Stimuli-Responsive Multilayers Based on Thiolated Polysaccharides

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Stimuli-Responsive Multilayers based on Thiolated Polysaccharides Affect Fibroblast Cell Adhesion Pegah Esmaeilzadeh, Alexander Köwitsch, Andrea Liedmann, Matthias Menzel, Bodo Fuhrmann, Georg Schmidt, Jessica Klehm, and Thomas Groth ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19022 • Publication Date (Web): 22 Feb 2018 Downloaded from http://pubs.acs.org on February 24, 2018

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ACS Applied Materials & Interfaces

Stimuli-Responsive Multilayers based on Thiolated Polysaccharides Affect Fibroblast Cell Adhesion Pegah Esmaeilzadeh1,2, Alexander Köwitsch1, Andrea Liedmann1, Matthias Menzel3, Bodo Fuhrmann2,4, Georg Schmidt2,4, Jessica Klehm3, Thomas Groth1,2,* 1

Biomedical Materials Group, Institute of Pharmacy, Martin Luther University Halle Wittenberg, Heinrich Damerow Strasse 4, D 06120 Halle (Saale), Germany, Email: [email protected] * 2

Interdisciplinary Center of Material Research, Martin Luther University Halle-Wittenberg, Heinrich-Damerow-Strasse 4, 06120 Halle (Saale), Germany 3

Fraunhofer Institute for Microstructure of Materials and Systems IMWS, Walter-Hülse-Strasse 1, 06120 Halle (Saale), Germany

4

Institute of Physics, Martin Luther University Halle-Wittenberg, 06099 Halle (Saale), Germany

Abstract Control of biomaterials properties through stimuli-responsive polymeric platforms has become an essential technique in recent biomedical applications. A multilayer system of thiolated chitosan (t-Chi) and thiolated chondroitin sulfate (t-CS), consisting of 5 double layers ([t-Chi/tCS]5) was fabricated here applying a layer-by-layer coating strategy. To represent a novel class of chemically tunable nanostructures, the ability to cross-link pendant thiol groups was tested by a rise from pH 4 during layer formation to pH 9.3 and a more powerful chemical stimulus by using chloramine-T (ChT). Following both treatments, the resulting multilayers showed stimulidependent behavior as demonstrated by their content of free thiols, wettability, surface charge, elastic modulus, roughness, topography, thickness and binding of fibronectin. Studies with human dermal fibroblasts further demonstrated the favorable potential of the ChT-responsive multilayers as a cell-adhesive surface, when compared to pH-induced cross-linking. Since [tChi/t-CS]5 multilayer system is responsive to stimuli such as pH and redox environment, multilayer systems with disulfide bond formation may help to tailor interaction with cells, film degradation and controlled release of bioactive substances like growth factors in a stimuliresponsive manner useful in future wound healing and tissue engineering applications. Keywords: polyelectrolytes, multilayers, stimuli-responsive biomaterials, thiolated chitosan, thiolated chondroitin sulfate, fibronectin adsorption, fibroblast adhesion.

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Introduction.

In blurring the boundary between traditional inanimate materials and living organisms, “smart” or “intelligent” models have been studied as significant developments in the science and application of materials.1 Communicative and response-driven polymeric systems2 which belong to the category of smart materials, are capable of monitoring the surrounding environment and initiating a line of communication to the molecular world that has hitherto been unavailable. The unique capabilities of synthetic stimuli-responsive platforms such as polyelectrolyte multilayers present some fascinating opportunities to explore relationships between interfacial material properties and the behavior of cells in terms of adhesion, spreading, migration, proliferation, and differentiation.3 Cells are complex and sensitive structures adaptable to a variety of external stimuli ranging from chemical, and physical cues presented by their environment. Therefore, dynamic changes of the environment are frequently addressed in novel synthetic cell carrier microenvironments, imitating the rudimentary aspect of life.4,5 Advances in the synthesis and characterization of stimuli-responsive models for medical use and their “tool-box” of triggers were summarized in several recent review articles.6,7 The benefits of adopting the layer-by-layer (LbL) technique using positively and negatively charged polyelectrolytes for constructing a stimuli-responsive thin multilayer films are growing a system that is structure-controlled and molecularly well blended with the ability to modulate surface properties (composition, morphology, and physicochemical properties) for a specific application, and the ease of assembly.8 Thus, LbL technique can offer compliant arrangements of matters for generating a programmable response of their functional properties such as wettability9, charge10, stiffness11, or topography12 when an external condition is varied. The present paper describes a pro-active structural platform towards this goal. Glycosaminoglycans (GAGs), a family of polysaccharides, are generally regarded as the materials of choice for well-known and vast innovative possibilities of biomaterial designs, not only because they provide affordable structural components, but participating in and regulating many cellular events and physiological processes, such as cell proliferation and differentiation, cell-cell and cell-matrix interactions.13,14 The advancements achieved to date using chemically modified-GAGs have led to more improvements of their cross-linking reactions with other molecules.15 Keeping this in mind, we recently initiated a study on the partial conjugation of


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chondroitin sulfate (CS), a sulfated-GAG, with DTPHY (3, 3΄-dithiobis (propanoic hydrazide)) and accordantly chitosan (Chi) with DTP (3, 3΄-dithiodipropionic acid) to synthesize base materials i.e. t-CS and t-Chi for formation of multilayers with biocompatible and cell-adhesive properties.16 Chi is a biocompatible polysaccharide with anti-inflammatory and anti-infective properties that makes it attractive for a large variety of medical applications.17 Thus, the application of both polysaccharides in multilayer formation should provide a surrounding to control biological processes. In addition, the presence of free thiols in both polyelectrolytes provides the system with a redox-responsible element that can be used for intrinsic cross-linking by disulfide bond formation, but in a reversible manner. Hence, the assembly of t-Chi and t-CS polyelectrolytes may provide a multi-functional and stimuli-responsive environment possessing reactive and adaptive features that are interesting for controlled release of growth factors, but also reversible effects on cell adhesion and spreading that may affect their subsequent growth and differentiation. This report demonstrates the properties of the t-Chi/t-CS multilayer system that contains both pH-sensitive groups (amino groups in Chi and carboxyl and sulfate groups in CS) and redoxresponsive elements (free thiols in both molecules). The latter were addressed by an oxidative stimulus either exerted by exposure of multilayers to pH 918 or a chemical treatment with Chloramine-T (ChT)19. We then studied the physical properties of multilayers before and after these treatments and studied the effect of multilayer oxidation on protein adsorption and cell behavior to obtain information about the bioactivity of the system. Results are reported herein.

