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Feb 17, 2017 - One prominent cause of implant failure is infection; therefore, research is focusing on developing surface coatings that render the sur...
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Alternating current electrophoretic deposition for the immobilization of antimicrobial agents on titanium implant surfaces Annabel Braem, Katrijn De Brucker, Nicolas Delattin, Manuela Sonja Killian, Maarten B.J. Roeffaers, Tomohiko Yoshioka, Satoshi Hayakawa, Patrik Schmuki, Bruno P.A. Cammue, Sannakaisa Virtanen, Karin Thevissen, and Bram Neirinck ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16433 • Publication Date (Web): 17 Feb 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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Alternating current electrophoretic deposition for the immobilization of antimicrobial agents on titanium implant surfaces Annabel Braem1*, Katrijn De Brucker2, Nicolas Delattin2, Manuela S. Killian3, Maarten B.J. Roeffaers4, Tomohiko Yoshioka5, Satoshi Hayakawa5, Patrik Schmuki3, Bruno P.A. Cammue2,6, Sannakaisa Virtanen3, Karin Thevissen2, Bram Neirinck1 1

KU Leuven Department of Materials Engineering (MTM), Kasteelpark Arenberg 44 box 2450, 3001 Heverlee, Belgium.

2

KU Leuven Centre of Microbial and Plant Genetics (CMPG), Kasteelpark Arenberg 20, box 2460, 3001 Heverlee, Belgium.

3

Department of Materials Science and Engineering, Chair for Surface Science and Corrosion, Friedrich-Alexander-University of Erlangen-Nuremberg, Martensstrasse 7, 91058 Erlangen, Germany. 4

KU Leuven Center for Surface Chemistry and Catalysis, Kasteelpark Arenberg 23, 3001 Leuven, Belgium.

5

Biomaterials Laboratory, Graduate School of Natural Science and Technology, Okayama University, 3-1-1, Tsushima, Kita-ku, Okayama, 700-8530, Japan.

6

Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Ghent, Belgium. 1 ACS Paragon Plus Environment

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KEYWORDS electrophoretic deposition; alternating current; titanium; biofunctionalization; caspofungin; antifungal activity; biofilm prevention

ABSTRACT One prominent cause of implant failure is infection, therefore research is focusing on developing surface coatings that render the surface resistant to colonization by microorganisms. Permanently attached coatings of antimicrobial molecules are of particular interest because of the reduced cytoxicity and lower risk of developing resistance compared to controlled release coatings. In this study, we focus on the chemical grafting of bioactive molecules on titanium. To concentrate the molecules at the metallic implant surface, we propose electrophoretic deposition (EPD) applying alternating current (AC) signals with an asymmetrical wave shape. We show that for the model molecule bovine serum albumin (BSA), as well as for the clinically relevant antifungal lipopeptide caspofungin (CASP), the deposition yield is drastically improved by superimposing a DC offset in the direction of the high-amplitude peak of the AC signal. Additionally, in order to produce immobilized CASP coatings, this experimental AC/DC-EPD method is combined with an established surface activation protocol. Principle component analysis (PCA) of time-of-flight secondary ion mass spectrometry (ToF-SIMS) data confirm the immobilization of CASP with higher yield as compared to a diffusion-controlled process, and higher purity than the clinical CASP starting suspensions. Scratch testing data indicate good coating adhesion. Importantly, the coatings remain active against the fungal pathogen C. albicans as shown by in vitro biofilm experiments. In summary, this paper delivers a proof-of-concept for the application of ACEPD as a fast grafting tool for antimicrobial molecules without compromising their activities.

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Introduction Implantation of a medical device in the body entails an inherent risk of infection. Due to its compulsory biocompatibility and the repression of the local immune response at the trauma site (locus minoris resistentiae), an implant surface is extremely prone to microbial attachment.1,

2

Subsequent biofilm formation protects these microbes (bacteria and fungi)

from stressful environmental conditions (pH variation, antimicrobial substances). As such, microbial biofilms are highly tolerant to antimicrobial treatment and are thought to be responsible for recalcitrant peri-implant infections despite aggressive systemic antibiotic treatment.3, 4 Modern asepsis practice and preventive peri-operative antimicrobial treatments have only slightly reduced peri-implant infection rates.5 A successful integration in the host tissue can protect the implant surface from colonization by microbial pathogens as the host cells will outcompete microbes from the available binding sites (“race for the surface”).6, 7 As such, improving the colonization of a biomaterial surface by host tissue cells is a valuable first approach to combating infections. This can be achieved either by altering the surface topography or by a chemical modification of the surface.8,

9

However, care has to be taken as surface modifications can also increase the colonization by micro-organisms due to an enlarged surface area.10 Therefore, significant efforts are focused on the development of anti-infective implant surfaces, which are generally categorized as either active antimicrobial surfaces, locally releasing antimicrobial agents, or passive antiadhesive surfaces, repelling microbial pathogens or killing/inactivating them upon contact.11, 12

While a localized delivery of antimicrobials at the target site has obvious advantages over a

systemic drug administration (such as an improved control over toxicity and bioavailability of the dose, no systemic drug exposure,…),13 high local concentrations of antimicrobials are only sustained over a short period of time (i.e. a few days). In this way, only early postsurgical implant infections can be treated efficiently. Moreover, there is a risk of re3 ACS Paragon Plus Environment

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infection and resistance development when antimicrobial concentrations drop to subinhibitory levels.5 Therefore, increasing interest is directed to the covalent immobilization of antimicrobials to the implant surface. It is hypothesized that such non-releasing coatings can eliminate infections at the implant surface over a prolonged period of time while decreasing the toxicity and resistance development risks compared to releasing coatings.11, 12 Studies of vancomycin covalently bound on titanium have shown its in vitro antimicrobial activity against S. epidermidis, which was preserved during several re-challenges, while there was no observable effect on osteoblasts. These results were confirmed in vivo in an osteomyelitis model.2 Similarly, the minimal toxicity and long-term stability profiles of immobilized antimicrobial peptides points to their potential for clinical applications.3 Up to now, covalent anchoring of biomolecules is usually accomplished by chemical grafting, i.e. activation of the surface using coupling agents (e.g. silanes) followed by immersion of the substrate in the biomolecule suspension.14-20 This approach requires long dipping times (several hours up to days) as the immobilization rate depends on the passive diffusion of biomolecules to the surface. Alternatively, an established coating technique for inorganic biomaterials is electrophoretic deposition (EPD), a colloidal processing route based on the electrophoresis of charged entities in suspension under the influence of an external electric field.21-23 This low-cost, low-temperature, high purity process allows to coat complex shapes with a wide range of materials and coating thicknesses. EPD research has traditionally been focused on applications based on organic solvents as the voltages used typically lead to electrolysis in aqueous systems, resulting in gas bubble formation, pH shifts at the electrodes and Joule heating. However, environmental concerns and a growing interest for biotechnological processing has triggered the development of alternative approaches for EPD from aqueous suspensions.24 As such, the use of pulsed direct current (DC) or alternating current (AC) fields in EPD has been suggested as this allows decreasing the water electrolysis 4 ACS Paragon Plus Environment

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and concomitant bubble formation and pH shifts.24-26 Initial results showing defect-free deposits for inorganic particles under these conditions have spiked the interest for the ACEPD of biomolecules and cells.27 The technique has been successfully applied for the deposition of enzymes, such as glucose oxidase, for biosensor applications or for the manipulation of bacterial cells, such as Staphylococcus aureus and Escherichia coli.28, 29 With this paper, we aim to establish a proof-of-concept for the use of AC-EPD as a fast immobilization process for grafting antimicrobial agents on titanium (Ti) implant surfaces. To begin with, the effect of various key AC-EPD parameters on the process performance is explored using bovine serum albumin (BSA), a readily available model protein with well characterized structure. Next, the optimized AC-EPD approach is implemented in the tethering process of the clinically relevant antifungal lipopeptide drug caspofungin (CASP) on Ti. Finally, the resulting CASP-based coatings were tested in vitro for their activity to withstand biofilm formation of the fungal pathogen Candida albicans (C. albicans), the main human fungal pathogen in the oral cavity.30, 31

