Biomacromolecules 2009, 10, 907–915
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Resistance of Polysaccharide Coatings to Proteins, Hematopoietic Cells, and Marine Organisms Xinyu Cao,† Michala E. Pettit,‡ Sheelagh L. Conlan,§ Wolfgang Wagner,| Anthony D. Ho,| Anthony S. Clare,§ James A. Callow,‡ Maureen E. Callow,‡ Michael Grunze,† and Axel Rosenhahn*,† Applied Physical Chemistry, University of Heidelberg, 69120 Heidelberg, Germany, School of Biosciences, University of Birmingham, B15 2TT, U.K., School of Marine Science and Technology, Newcastle University, Newcastle upon Tyne, NE1 7RU, U.K., and Department of Medicine V, University of Heidelberg, 69115 Heidelberg, Germany Received December 8, 2008; Revised Manuscript Received February 5, 2009
The interaction of covalently coupled hyaluronic acid, alginic acid, and pectic acid with proteins, cells (hematopoietic KG1a and Jurkat cells), and marine organisms (algal zoospores and barnacle cypris larvae) is compared. In contrast to cells and proteins for which such polysaccharide coatings are known for their antiadhesive properties, marine algal spores and barnacle cyprids were able to colonize the surfaces. Of the three polysaccharides, hyaluronic acid showed the lowest settlement of both UlVa zoopores and barnacles. Photoelectron spectroscopy reveals that the polysaccharide coatings tend to bind bivalent ions, such as calcium, from salt water. Such pretreatment with a high salinity medium significantly changes the protein and hematopoietic cell resistance of the surfaces. Complexation of bivalent ions is therefore considered as one reason for the decreased resistance of polysaccharide coatings when applied in the marine environment.
1. Introduction Representatives of all the phyla living in aquatic environments, from bacteria, through lower plants (algae) to invertebrate animals, use sticky materials with permanent or temporary adhesive capabilities at some point in their life cycle. For example, in a naturally turbulent marine environment, larvae of invertebrates and spores of algae need to quickly find and bind to a surface in order to complete their life histories. For an adhesive to bind to other surfaces it has to “wet” that surface, and whether it does so will depend on the competition between the adhesive and water interacting with the surface. The interaction is complex, involving the displacement of water from the adhesive and the surface, intermolecular adhesive bonding between the substrate and adhesive, and cohesive intramolecular cross-linking.1 It follows that one method for achieving fouling resistance is to create surfaces with properties that resist displacement of water, as is has been shown, for example, in synthetic hydrogels such as polyHEMA or PEO/PEG. A different approach uses the intrinsic hydrophilicity of natural macromolecules to achieve the same effect, e.g., polyanionic polysaccharides, which have been investigated by Morra et al.2-4 These biopolymers have been used as model systems in which to investigate structure-properties relationships in the adhesion of mammalian cells and certain classes of bacteria to substrates and are considered to exhibit “non-fouling properties”.2 As yet there have been no studies on the ability of such polysaccharides to perturb adhesion of biofouling organisms in marine environments, although carbohydrates have been implicated, e.g., in the settlement of barnacle cypris larvae * Corresponding author. E-mail:
[email protected]. † Applied Physical Chemistry, University of Heidelberg. ‡ University of Birmingham. § Newcastle University. | Department of Medicine V, University of Heidelberg.
(cyprids). Gregarious settlement behavior, for example, is modulated by a large glycoprotein, the settlement-inducing protein complex (SIPC) (see, e.g., refs 5 and 6). Although the involvement of the carbohydrate moiety of SIPC in gregariousness was originally dismissed,7 glucose/mannose residues in the SIPC have since been implicated through the inhibitory effect of lentil lectin on settlement.8 Similar conclusions can be drawn with respect to the stimulatory effect of the diatom NaVicula ramosissima9 and the bacterium Pseudomonas aeruginosa10 on settlement of barnacle cypris larvae. In contrast, galactosebinding lectins reversed the inhibitory action of two bacteria.10 When tested in solution, mannose and glucose inhibited the temporary adhesion of cyprids.11 This effect was attributed to binding of the monosaccharides to polar groups in the temporary adhesive, which shares at least one epitope with the SIPC.12 Sugars may, therefore, affect cyprid settlement by modulating chemical cue recognition or by physically inhibiting adhesion. In the only study of the effect of polysaccharides, xanthan gum, agarose, and alginic acid, which were allowed to partition onto polystyrene from solution, it was found that all inhibited settlement of cyprids relative to an untreated polystyrene control,13 as did hydrogels of both chitosan and alginic acid.14 It is difficult to generalize the effects of carbohydrates, other than that they are important to settlement, because the aforementioned studies have used different barnacle species that are known to differ with respect to other modulators of settlement. In the present study we focus on three different acidic polysaccharides known to play an important role in biological adhesion and for the buildup of cell boundaries: hyaluronic acid (HA), alginic acid (AA), and pectic acid (PA) (Figure 1). These hydrophilic, negatively charged molecules have a high affinity for water, and HA, for example, is an important component of the extracellular matrix secreted by adhering cells.15 They are large, linear glycosaminoglycanes, which contain repeating units of the disaccharide D-N-acetylglucosamine-β-D-glucuronic acid.