Materials and Methods section.

Materials. Native chondroitin sulfate A (from bovine trachea, Mw ~25 kDa, sulfation degree of 0.8), was provided by VWR International GmbH (Dresden, Germany). Native low molecular weight chitosan (Mw ~62 kDa, 84.6% degree of deacetylation) was received from Heppe Medical Chitosan GmbH (Halle, Germany). Chloramine-T trihydrate (ChT) and Whatman® syringe filters (cellulose acetate ø30mm, 0,2µm) were purchased from Diagonal GmbH & Co. KG (Münster, Germany). Triton X-100 and paraformaldehyde were obtained from SigmaAldrich Chemie GmbH (Taufkirchen, Germany). Phosphate buffered saline (PBS) was prepared according to the following formulation: 2.7 mmol L−1 KCl, 137 mmol L−1 NaCl, 1.4 mmol L−1 KH2PO4, 4.3 mmol L−1 Na2HPO4 × 2H2O, pH 7.4.



Formation of [t-Chi/t-CS]5 multilayers. The synthesis of thiolated chitosan (t-Chi, ≈13.7% thiol content) and thiolated chondroitin sulfate (t-CS, ≈32.5% thiol) and the formation of the thiolated polyelectrolyte multilayers (PEMs) were described previously.16 Solutions of t-Chi and t-CS (each 1 mg mL-1 in 150 mmol L-1 NaCl solution, pH 4) were prepared. The t-Chi solution was allowed to adsorb onto the defined substrata (either glass or Si wafer, mentioned later for each section) for 20 min at room temperature under gentle shaking, to form the first polycation layer. The adsorption process was terminated by washing with 150 mmol L-1 NaCl (pH 4) for 5 min in triplicate. Thereafter, substrata were incubated in t-CS solution for the second polyanion layer similarly for 20 min followed by washing with NaCl (150 mmol L-1, pH 4, 3 × 5 min). This bilayer sequence (a bilayer consists of a first layer of Chi and a second layer of CS) was repeated until five bilayers were fabricated. Finally, the samples were washed briefly with Milli-Q water to avoid salt crystallization, dried and kept under a laminar flow hood, protected from light. All polyelectrolyte solutions as well as the washing solution were filtered through a 0.2 µm-pore-size membrane (Whatman) before use. Oxidation of PEMs. For the oxidation procedures two different methods were applied. A method of oxidation of thiols by high pH was performed here by incubation of multilayers in 150 mmol L-1 NaCl solution of pH 9.3 for 1.5 h or 4.5 h followed by washing with NaCl (150 mmol L-1, pH 4) for 5 min twice and a brief final wash with Milli-Q water. The pH 9.3 will be abbreviated as pH 9 throughout this manuscript. The second method was based on treatment of multilayers with ChT as published recently19. Here, multilayers were immersed in ChT solution (5 mmol L-1, in filtered 150 mmol L-1 NaCl solution, pH 4) for 90 s, followed by washing using 150 mmol L-1 NaCl (pH 4) for 5 min, 5 times and briefly washing with Milli-Q water. In this study “+pH 9, 1.5 h” and “+pH 9, 4.5 h” stand for pH-mediated oxidation of [t-Chi/t-CS]5 multilayer at pH 9.3, 1.5 h or 4.5 h incubation and “+ChT” stands for the oxidation induced by ChT. Chemical and mechanical characterization of multilayers. Measurement of thiol content. We employed Ellman`s assay to measure the thiol content16 of the multilayers before and after treatment with oxidative stimuli. Briefly, the LbL deposition protocol of the [t-Chi/t-CS]n was performed in disposable polystyrene cuvettes (Carl Roth GmbH, Karlsruhe, Germany). To increase assay sensitivity, the number of double layers was


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selected as n=10. Afterwards, the oxidation and washing steps were applied in both reference (empty cuvette without multilayers) and cuvettes coated with multilayers. Then, Ellman`s reagent (5, 5΄-dithiobis-(2-nitrobenzoic acid or DTNB) in phosphate buffer [0.1 mol L-1, pH 7.5] was added to the cuvettes, which were then shaken in the dark at room temperature for 1 h (Heidolph, polymax 1040, at 40 rpm, Schwabach, Germany). Finally, the total thiol concentration of the multilayers was quantified with a UV–Vis spectrophotometer (Specord 200, Analytik Jena AG, Jena, Germany) at a wavelength of 412 nm.16 All tests were done in triplicate. Surface wettability. The surface wettability of the various multilayer models on glass substrates was assessed by static contact angle measurements using fresh Milli-Q water at room temperature in an OCA 15+ device (Dataphysics GmbH, Filderstadt, Germany). The measurement was performed in triplicate for each condition applying the sessile drop method. 5 droplets of 0.5 µL ultrapure water with a flow rate of 2 µL s-1 were applied to each sample and 10 consecutive values were considered using the Ellipse fitting method. The samples were equilibrated at ambient conditions for 24 to 48 h prior to the measurements. Surface potential. The zeta-potential of multilayers was measured with the SurPASS electrokinetic analyzer (Anton Paar, Graz, Austria). A glass coverslip (10 × 20 mm2) was coated with the multilayers and mounted on the adjustable gap cell with double-sided tape. 1 mmol L−1 potassium chloride (Carl Roth GmbH) solution was applied as model electrolyte, and 0.1 mol L−1 sodium hydroxide was used for pH titration from pH 3.0 to pH 10.0 (acid-to-base pH titration). A flow rate of 100−150 mL min−1 at a maximum pressure of 300 mbar was adjusted to determine the zeta-potential using the streaming current. Each measurement was done in triplicate. Surface topography and E modulus. For Atomic Force Microscopy (AFM) (nanowizard® II, JPK-Instruments, Berlin, Germany), the multilayers were prepared on glass substrates in duplicate. Topographical images were recorded using Silicon Nitride Cantilevers (SNL, Bruker Nano Inc., Santa Barbara, USA) in intermittent contact mode in a standard liquid cell (JPKInstruments) with 150 mmol L-1 NaCl (pH 4). For force map spectroscopy experiments, standard silicon nitride cantilevers (MLCT, Bruker Nano Inc.) were employed. All cantilever for force mapping experiments were inspected by scanning electron microscopy before and after experiments to exclude any significant changes of tip geometry and to determine tip radii for later calculation of elastic modulus (E). Force-constant calibration was carried out by the thermal