Materials and methods Materials and chemicals The substrate material used for the optimization and characterization of the AC-EPD process was commercially pure Ti grade 2 and Ti6Al4V alloy grade 5 (sheet as rolled, thickness 1 mm, Salomon’s Metalen). Electrodes of reproducible size (9 x 9 mm) were obtained from the sheet by laser cutting (Lasertek). In order to achieve a reproducible surface chemistry, all electrodes were etched in a 4 wt% HF (40%, Riedel-de Haën) and 20 wt% HNO3 (65% p.a., Chem-Lab nv) aqueous solution during 60 s followed by extensive rinsing in demineralized water and technical ethanol (Disolol®, Chem-Lab nv). Next, the dried electrodes were treated in an autoclave for 1 h at 120°C. Substrates were stored in a desiccator until further use. For the biofilm growth assay, a clinically relevant Ti implant surface was 5 ACS Paragon Plus Environment

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prepared by beadblasting with Al2O3 particles followed by acid etching of machined Ti disks (grade 2, t = 2 mm, Ø = 10 mm, Biotech Dental). Solutions

of

3-aminopropyltriethoxysilane

(APTES,

99%,

Sigma-Aldrich)

and

hexamethylene diisocyanate (HMDI, 98%, Sigma-Aldrich) were prepared in isopropyl alcohol (IPA, 99+%, Chem-Lab nv). BSA (>98%, Sigma-Aldrich), fluorescein isothiocyanate conjugate BSA (FITC-BSA, A9771, Sigma-Aldrich) and CASP (Cancidas, Merck) suspensions were prepared fresh in ultrapure MilliQ water (MQ, Merck Millipore) for each experiment. The isoelectric point (IEP) of CASP was determined using the Marvin package (version 6.2.2) from ChemAxon (http://www.chemaxon.com). Additionally, a bicinchoninic acid kit (BCA, MicroBCATM Protein Assay Kit, Thermo Fisher Scientific) and a Bradford protein assay (Pierce™ Coomassie (Bradford) Protein Assay Kit, Thermo Fisher Scientific) were obtained for characterisation.

Surface functionalisation In order to covalently couple the AC-EPD deposited CASP molecules to the Ti, the substrate was functionalized prior to AC-EPD by silanization followed by grafting with isocyanate groups. Silanization was obtained by immersing the disks in a 5 vol% APTES solution in IPA for 1 h while gently shaking using an orbital shaker. Subsequently, the disks were removed from the solution and rinsed by dipping in MQ followed by drying at 100°C for approximately 1 h. The dried samples were stored in a desiccator until further use. Prior to AC-EPD, the silanized samples were immersed in a 4 vol% HMDI solution in IPA for a few seconds. Afterwards, excess solution was removed by gently dipping the samples in MQ. Because of the high reactivity of HMDI with ambient water, the samples were immediately processed further by AC-EPD.

Alternating current electrophoretic deposition (AC-EPD) A schematic representation of the experimental setup is given in figure 1. Disposable cuvettes (polystyrene, 1 ml, VWR) served as the deposition cell in which electrodes were 6 ACS Paragon Plus Environment

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placed vertically at a fixed distance of 8 mm using a PTFE spacer. AC-EPD was carried out under ambient conditions using either voltage- or current-controlled conditions. The signal was generated using a high resolution digital-to-analog signal output module (NI9269, National Instruments) coupled to a bipolar amplifier (PZD 700M/S, Trek inc). An asymmetric triangular AC-signal with a 50 Hz frequency and an asymmetry of 3, was applied,27 either with or without a DC offset in the direction of the high-amplitude phase. Current, voltage and temperature of the suspension were instantaneously monitored during the experiments using an analog-to-digital acquisition module (NI9223, National Instruments) linked to the monitor channels of the operational amplifier and a multifunctional acquisition module (NI9219, National Instruments) coupled to a shielded K-type thermocouple inserted into the deposition cell. After deposition, the electrodes were removed from the cuvette and gently rinsed to remove unsettled protein molecules by dipping in MQ followed by rinsing in IPA. The rinsed samples were dried at room temperature and stored in a refrigerator at approximately 4°C until further use.

Coating characterization Scanning electron microscopy (SEM) The surface of both coated and non-coated samples was examined by scanning electron microscopy (SEM, Nova NanoSEM 450, FEI) with associated energy dispersive X-ray spectroscopy (EDX, EDAX) operated at standard high-vacuum settings. In order to avoid beam damage of the organic coatings, low-energy imaging was performed by applying a 3-4 keV stage (and sample) bias field, resulting in an effective landing energy of 0.5 keV. For surface profile measurements, 3 SEM images taken at the eucentric height with -10°, 0° and 10° tilting angle, respectively, were reconstructed using the MeX software package (Alicona Imaging GmbH).

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Fluorescence microscopy Deposits produced using FITC-BSA were examined by means of confocal laser scanning microscopy (CLSM, Fluoview FV1000, Olympus) equipped with an UPLSAPO objective (10x magnification, 0.40 numerical aperture) and an DM405/488 excitation dichroic mirror. Samples were excited using an Ar laser at an excitation wavelength of 488 nm. Stacked images (in the z-direction) were acquired with an image size of 512 x 512 pixel² and pixel size of 2.485 x 2.485 µm². Micro bicinchoninic acid (BCA) protein assay A BCA formulation for the colorimetric detection and quantitation of the total protein content was applied. In a 24-well plate, the Ti surfaces were incubated with 700 µl of the BCA reagent for 1.5 h at 60°C applying a plate shaker to ensure continuous mixing of the working reagent. Afterwards samples were analyzed by UV spectrophotometry at 562 nm with a Synergy Mx multi-mode microplate reader (Biotek). The absorbance readings were corrected for the absorbance reading of a pristine Ti surface incubated with the working reagent before determining the biomolecule concentration based on a simultaneously prepared standard series. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) Analysis of the chemical composition of the coatings was carried out using ToF-SIMS (TOF.SIMS 5, ION-TOF GmbH). The samples were irradiated over a 500 x 500 µm² area using a pulsed 25 keV bismuth (Bi3+) cluster ion beam. Positive and negative ion mass spectra were recorded in high resolution mode. During measurement, the primary ion dose density (PIDD) was kept constant at 5 x 1011 ion/cm² in order to ensure static conditions. Fragment ions from 1 up to 800 amu were used for analysis. The Ionspec V4.1 software (ION-TOF GmbH) was used to calibrate all spectra and generate peak lists that could be further processed. Peaks were chosen with the automated peak search option. Principle component analysis (PCA) was performed using the NESAC/BIO MVA Toolbox (Spectragui v2.7 8 ACS Paragon Plus Environment

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standalone,

https://www.nb.engr.washington.edu/mvsa/nbtoolbox)

in

MATLAB

(MathWorks). Spectral data sets were normalized to the total intensity of all selected peaks, square root transformed and mean centered prior to PCA. Fourier transform infrared reflection absorption spectroscopy (FTIR-RAS) The secondary structure of BSA deposited on Ti was analyzed on an FTIR spectrometer (Thermo Nicolet NEXUS 470 FTIR) equipped with a Smart SAGA system (Thermo Nicolet). Using a mask with a window (5 mm in diameter), samples were scanned between 4000 and 650 cm-1 with a resolution of 4 cm-1 and 256 scans were averaged for each spectrum. The secondary structure was quantified through Fourier-self deconvolution of the amide I region (1700 - 1600 cm-1) using the OMNIC software (version 8.3, Thermo Fisher Scientific). The resolution factor, K, and the half-width of the unresolved bands were set to 3.0 and 25 cm-1, respectively. The overlapping bands in the deconvoluted band region were then fitted using the Gaussian function. From the areas of the resolved bands, the secondary structure content was calculated. Scratch adhesion test Evaluation of the coating adhesion was done by scratch testing (Revetest®, CSM Instruments) in a clean environment at ambient temperature. Samples were fixed on the sample holder using super glue applying pressure on the sample edges for 1 min. A tip with spherical indenter (chrome-steel ball, Ø =800 µm) was moved along the coating surface over a 5 µm distance and applying a progressive normal load at a rate of 20 N/min from 3 to 23 N. Afterwards the scratch tracks were evaluated by SEM.

