Cyclic Redox-Mediated Switching of Surface Properties of Thiolated

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Biological and Medical Applications of Materials and Interfaces

Cyclic Redox-Mediated Switching of Surface Properties of Thiolated Polysaccharide Multilayers and Its Effect on Fibroblast Adhesion Pegah Esmaeilzadeh, Matthias Menzel, and Thomas Groth ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018

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ACS Applied Materials & Interfaces

Cyclic Redox-Mediated Switching of Surface Properties of Thiolated Polysaccharide Multilayers and Its Effect on Fibroblast Adhesion Pegah Esmaeilzadeh1,2, Matthias Menzel3, 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 for 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

Abstract Advanced technologies for controlled cell adhesion and detachment in novel bio-interface designs profit from stimuli-responsive systems able to react to their environment. Here a multilayer system made of thiolated chitosan (t-Chi) and thiolated chondroitin sulfate (t-CS) was constructed, with the potential of switchable inter- and intramolecular thiol/disulfide interactions representing a redox-sensitive nanoplatform. Owing to the formation and cleavage of inherent disulfide bonds by oxidation (Oxi) and reduction (Red), surface properties of the multilayer can be controlled towards protein adsorption/desorption and cell adhesion in a reversibly manner. Oxidation of thiols by chloramine-T (ChT) promotes fibronectin (FN) adsorption and fibroblast cell adhesion, while the reduction by tris (2-carboxyethyl) phosphine (TCEP) reverses these effects leading to low FN adsorption and little cell adhesion and spreading. These effects on the biological systems are related to significant changes of wetting properties, zeta potential and mechanical properties of these multilayer films. The system presented may be useful for biomedical applications as responsive and obedient surfaces in medical implants and support tissue regeneration. Keywords: redox-responsive multilayers; instructive biomaterials; thiolated polysaccharides; fibronectin adsorption; fibroblast adhesion.

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1. Introduction. The control of protein adsorption on and subsequent cell interaction with biomaterials surfaces are of great importance for the function of implant materials in diagnostic and therapeutic applications, because they are related to inflammation,1 wound healing 2 and tissue regeneration3. Cell adhesion as fundamental process during implant-tissue interaction represents a dynamic process dependent on chemical

4

and mechanical

5

that surface wetting properties and surface potential

cues. In fact, it has been shown previously 6,7

or mechanical properties of materials 8 are

effective modulators of adhesion and subsequent fate of cells. Now, researchers have taken this concept of cell behavior management a step further by creating a ‘dual-mode’ system that can provide both active (on) and inactive (off) states such as in areas of adhesiveness and bioinertness.9 Biomaterials for controlled attachment/detachment of cells have been achieved by thermo-responsive,10 electroactive,11 photosensitive,12 pH-responsive,13 enzymatic cleavable,14 host-guest chemistry-dependent

15

and magnetite16 modified surfaces. However, existing smart

surface technologies can be particularly limited by their lack of reproducible and reliable switching performance over multiple cycles.17 This scenario represents an opportunity for switchable and reversible materials changing cell adhesion, which has been recognized as a crucial event in biomaterials applications,18 but also in a wide range of diseases including arthritis, cancer, osteoporosis, atherosclerosis, asthma, or multiple sclerosis

19,20

. A smart system that can be suggested for this purpose are “thiol-based

redox switches”, which grant both change of chemical and mechanical properties to control protein adsorption, cell adhesion, growth and function.21 A synthetic polymeric thiol reservoir can equip us with reversible inter- and intramolecular thiol–disulfide exchanges, which are kept running smoothly in nature.22 Evidences of the significance of disulfide bond formation - a reaction typically considered to be stabilizing and structure forming - regarding cellular interfacial performances were formerly presented.23 This view can be substantiated by constructing a redox-responsive polyelectrolyte multilayer system. Only a few attempts, however, have been performed using a ‘dual-mode’ redoxresponsive polyelectrolyte multilayer directly for cell-based applications.24 A layer-by-layer charged polyelectrolyte model closely matches the design of a particular functionality required to interface, imposing a defined cell program as it is well-documented by other researchers.25,26