noise method.20 A force-map of an area of 3 x 3 µm2 was recorded for each sample with a lateral resolution of 256 x 256 pi2. Each pixel (pi) contains a single spectroscopy force curve including information about stiffness. E moduli were calculated from each single force curve according to an advanced Hertzian model for spherical indenter geometry.21 JPK Data Processing V5.0.85 and Gwyddion software V2.49 were utilized for data post-processing and for E modulus analysis. Roughness parameters were determined according to DIN EN ISO 4287/4288. Thickness measurements. The instrument employed in this study was a spectroscopic ellipsometer (M-2000V, J.A. Woollam Company, Lincoln, NE, USA) equipped with WVase32 software. The film thickness and the refractive index (An) of films measured under dry and wet conditions were fitted from the measured ellipsometric parameters ∆ and ψ using a Cauchy model for the optical properties of the polymer film: n (λ) =An+ Bn/ λ2 + Cn/ λ4. Here, the negligible Bn and Cn were considered as zero. The measurements in dry states were carried out at incident angles of 55◦ to 70◦ within a wavelength range of λ= 375 to 1000 nm. The initial substrate was a silicon wafer with a native oxide layer. Using samples in duplicate, data were obtained from 5 different points on each sample. The measurements in wet states were done in a fluid cell (0.5 mL Liquid CellTM, Woollam) at a fixed angle of incidence of 70˚. The delta offset, generated by the glass windows of the cell were firstly determined and later subtracted from the raw ellipsometric data in the WVase32 software. To study the pH-stimuli and ChT-stimuli behaviors, the stimulus solution (refer to oxidation of PEMs procedure) was injected into the fluid cell with the initially adsorbed [t-Chi/t-CS]5 system, followed by a washing step using filtered 150 mmol L-1 NaCl (pH 4) to remove residual chemicals. The wet thickness experiments were performed twice for each sample. Fibronectin adsorption/desorption. Human plasma fibronectin (FN) (YO Proteins, Huddinge, Sweden) was labelled with fluorescein isothiocyanate (FITC) according to a protocol published previously.22 The multilayers were prepared directly in black 96well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) for studying FN adsorption, using FITC-FN solution concentration of 20 µg mL–1 in PBS of pH 7.4. Plates were incubated with 100 µL of FITC-FN for 4 h at 37˚C, covered with Parafilm and Al-foil. Upon termination of adsorption, the 100 µL supernatants were transferred into new black 96-well plates and the fluorescence was measured using a plate reader (FLUOStar Optima, BMG Labtech GmbH, Offenburg, Germany) set at 485


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nm excitation/ 520 nm emission wavelength (PBS solution was the blank). In parallel, the multilayer-coated wells were carefully rinsed with 200 µL PBS, twice, each for 5 min. Thereafter, the PBS solution was removed and the multilayer-coated wells were incubated with 1 mol L-1 sodium hydroxide solution (NaOH) at room temperature for 2 h, to desorb FITC-FN. Subsequently, fluorescence intensities of the resulting supernatants were quantified with the fluorescence plate reader (NaOH solution was the blank). Each experiment was conducted with 8 replicates. Studies on adhesion of human dermal fibroblasts. Cell culture. Human dermal fibroblasts (HDFs, Promocell, Heidelberg, Germany) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Biochrom, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS, Biochrom) and 1% antibiotic-antimycotic solution (Penicillin/Streptomycin/Amphotericin B, AAS, Promocell) at 37 °C in a humidified 5% CO2/95% air atmosphere (NuAire, MN, USA). The cells were harvested using 0.25% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA, Biochrom) at 37 °C for 5 min. Trypsin was inactivated by addition of serum-containing DMEM. After centrifugation at 250 rpm at room temperature for 5 min, the cells were resuspended in pure DMEM. Cell adhesion studies. Before performing the LbL process, cleaned16 round glass cover slips were sterilized with 70% ethanol in water for 10 min, followed by rinsing with NaCl solution (0.15 mol L-1, pH 4, sterilized through 0.2 µm-pore-size membrane filter) twice. Then, LbL procedure was performed as aforementioned, followed by the oxidation step (pH 9 or ChT). All glass slides were dried and kept under a sterile hood. Cell adhesion was performed comparatively on FN pre-coated and on non-coated multilayers in the presence of 10% FBS. Therefore, one-half of the samples was pre-coated with 20 µg mL−1 FN (YO Proteins) at 37 °C for 4 h, while the other half remained uncoated. Cell adhesion was studied seeding cells at a density of 25,000 cells mL−1 in DMEM on FN pre-coated multilayers and in DMEM+10% FBS on uncoated samples for 24 h. Cellular structures (e.g. filamentous actin, nucleus) were then visualized using fluorescence stains and confocal laser scanning microscopy (CLSM). Briefly, after 24 h of incubation the medium was carefully removed and samples were washed once with PBS. The adherent cells were fixed with 4% paraformaldehyde in PBS solution at room temperature for 15 min, then washed 3 times with PBS, each for 5 min. After 10 min of