In vitro Candida albicans biofilm growth assay Disks were incubated overnight in fetal bovine serum (FBS, Gibco®, Life Technologies Europe, Paisley, UK) at 37°C. In parallel, C. albicans SC5314 was grown overnight in YPD (1% yeast extract, 2% peptone and 2% dextrose) at 30°C. Silicone tubes (VWR international, reference 228-0730) were cut in fragments of 1.5 cm and sterilized by submerging into 9 ACS Paragon Plus Environment

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ethanol. Disks were washed twice with phosphate buffered saline (PBS, 8 g/l NaCl, 0.291 g/l KCl, 1.44 g/l Na2HPO4 and 0.24 g/l KH2PO4, pH 7.4, VWR International) to remove excess of FBS and inserted into the tubes, to exclude sides and bottom of the disks (preventing attachment of the fungal cells to the non-coated sides of the disks). Afterwards, tubes and inserted disks were wrapped in parafilm. The overnight cultures of C. albicans were diluted in RPMI 1640 medium (Sigma-Aldrich) until 5 x 104 cells/ml and 500 µl of this suspension were added to the tubes containing the disks. After an incubation phase for 90 min at 37°C, disks were washed twice with PBS to remove non-adherent cells. Subsequently, 500 µl of fresh RPMI were added and disks were incubated for 24 h at 37°C. The disks were washed twice with PBS before removal of the parafilm and silicone tubes and were placed in falcon tubes containing 2 ml PBS and cells were removed from the disks by performing the following procedure three times: scraping, followed by sonication (10 min) and vortexing. The resulting cell suspensions were diluted in PBS and plated on YPD plates. Upon 24 h incubation at 30°C, the number of colony forming units/disk was determined and this for 3 independent experiments, each consisting of at least 3 technical repeats. Statistical analysis was performed using a student’s t-test and differences were considered significant if p < 0.05. Representative biofilms were visualized by SEM (XL30-FEG, FEI) operated at standard highvacuum settings at 10 mm working distance and 10 keV accelerating voltage. Samples for SEM analysis were prepared as described elsewhere.32 Samples were carefully washed in PBS to remove non-adherent cells and fixated with glutaraldehyde (2,5 % in a cacodylate buffer), followed by dehydration in a series of increasing ethanol concentrations. Samples were dried and coated with Au-Pd using a sputtering device (Edwards S150) in order to produce a thin conductive film on the surface.

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Results AC-EPD of BSA Initial experiments to verify the potential of AC-EPD for the deposition of biomolecules on metallic substrates were conducted using Ti6Al4V electrodes and the fluorescently FITClabelled model protein BSA. Electrodes were treated for 40 min in a 5 mg/ml FITC-BSA suspension in MQ (pH 6). AC-EPD samples were prepared using an asymmetrical biphasic triangular AC-signal as shown in Figure 1 with a peak-to-peak voltage, Vp-p, of 100 V/cm both with and without an additional DC offset of 2 V/cm (FITC-AC 40’ resp. FITC-AC/DC 40’). These specific AC and DC field strength values were selected as a compromise to obtain sufficiently high BSA deposition rates, while still avoiding denaturation of BSA due to heating or electrolysis. We found that limiting AC signals to 100 V/cm (corresponding to a manageable 20 mA current) allowed keeping the Joule heating to levels below 2°C during the course of a single experiment. Furthermore, using a DC offset of 2 V/cm (or 1.6 V absolute), water electrolysis does not occur, thus preventing pH shifts at the electrodes. As a reference, Ti6Al4V electrodes were immersed in a fresh FITC-BSA suspension for the same duration as the AC-EPD treatment (FITC-dip 40’), in order to examine the physical adsorption of the biomolecule to the metal surface. Additionally, one set of electrodes was exclusively treated using a DC signal of 2 V/cm (FITC-DC). Qualitative evaluation of the deposits was done by CLSM imaging (figure 2). The height of the fluorescence signal in the z-direction was taken as a measure for the coating thickness. While no fluorescence signals can be observed for the immersed and DC-treated reference samples (figure 2a and 2d respectively), AC-EPD results in a homogeneous deposit of FITCBSA on the electrode coupled as the anode during the high-amplitude phase of the AC-signal (figure 2b and 2e). Without the DC offset, the coating thickness is about 8 µm, but the thickness is considerably increased to about 25 µm when a DC offset 2 V/cm is superimposed on the of AC-field, leading to shrinkage cracks upon drying of the coating. No detectable 11 ACS Paragon Plus Environment

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deposits were observed on the counter electrodes (figure 2c and 2f). Similarly, a micro BCA protein assay revealed that, for a clinically relevant beadblasted Ti surface, EPD in an asymmetric 100 Vp-p/cm AC field with a 2 V/cm DC offset for 30 min with a 1 mg/ml FITCBSA suspension resulted in a coating with a surface density of about 3 nmol/cm², while no detectable amount could be observed on the surface after immersion. For the deposition of non-labelled BSA, higher amplitude fields were required in order to reproduce comparable current levels as for FITC-BSA, as in EPD deposition rates are linked to the current rather than field strength.33 As a result, EPD was carried out with a 1000 Vpp/cm

asymmetric AC-field with a 5 V/cm DC offset for 5 min in a 1 mg/ml BSA suspension

in MQ (pH 6). These conditions led to a homogeneous coating on the electrode coupled as the anode (BSA-AC/DC anode 5’) during the high-amplitude phase of the AC-signal. This is demonstrated by a smooth cover-up of the characteristic surface features (roughness, grain boundaries) on the pristine Ti6Al4V electrode, but not on BSA-AC/DC anode 5’ (figure 3). The organic nature of the surface layer was also confirmed by EDX point analyses (Supporting Information, figure S1). No coating was observed on the counterelectrode (BSAAC/DC cathode 5’) or after immersion (BSA-dip 5’) in a fresh BSA suspension for the same period of time, as visualized by SEM (figure 3). This was confirmed by a Bradford protein assay (Supporting Information, figure S1) as well. For these strong electric fields in a voltage-controlled mode, an increase in current amplitude could be observed over time (results not shown), which was accompanied by heating at the electrodes. To limit the temperature increase, further experiments were conducted in a current-controlled mode. To this end, the same current level as measured at the start of the voltage-controlled experiments was enforced, i.e. a 20 mA AC field superimposed with a 0.1 mA DC offset. Under these conditions, it could be observed that the suspension temperature levelled off at about 37°C. For the subsequent experiments, electrodes were 12 ACS Paragon Plus Environment

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treated in a 1 mg/ml BSA suspension during 30 min either by immersion (BSA-dip 30’) or AC-EPD (BSA AC/DC anode 30’, BSA AC/D cathode 30’). In order to confirm the presence of BSA on the Ti6Al4V electrodes and to probe its molecular structure following different processing parameters, a multivariate ToF-SIMS analysis was performed (figure 4). Sets of 3 positive mass spectra were collected from distinct areas on the sample surface and compared using PCA. Relationships between the mass spectra are visualized by scores plots of the principle components (PC) that indicate the greatest variance within the dataset, while loadings plots elucidate which mass fragments are included in the PCs and to which samples these correspond. Only peaks above a 0.1 threshold loading are considered. The scores plot on PC1 and PC2 (figure 4a) shows two well separated data clusters along PC1 (65% of data variance), indicating a different surface chemistry for BSA-dip and BSAAC/DC samples (anode as well as cathode). The PC1 loadings plot (figure 4c) shows that especially Ti containing fragment ions load positively, meaning that those are more intense in the spectra of the BSA-dip surface, while several specific BSA fragments, such as signals at m/z = 70, m/z = 84 and m/z = 110 corresponding to C4H8N+ (Pro/Arg), C5H10N+ (Lys) and C5H8N3+ (His/Arg), respectively 34, load negatively and are more prominent on BSA-AC/DC samples. When comparing the surface of the electrodes that served as anode for 2 min (BSA-AC/DC anode 2’) or 30 min (BSA-AC/DC anode 30’) or as cathode for 30 min (BSA-AC/DC cathode 30’) during the high-amplitude peak of AC-EPD, the PC1/PC2 scores plot (figure 4b) shows a distinctive difference depending on polarity along PC1 (70% of data variance) and process time for both anodes along PC2 (23% of data variance). A tight clustering of the individual data points indicates a high degree of uniformity of the surface compositions within the three samples. The PC1 loadings plot in figure 4d indicates that shorter organic fragment 13 ACS Paragon Plus Environment

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ions correlate with the cathode surface, while longer fragments can be found on anodic samples. The PC2 loadings plot shows that Ti containing fragment ions load positively, correlating with BSA-AC/DC anode 2’ and BSA-AC/DC cathode 30’, while protein fragments load negatively, meaning these are especially present on BSA-AC/DC anode 30’ (figure 4e). This confirms a selective deposition of BSA on anode and an increased yield of deposition with process time. The possible conformational changes of BSA upon AC-EPD were evaluated using FTIRRAS in order to compare the secondary structure content for BSA-AC/DC anode 30’ deposits with an adsorbed BSA reference sample obtained by dropcoating, i.e. deposition of consecutive drops of the BSA suspension followed by drying. Figure S2 in the Supporting Information presents the (baseline corrected) FTIR-RAS spectra and the Fourier-self deconvoluted spectra for the amide I region, while the quantitative analysis of the secondary structure content for BSA is summarized in table 1. An α-helix band area of 14% was detected for BSA-AC/DC anode 30’ compared to an 20% band area for the adsorbed BSA reference, indicating only a slightly different conformation for the AC-EPD based BSA coating. It should be noted that BSA-dip 30’ and BSA-AC/DC cathode 30’ samples could not be analyzed as the amount of adsorbed BSA was either below the detection limit of the FTIRRAS device or too low for subsequent Fourier-self deconvolution analysis, respectively. This again confirms the improved deposition yield of BSA on Ti6Al4V using AC-EPD.