10.1021/bm8014208 CCC: $40.75 2009 American Chemical Society Published on Web 03/26/2009
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Figure 1. Polysaccharides used as surface coating: alginic acid (AA), hyaluronic acid (HA) and pectic acid (PA). As majorly containing “high-M” alginates were used, only the M unit is shown as an example for AA.
Hyaluronans are negatively charged at a physiological pH of 7.4, and their viscosity in solution behaves as a polyelectrolyte.15 Surface grafted hyaluronan layers are hydrophilic and can swell within seconds, absorbing water to 2.4-fold their initial thickness.16 Because of their water affinity and charge repulsion, hyaluronans are considered as biological lubricants.15,17 Disease processes that exhibit aberrant cell behavior, such as cancer and artherosclerosis, involve altered hyaluronan-cell interactions.18 Immoblilized HAs form surfaces resistant to the adhesion of mammalian cells such as L929 fibroblasts.2 Meshes coated with HA are resistant to attachment of cells as tested with implants in the rabbit model.4 Covalently coupled to surfaces, HA significantly reduces bacterial adhesion for Staphylococcus epidermidis and Escherichia coli compared to glass surfaces.4 AA and PA are more common as constituents within plant cells. Alginate is a natural polymer known as a major component of the extracellular polymeric substance (EPS) in bacterial biofilms19 and the cell walls and mucilages of brown seaweeds. It is composed of two sterically different repeating units: (1-4)RL-guluronate (G unit) and (1-4)β-D-mannuronate (M unit). In our study, straight-chain alginate mostly containing mannuronate units, or “high M” alginate were used, which is why only the M units are shown in Figure 1. PA, or pectin, is one of the major plant cell-wall polysaccharides.20 The PA studied in this work consists of linear chains of 1,4-linked R-galacturonic acid. All three polysaccharides play an important role in adhesion and for the buildup of cell boundaries and therefore fulfill important functions in biology. On this basis it is noticeable that immoblilized or copolymerized21 polysaccharides form cellresistant surfaces. As negatively charged hydrogels that strongly bind water, polysaccharides fulfill the above-described criteria for the inhibition of Biofilm formation and protein resistance.22-29 The goal of our work presented here was to evaluate whether the effects of polysaccharides in cell biology and tissue engineering apply also to the marine environment. Furthermore, we have used human hematopoietic cell lines (KG1a and Jurkat cells) as a reference and to analyze the adhesion of mammalian cells with potential applications for biomaterial research. Gaining a clearer understanding of fundamental aspects of settlement (attachment) and adhesion is aimed at informing the development and understanding performance of new environmentally benign coating technologies for the marine environment.30,31 The three types of polysaccharide-coated surfaces (HA, AA, and PA) were tested toward protein adsorption (Fib, Lys, PK, and BSA), mammalian cell adhesion (hematopoietic KG1a and Jurkat cells), the settlement and adhesion of spores of the green alga UlVa linza and the settlement of cyprids of the barnacle Balanus amphitrite. The marine fouling organisms used in this study were chosen because of their relevance in biofouling research. UlVa linza is
a green macroalga that reproduces by the production of vast numbers of microscopic, “naked” (i.e., without a cell wall) zoospores, 5-7 µm in length, which swim through the water using four flagella. In order to complete their life cycle, the zoospores need to locate a surface, settle on it, and then firmly adhere to it. On locating a suitable surface, the zoospore undergoes “settlement” and permanent attachment, involving loss of motility and secretion of adhesive, which anchors the spore to the substratum.32 The UlVa spore adhesive is a polydisperse, self-aggregating hydrophilic glycoprotein, resembling the group of hydroxyproline-rich extracellular matrices of both plants and animals.32 Swimming zoospores settle in response to a number of physical surface cues, including wettability,26 topography33 and charge.34 Once settled, the adhesion of the attached spores is also influenced strongly by surface energy26 and surface friction.35 Balanus amphitrite (Amphibalanus amphitrite36) is a cosmopolitan, tropical/subtropical barnacle. The