permeabilization using 0.1% (v/v) Triton X-100 followed by PBS rinsing (3 times, each 5 min), the nonspecific binding sites were blocked with 1% (w/v) bovine serum albumin solution (BSA) in PBS at room temperature for 2.5 h. The order of staining was designed as follows: a) Phalloidin CruzFluor™ 555 conjugate (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany) for staining filamentous actin b) TO-PRO™-3 (1:400, Invitrogen, Darmstadt, Germany) for nuclei staining. Both dyes were diluted in 1% (w/v) BSA in PBS and cells were incubated in each solution for 30 min at room temperature. After each staining step, we performed PBS rinsing (3 times, each time 5 min). Subsequently, all samples were briefly dipped into ultrapure water and mounted to object holders using Mowiol 4-88 (Calbiochem, Darmstadt, Germany) containing 25 mg mL−1 1,4diazabicyclo[2.2.2]octane (DABCO, Carl Roth GmbH). The samples were then examined with CLSM (LSM 710, Carl Zeiss, Oberkochen, Germany) applying 10x (cell count), 20x (cell size), and oil-immersion 40x (cell morphology) objectives, respectively. Qualitative image analysis was performed with Zeiss efficient navigation (ZEN 2011) software, while quantitative data of cell parameters, such as cell count, cell area and cell aspect ratio were determined with ImageJ (version 1.50e). The micrographs with different magnification of fibroblasts were obtained from two replicates of each sample. Cell viability. The viability of human dermal fibroblast cells was quantified with QBlue cell viability assay kit (Bio-Chain, Newark, USA) to determine any cytotoxic effects of the multilayers. For cell seeding, 1 mL cell suspension (75,000 cells mL-1) in serum-containing medium (DMEM+10%FBS+1% Pen/Strep/Amphotericin B) was seeded on each sample and incubated at 37°C in a humidified 5%CO2/95% air atmosphere for 24 h. Then, the medium was removed and the cells were washed with PBS once. Subsequently, 150 µL/well of pre-warmed colorless DMEM containing 10% QBlue reagent was added and incubated at 37 °C for another 3 h. Finally, 100 µL of the supernatant from each well was transferred into a black 96-wellplate and the fluorescence intensity values were read out at an excitation wavelength of 544 nm and emission wavelength of 590 nm using a fluorescence plate reader. QBlue solution (10% in DMEM) without cells represented a blank value. The assay was performed with three replicates of each sample.


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Cell proliferation. For tracking the proliferation of cells after 72h in serum containing medium (DMEM+10%FBS+1% Pen/Strep/Amphotericin B), phase-contrast microscopy (Leica EC3, Wetzlar, Germany) using a 10x objective was used to take survey images of triplicates per sample for assessment of surface coverage. A phase-contrast microscope equipped with DMIL camera and image analyzing software LAS EZ (Leica application Suite, V. 2.0.0) was used for this purpose. Statistical Analysis. The final mean values ± the standard deviation (SD) or box plots were calculated for all data. Statistical comparisons were performed using Origin software with oneway analysis of variance (ANOVA), evaluated by post-hoc Tukey’s test. Statistical significance was indicated by *, P was taken as ≤ 0.05.

Results.

Studies on multilayer thickness and surface properties The LbL assembly method was applied here for the coating of solid surfaces with 10 layers of semi-synthetic polyelectrolytes alternating t-Chi and t-CS (Fig 1), carrying out at pH 4 to allow for ion paring between the positively charged amino groups of Chi and negatively charged carboxyl and sulfate groups of CS.23

Fig 1. Structure of thiolated chitosan (t-Chi) and thiolated chondroitin sulfate (t-CS). The highlighted groups show interaction sites for ion pairing (blue circles, -NH2 in Chi, -COOH and HO3SO in CS) and disulfide bond formation (red circles, -SH in both molecules).



The thiol quantity of the [t-Chi/t-CS]5 system was measured with Ellman`s reagent, which is quantitatively converting one thiol into one optical active derivative, having the strongest absorbance at 412 nm. The absorbance values of Ellman`s assay are presented in Fig 2. The reaction solutions corresponding to the [t-Chi/t-CS]5 exhibited an average absorbance of A≈ 0.21 at 412 nm. An average of A≈ 0.19 for +pH 9 (1.5 h), A≈ 0.13 for +pH 9 (3 h), A≈ 0.08 for +pH 9 (4.5 h) and A≈ 0.07 for +pH 9 (6 h) was detected. For the solutions corresponding to the [t-Chi/tCS]5 oxidized by ChT, a much lower value (A ≈ 0.03) was obtained in contrast to that of the control sample (A≈ 0.20). There was no significant difference observed between 4.5 h and 6 h reaction times for pH 9.3 treatment. Thus, the +pH 9, 1.5 h and +pH 9, 4.5 h were chosen as pHcondition for oxidation of thiol groups in the following experiments. Here, the results suggest an almost complete absence of thiol groups in +ChT system, followed by +pH 9, 4.5h. The surface wettability is an important parameter in characterizing biomaterials because wetting properties determine protein adsorption and cell attachment.24 Figure 3 shows a static water contact angle (WCA) of about 13° for the initial [t-Chi/t-CS]5 system, using the sessile-drop method. Hence, the thiolated film possessed a high wettability and can be considered as a hydrophilic coating. The surface modifications with pH 9.3 resulted in slightly increased WCA, but yet WCA values were lesser than 20°. By contrast, the multilayers exposed to +ChT gives rise to a WCA value of 49°, which was statistically significant.


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Fig 2. Results of Ellman`s test to detect free thiols. The figure shows the absorbance analysis of the designed multilayers representing the amount of free thiols. The initial [t-Chi/t-CS]n system (n=10 double layers) stimulated with pH 9.3 (monitored at different times of oxidation: 1.5h, 3h, 4.5h and 6h) and chloramine-T (ChT, oxidation time: 90s). t-: thiolated. Chi: chitosan. CS: chondroitin sulfate. (+) stimulated with. Error bars represent means ±SD, triplicate samples. (*): statistically significant difference (p≤0.05).

Fig 3. Water contact angles of the initial [t-Chi/t-CS]5 multilayers and those treated with pH 9 (monitored at 1.5h and 4.5h times of oxidation) and chloramine-T (ChT). Optical images show spreading of a 0.5 µL droplet of ultrapure water on the multilayer films. (+): stimulated with. Means ±SD, triplicate samples. (*): statistically significant p ≤ 0.05.