AC-EPD of CASP In a next step, we used AC-EPD to immobilize the antifungal lipopeptide drug CASP on Ti surfaces. Based on the previous results, an AC-signal with a net DC component was selected and a current-controlled mode was applied in order to avoid extensive heating of the suspension. Additionally, the metal surface was silanized using APTES and activated with

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isocyanate groups using HMDI prior to the AC-EPD process, as described above, to establish covalent bonding of the protein to the Ti surface. Using an amplitude of 10 mA superimposed with a mild DC offset of 0.1 mA during 10 min in a suspension of 1 mg/ml CASP in MQ, a selective deposition is achieved at the electrode coupled as cathode during the high-amplitude peak (CASP-AC/DC cathode 10’). SEM top view images in figure 5 show that the Ti electrode surface following immersion in a CASP suspension (CASP-dip 10’) does not show any difference with the pristine surface after functionalization (Ti + APTES + HMDI). Similarly, the surface of the anode during the highamplitude peak (CASP-AC/DC anode 10’) was not significantly altered after AC-EPD for 10 min. On the other hand, a thick network of lipopeptide aggregates covering the whole electrode surface could be observed on CASP-AC/DC cathode 10’. In order to determine the coating thickness, the protein layer was scratched using a blunt instrument exposing the underlying substrate. The height difference between the coating surface and substrate was measured by SEM-based surface profilometry and amounted to approximately 10 µm (figure 5e). A micro BCA protein assay confirmed the selective deposition of a proteinaceous layer at the cathode surface when compared to the anode. Functionalized Ti, either after silanization (Ti + APTES) or silanization combined with isocyanate activation (Ti + APTES + HMDI), showed similar absorbance levels as the pristine Ti material (Ti) (Figure S3 in the Supporting Information). The surface chemistry of the Ti electrodes after various processing steps of CASP coating was verified by ToF-SIMS analysis (figure 6). In order to identify characteristic CASP signals, an adsorbed CASP reference sample (CASP reference) was included. The scores plot indicates a distinctively different surface chemistry after each functionalization step (figure 6a). Substrate signals, at m/z = 48, m/z = 64 and m/z = 81 corresponding to Ti+, TiO+ and 15 ACS Paragon Plus Environment

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TiOOH+, respectively, and APTES related signals, at m/z = 28, m/z = 45 and m/z = 79 corresponding to Si+, SiOH+ and SiH5NO2+, respectively, load negatively on PC1 and relate to Ti and Ti+APTES, while HMDI-related signals, at m/z = 55, m/z = 98 and m/z = 143 corresponding to C4H7+, C6H12N+ and C7H15N2O+, respectively, load negatively and are present in Ti+APTES+HMDI, but also in CASP-dip 10’ and CASP-AC/DC cathode 10’ (figure 6b). The PC2 loadings plot distinguishes CASP signals, at m/z = 72, m/z = 74, m/z = 84 and m/z = 86 corresponding to C3H6NO+, C3H8NO+, C4H6NO+ and C4H8NO+, respectively,35 relating to CASP reference, CASP-dip 10’ and CASP-AC/DC cathode 10’ (figure 6c). Other negatively loaded signals in PC2 at m/z = 205 and m/z = 365, corresponding to additives present in the clinical CASP suspension, i.e. mannitol and sucrose conjugated with Na+, respectively, are only found on the CASP reference sample. When comparing APTES + HMDI functionalized Ti electrodes that were subsequently coated with CASP either by AC-EPD (CASP-AC/DC anode 10’ resp. cathode 10’) or dipcoating (CASP-dip 10’), the surface chemistry appears significantly different (figure 7a). Signals that load positively in PC1 and correspond to CASP-AC/DC anode 10’ relate to either APTES (SiH4N3+ at m/z = 74), HMDI (C6H12N+ at m/z = 98 and C7H15N2O+ at m/z = 143), but mainly to fragments from mannitol, which was present in the clinical CASP starting suspension, bound to APTES at m/z = 331, m/z = 357, m/z = 403 and m/z = 417 (figure 7c). On the other hand, differences between CASP-dip 10’ and CASP-AC/DC cathode 10’, which are seen along PC2, correspond to a higher intensity of HMDI signals, C3H7+ at m/z = 43, C4H9+ at m/z = 57, C5H9+ at m/z = 69 and C5H11+ at m/z = 71, in the CASP-dip 10’ sample (figure 7d). Furthermore, when applying AC-EPD of CASP (same conditions) on electrodes that are not functionalized (CASP-AC/DC no APTES + HMDI) or only pretreated with APTES (CASP-AC/DC no HMDI), again a different surface chemistry is found compared to 16 ACS Paragon Plus Environment

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APTES+HMDI functionalized electrodes (figure 7b). While the loadings plot on PC1 (figure 7e) highlights HMDI (C6H12N+ at m/z = 98 and C7H15N2O+ at m/z = 143) and CASP (C4H8NO+ at m/z = 86) signals on fully functionalized electrodes (CASP-AC/DC cathode 10’), the non-fully pre-treated samples’ signals correspond mainly to the Ti substrate (Ti+ at m/z = 47, TiO+ at m/z = 64 and TiOOH+ at m/z = 81) or APTES (SiOH+ at m/z = 45). Overall, the results confirm that CASP was selectively deposited on the cathode during AC/DC-EPD, while impurities originating from the clinical starting suspension were mainly found on the anode. Coatings obtained by dipcoating or AC/DC EPD on non-fully pre-treated substrates do not show the same yield of CASP.

Scratch adhesion testing To assess the adhesion of AC-EPD CASP coatings to the Ti substrate, CASP-AC/DC cathode 10’ samples were scratched using a progressively loaded stylus. Representative SEM micrographs of a scratch track are shown in Figure 8. At the starting load of 3 N, damage of the coating is already initiated, indicated by the dashed lines in figure 8a. While linearly increasing the normal load (left to right), the damage zone is expanded. Importantly, damage occurs within the coating (cohesive failure) and not at the coating/substrate interface (adhesive failure), over the whole loading range. This is confirmed by the remaining contiguous protein layer within the damage zone (figure 8b). Even at the maximum applied normal load of 23 N, delamination of the coating does not extend to the substrate, suggesting good coating adhesion.

Antibiofilm activity testing To determine the in vitro antibiofilm activity of CASP-functionalized Ti electrodes relative to pristine Ti (control Ti), C. albicans cells were diluted in RPMI and added to the FBSpretreated control Ti, CASP-AC/DC cathode 10’ and CASP-dip 10’ samples. Upon an adhesion phase of 90 min and 24 h of biofilm formation at 37°C, the latter was quantified by colony forming unit (cfu) counting and, in parallel, visualized via SEM (figure 9). 17 ACS Paragon Plus Environment

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Significantly (p < 0.05) less colonization of fungal cells was observed on CASP-AC/DC cathode 10’ and CASP-dip 10’ discs compared to untreated control Ti discs (figure 9a), as measured by cfu counting. This was also evidenced by SEM biofilm visualization, indicating a thick network of hyphae on control Ti (figure 9b), while on CASP-AC/DC cathode 10’ and CASP-dip 10’only limited amounts of C. albicans hyphae and yeast cells are covering the surface (figure 9c-d).

