) Wang, B.; Liu, Z.; Xu, Y.; Li, Y.; An, T.; Su, Z.; Peng, B.; Lin, Y.; Wang, Q. J. Mater. Chem. 2012, 22, 17954−17960. (35) Vreuls, C.; Zocchi, G.; Garitte, G.; Archambeau, C.; Martial, J.; Van de Weerdt, C. Biofouling 2010, 26, 645−656. (36) Jeffers, F.; Fuell, C.; Tailford, L. E.; MacKenzie, D. A.; Bongaerts, R. J.; Juge, N. Carbohydr. Res. 2010, 345, 1486−1491. (37) Shi, L.; Miller, C.; Caldwell, K. D.; Valint, P. Colloids Surf., B 1999, 15, 303−312. (38) Lundin, M.; Sandberg, T.; Caldwell, K. D.; Blomberg, E. J. Colloid Interface Sci. 2009, 336, 30−39. (39) Crouzier, T.; Beckwitt, C. H.; Ribbeck, K. Biomacromolecules 2012, 13, 3401−3408. (40) Brockhausen, I.; Schachter, H.; Stanley, P. O-GalNAc Glycans. In Essentials of Glycobiology, 2nd ed.; Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., Etzler, M. E., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2009. (41) Lichter, J. A.; Rubner, M. F. Langmuir 2009, 25, 7686−7694. (42) Xu, L.; Selin, V.; Zhuk, A.; Ankner, J. F.; Sukhishvili, S. A. ACS Macro Lett. 2013, 2, 865−868. (43) Soltwedel, O.; Nestler, P.; Neunnann, H.-G.; Passvogel, M.; Koehler, R.; Helm, C. A. Macromolecules 2012, 45, 7995−8004.

ASSOCIATED CONTENT

S Supporting Information *

Additional figures and tables as described in the text (Figure S1−S7, and Table S1−S2). This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions #

These authors contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported in part by the MRSEC Program of the National Science Foundation under award number DMR 0819762. R. Polak is supported by FAPESP (São Paulo Research Foundation) Fellowships 2010/51066-3 and 2011/ 21326-6 and R. Lim by the Chyn Duog Shiah Memorial Fellowship. T. Crouzier is supported by Marie Curie International Outgoing Fellowship “BIOMUC”. We would like to thank Professor Darrell Irvine of Biological Engineering and Materials Science and Engineering for the use of cell culture facilities and the confocal microscope.

■

REFERENCES

(1) Ono, S. S.; Decher, G. Nano Lett. 2006, 6, 592−598. (2) Lee, H.; Sample, C.; Cohen, R. E.; Rubner, M. F. ACS Macro Lett. 2013, 2, 924−927. (3) Swiston, A.; Gilbert, J.; Irvine, D.; Cohen, R.; Rubner, M. Abstr. Pap.Am. Chem. Soc. 2010, 240. (4) Swiston, A. J.; Cheng, C.; Um, S. H.; Irvine, D. J.; Cohen, R. E.; Rubner, M. F. Nano Lett. 2008, 8, 4446−4453. (5) Swiston, A. J.; Gilbert, J. B.; Irvine, D. J.; Cohen, R. E.; Rubner, M. F. Biomacromolecules 2010, 11, 1826−1832. (6) Doshi, N.; Swiston, A. J.; Gilbert, J. B.; Alcaraz, M. L.; Cohen, R. E.; Rubner, M. F.; Mitragotri, S. Adv. Mater. 2011, 23, H105−H109. (7) Lutkenhaus, J. L.; Hrabak, K. D.; McEnnis, K.; Hammond, P. T. J. Am. Chem. Soc. 2005, 127, 17228−17234. (8) Dubas, S. T.; Farhat, T. R.; Schlenoff, J. B. J. Am. Chem. Soc. 2001, 123, 5368−5369. (9) Jiang, C. Y.; Markutsya, S.; Pikus, Y.; Tsukruk, V. V. Nat. Mater. 2004, 3, 721−728. (10) Jiang, C. Y.; Markutsya, S.; Shulha, H.; Tsukruk, V. V. Adv. Mater.. 2005, 17, 1669−+. (11) Jiang, C. Y.; Tsukruk, V. V. Soft Matter 2005, 1, 334−337. (12) Jiang, C. Y.; Tsukruk, V. V. Adv. Mater. 2006, 18, 829−840. (13) Zimnitsky, D.; Shevchenko, V. V.; Tsukruk, V. V. Langmuir 2008, 24, 5996−6006. (14) Ma, Y.; Sun, J.; Shen, J. Chem. Mater. 2007, 19, 5058−5062. (15) Kozlovskaya, V.; Baggett, J.; Godin, B.; Liu, X. W.; Kharlampieva, E. ACS Macro Lett. 2012, 1, 384−387. (16) de Vos, W. M.; de Keizer, A.; Stuart, M. A. C.; Kleijn, J. M. Colloids Surf., A 2010, 358, 6−12. (17) Wang, B. Z.; Tokuda, Y.; Tomida, K.; Takahashi, S.; Sato, K.; Anzai, J. Materials 2013, 6, 2351−2359. (18) Chkhalo, N. I.; Drozdov, M. N.; Gusev, S. A.; Kluenkov, E. B.; Lopatin, A. Y.; Luchin, V. I.; Salashchenko, N. N.; Shmaenok, L. A.; Tsybin, N. N.; Volodin, B. A. Freestanding multilayer films for application as phase retarders and spectral purity filters in the soft Xray and EUV ranges. In Euv and X-Ray Optics: Synergy between Laboratory and Space II; Hudec, R., Pina, L., Eds.; SPIE: Bellingham, WA, 2011; Vol. 8076. 3098

dx.doi.org/10.1021/bm5006905 | Biomacromolecules 2014, 15, 3093−3098