settlement-stage cypris larva (cyprid), which measures approximately 500 µm in length, is highly specialized in its settlement strategies. Cyprids use their paired attachment discs to attach temporarily while exploring surfaces (e.g., ref 37). The relative contributions of the “hairy” appendages and a proteinaceous secretion to adhesion by “dry” and “wet” adhesion, respectively remain unclear.38,39 Although the so-called “temporary adhesive” remains to be fully characterized, a structural and functional relation to SIPC (an R2 macroglobulin-like molecule) has been proposed.12 Numerous physicochemical factors modulate cyprid settlement:40 biogenic chemical cues being particularly important to gregarious settlement41,42 and the outcome of interactions with biofilms.43
2. Experimental Section Materials. All chemicals, such as ethanol, 3-aminopropyltriethoxysilane (APTES), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC), N-Hydroxysuccinimide (NHS), bovine serum albumin (BSA), fibrinogen (Fib), lysozyme (Lys), pyruvate kinase (PK), HA, AA, PA, and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich (Munich, Germany). Deionized water was purified with a Milli-Q Plus system (Millipore, Schwalbach, Germany). PBS buffer was used at pH 7.4 and a concentration of 0.01 M. HEPES buffer, and microscope glass slides were obtained from Carl Roth (Karlsruhe, Germany). Preparation of the Polysaccharide Coatings. The reaction for building up the polysaccharide films is schematically shown in Figure 2. Glass slides were cleaned and activated with “piranha solution” (H2SO4/H2O2 ) 3:1). APTES monolayers were coupled to the hydrophilic glass slides by chemical vapor deposition (CVD). Polysaccharides were coupled to the APTES monolayers following literature protocols.44 Therefore, 0.1 M EDC and 0.05 M NHS were dissolved in 10 mM HEPES buffer solution (pH 6-7). The corresponding polysaccharide
Polysaccharide Resistance in Marine Organisms
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Figure 2. Scheme for the covalent immobilization of polysaccharides on glass substrates. Table 1. Properties of the Proteins for the Adsorption Test45
protein
source
molecular weight (kDa)
BSA Fib Lys PK
bovine serum bovine plasma chicken egg white rabbit muscle
66 340 14.7 237
isoelectric point
net charge at pH 7.4
5.60 R 7.73 β 8.66 γ 5.47 9.32 7.60
+ +
was added to this solution at a concentration of 1 mg/mL. After 20 min, the APTES-functionalized glass slides were immersed into the activated polysaccharide solutions for 16 h at room temperature. In order to remove physically absorbed molecules, the slides were first rinsed and then immersed into Millipore water (MW) while shaking on a vibration table for 5 days. The water was exchanged every day. Surface Characterization. Sessile drop (Millipore) water contact angles were measured under ambient conditions. The reported values are the average of three measurements taken for different samples with the tip not being in contact with the droplet. To determine the thickness of the different polysaccharide coatings on silicon wafers, ellipsometry measurements were performed with a fixed angle M-44 spectral ellipsometer (J. A. Woollam Co., Inc.) operating in the wavelength range between 280 and 800 nm. The organic film was modeled as a single Cauchy layer. APTES and the final polysaccharide film quality and thickness was furthermore analyzed by X-ray photoelectron spectroscopy (XPS) using a Leybold-Heraeus MAX 200 X-ray photoelectron spectrometer with an aluminum anode as X-ray source (KR ) 1486.4 eV). The thickness was determined by the attenuation of the Si2p XPS signal from glass substrate, using Lambert-Beer’s law and by the intensity of the C1s signal. For the latter, a cross-calibration with thicknesses obtained by spectral ellipsometry on coated silicon wafers was done. Protein Affinity Test. Protein binding affinity of surfaces coated with the three polysaccharides was tested toward BSA (from bovine serum; g96%), Fib (from bovine plasma, 55-70% protein, g90% clottable protein), Lys (from chicken egg white, 85%, 50 000 units/ mg protein), and PK (from rabbit muscle, 400-800 units/mg protein). The four chosen proteins have different molecular weights and net charges in PBS buffer solution (Table 1). The isoelectric point and charge at pH 7.4 were obtained from the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss institute of bioinformatics.45 1-Dodecanethiol