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Such model can provide sufficient level of detail of the molecular arrangement to define precisely the orchestrated matrix,27 which aids in manipulation of the functions and physicochemical properties involved.28 In this study, the target naturally 29 derived cell instructive multilayer system is comprised of 5 double layers of semi-synthetic biopolymers alternating thiolated chitosan (t-Chi, a positively charged polysaccharide) and thiolated chondroitin sulfate (t-CS, a negatively charged glycosaminoglycan), abbreviated as [t-Chi/t-CS]5 .30,31 We introduce here stepwise oxidation with chloramine-T (ChT)

31

and reduction with tris (2-carboxyethyl) phosphine (TCEP)

32

to

demonstrate a switchability of the system. Thereby, the use of ChT results in oxidation of thiols and disulfide linkages (S-S) 31, which later reduce by exposure to TCEP to free thiols (-SH). The resulting changes after oxidation and reduction in surface and intrinsic properties of multilayers are shown in terms of zeta potential, mechanical properties and the bioactivity regarding adsorption of fibronectin and adhesion of human fibroblasts. Results are reported herein.

Materials and Methods section. Materials. Native chondroitin sulfate A (from bovine trachea, Mw ~25 kDa, sulfation degree of 0.8) provided by VWR International GmbH (Dresden, Germany) and native low molecular weight chitosan (Mw ~62 kDa, 84.6% degree of deacetylation) by Heppe Medical Chitosan GmbH (Halle, Germany) were used as polyelectrolytes in this research. Chloramine-T trihydrate (ChT) purchased from Diagonal GmbH & Co. KG (Münster, Germany) and Tris-(2carboxyethyl)-phosphin hydrochlorid (TCEP) from Carl Roth GmbH (Karlsruhe, Germany) were employed as redox-agents. 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. Oxidation and reduction of [t-Chi/t-CS]5 multilayers. Synthesis and characterization of thiolated chitosan (t-Chi, ≈13.7% thiol content) and thiolated chondroitin sulfate (t-CS, ≈32.5% thiol) and subsequent formation and characterization of the 5 double layered thiolated polyelectrolyte multilayer films were described in detail in our previous publications.30,

31

Herein, the study particularly focuses on the applied method of oxidation of thiols by



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chloramine-T (ChT),31 wherein the multilayers were immersed in ChT solution (5 mmol L-1, in filtered 150 mmol L-1 NaCl solution, pH 4) for 90 s, and a method of reduction of thiols by Tris(2-carboxyethyl)-phosphin hydrochlorid (TCEP), during which the multilayers were immersed in TCEP solution (5 mmol L-1, in filtered 150 mmol L-1 NaCl solution, pH 4) for 30 min. Both methods were followed by washing using filtered 150 mmol L-1 NaCl (pH 4) for 5 min, 5 times and briefly washing with Milli-Q water. For filtration of solutions we used Whatman® syringe filters (cellulose acetate ø30mm, 0,2µm, Diagonal GmbH & Co. KG, Münster, Germany), accordingly. The oxidation and reduction steps that are party done in sequence will be abbreviated in the following sections by Oxi and Red, receptively. If several oxidation and reduction steps were carried out then it was labeled either (Oxi/Red)n or (Red/Oxi)n with n for the number of steps and which process was started first. Measurements of thiol content of multilayers. Using UV–Vis spectrophotometer (Specord 200, Analytik Jena AG, Jena, Germany) at a wavelength of 412 nm, Ellman`s reagent [5,5′dithiobis(2-nitrobenzoic acid)] in phosphate buffer (0.1 mol L-1, pH 7.5) was applied to label the thiol fraction of the triggered-multilayers by redox stimuli, as explained previously.30,