Fig 4. Zeta potential of the [t-Chi/t-CS]5 multilayer upon oxidation by pH 9,1.5h and pH 9,4.5h versus chloramine-T (ChT). Measurements were performed in 1 mmol L−1 KCl electrolyte via an acid-to-base pH titration, triplicate samples. (+) stimulated with. Zeta potential is a reflection of type of ionic species and number of charges at the surface of materials and dependent on the ionic strength of the electrolyte solution. Thereby, changes of zeta potential of biomaterials can result in different cellular responses.25,26 Fig 4 shows the change of zeta potential during the titration from acidic to basic pH for the different multilayers. The arrows highlight the initial pH (4) during multilayer formation, physiological pH (7.4) and the oxidizing stimulus (pH 9.3). Little difference in zeta potential in dependence on the pH during titration was observed between the original [t-Chi/t-CS]5 multilayer and after treatment with pH 9.3, except for a lower potential at basic pH values for the treatment +pH 9, 1.5h. For the latter conditions, the point of zero charge (PZC) of these multilayers was around a pH of 3.5, while the most negative zeta potential at basic pH values was below -60 mV. A striking difference was observed when the multilayers were exposed to ChT, which increased the zeta potential at basic pH values above -60 mV, while no PZC could be observed because the zeta potential was -37 mV at a pH 3, at which the titration was started. The surface topography of multilayers is visualized in AFM height images captured in 150 mmol L-1 NaCl (pH 4) solution (Fig 5-a). The images of [t-Chi/t-CS]5 multilayers show a surface texture with randomly distributed spherical globules. The oxidation of the films under +pH 9


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stimuli resulted in a similar surface texture. A greater change was observed after +ChT treatment, as the globular-like structures became visibly smaller than those on the other samples. Along with the topographical images, two roughness parameters Ra (roughness average) and Rq (root mean squared roughness) were assessed to indicate possible changes in surface roughness quantitatively (Fig 5-a). The initial multilayer before any treatments showed a Ra and Rq of 1.98±0.11 nm and 2.56±0.18 nm, respectively. We also report a Ra:1.65±0.11 nm and Rq:2.07±0.15 nm for +pH 9, 1.5h treatment, and a Ra≈ 1.62±0.08 nm and Rq≈2.18±0.10 nm for +pH 9, 4.5h. In contrast, the +ChT treatment was related to have a lower Ra and Rq (Ra: 0.98±0.09, Rq: 1.25±0.12).

Ra= 1.98 ± 0.1 (nm) Rq= 2.56 ± 0.2 (nm)

Ra= 1.65 ± 0.1 (nm) Rq= 2.07 ± 0.1 (nm)

Ra= 1.62 ± 0.1 (nm) Rq= 2.18 ± 0.1 (nm)

Ra= 1.00 ± 0.1 (nm) Rq= 1.25 ± 0.2 (nm)

Fig 5. (a) Topographical images of multilayers using Intermittent Contact Mode in NaCl solution. Roughness average (Ra) and root mean square roughness (Rq) for multilayer systems derived from Atomic Force Microscopy in NaCl solution are shown below the images. (b) Distribution curves of E moduli demonstrate a force-map of an area of 3 x 3 µm2 for each sample



with a lateral resolution of 256 x 256 pi2. The whole experiment was performed in duplicate. ChT: chloramine-T. (+): stimulated with. The mechanical properties of multilayers before and after oxidation were also investigated using AFM in 150 mmol L-1 NaCl (pH 4) solution, too. Elastic modulus (E) can be used to characterize the mechanical properties of polyelectrolyte multilayers, whose elasticity were tuned by varying the cross-linking degree in recent work of other groups, as well.27 From the E distribution graphs (Fig 5-b), the mean E modulus of the [t-Chi/t-CS]5 multilayer was ∼110 kPa. The mean E modulus after +pH 9, 1.5h treatment was ∼124 kPa, and ∼130 kPa after +pH 9, 4.5h treatment. However, in contrast to this, the treatment with ChT resulted in a lowering of E modulus to ∼76 kPa. The thickness of the multilayers was measured with ellipsometry in dry and wet conditions and is shown in Fig 6. As can be seen the initial system [t-Chi/t-CS]5, measured in air, exhibited a dry thickness of ≈12 nm. The pH-oxidized multilayers showed a minor decrease in dry thickness, as the +pH 9, 1.5h and + pH 9, 4.5h treatment yielded multilayers with a thickness of ≈11 nm. Further, the initial film thickness was reduced by 2 nm after treatment with ChT, having a dry thickness of ≈10 nm. After immersing the multilayers in 150 mmol L-1 NaCl, the thickness of the [t-Chi/t-CS]5 multilayers increased by approximately 7 nm to attain ≈19 nm, showing the highly hydrated nature of the thiolated multilayers as suggested by WCA results as well. The multilayers treated with pH 9, 1.5h and pH 9, 4.5h showed an enhanced thickness of ≈23 nm and ≈22 nm, respectively. In opposite, by employing ChT, a wet thickness of about 18 nm only, was measured.


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Fig 6. The calculated dry thickness and wet thickness (swelling capacity) of initial [t-Chi/t-CS]5 system compared to the triggered models by pH 9 (1.5h and 4.5h) and chloramine-T (ChT) studied with ellipsometry. The wet thickness examines at the aqueous NaCl/silica interface. (+): stimulated with. Error bars represent ±SD, thickness measurements were done twice for each sample. Studies on fibronectin adsorption and fibroblast adhesion Protein adsorption is one of the first events during interaction of biomaterial surfaces with the biological environment.28 FN adsorption on multilayers was studied using FITC-labelled FN as done in previous studies.29 Here, first the depletion of FITC-FN from supernatant solution reiterating the adsorption of the protein on the multilayers was studied (Fig 7-left). The results show little difference between FITC-FN content in the supernatants, indicated by similar fluorescence intensities after treatment with +pH 9, for 1.5h and 4.5h in comparison to the nontreated multilayer film. By contrast a significant drop in fluorescence indicating strong adsorption of FITC-FN was observed after exposure of multilayers to ChT. FITC-FN was released after washing of wells by incubation with 0.1 mol L-1 NaOH solution (Fig 7-right). Compared to the initial multilayer, the +pH 9, 1.5h and +pH 9, 4.5h treatments caused significantly higher fluorescence indicating that more FITC-FN could be desorbed compared to the [t-Chi/t-CS]5 multilayer. By contrast, no difference in fluorescence was found for ChT-



treated multilayers compared to the original system, which was strikingly different to the finding of the analysis of supernatants.

