Cellular interactions on nano-structured polyelectrolyte multilayers

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Colloids and Surfaces A: Physicochem. Eng. Aspects 313–314 (2008) 526–529

Cellular interactions on nano-structured polyelectrolyte multilayers Sung Yun Yang ∗ , Ju-Yong Seo Department of Polymer Science and Engineering, Chungnam National University, Gung-Dong 220, Yuseong-Gu, Daejeon, Republic of Korea Received 30 October 2006; accepted 28 April 2007 Available online 2 June 2007

Abstract Cellular interactions onto the polymeric surfaces which prepared with polyelectrolytes by layer-by-layer process using H-bonding interactions were studied. Hydrogen-bonded multilayers containing polyacrylamide (PAAm) that assembled with weak polyelectrolytes, such as poly(acrylic acid) (PAA) or polyaspartic acid (PASA) were investigated for their surface–cell interactions. The assembled films were lightly crosslinked thermally in order to render them stable in a physiological environment. Both PAA/PAAm and PASA/PAAm multilayers were found to exhibit a high resistance to the adhesion (cytophobicity) of mammalian cells (epithelial cell and blood cell), even with only a few nanometer-thick coating. PASA is a biodegradable polymer; therefore, the unmodified form may not be stable as a surface coating. However, the well-blended PASA/PAAm multilayer films exhibit the cell-adhesion resistance property without a significant film deformation at least more than a week. More interestingly, one additional coating of polycationic layer rendered the cell-resistant surface into the cell-adhesive surface. Using the reactive carboxylic acid groups remaining after the multilayer assembly, antibacterial nanoparticles were also synthesized. © 2007 Elsevier B.V. All rights reserved. Keywords: Polyelectrolyte multilayer; H-bonding; Cell-adhesion; Biodegradable polymer; Cytophobic; Antibacterial coating; Layer-by-layer

1. Introduction Most of the natural biopolymers including DNA, protein and polysaccharide are polyelectrolytes that are positively or negatively charged macromolecules. Conformational change of biopolymer, which is lead by the repulsive/attractive interactions along the polymer chain, is extremely important to perform their bioactivities. Some synthetic polymers also alter their chain conformations upon their environmental changes [1]. Weak polyelectrolytes, which have a different charge density according to their solution pH condition, are these cases [2,3]. For example, poly(carboxylic acid)s usually form compact coils at low pH, and as the solution pH arises, the polymers have extended chain conformations due to the increasing repulsion between the same charges [4]. Layer-by-layer deposition (LbL) is a versatile technique whereby ultrathin films are assembled from the repetitive, sequential adsorption of oppositely charged polyelectrolytes from dilute aqueous solution [5]. It allows nano-scale control

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over the thickness, composition, and molecular structure of the deposited multilayer film [6]. Due to the unique property of the weak polyelectrolyte, conformational change described above, the layer-by-layer process using weak polyelectrolytes may able to tuning the film properties by simple changing the pH of the dipping condition [7]. Moreover, the functional groups which remained reactive after the film deposition allow further chemical reactions such as the polymer micro-contact printing [8] and the selective crosslinking (micropatterning) [9,10]. Controlling the interactions of proteins and cells with the surface is critically important in many biomedical applications including biomedical devices, because most of cellular activities such as adhesion, growth rate, motility, differentiation and apoptosis are surface-mediated [11]. Due to the import advantages of polyelectrolyte multilayer films discussed above, we and others have chosen the layer-by-layer technique to prepare various polymeric surfaces to study cellular responses on the surfaces [10,12–15]. In the previous studies, we have found that the H-bonded multilayers containing poly(acrylamide) (PAAm) coating on flat surfaces [10] and colloidal particles [16] exhibited the unprecedented resistance toward the fibroblast cell adhesion. This multilyer films also showed evidence of good repelling properties of some protein adsorptions (bio-inertness)

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[10]. We here extend the study for cell-resistant surfaces using the H-bonded multilayer systems including biopolyelectrolytes. This new system may benefit the additional biocompatibility of the bio-inert multilayer coatings. Further understanding of the H-bonded multilayers might lead to new opportunities for fabricating biosensor arrays and cell-based drug screening devices.

conditions. The multilayer films were thermally crosslinked at 90 ◦ C for 12 h. Details concerning the assembly and crosslinking of these multilayer films can be found in a previous paper [9].