31

To

improve assay sensitivity, the selected number of layers was n=20. All tests were conducted in triplicate. Zeta-potential measurements. The analysis of the charged solid/liquid interface was carried out with a SurPASS electrokinetic analyzer (Anton Paar, Graz, Austria), while 1 mmol L-1 potassium chloride (Carl Roth GmbH) solution as model electrolyte flows through the measuring cells with a flow rate of 100-150 mL min-1 at a maximum pressure of 300 mbar. To create a streaming potential between two identical surfaces of each condition mounted in parallel, the pH of the electrolyte changed by 0.1 mol L-1 sodium hydroxide from pH 3.0 to pH 10.0 to assess an acidto-base pH titration. Each test was done in triplicate. Study of mechanical properties of multilayers. Force map spectroscopy experiments of the multilayers (deposited on glass substrates in duplicate) were performed using Atomic Force Microscopy (AFM) (nanowizard® II, JPK-Instruments, Berlin, Germany). Here, a standard silicon nitride cantilever (MLCT, Bruker Nano Inc. Santa Barbara, USA) in a standard liquid cell (JPK-Instruments) containing filtered 150 mmol L-1 NaCl (pH 4) was employed. Realizing the importance of the unchanged tip state plus determining tip radii for later calculation of Elastic


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modulus, all cantilevers were inspected by Scanning Electron Microscopy before and after experiments. Employing the thermal noise method for the force-constant calibration,31 the forcemaps were acquired over an area of 3 x 3 µm2 for each sample with a lateral resolution of 256 x 256 pi2, wherein each pixel (pi) contains a single spectroscopy force curve. Fitting each of the force curve to an advanced Hertzian model for the corresponding spherical indenter geometry 31 gives quantitative measurements of material stiffness (Elastic moduli), using JPK Data Processing V5.0.85 and Gwyddion V2.49 software. Measurement of fibronectin adsorption. [t-Chi/t-CS]5 multilayers and its various stimulated models were directly produced in black 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany). Preparing FITC-labeled

31

human plasma fibronectin (FN, YO Proteins, Huddinge,

Sweden) solution concentration of 20 µg mL-1 in PBS of pH 7.4, all the multilayers were simultaneously incubated with 100 µL of FITC-FN for 4 h at 37˚C, covered with Parafilm and Al-foil. Then, the culture supernatants were transferred into new black 96-well plates to quantify the fluorescence intensity (FI) with a plate reader (FLUOStar Optima, BMG Labtech GmbH, Offenburg, Germany), reiterating the measure of adsorbed FITC-FN on ultra-thin multilayers. To this purpose, the excitation filter was set at 485 nm, the emission filter at 520 nm and the gain at 1300 (PBS solution was the blank). Each experimental design involves using 8 repeats. Adhesion studies of human dermal fibroblasts (HDFs). Cell culture. Using Dulbecco’s modified Eagle’s medium (DMEM, Biochrom, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS, Biochrom) and 1% antibioticantimycotic solution (Penicillin/Streptomycin/Amphotericin B, AAS, Promocell), HDFs (Promocell, Heidelberg, Germany) were grown at 37 °C in a humidified 5% CO2/95% air atmosphere

(NuAire,

MN,

USA).

With

applying

0.25%

trypsin/0.02%

ethylenediaminetetraacetic acid (EDTA, Biochrom) cells were detached at 37 °C for 5 min and with a subsequent addition of serum-containing DMEM the trypsin was inactivated. The cell suspension was then centrifuged at 250 rpm at room temperature for 5 min, and cells resuspended in pure DMEM. Cell viability studies. To learn any cytotoxic effects of the treated multilayers, the viability of HDFs cells was measured with QBlue cell viability assay kit (Bio-Chain, Newark, USA). After seeding 1 mL cell suspension (75,000 cells mL-1) in serum containing medium



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(DMEM+10%FBS+1% Pen/Strep/Amphotericin B) on each sample and incubation at 37°C in a humidified 5%CO2/95% air atmosphere for 24 h, the medium was carefully removed and the cells were rinsed with PBS, once. Adding 150 µL/well of pre-warmed colorless DMEM containing 10% QBlue reagent, the samples incubated at 37 °C for an additional 3 h. At the end, 100 µL/well of the supernatant was transferred into a black 96-wellplate and the FI values were read out @ 544 nm excitation and 590 nm emission, applying a fluorescence plate reader. QBlue solution (10% in colorless DMEM) without cells was used as blank, and 3 replicates per sample were analyzed. Cell adhesion studies. The LbL process was applied on cleaned and sterilized