Discussion In this work, we demonstrate the applicability of AC-EPD for the deposition of biologically active biomolecules onto metallic substrates in order to accelerate generally diffusioncontrolled immobilization processes. Using the model molecule BSA, we show that the combined effect of AC signals with a mild DC offset strongly increases the deposition yield during EPD on one of both electrodes. When applied to the clinically relevant antifungal lipopeptide drug CASP, AC-EPD in combination with a surface functionalization leads to a rapid selective formation of CASP coatings. Moreover, AC-EPD allows to separate the CASP molecules from the sugar impurities present in the clinical starting suspension leading to highpurity coatings that remain active against the fungal pathogen C. albicans. Several recent review papers have highlighted the advantages of using AC fields for EPD from aqueous media.24, 26, 36 For AC signals at sufficiently high frequencies, the gross of the current will flow through the double layer capacitance at the electrode-electrolyte interface rather than through the electrochemical reactions at the electrode surface, because of the high resistance for Faradaic current flow associated with these reactions.26,

27

This effectively

suppresses the electrolysis of water and the concomitant gas bubble formation and electrode pH shift, opening up new perspectives for the processing of biological entities from aqueous suspensions using EPD. 18 ACS Paragon Plus Environment

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In AC fields, the direction of the electrical charge is periodically reversed. For symmetrical signals (such as the commonly applied sinusoidal wave), the negative and positive half cycles are equal in amplitude. Therefore, charged particles are expected to move forward during the positive half cycle with the same distance as they move back during the negative half cycle. This will result in an oscillation of the charged particle around a fixed spot in the suspension and, in theory, no net electrophoresis will occur. However, AC fields with an asymmetric wave shape (such as in figure 1) and sufficiently high field strengths are hypothesized to establish a net drift of particles. This is due to the non-linear relationship between the particle velocity and the electric field strength which causes a particle to travel a longer distance during the high-amplitude half cycle than it will move back during the low-amplitude half cycle, resulting in a net migration of particles towards one of both electrodes as for DC fields.24,

37

Indeed, in line with earlier work on other macromolecules, our results for the

model molecule FITC-BSA (figure 2) confirm an improved deposition yield when applying an AC signal without a net DC component.28 Additionally, we show that the deposition selectively occurs at the electrode coupled as anode during the high-amplitude phase of the AC-signal and not at the counter electrode. The IEP of FITC-BSA has been reported to be around 4.7,38 indicating the molecule has a net negative charge at the observed pH of 6.0. This validates the hypothesis that for asymmetric AC-EPD the molecules migrate and deposit on the electrode of opposite charge during the high-amplitude half cycle, similar to a conventional DC-EPD process. However, besides the customary AC signal without a net DC component (i.e. when the time/amplitude surface area of the negative and positive half cycles are equal),28, 29, 39 the combination of an AC-signal with a DC offset in the direction of the high-amplitude peak was also considered here (figure 1). Importantly, the deposition yield is significantly enhanced when a DC offset is superimposed on the AC-signal in the same direction as the high-amplitude half cycle. Even if the relatively small DC offset of 2 V/cm 19 ACS Paragon Plus Environment

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does not result in a detectable adhering deposit when applied separately, it has a synergistic effect on the deposition yield during asymmetric AC-EPD, leading to a ~6 fold increase in coating thickness. This high deposition yield obtained by AC/DC-EPD is also confirmed by an estimated surface density of 3 nmol/cm² (or 200 µg/cm²), whereas a monolayer of BSA is only expected to yield a surface density in the range of 5 pmol/cm² (or 350 ng/cm²).40 We suspect that the relatively weak DC-field is in itself capable of instigating electrophoresis of the molecules towards the electrode. However, due to the strong and thick double layer formed at the electrode during DC-polarization, the molecules - which are only partially negatively charged - cannot reach the electrode surface itself. On the other hand, the formation of a thin deposition in an asymmetric AC-field without DC-component suggests that the application of a strong AC-field prevents this double layer from forming, or allows the molecules to bypass it. As a result, a composed AC/DC field, which applies the high mobility of molecules in a DC-field in the absence of a double layer (due to the AC-field), would allow most of the gathered molecules to reach the electrode and deposit on the surface. In line with the results obtained for FITC-BSA, non-labelled BSA, having an IEP of 4.9 and hence also negatively charged at the natural pH 6.0,38 also deposits on the electrode coupled as anode during the high-amplitude half cycle (figure 3). However, it can be noted that a decrease in suspension concentration and deposition time must be matched to an increased field amplitude and this leads to a local heating at the electrode due to the high current density obtained. This overheating is not observed when conducting the experiments in a currentcontrolled mode, yet the selective deposition on the high-amplitude phase anode is maintained. Analysis of the surface chemistry using PCA of ToF-SIMS spectra corroborates these observations by confirming the presence of BSA fragments on AC/DC electrodes, which cover up Ti substrate signals that are clearly exposed on dipped samples (figure 4). Furthermore, the coating thickness increased with anodic polarity and deposition time as 20 ACS Paragon Plus Environment

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indicated by the reduced detection of Ti substrate signals for these samples. Concerning the conformation of BSA, FTIR-RAS data showed a small reduction of the α-helix band area for the BSA-AC/DC anode 30’ compared to the adsorbed BSA reference, indicating at the ACEPD process somewhat affecting the secondary structure of BSA (Table 1). This may be caused by gas bubble formation at the electrodes, implying that electrolysis still takes place to some extent and that the magnitude of the DC component should be reduced. However, in a recent paper by our group, we have shown that BSA coatings applied by means of AC-EPD not only act as a barrier coating limiting metal ion release to the environment, but also increase the protein adsorption and calcium phosphate formation at the surface, which are two essential steps in the osseointegration process. This was observed under physiological as well as inflammatory conditions and proves that BSA coatings maintain their bioactivity upon ACEPD, despite the small conformational changes.41 Nevertheless, the results presented for the model molecule BSA show that the obtained biomolecule coatings do not emerge from physical adsorption, but are a result of the electrophoresis instigated by the applied asymmetric AC-EPD fields with a net DC component. Although gentle rinsing does not remove the BSA coatings, the molecules are most probably not permanently attached to the Ti surface. This instability problem after AC-EPD was also observed by Ammam et al., where a polyurethane coating was introduced to fix glucose oxidase deposits onto a Pt wire.28 In view of a clinically relevant anti-infective coating with improved durability, we propose to tether the envisaged CASP coatings by covalent linking. We have recently shown that a chemical grafting process based on silanization followed by implementing HMDI spacer molecules is a promising approach to couple a variety of antimicrobial compounds, CASP among others, to Ti.14, 20 Not only did the proposed immobilization route preserve the antifungal activity of CASP both in vitro and in vivo, it was also shown that the osseointegration potential of such CASP-coated Ti substrates 21 ACS Paragon Plus Environment

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was not jeopardized.14 In the current paper, we show that combining this established surface activation protocol with the current-controlled AC/DC-EPD signals significantly accelerates the immobilization procedure of CASP. Moreover, while in our previous work pure CASP starting solutions were required to obtain high-purity CASP coatings, this AC/DC-EPD procedure allows to use less pure sugar-containing starting solutions, as the CASP molecule can be separated from the additives by AC-EPD. It could be observed by SEM and micro BCA protein assay that a proteinaceous deposit is produced, which is not the case when simply immersing the functionalized Ti substrates in the CASP solution for the same amount of time (figure 5). At a neutral pH, which is below the IEP of 9.3, CASP is positively charged and deposits selectively on the electrode coupled as cathode during the high-amplitude half cycle with a deposit thickness of approximately 10 µm. PCA of the ToF-SIMS spectra reveals that the surface chemistry after AC-EPD or simple immersion in a CASP solution shows clear similarities. Signals from the Ti substrate or silane linker have been covered up and replaced by HMDI spacer and CASP related signals. These HMDI signals are more prominent on the CASP-dip 10’ sample, indicating a reduced CASP coating thickness compared to CASPAC/DC cathode 10’ samples (figure 7d). Importantly, these CASP coatings do not contain sugar additives that are present in the starting suspension. Instead, these additives are found on the (functionalized) Ti electrode serving as anode during the high-amplitude half cycle (figure 7c) indicating the CASP molecules can be separated from the sugar additives in the starting suspension by means of AC-EPD. Furthermore, ToF-SIMS results also show that the two-step functionalization (i.e. APTES + HMDI) of the electrodes prior to AC-EPD is recommended, as the CASP yield decreases when no functionalization or only APTES is applied on the Ti surface (figure 7e). Since Ti is commonly used for bone implant applications which often undergo high insertion forces upon implantation, the mechanical stability of the coatings is crucial to 22 ACS Paragon Plus Environment