Fig 7. Fluorometric determination of the FITC conjugates of Fibronectin (FN). Left: depletion of FITC-FN from supernatant solution corresponds to the adsorption of the protein on the multilayers. Grain :1300. Right: FITC-FN was desorbed after washing of wells and incubation with 0.1 mol L-1 NaOH solution. Gain: 2000. ChT: chloramine-T. (+): stimulated with. Means ±SD of 8 samples per condition. (*): statistically significant. Human dermal fibroblast cells were allowed to attach on the different multilayers samples either serum-free after pre-coating with FN or in the presence of 10% FBS. Cells were cultured then for 24 h to show the effect of the differently treated multilayers on cell adhesion. Fig 8-a shows confocal images of cells stained for actin and nuclei at a lower magnification to permit an overview. Cell attachment on the untreated [t-Chi/t-CS]5 multilayer was considered as a control to judge the effect of the different oxidation methods on cell adhesion. Results of cell adhesion studies are shown in Fig 8-b. The studies yielded average values of 13± 7, 23± 5, 30± 9, 38± 8 cells mm-2 of [t-Chi/t-CS]5, +pH 9, 1.5h, +pH 9, 4.5h, and +ChT treated multilayers for fibroblast adhesion in the presence of FBS. This means with respect to the initial system, +pH 9, 1.5h, +pH 9, 4.5h and +ChT treatment increased the cell number by about 1.7-fold, 2.3-fold, and 2.9-fold, respectively. Attachment of fibroblasts on the FN-coated systems was enhanced in comparison to


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the use of serum only. Fibroblast adhesion was found to be of 34± 14, 40± 14, 48± 15, and 101± 15 cells mm-2 found for [t-Chi/t-CS]5+FN, +pH 9, 1.5h+FN, +pH 9, 4.5h+FN, and +ChT+FN surfaces, respectively (see Fig 8b again). Viability of cells after exposure to the different multilayers was tested with QBlue assay. Results of this study show clearly that oxidation of [t-Chi/t-CS]5 multilayer did not exert any adverse effect because the higher fluorescence intensities that are related to the conversion of the QBlue reagent into a fluorescent product by metabolically active cells indicate the absence of toxicity. In addition, metabolic activity was also related to the previous number of adherent cells described above. These findings were confirmed by studies on cell proliferation after 72h done by phase contrast microscopy studying surface coverage with cells (Fig 8-d). Here the +ChT system, exhibits the highest surface coverage, which shows that the growth of fibroblasts was promoted by oxidation of multilayers particularly through ChT, but also after their treatment at pH 9 for 4.5h when compared to the initial [t-Chi/t-CS]5 multilayer.




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Fig 8. a) Representative confocal images of fibroblast cells cultured on the different systems (for 24h) in absence and presence of human plasma fibronectin (+FN) at concentration of 20 µg mL−1. The cells stained with Phalloidin CruzFluor™ 555 conjugate (red) and TO-PRO3 (blue) dyes. Magnification: 10x. Scale bar: 50 µm. b) Analysis of the attached cell numbers per square mm for each of the conditioned surfaces. Results represent duplicate sets c) Viability of human dermal fibroblasts after having performed the QBlue assay on samples after 24 h incubation. The pink color medium in QBlue test stands for a high metabolic activity of cells. Results obtained from triplicate sets. d) Representative images of fibroblasts after 72h culturing on each system using phase-contrast mode. Magnification: 10x. Scale bar: 100 µm. ChT: chloramine-T. (+): stimulated with. Error bars represent ±SD. (*): statistically significant.



As cell spreading is a different marker than cell adhesion, we studied how these multilayers affected morphology, spreading and shape of fibroblasts with and without FN pre-coating after incubation for 24h (Fig 9). Fibroblasts plated on [t-Chi/t-CS]5 multilayer in serum-containing medium (no FN) showed signs of beginning spreading and polarization. Fibroblasts plated on pH 9-treated samples (pH 9, 1.5h and 4.5h) spread only little and had a more stellate morphology. By contrast, treatment of multilayers with +ChT resulted in a large increase of cell size and strong polarization of cells, which acquired a typical triangular fibroblast morphology. The precoating of surfaces with FN promoted a more elongated cell shape with more prominent stress fibers on all the samples except pH 9, 1.5+FN (Fig 9-a, second row). Immune fluorescence images were also used to quantify cell area (Fig 9-b) and aspect ratio (Fig 9-c) of all samples. As can be seen in Fig 9-b for multilayers [t-Chi/t-CS]5, +pH 9, 1.5h and +pH 9, 4.5h, fibroblasts plated in serum-containing medium did not show differences of the cell area. However, cells seeded on +ChT treated multilayers were significantly larger, wherein 50% of the observed cells were at their area between 2800 and 4300 µm2. The median values are ~3400 µm2 for the +ChT versus ~1400 µm2 for the initial [t-Chi/t-CS]5 multilayer, which corresponds to a 2.4 fold increase in cell size. The green boxes represent the multilayers covered by FN. Pre-coating of multilayers with FN caused a distinct increase in the spreading of cells on all substrata except +pH 9, 1.5h+FN, wherein the lowest median value (~1100 µm2) was found among all the conditions (Fig 9-b). Fibroblasts plated on +ChT+FN multilayers, however, had median cell sizes of 3900 µm2. Fig 9c displays the aspect ratio of fibroblast that is related to polarization of cells. The results show that among no FN pre-coated samples only the oxidation of multilayers with ChT caused an increase in cell polarization, while fibroblasts plated on multilayers treated by exposure to pH 9 remained rather round, which later was slightly different compared to the initial [t-Chi/t-CS]5 multilayer. In addition, the pre-coating of multilayers with FN had little effect on cell shape compared to the culture in the presence of serum, except in +ChT+FN.