31

round glass

cover slips, followed by the planned modification steps either ChT or TCEP. The samples were washed briefly with Milli-Q water to prevent salt crystallization. Then, the water was aspirated and plates were kept under a sterile laminar flow hood, sheltered from light. In a comparative manner, only half the samples were pre-coated with 20 µg mL-1 FN at 37 °C for 4 h, while the other half remained uncoated. Cell adhesion was assayed on FN pre-coated multilayers and on non-coated multilayers in the presence of 10% FBS through seeding 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. Using fluorescence staining and confocal laser scanning microscopy (CLSM), filamentous actin, nucleus, and vinculin were then visualized. Briefly, after 24 h of incubation the medium was carefully removed and samples were rinsed with PBS, once. Using 4% paraformaldehyde in PBS solution, the adherent cells were fixed at room temperature for 15 min, then washed 3 times with PBS, each for 5 min. Permeabilization was then conducted through applying 0.1% (v/v) Triton X-100 for 10 min, followed by PBS rinsing (3 times, each 5 min). The nonspecific binding sites were further blocked using 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) primary mouse antibody raised against vinculin (1:50, Santa Cruz Biotechnology Int., Heidelberg, Germany) b) goat anti-mouse IgG, Fc-Cy2, MinX Hu,Bo,Rb (1:100, Dianova, Hamburg, Germany) for detection of the target primary antibody. c) Phalloidin CruzFluor™ 555 conjugate (1:1000, Santa Cruz Biotechnology Int.) for staining filamentous actin and d) TO-PRO™-3 (1:400, Invitrogen, Darmstadt, Germany) for staining nucleus. All antibodies and dyes were diluted in 1% (w/v) BSA in PBS and cells were incubated in each solution for 30 min at room temperature. PBS washing (3 times, each 5 min) was performed after each staining step.


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Afterwards, all samples were briefly dipped in ultrapure water and mounted to object holders employing Mowiol 4-88 (Calbiochem, Darmstadt, Germany) containing 25 mg mL-1 1,4diazabicyclo[2.2.2]-octane (Carl Roth GmbH & Co. Kg, Karlsruhe, Germany). For exploring the samples with CLSM (LSM 710, Carl Zeiss, Oberkochen, Germany) we applied 10x (cell count), and oil-immersion 40x (cell morphology) objectives. The Zeiss efficient navigation (ZEN 2011) software, and ImageJ (version 1.50e) were used for qualitative image analysis and quantitative cell count, respectively, where results represent 3 replicates per sample. Cell proliferation studies. At 72 h after cell plating, live monitoring of cell proliferation in serum containing medium (DMEM+10%FBS+1% Pen/Strep/Amphotericin B) was performed through a 10x objective of a phase-contrast microscopy (Leica EC3, Wetzlar, Germany) equipped with DMIL camera and image analyzing software LAS EZ (Leica application Suite, V. 2.0.0) to take survey images of triplicates per sample for estimation of the surface coverage. Statistical Analysis. While the final mean value ± the standard deviation (SD) were calculated for the data sets, statistical comparisons were done in Origin software employing Kruskal-Wallis ANOVA calculator. Statistical significance was marked by *, and P was taken as ≤ 0.05. 2. Results and discussion. Aiming to design a redox-sensitive multilayer system, the individual layer constituents represented thiolated chitosan (t-Chi) alternated by thiolated chondroitin sulfate (t-CS) with side groups, that permit ion paring (-NH2 in Chi, -COOH and –HO3SO in CS) and covalent inter- and intramolecular bond formation (-SH in both) as demonstrated in our previous studies.30,31 Intrinsic cross-linking of thiols and splitting of disulfide bonds of [t-Chi/t-CS]5 multilayers was achieved by treatment with chloramine-T (ChT) and tris (2-carboxyethyl) phosphine (TCEP), respectively, studying the individual steps of oxidation-reduction responses in a dynamic and reversible approach. The redox-dependent changes of free thiol content, surface properties and mechanics of the multilayer system were investigated using UV–Vis photometry, zeta potential measurements and atomic force microscopy (AFM). The response of the biological system was evaluated by fluorimetric protein adsorption measurements, and cell adhesion and growth studies with human fibroblasts.