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clinical success. Therefore, we evaluated the coating adhesion by scratch resistance testing. Although damage can be observed to some extent, AC-EPD CASP coatings do not completely detach from the surface at normal loads up to 23 N, suggesting good coating adhesion. In comparison, for hydroxyapatite coatings on Ti for use in orthopaedic implants, normal loads below 15 N have been reported to lead to complete coating delamination.42, 43 Finally, because preservation of the biological activity is another indispensable requisite for clinical biomolecule tethering processes, we addressed the antifungal activity of the experimental CASP coated Ti substrates. It is demonstrated that these surfaces strongly affected the amount of C. albicans biofilm formation and its structural integrity compared to non-functionalized Ti (figure 9). Overall, the presented results indicate that AC-EPD can be a valuable tool to increase the immobilization rate of biomolecules on metal implant surfaces. Additionally, the versatility towards its use with various molecules holds a promise of a hybrid approach incorporating both antibacterial/antifungal and osteostimulatory compounds in one coating. However, further research addressing the biocompatibility with host cells and osseointegration potential of such AC-EPD based coatings is required to further validate the clinical usefulness of the method.14, 44

Conclusions In order to suppress artifacts related to water electrolysis (bubble formation, pH shift) during the EPD of biomolecules from aqueous systems, high-frequency AC fields with an asymmetrical triangular waveform were used instead of DC fields. Depending on their surface charge, BSA or CASP biomolecules deposited on the electrode having an opposite sign during the high-amplitude half cycle. Superimposing a mild DC offset in the direction of the high-amplitude peak drastically improved the deposition yield. The electrophoresis effect during AC-EPD was then used to actively concentrate the clinically relevant antifungal 23 ACS Paragon Plus Environment

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lipopeptide CASP at the surface activated Ti electrode. In combination with a two-step surface activation of the electrode, incorporating APTES linker and HMDI spacer molecules, tethered CASP coatings were produced with a higher yield as compared to a diffusioncontrolled immobilization process. The experimental coatings showed a higher purity than the clinical CASP starting suspension, adhered well to the Ti substrate and remained active in vitro against the fungal pathogen C. albicans. As such, a proof-of-concept for the application of AC-EPD as a fast tool for grafting antimicrobial agents on Ti implant surfaces was delivered.

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Associated content Supporting information The following content is available free of charge: EDX analysis, Bradford protein assay and original FTIR-RAS spectra for BSA adsorbed on Ti6Al4V electrodes, microBCA protein assay for CASP coatings on Ti electrodes (PDF)

Author information Corresponding author * E-mail: [email protected]

Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest

Acknowledgements The research leading to these results has received funding from the European Commission’s Seventh Framework Program (FP7/2007-2013) under the grant agreement COATIM (project n° 278425), the Industrial Research Fund of KU Leuven by the knowledge platform IOF/KP/11/007 and the Flemish government via the Hercules Foundation (project ZW09-09). A.B., B.N., K.T. and N.D. acknowledge the receipt of a travel grant from FWO-Vlaanderen (K229615N), a postdoctoral grant from FWO-Vlaanderen (1.2.B62.12N) with Krediet aan Navorser (1505213N) and a YouReCa Junior Mobility grant (JUMO/14/024), a mandate from the Industrial Research Fund (IOFm/05/022, KU Leuven), and a predoctoral grant from IWTVlaanderen (IWT101095), respectively. M.S.K. and P.S. acknowledge the DFG (research unit FOR 1878) for funding. T.Y. acknowledges a travel fund from "the program for promoting the enhancement of research universities" of the Ministry of Education, Culture, Sports, 25 ACS Paragon Plus Environment

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Science and Technology, Japan. The authors thank Dan Graham, Ph.D., for developing the NESAC/BIO Toolbox used in this study and NIH grant EB-002027 for supporting the toolbox development. The funders had no involvement in study design; collection, analysis and interpretation of data; writing of the report; neither in the decision to submit the article for publication.

References (1) Campoccia, D.; Montanaro, L.; Arciola, C. R. The Significance of Infection Related to Orthopedic Devices and Issues of Antibiotic Resistance. Biomaterials 2006, 27 (11), 2331-2339. (2) Hickok, N. J.; Shapiro, I. M. Immobilized Antibiotics to Prevent Orthopaedic Implant Infections. Adv. Drug Delivery Rev. 2012, 64 (12), 1165-1176. (3) Costa, F.; Carvalho, I. F.; Montelaro, R. C.; Gomes, P.; Martins, M. C. Covalent Immobilization of Antimicrobial Peptides (Amps) onto Biomaterial Surfaces. Acta Biomater. 2011, 7 (4), 1431-1440. (4) Fux, C. A.; Costerton, J. W.; Stewart, P. S.; Stoodley, P. Survival Strategies of Infectious Biofilms. Trends Microbiol. 2005, 13 (1), 34-40. (5) Campoccia, D.; Montanaro, L.; Speziale, P.; Arciola, C. R. Antibiotic-Loaded Biomaterials and the Risks for the Spread of Antibiotic Resistance Following Their Prophylactic and Therapeutic Clinical Use. Biomaterials 2010, 31 (25), 6363-6377. (6) Pham, V. T.; Truong, V. K.; Orlowska, A.; Ghanaati, S.; Barbeck, M.; Booms, P.; Fulcher, A. J.; Bhadra, C. M.; Buividas, R.; Baulin, V.; Kirkpatrick, C. J.; Doran, P.; Mainwaring, D. E.; Juodkazis, S.; Crawford, R. J.; Ivanova, E. P. "Race for the Surface": Eukaryotic Cells Can Win. ACS Appl. Mater. Interfaces 2016, 8 (34), 22025-31. (7) Perez-Tanoira, R.; Han, X.; Soininen, A.; Aarnisalo, A. A.; Tiainen, V. M.; Eklund, K. K.; Esteban, J.; Kinnari, T. J. Competitive Colonization of Prosthetic Surfaces by Staphylococcus Aureus and Human Cells. J. Biomed. Mater. Res., Part A 2017, 105 (1), 62-72. (8) Kutty, M. G.; De, A.; Bhaduri, S. B.; Yaghoubi, A. Microwave-Assisted Fabrication of Titanium Implants with Controlled Surface Topography for Rapid Bone Healing. ACS Appl. Mater. Interfaces 2014, 6 (16), 13587-93. (9) Braem, A.; Chaudhari, A.; Vivan Cardoso, M.; Schrooten, J.; Duyck, J.; Vleugels, J. Peri- and Intra-Implant Bone Response to Microporous Ti Coatings with Surface Modification. Acta Biomater. 2014, 10 (2), 986-95. (10) Braem, A.; Van Mellaert, L.; Mattheys, T.; Hofmans, D.; De Waelheyns, E.; Geris, L.; Anne, J.; Schrooten, J.; Vleugels, J. Staphylococcal Biofilm Growth on Smooth and Porous Titanium Coatings for Biomedical Applications. J. Biomed. Mater. Res., Part A 2014, 102 (1), 215-24. (11) Goodman, S. B.; Yao, Z.; Keeney, M.; Yang, F. The Future of Biologic Coatings for Orthopaedic Implants. Biomaterials 2013, 34 (13), 3174-3183. (12) Zhao, L.; Chu, P. K.; Zhang, Y.; Wu, Z. Antibacterial Coatings on Titanium Implants. J. Biomed. Mater. Res., Part B 2009, 91 (1), 470-480. (13) Lyndon, J. A.; Boyd, B. J.; Birbilis, N. Metallic Implant Drug/Device Combinations for Controlled Drug Release in Orthopaedic Applications. J. Controlled Release 2014, 179, 63-75. (14) Kucharikova, S.; Gerits, E.; De Brucker, K.; Braem, A.; Ceh, K.; Majdic, G.; Spanic, T.; Pogorevc, E.; Verstraeten, N.; Tournu, H.; Delattin, N.; Impellizzeri, F.; Erdtmann, M.; Krona, A.; Lovenklev, M.; Knezevic, M.; Frohlich, M.; Vleugels, J.; Fauvart, M.; de Silva, W. J.; Vandamme, K.; Garcia-Forgas, J.;