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Fig 9. a) Representative cell morphological properties for the different systems in absence and presence of human plasma fibronectin (+FN) at concentration of 20 µg mL−1. After 24 h of incubation the F-actin (filamentous) components of the cytoskeleton and nuclei were labeled using Phalloidin CruzFluor™ 555 conjugate dye (red) and TO-PRO3 dye (blue), respectively. Magnification: 40x. Scale bar: 50 µm. Box plots showing cell area (b) and cell aspect ratio (c) analyses are derived from confocal micrographs of two replicates per sample. The bottom of each box is the 25th percentile, the top is the 75th percentile, and the line in the middle is the 50th percentile. The spread of the data is shown by whiskers above and below each box. The mean score for a group is indicated by a □ sign and the outside values are marked by × signs. Spreading out the blue dots on the left side of the boxes helps to find multiple occurrences of a given score. ChT: chloramine-T. (+): stimulated with.

Discussion.

Stimuli-responsive multilayers made of polysaccharides have attracted considerable interest during the last years.30 Particularly, the control of protein adsorption and cell adhesion was achieved by different cross-linking strategies from the use of small bivalent cross-linker31 to intrinsic cross-linking obtained for example by imine32 or disulfide bond formation33, which later provides the systems also with redox-dependent stimuli-responsive behavior.34 Previous work of the authors has shown that multilayers can be formed from thiolated chitosan (t-Chi) and


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thiolated chondroitin sulfate (t-CS) as biocompatible polyelectrolytes that provide not only charged species for ion pairing to make multilayers by the LbL approach, but has also the potential for intrinsic cross-linking by oxidation of thiols and formation of disulfide bonds16. Hence, mechanical and surface properties of multilayers might change after oxidation, which will result in changes of cell adhesion. Two protocols from literature were modified for the present study including oxidation of thiols by high pH18 or exposure to the oxidant chloramine-T (ChT)19.

Studies on thiol content, multilayer thickness and surface properties As described in our previous work, a small fraction of primary amines (13.7%) of Chi and carboxylic groups (32.5%) of CS were modified with thiol groups, preserving their charged nature for use in LbL assembly, while integrating the beneficial cross-linking option35 of disulfides into LbL platforms. The significant decrease of thiol concentrations detected with Ellman`s reagent (Fig 2) after exposure to pH 9.3 confirmed the oxidation of thiols, which was increasing with time of exposure. Ellman`s test showed that thiol concentration was lowest after use of ChT, demonstrating that ChT had the strongest oxidative potential toward the [t-Chi/tCS]5 system. Oxidation of free thiols may lead also to formation intra- and intermolecular disulfide bonds. However, a direct spectroscopic detection of the small fraction of disulfide bonds was not possible because there is actually a slight difference in the range where sulfide/disulfide is detected compared to free thiol groups as also found in a previous work of Castner36 and Moulder et al

37

. Hence, other methods were used to detect the effect of the

oxidation procedures on intrinsic and surface properties of multilayers that could affect protein adsorption and cell adhesion. Wetting properties and surface potential are important determinants of protein adsorption and cell adhesion.26 Water contact angle (WCA) measurements showed that [t-Chi/t-CS]5 multilayer were quite hydrophilic due to the presence of ionic groups (NH3+, COO-, OSO3-) and polar groups like OH with contact angles close to 10°. The exposure of multilayers to pH 9.3 led to slight but significant increase of WCA to about 20°, which indicates some chemical changes that might be related to the oxidation of the thiols. A more dramatic change was observed after the treatment with ChT when the WCA increased to values of about 50°, which was not expected. This was a first hint that the oxidation with ChT may not only lead to decreased thiol content and



enhanced disulfide formation as observed in a previous work19, but that ChT may undergo further side reactions with amino groups38, 39 leading to a loss of primary amines and possibly a permanent incorporation of ChT into the multilayers because of its highly reactive nature.40 Because of the aromatic ring, the presence of ChT in and on multilayers could increase WCA considerably. A further hint about the different nature of oxidation processes by pH 9.3 and ChT treatment was obtained by streaming potential measurements. Here, titration curves of the [tChi/t-CS]5 multilayer before and after treatment with pH 9.3 showed a very similar behavior, which is typical for swollen surface layers with zwitterionic character meaning that in the low pH region the polycation Chi dominates leading to positive zeta potentials, while the polyanion CS rules in the basic pH region leading to negative surface potentials.41 By contrast, the strong decrease of zeta potential in the acidic region after treatment with ChT therefore indicate a loss of the primary amines of chitosan. Apparently, the treatment with ChT is reducing the number of primary amines, which can proceed through displacement of chlorine from the reactive nitrogenchlorine species of the benzene ring in ChT.38,39 According to literature,38 the mechanism for reaction of ChT with amines is a chlorination reaction where an electrophilic chlorine is attacking the nucleophilic amine. The result is a much less nucleophilic chloramine because electrons are much more broadly distributed over N-Cl than N-H. Figure 10 shows a possible scenario of the additional effects of ChT by covalent and adsorptive interaction with [t-Chi/tCS]5 multilayers.

Fig 10. Illustration of the potential side reaction of chloramine-T (ChT) beside the oxidation of thiols and formation of disulfide bonds with a chlorination of primary amino groups of chitosan and an adsorptive binding inside multilayers.


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Oxidation of thiols that may lead to disulfide formation inside multilayers, which was expected to increase the E modulus of multilayers as observed in other studies using small bivalent crosslinker chemistries.42 In fact, such changes in direction of increased stiffness of multilayers were observed after the treatment with pH 9.3 showing higher E moduli than the original multilayer. Standing in contrast, a drop in E modulus (from 110 to 76 kPa) was observed between samples tested in the untreated and ChT-stimulated states. One might conclude here that ChT is less effective in formation of disulfide bond formation because E modulus was raised in the work of other authors.43 In a further work, Wang et al. used 12 bilayers of poly-L-lysine (PLL) and thiolmodified hyaluronan, where ChT obviously enhanced the stiffness.19 However, we noticed that Ellman results confirmed almost a complete absence of thiol groups in +ChT treated samples. Evidence from zeta potential measurements showed a decrease of zeta potential in the acidic range that indicates a chemical reaction of ChT with the amino groups of chitosan. This would reduce the number of primary amines that can undergo ion pairing with acidic groups from thiolated chondroitin sulfate and thus could cause the softening of multilayers due to reduced intermolecular cross-linking. Ellipsometry data show that the films possessed a thickness between 10 to 23 nm with little differences among the dry films although thickness was lowest for films treated with ChT, which indicates a compaction of multilayers possibly by hydrophobic interactions due to the incorporation of ChT molecules. This may also relate to the finding that these multilayers (+ChT) swelled least when immersed into NaCl solution because of a less hydrophilic nature of the films as found also in contact angle measurements. By contrast, the treatment of the [t-Chi/t-CS]5 system with pH 9.3, revealed a higher swelling of these multilayers compared to the initial film or those oxidized by ChT. Previous studies with similar multilayer films have shown substantial swelling of polyelectrolyte multilayers with weak acidic and basic groups at high pH regions above pH 9 that were only partially reversible.44