monolayers (alkane thiol self-assembled monolayers (SAMs)) were used as a non-protein-resistant reference for Fib and Lys adsorption. In BSA, Fib, and Lys assays, 1 mg/mL protein solutions in PBS buffer (0.01M, pH 7.4) were prepared. In PK assay, the protein concentration was 80 units/mL. The protocol for the affinity test was following earlier work.46 Therefore, the samples were immersed in 5 mL PBS buffer solution for 5 min, and then 10 mL of the protein solution were added. After 2 hours, the solution was diluted with 400 mL of MW, rinsed with MW, and dried in a stream of nitrogen. The amount of adsorbed protein was determined by averaging spectral ellipsometric thicknesses at three points on each sample. Settlement and Adhesion Assays for Zoospores of UlWa. Test surfaces were equilibrated in Tropic Marin artificial seawater (ASW) for 1 h prior to the start of the assay. Zoospores were released from
fertile plants of UlVa linza and prepared for settlement and adhesion experiments as described previously.47 Ten milliliters of freshly released spores in ASW (1.5 × 106 spores per mL) were added to individual compartments of a sterile Quadriperm dishes each containing a test surface. Six replicates of each test sample were immersed simultaneously. The slides were incubated in darkness for 45 min and then washed gently in ASW to remove unsettled, i.e., motile spores. Three replicates were used to determine the number of settled (attached) spores. Spores were fixed in 2.5% glutaraldehyde in ASW, washed in deionized water, and dried. Spore counts were taken using a Kontron 3000 image analysis system attached to a Zeiss epifluorescence microscope. Spores were visualized by autofluorescence of chlorophyll, and counts were recorded for 30 fields of view on each replicate slide as described by Callow et al.48 To determine the adhesion strength of attached spores, the remaining three replicates were exposed to a wall shear stress of 53 Pa in a calibrated water channel using methods previously described.49 The number of spores remaining after flow was compared to the unexposed samples. Acid-washed glass (AWG) slides were included in all assays as laboratory standards. Settlement Assay with Barnacle (Balanus amphitrite) Cyprids. Cypris larvae were cultured as described previously.50 They were then aged at 6 °C for 3 days prior to use. Slides of the different polysaccharides and AWG (a standard against which viability and behavior of larvae was determined) were conditioned by immersion in a tank of ASW (Tropic Marin) for 1 h. Excess water was removed by blotting the edge of the slides with an absorbent paper towel. One milliliter of ASW, containing 20 cyprids, was then pipetted onto each slide (n ) 6), which was contained within the well of a Quadriperm dish. The cyprids were incubated in the dark at 28 °C for 48 h with settlement enumerated at 24 and 48 h. Data were analyzed using Kruskal-Wallis (K-W) and post hoc Dunn’s tests to determine significance (R level of 0.05) between means. All statistical procedures were done on GraphPad Prism version 5.0. AWG slides were included in all assays as laboratory standards. Adhesion Assay with Cultured Hematopoietic Cells. Cell adhesion was analyzed using a novel adhesion assay based on gravity force as described before.51,52 In brief, a leak-proof silicone gasket with eight wells (9 mm in diameter and 1 mm in depth) was fixed on the polysaccharide surface. A suspension of KG1a or Jurkat cells in RPMI1640 culture medium was stained with the fluorescent membrane dye PKH26, and 10 000 cells were added into each well. Then the cells were kept in the incubator (with 5% CO2) at 37 °C for 1 h. After that, a glass coverslip was put on top of the silicone gasket to cover the cell suspension. A “sandwich” system with glass/cells/polysaccharide was setup in this way. Then the “sandwich” system was turned up-side down, and the cells that did not adhere on the polysaccharide surfaces fell down to the glass surface by gravity. The cells were incubated for another 15 min. Finally, samples were investigated under a microscope, and photos were taken from adherent and nonadherent cells for quantitative analysis. Coverslips were included in all assays as laboratory standards.
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Table 2. Sessile Drop Contact Angles of the Different Polysaccharide Coatings surfaces
sessile water drop contact angle
APTES AA HA PA
53-63° 20.4 ( 1.8°