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Cammue, B. P.; Michiels, J.; Van Dijck, P.; Thevissen, K. Covalent Immobilization of Antimicrobial Agents on Titanium Prevents Staphylococcus Aureus and Candida Albicans Colonization and Biofilm Formation. J. Antimicrob. Chemother. 2016, 71 (4), 936-945. (15) Sevilla, P.; Vining, K. V.; Dotor, J.; Rodriguez, D.; Gil, F. J.; Aparicio, C. Surface Immobilization and Bioactivity of Tgf-Beta1 Inhibitor Peptides for Bone Implant Applications. J. Biomed. Mater. Res., Part B 2016, 104 (2), 385-394. (16) Zhou, L.; Lai, Y.; Huang, W.; Huang, S.; Xu, Z.; Chen, J.; Wu, D. Biofunctionalization of Microgroove Titanium Surfaces with an Antimicrobial Peptide to Enhance Their Bactericidal Activity and Cytocompatibility. Colloids Surf., B 2015, 128, 552-560. (17) Godoy-Gallardo, M.; Mas-Moruno, C.; Fernandez-Calderon, M. C.; Perez-Giraldo, C.; Manero, J. M.; Albericio, F.; Gil, F. J.; Rodriguez, D. Covalent Immobilization of Hlf1-11 Peptide on a Titanium Surface Reduces Bacterial Adhesion and Biofilm Formation. Acta Biomater. 2014, 10 (8), 3522-3534. (18) Killian, M. S.; Schmuki, P. Influence of Bioactive Linker Molecules on Protein Adsorption. Surf. Interface Anal. 2014, 46 (S1), 193-197. (19) Holmberg, K. V.; Abdolhosseini, M.; Li, Y.; Chen, X.; Gorr, S. U.; Aparicio, C. Bio-Inspired Stable Antimicrobial Peptide Coatings for Dental Applications. Acta Biomater. 2013, 9 (9), 8224-8231. (20) Gerits, E.; Kucharikova, S.; Van Dijck, P.; Erdtmann, M.; Krona, A.; Lovenklev, M.; Frohlich, M.; Dovgan, B.; Impellizzeri, F.; Braem, A.; Vleugels, J.; Robijns, S. C.; Steenackers, H. P.; Vanderleyden, J.; De Brucker, K.; Thevissen, K.; Cammue, B. P.; Fauvart, M.; Verstraeten, N.; Michiels, J. Antibacterial Activity of a New Broad-Spectrum Antibiotic Covalently Bound to Titanium Surfaces. J. Orthop. Res. 2016, 34 (12), 2191-2198. (21) Boccaccini, A. R.; Keim, S.; Ma, R.; Li, Y.; Zhitomirsky, I. Electrophoretic Deposition of Biomaterials. J. R. Soc. Interface 2010, 7 Suppl 5, S581-613. (22) Braem, A.; Mattheys, T.; Neirinck, B.; Schrooten, J.; Van der Biest, O.; Vleugels, J. Porous Titanium Coatings through Electrophoretic Deposition of Tih2 Suspensions. Adv. Eng. Mater. 2011, 13 (6), 509-515. (23) Afshar-Mohajer, M.; Yaghoubi, A.; Ramesh, S.; Bushroa, A. R.; Chin, K. M. C.; Tin, C. C.; Chiu, W. S. Electrophoretic Deposition of Magnesium Silicates on Titanium Implants: Ion Migration and Silicide Interfaces. Appl. Surf. Sci. 2014, 307, 1-6. (24) Neirinck, B.; Van der Biest, O.; Vleugels, J. A Current Opinion on Electrophoretic Deposition in Pulsed and Alternating Fields. J. Phys. Chem. B 2013, 117 (6), 1516-1526. (25) Seuss, S.; Boccaccini, A. R. Electrophoretic Deposition of Biological Macromolecules, Drugs, and Cells. Biomacromolecules 2013, 14 (10), 3355-3369. (26) Ammam, M. Electrophoretic Deposition under Modulated Electric Fields: A Review. RSC Adv. 2012, 2 (20), 7633–7646. (27) Neirinck, B.; Fransaer, J.; Van der Biest, O.; Vleugels, J. Aqueous Electrophoretic Deposition in Asymmetric Ac Electric Fields (Ac–Epd). Electrochem. Commun. 2009, 11 (1), 57-60. (28) Ammam, M.; Fransaer, J. Ac-Electrophoretic Deposition of Glucose Oxidase. Biosens. Bioelectron. 2009, 25 (1), 191-197. (29) Neirinck, B.; Van Mellaert, L.; Fransaer, J.; Van der Biest, O.; Anné, J.; Vleugels, J. Electrophoretic Deposition of Bacterial Cells. Electrochem. Commun. 2009, 11 (9), 1842-1845. (30) Meurman, J. H.; Siikala, E.; Richardson, M.; Rautemaa, R., Non-Candida Albicans Candida Yeasts of the Oral Cavity. Formatex: Badajoz, Spain, 2007; Vol. 1. (31) Chandra, J.; Kuhn, D. M.; Mukherjee, P. K.; Hoyer, L. L.; McCormick, T.; Ghannoum, M. A. Biofilm Formation by the Fungal Pathogen Candida Albicans: Development, Architecture, and Drug Resistance. J. Bacteriol. 2001, 183 (18), 5385-5394. (32) De Brucker, K.; Tan, Y.; Vints, K.; De Cremer, K.; Braem, A.; Verstraeten, N.; Michiels, J.; Vleugels, J.; Cammue, B. P. A.; Thevissen, K. Fungal Β-1,3-Glucan Increases Ofloxacin Tolerance of Escherichia Coli in a Polymicrobial E. Coli/Candida Albicans Biofilm. Antimicrob. Agents Chemother. 2015, 59 (6), 3052-3058.

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(33) Van der Biest, O.; Vandeperre, L. Electrophoretic Deposition of Materials. Annu. Rev. Mater. Sci. 1999, 29, 327-352. (34) Wald, J.; Müller, C.; Wahl, M.; Hoth-Hannig, W.; Hannig, M.; Kopnarski, M.; Ziegler, C. TofSims Investigations of Adsorbed Proteins on Dental Titanium. Phys. Status Solidi A 2010, 207 (4), 831836. (35) Griesser, S. S.; Jasieniak, M.; Coad, B. R.; Griesser, H. J. Antifungal Coatings by Caspofungin Immobilization onto Biomaterials Surfaces Via a Plasma Polymer Interlayer. Biointerphases 2015, 10 (4), 04A307. (36) Chávez-Valdez, A.; Boccaccini, A. R. Innovations in Electrophoretic Deposition: Alternating Current and Pulsed Direct Current Methods. Electrochim. Acta 2012, 65, 70-89. (37) Stotz, S. Field Dependence of the Electrophoretic Mobility of Particles Suspended in LowConductivity Liquids. J. Colloid Interface Sci. 1978, 65 (1), 118-130. (38) Bingaman, S.; Huxley, V. H.; Rumbaut, R. E. Fluorescent Dyes Modify Properties of Proteins Used in Microvascular Research. Microcirculation 2003, 10 (2), 221-231. (39) Neirinck, B.; Singer, F.; Braem, A.; Virtanen, S.; Vleugels, J. Alternating Current Electrophoretic Deposition of Bovine Serum Albumin onto Magnesium. Key Eng. Mater. 2015, 654, 139-143. (40) Thourson, S. B.; Marsh, C. A.; Doyle, B. J.; Timpe, S. J. Quartz Crystal Microbalance Study of Bovine Serum Albumin Adsorption onto Self-Assembled Monolayer-Functionalized Gold with Subsequent Ligand Binding. Colloids Surf., B 2013, 111, 707-12. (41) Höhn, S.; Braem, A.; Neirinck, B.; Virtanen, S. Albumin Coatings by Alternating Current Electrophoretic Deposition for Improving Corrosion Resistance and Bioactivity of Titanium Implants. Mater. Sci. Eng., C 2017, 73, 798-807. (42) Surmenev, R. A. A Review of Plasma-Assisted Methods for Calcium Phosphate-Based Coatings Fabrication. Surf. .Coat. Technol. 2012, 206 (8-9), 2035-2056. (43) Rohanizadeh, R.; LeGeros, R. Z.; Harsono, M.; Bendavid, A. Adherent Apatite Coating on Titanium Substrate Using Chemical Deposition. J. Biomed. Mater. Res., Part A 2005, 72 (4), 428-38. (44) Coad, B. R.; Griesser, H. J.; Peleg, A. Y.; Traven, A. Anti-Infective Surface Coatings: Design and Therapeutic Promise against Device-Associated Infections. PLoS Pathog. 2016, 12 (6), e1005598.

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Figures

Figure 1. Schematic representation of the AC-EPD setup. An asymmetrical biphasic triangular waveform is generated at a 50 Hz frequency, amplified and delivered to the EPD cell, while the voltage and current are instantaneously monitored. To ensure that the AC signal is balanced, the amplitude asymmetry, i.e. the ratio of the largest and smallest phase amplitude, and duration asymmetry, i.e. the ratio of the longest and shortest phase duration, are equal (and set to 3 in this case). Additionally, a DC offset in the direction of the highamplitude phase is applied. As deposition cell, 1 ml disposable cuvettes are used and electrodes are placed vertically at a distance of 8 mm.