FN adsorption and adhesion studies with fibroblasts. Protein adsorption analysis can provide first information about cell adhesive properties of biomaterials surfaces45 and is an important parameter since, cells require the presence of ligands for integrins (a family of cell surface receptors) for a firm attachment, growth and survival.28 FN, which is synthesized by many cells from connective tissue and contained also in blood plasma



and serum46 was used here after labelling with fluorescein (FITC-FN) to monitor how the oxidation of multilayers affects adsorption of proteins that interact with glycosaminoglycans like CS due to FN heparin-binding domains47 and with integrin cellular adhesion receptors48. The studies on depletion of FITC-FN from supernatant solution due to its adsorption on the multilayers found little differences between the [t-Chi/t-CS]5 before and after oxidation by pH 9.3, but a significant drop when exposed to ChT-oxidized multilayers. Studies on wetting properties revealed that multilayers treated with ChT had much higher water contact angles, which is also related to more protein adsorption as found in many other studies.49 Moreover the desorption experiment performed with NaOH showed then that oxidation of multilayers by exposure to pH 9 is increasing the adsorption of FITC-FN in an oxidation time-dependent manner because significantly more FITC-FN was released from pH 9, 4.5h multilayers. In contrast to the depletion of FITC-FN only a small fraction of the adsorbed protein was desorbed from +ChT and the level of protein recovery remained similar to that of the initial film. Reasons for the inability to mobilize FITC-FN can be due to hydrophobic interactions with ChT inside the multilayers in similar manner like the FITC-FITC interaction described recently by Prokopovic et al.50 and are also well in line with a previous work51 that demonstrated that NaOH is not able to release FITC-FN completely from polyelectrolyte multilayers composed of collagen I and CS. The apparently higher quantity of FN adsorbed on ChT-oxidized multilayers may also affect its conformation because denser packing stabilizes or enhances the cell-adhesive properties of the molecules as found in other studies.52 Initial adhesion and spreading of cells on biomaterials is related to organization of integrins in focal adhesions and signal transduction processes that control expression of genes responsible for cell growth and differentiation.53 Hence, it can be helpful to predict the performance of biomaterials in clinical settings. Surface chemistry, wetting properties, surface potential, topography and mechanical properties of biomaterials affect protein adsorption and cell adhesion.3 A first important finding was that the adhesion of fibroblast on multilayers pre-coated with FN, but also in absence of FN but with addition of 10% serum was related to the findings on FITC-FN adsorption. On surface that adsorbed more FITC-FN, also more fibroblasts were detected. A further important finding was made by the QBlue assays which detects viable cells showing that none of the treatments had any toxic effects because signal intensities of the QBlue were well related to the number of cells counted from microscopy images. The multilayer films


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studied here were highly hydrophilic as found for the initial [t-Chi/t-CS]5 multilayer, but also after treatment with pH 9.3. These multilayers had also very similar zeta potentials, but differed slightly in E moduli because the treated films possessed higher E moduli. Normally, one would expect more cell adhesion and spreading on stiffer films, which holds for the number of adhering cells, but fibroblast spreading and polarization was even more developed on the initial [t-Chi/tCS]5 multilayer. We found in our previous work that thiolated multilayer films possessed a superior biocompatibility when compared to their non-thiolated precursor molecules.16 Since, oxidation by pH 9.3 exposure reduced the quantity of free thiols as shown with Ellman`s test, this might be related to slightly lower cell spreading and lesser polarization of cells, particularly when compared to FN pre-coated multilayers. Indeed, highest adhesion, spreading and polarization of cells was observed on [t-Chi/t-CS]5 multilayers oxidized with ChT. This was maybe contradictory to the observed softening of multilayers, although the decrease of about 34 kPa was not that small, but well related to the decreased wettability with WCA of about 50°, which is close to the so-called biocompatible zone as described in previous work.49 Hence, although ChT treatment did not yield the expected increase in E modulus as it was expected from the anticipated increase of intermolecular disulfide formation, the change in surface properties had obviously a promoting effect on the cell adhesive properties of the multilayers. Similar findings were made recently with other multilayer films based on poly-L-lysine and hyaluronan treated by small bivalent cross-linker showing that changes in physical properties seem to be of similar significance like the mechanical properties of surfaces.54 Conclusions. Overall, the [t-Chi/t-CS]5 system can provide a useful platform for responsive multilayer systems that may be tailored towards protein adsorption and cell adhesion by different oxidative stimuli useful for controlled drug or growth factor delivery, but also as implant coating or tissue engineering applications. The cross-linking by different actuating functions, such as alkaline pH or ChT changes not only mechanical and surface properties, but has also strong effects on cell adhesion and spreading, which was related to the adsorption of the extracellular matrix protein FN. On the other hand, the reduction of disulfide bonds by mild reducing agents may be also applicable during in vivo application of such films or coatings, reversing effects of cross-linking leading to lowered cell adhesion or supporting the release of bioactive factors like cytokines,



which can be useful for wound healing and other medical applications of films and scaffolds. In a future publication, we will demonstrate the adaptability of the multilayer systems based on tChi and t-CS towards reversibly switchable protein adsorption/desorption and cell attachment/detachment.

Acknowledgements The technical assistance of Mrs. Marlis Porobin performing the zeta potential measurements is highly appreciated. This work was part of the High Performance Center Chemical and Biosystems Technology Halle/Leipzig and supported by the European Regional Development Fund (ERDF) and Deutsche Forschungsgemeinschaft grant Gr1290/10-1 and Gr1290/11-1. References [1]

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Graphical Abstract


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Stimuli-Responsive Multilayers Based on Thiolated Polysaccharides

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