2. Experiment

Two hundred and ninety-three epithelial cells were cultured on tissue culture polystyrene (TCPS) in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 ◦ C in a humidified atmosphere of 95% air and 5% CO2 . The pH of the medium was adjusted to 7.4. For attachment and proliferation assays, cells were removed from their growth surface by trypsin and then spun down in a centrifuge at ∼1000 rpm for ∼5 min. The cells were then resuspended in fresh media, mixed in a 1:1 ratio with 0.4% trypan blue (Sigma) and counted with a hemocytometer with trypan blue exclusion to determine cell viability prior to seeding. The 293 cells were seeded onto the sterilized multilayer-coated slide glasses containing TCPS dishes at a population density of ∼37,000 cells/cm2 for cell-adhesion investigations. Prior to cell adhesion studies, the multilayers were sterilized by spraying with 70% ethanol. An Olympus inverted phase contrast microscope was used for all experiments to capture images of the cell density, morphology, and spreading on the multilayer surfaces over several weeks. The media was exchanged every other day, and cells were photographed daily.

2.1. Materials Weak polyelectrolytes including poly(allylamine hydrochloride) (PAH) (Mw = 60,000), poly(acrylic acid) (PAA) (Mw = 90,000) and polyacrylamide (PAAm) (Mw = 5,000,000) were purchased from Polysciences Inc. Poly(aspartic acid) sodium salt (PASA) (Mw = 15,000–50,000) was purchased from Sigma–Aldrich. Silver acetate and borane-dimethylamine complex were obtained from Aldrich Chemical and used without the further purification. Hydrochloric acid (HCl) and potassium hydroxide (KOH) aqueous solutions were used to adjust the pH of the dipping solution. De-ionized water (∼18 M cm) was used for the preparation of all aqueous solutions and rinsing water. 2.2. Substrate preparation Slide glasses or silicon substrates were washed ultrasonically with detergent (Microsol® ) for 15 min followed by rinsing with de-ionized water. This cleaning step was repeated at least three times and then the substrates were blown-dried with N2 gas. In the case of silicon substrates were treated with plasma cleaner prior to the multilayer deposition. 2.3. Multilayer film formation All polymer solutions were prepared with deionized water (0.01 M, based on the repeating unit molecular weight) without addition of salt. The pH of the rinsing baths as well as the polymer solutions was adjusted to 3.0 with dilute HCl (0.01 M). All polymer solutions were filtered with 0.45 ␮m-pore size cellulose nitrate filters. The layer-by-layer process was carried out via the use of an automatic dipping machine (Inc.) or manual dipping. The substrates (tissue culture glass slide for cell study or silicon slide for other characterization) were first immersed into a PAH solution (0.01 M, pH 3.0) for 15 min followed by rinsing with pH 3.0 water. This step introduces a primer/adhesion layer for the subsequent PAA and PAAm layers. Similar results were obtained without the use of the PAH primer layer. The substrates were then dipped into a polyacid (either PAA or PMA) solution followed by three rinses (2, 2 and 1 min in separate bins) and then dipped into a PAAm solution followed by the same rinsing procedure. Deposition of a PAA layer and a PAAm layer completes one cycle, which is referred as a single bilayer formation. This dipping cycle was repeated until the multilayer was produced. In the case of using PASA, the PAA solution was replaced with the PASA solution with the same concentration. All the dipping steps for PASA/PAAm system were identical to the PAA/PAAm

2.4. Cell culture

2.5. Nanoparticle synthesis The deposited multilayer film was immersed into the 5 mM silver acetate solution for 5 min followed by rinsing with deionized water for 2 min. The rinsing step was repeated at lest two times. The metal ion absorbed film was then immersed into the reduction bath (5 mM borane–dimethylamine complex aqueous solution) for 5 min. 2.6. Film characterization The multilayer film properties were measured by various instruments including Perkin-Elmer Lam BDA 35 UV-Vis spectrometer, J.A. Woollam Co. ellipsometry and contact angle analyzer. Nanoparticles were characterized by a transmission electron microscopy (TEM). 3. Results and discussion The multilayer of poly(acrylamide) and poly(acrylic acid) or poly(aspartic acid) were successfully prepared on glass slides and silicon substrates by hydrogen bonding interactions. Fig. 1 shows the chemical structures of the polyelectrolytes and the possible H-bonding structure between polyacid and polyamide. These films were assembled at pH 3.0/3.0 for both dipping solutions and the rinsing steps also were kept at the same low pH condition. Otherwise, the multilayer could not form due to the ionization of the carboxylic acid groups that supposed to be the H-bonding partners.

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Fig. 1. The chemical structures of the weak polyelectrolytes used in this study: (a) poly(acrylic acid) (PAA) and (b) poly(aspartic acid) (PASA). (c) H-bonding of polyacid and PAAm.