Since about 14% of -NH2 in Chi and 33% of -COOH in CS were conjugated with thiol groups,30 Ellman`s reagent was used to detect changes in content of free thiols of multilayers during oxidation-reduction steps by determining the UV-Vis absorbance of Ellman solution exposed to multilayers at 412 nm. According to Fig. 1, non-treated [t-Chi/t-CS]10 multilayers (as reference) exhibited an average absorption of A≈ 0.23 at 412 nm, whereas the average absorbance values of oxidized system (ChT, A≈ 0.04) and reduced system (TCEP, A≈ 0.38) were significantly lower and higher than the reference, respectively. Similarly, the stepwise oxidation and reduction (Oxi/Red)1, A≈ 0.31 yielded statistically higher absorbance values than [t-Chi/t-CS]10 and also initial oxidation with ChT. By contrast, the absorbance of the stepwise reduction and oxidation (Red/Oxi)1, A≈ 0.05 was statistically lower than the [t-Chi/t-CS]10 and TCEP state. These observations confirmed the thiol oxidation potential of ChT versus the disulfide reduction potential of TCEP. Hence, during oxidation-reduction steps (Oxi/Red)1, intra- and intermolecular disulfide bonds formed after exposure to ChT can be reduced to free thiols again using TCEP. It was also possible to re-establish disulfide bonds after reduction with TCEP if these multilayers were exposed again to ChT (Red/Oxi)1. The assumption of a reversibility of redox reaction was supported by the non-significant statistically difference between TCEP and (Oxi/Red)1, also ChT and (Red/Oxi)1 samples.

Figure 1. UV-Vis absorbance analysis following the Ellman`s test for quantification of thiols in multilayers. The [t-Chi/t-CS]n systems were subjected to ChT oxidant and TCEP reductant. t-


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Chi: thiolated chitosan. t-CS: thiolated chondroitin sulfate. n=10: number of double layers. ChT: Chloramine-T. TCEP: tris (2-carboxyethyl) phosphine. Error bars express ±SD, for triplicate samples. (*): statistically significant, where statistically non-significant differences correspond to reversibility of the redox reactions distinguished from the results in the early TCEP and ChT states. Molecular structure illustrations include: C: gray atoms, O: red atoms, S: orange atom, N: blue atom, Na: green atom, Cl: violet atom, H: white atoms and P: yellow atom.

Figure 2. Zeta potential measured vs. pH for multilayers after oxidation-reduction steps either (A) (Oxi/Red)1 or (B) (Red/Oxi)1. Measurements were done in a 1 mmol L−1 KCl electrolyte with stepwise change of pH, for triplicate samples.

Cell-carrier platforms with stimuli-responsive properties for control of cell adhesion and subsequent processes (e.g. apoptosis 33) after alterations of extracellular conditions (e.g. in tumor microenvironments

34

) should react to changes of charge densities and wetting properties of

biomaterial surfaces. To examine the pH-dependent change of surface charge density, Fig. 2 shows zeta potentials measured versus pH of 1 mmol L-1 KCl solutions. The zeta potentials of multilayers were measured after treatment with ChT and TCEP. Zeta potential decreased with increase of pH from acidic to pH7, which is related to deprotonation of amine functionalities of Chi, where complete protonation occurs at pH values below 3.4.35 At pH 3.4, multilayers after reducing steps with TCEP or (Oxi/Red)1 possessed a point of zero charge (PZC), while no PZC was observed for the oxidized state with ChT and (Red/Oxi)1. The range of zeta potentials at pH



4 demonstrates that the reduced state with TCEP shows the highest and the oxidized ChT the lowest zeta potential with -16 and -45 mV, respectively. The graphs in Fig. 2 demonstrate also the reversibility of redox reactions of multilayer during oxidation-reduction triggers regarding zeta potentials, as switching “(Oxi/Red)1” cycle yields almost the same potential curves like TCEP alone (Fig. 2A), and the “(Red/Oxi)1” reaction achieves nearly the same potential curves like ChT (Fig. 2B) showing that surface charge-switching successfully occurs through (Oxi/Red)1 cycles. These changes occur only in the acidic pH (pH

Cyclic Redox-Mediated Switching of Surface Properties of Thiolated

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