To examine how these multilayer thin films interacted with adherent mammalian cells, the films were deposited onto tissue culture glass dishes and thermally cross-linked. Heating of the deposited film at the temperature above 90 ◦ C provided the essential film stability at pH 7 that the cellular study was performed. Since the crosslinking density was kept low (about less than 10% of carboxylic acid groups), both multilayer systems were highly wettable after exposure to aqueous solutions as indicted by very low receding contact angles (below 10◦ ). The multilayer film thickness was measured by ellipsometry The PAA/PAAm multilayer film was found thicker than the PASA/PAAm multilayer at the same number of layer. In the cases of the 10 bilayer-films, the thicknesses of the PAA/PAAm and PASA/PAAm multilayers are 102 and 75 nm, respectively.

Cellular interactions on the multilayer films were studied by using embryonic kidney cells. Two hundred and ninetythree epithelial cells were seeded onto the sterilized multilayer samples and the cellular activity was followed by taking optical microscope images of the cells for at least 5 days after seeding. As an anchorage-dependent cell line, 293 cells are adherent to the bare glass surfaces as expected (Fig. 2(a)). However, in sharp contrast to what was observed with uncoated glass controls, slide coated with the PAAm-containing hydrogen-bonded multilayers displayed a high resistance to cell attachment. As summarized in Fig. 2(b, c, e and f), there was no significant top layer effect observed. In our film thickness control study of the cell resistance toward 293 cell attachment, surprisingly only one bilayer (<5 nm-thick) was sufficient to exhibit the cell-adhesion resistance (Fig. 3(b)). More interestingly, by addition of the single layer of poly(allylamine hydrochloride) (PAH), a cationic polyelectrolyte, onto the cell-resistant PAA/PAM film alter the coating as the cell-attractive surface (Fig. 3(c)). We found the excellent cell-adhesion resistant property in the initial study of white blood cell (Raw 264.7) with PAA/PAAm multilayers. More detailed study will be followed.

Fig. 2. Optical microscopic images of cells on the multilayer surfaces on 3 days-post seeding: cells on (a) bare glass (control), (b) (PAA/PAAm)10 film, (c) (PAA/PAAm)10.5 film, (d) the half-coated PAA/PAAm multilayer surface (the right half is coated area), (e) (PASA/PAAm)10 film and (f) (PASA/PAAm)10.5 film.

Fig. 3. Optical microscopic images of cells on the multilayer surfaces: cells on (a) bare glass (control), (b) (PAA/PAAm/PAA) three layered film and (c) PAH single layer addition to the film (b).

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multilayer films exhibit the excellent resistance toward mammalian cell-adhesion. The reactive carboxylic acid groups which remained after the film deposition may be used for the further chemical treatment, bio-coupling or antibacterial nanoparticle synthesis. This cell-adhesion resistant property of the H-bonded multilayers with the capability of silver nanoparticle synthesis might be highly beneficial to medical implant coatings, because blocking non-specific cellular adhesion to medical implants is extremely critical for keeping the device from malfunctions or immune responses [20]. Acknowledgements

Fig. 4. TEM image of the silver nanoparticle-embeded H-bonded PAA/PAAm multilayers (scale bar: 50 nm).

This work was supported by the Korea Research Foundation, Grant funded by Korean Government (D00004) and the research fund of Chungnam National University. The authors thank to Y. Bae of Prof. Choi’s group at the Department of Biochemistry for the assistance in cell culture. References

The H-boned multilayers are free acid-rich thin films, while the actively remaining carboxylic acid can be used for the further chemical reactions [10,13,16,17]. One of the practical usages of these free acid groups is a nanoparticle synthesis. Silver nanoparticles were synthesized by using the carboxylic acid groups in the H-bonded multilayers as the silver ion binding sites. The adsorbed silver ions later reduced by reducing agents to forming nano-sized particles (Fig. 4). The synthesized particles are about 5 nm in their diameters and very well dispersed within the polyelectrolyte film. Silver nanoparticles have well-known antibacterial activity. We and others found that the silver nanoparticle-embedded multilayer films exhibited profound results on the bacterial cell killing effect [18,19]. More detailed study of silver nanoparticle-embedded multilayers will be continued for the better understanding of the biocidal functions of metal–polymer hybrid systems. The H-bonded multilayers presented here have multifunctionality including bio-inert surface property will provide a great potential to many bioapplications. 4. Conclusion The multilayer containing PAAm with weak polyelectrolyte were assembled by layer-by-layer (LbL) technique using hydrogen bonding interactions. Both PAA/PAAm and PASA/PAAm

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