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Figure 2. Fluorescence micrographs of Ti6Al4V electrodes after FITC-BSA deposition. Samples were coated for 40 min in a 5 mg/ml suspension by means of (a) immersion, (b, c) EPD in an asymmetric 100 Vp-p/cm AC field as anode and cathode respectively during the high-amplitude peak, (d) EPD in an DC-field of 2 V/cm as anode, (e, f) EPD in an asymmetric 100 Vp-p/cm AC field with a superimposed 2 V/cm DC offset as anode and cathode respectively during the high-amplitude peak.

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Figure 3. SEM micrographs of Ti6Al4V electrodes after AC-EPD of BSA. (a) The pristine surface of a Ti6Al4V electrode is shown. Other samples were coated for 5 min in a 1 mg/ml BSA suspension by means of (b) immersion or (c, d) EPD in an asymmetric 1000 V/cm AC field with a superimposed 5 V/cm DC offset as anode and cathode respectively during the high-amplitude peak.

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Figure 4. PCA results of positive ToF-SIMS spectra of Ti6Al4V electrodes after ACEPD of BSA. (a) Scores plot on PC1 and PC2 with corresponding (c) PC1 loadings plot for positive mass spectra of Ti6Al4V electrodes after 30 min of immersion (BSA-dip 30’) or ACEPD, serving as anode resp. cathode during the high-amplitude peak (BSA-AC/DC anode 30’ 32 ACS Paragon Plus Environment

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resp. cathode 30’), in 1 mg/ml BSA suspensions. (b) Scores plot on PC1 and PC2 with corresponding loadings plots on (d) PC1 and (e) PC2 for positive mass spectra of BSAAC/DC anode 30’ resp. cathode 30’ compared to a Ti6Al4V electrode serving as AC-EPD anode during the high-amplitude peak for 2 min (BSA-AC/DC anode 2’) in a 1 mg/ml BSA suspension. AC-EPD was carried out in a current-controlled mode in an asymmetric 20 mA AC field superimposed with a 0.1 mA DC offset.

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Figure 5. SEM micrographs of Ti electrodes after CASP deposition. (a) The functionalized surface of a Ti electrode after silanization and isocyanate activation is shown. Other samples were coated for 10 min in a 1 mg/ml CASP suspension by means of (b) immersion or (c, d) EPD in an asymmetric 10 mA AC-field with a superimposed 0.1 mA DC offset as anode and cathode respectively during the high-amplitude peak. (e) Scratched surface of a CASP-AC/DC cathode 10’ sample and corresponding surface profile, showing the coating thickness.

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Figure 6. PCA results of positive ToF-SIMS spectra of Ti electrodes after the various processing steps of CASP coating. (a) Scores plot on PC1 and PC2 with corresponding (b) PC1 and (c) PC2 loadings plots for positive mass spectra of pristine Ti electrodes (Ti), after silanization (Ti + APTES), subsequent isocyanate activation (Ti + APTES + HMDI), and followed by 10 min of immersion (CASP-dip 10’) or AC-EPD, serving as cathode during the high-amplitude peak (CASP-AC/DC cathode 10’), in 1 mg/ml CASP suspensions. AC-EPD was carried out in a current-controlled mode in an asymmetric 10 mA AC field superimposed with a 0.1 mA DC offset. An adsorbed CASP reference on pristine Ti obtained by dropcoating was included (CASP reference). 35 ACS Paragon Plus Environment

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Figure 7. PCA results of positive ToF-SIMS spectra of functionalized Ti electrodes after AC-EPD of CASP. (a) Scores plot on PC1 and PC2 with corresponding (c) PC1 and (d) PC2 loadings plots for positive mass spectra of functionalized Ti electrodes (i.e. pretreated using 36 ACS Paragon Plus Environment

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APTES and HMDI) after 10 min of immersion (CASP-dip 10’) or AC-EPD, serving as anode resp. cathode during the high-amplitude peak (CASP-AC/DC anode 10’ resp. cathode 10’), in 1 mg/ml CASP suspensions. (b) Scores plot on PC1 and PC2 with corresponding (e) PC1 loadings plot for positive mass spectra of Ti electrodes serving as AC-EPD cathode during the high-amplitude peak for 10 min and that were either not functionalized prior to AC-EPD (CASP-AC/DC no APTES + HMDI), only functionalized using APTES (CASP-AC/DC no HMDI) or with both APTES and HMDI (CASP-AC/DC cathode 10’). AC-EPD was carried out in a current-controlled mode in an asymmetric 10 mA AC field superimposed with a 0.1 mA DC offset.

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Figure 8. Scratch adhesion test of AC-EPD CASP coatings on Ti. (a) Overview and (b) detailed SEM micrographs of the scratch tracks on CASP-AC/DC cathode 10’. Scratch tracks were 5 mm in length and representative images were taken at position 0 mm (left), 2.5 mm (middle) and 5 mm (right). Dashed lines indicate the damage zone.

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Figure 9. In vitro antibiofilm activity of CASP-coated Ti substrates. (a) Quantification of the number of C. albicans biofilm cells on CASP-AC/DC cathode 10’ and CASP-dip 10’, relative to control Ti, as measured by cfu counting. Data represent means ± standard errors from 3 independent experiments, each consisting of at least 3 technical repeats, and statistically significant (*p < 0.05) differences are indicated. (b-d) SEM visualization of biofilms developed after 24 h incubation at 37°C.

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Tables Table 1. FTIR-RAS analysis of the amide I band for BSA adsorbed on Ti6Al4V electrodes. The BSA secondary structure content (represented as band area) in the amide I region for a Ti6Al4V electrode after 30 min of AC-EPD, serving as anode during the highamplitude peak, in 1 mg/ml BSA suspension (BSA-AC/DC anode 30’) is compared to an adsorbed BSA reference sample. AC-EPD was carried out in a current-controlled mode in an asymmetric 20 mA AC field superimposed with a 0.1 mA DC offset, while the adsorbed reference sample was obtained by dropcoating of a BSA suspension on Ti6Al4V. FTIR-RAS spectra before and after deconvolution are presented in figure S2 in the Supporting Information. BSA AC-DC anode 30’ Wavenumber Band area (cm-1) (%) β-sheet or side chain 1619 13 β-sheet 1629 7 Random coil 1639 9 Random coil 1647 8 α-helix 1658 14 Random coil 1667 11 Random coil 1679 24 β-sheet 1695 16 Assignment

Adsorbed BSA reference Wavenumber Band area (cm-1) (%) 1620 7 1631 10 1641 9 1647 9 1659 20 1669 18 1681 16 1693 11

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TOC

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Figure 1. Schematic representation of the AC-EPD setup. An asymmetrical biphasic triangular waveform is generated at a 50 Hz frequency, amplified and delivered to the EPD cell, while the voltage and current are instantaneously monitored. To ensure that the AC signal is balanced, the amplitude asymmetry, i.e. the ratio of the largest and smallest phase amplitude, and duration asymmetry, i.e. the ratio of the longest and shortest phase duration, are equal (and set to 3 in this case). Additionally, a DC offset in the direction of the high-amplitude phase is applied. As deposition cell, 1 ml disposable cuvettes are used and electrodes are placed vertically at a distance of 8 mm. figure 1 104x49mm (300 x 300 DPI)

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Figure 2. Fluorescence micrographs of Ti6Al4V electrodes after FITC-BSA deposition. Samples were coated for 40 min in a 5 mg/ml suspension by means of (a) immersion, (b, c) EPD in an asymmetric 100 Vp-p/cm AC field as anode and cathode respectively during the high-amplitude peak, (d) EPD in an DC-field of 2 V/cm as anode, (e,f) EPD in an asymmetric 100 Vp-p/cm AC field with a superimposed 2 V/cm DC offset as anode and cathode respectively during the high-amplitude peak. figure 2 159x105mm (300 x 300 DPI)

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Figure 3. SEM micrographs of Ti6Al4V electrodes after AC-EPD of BSA. (a) The pristine surface of a Ti6Al4V electrode is shown. Other samples were coated for 5 min in a 1 mg/ml BSA suspension by means of (b) immersion or (c, d) EPD in an asymmetric 1000 V/cm AC field with a superimposed 5 V/cm DC offset as anode and cathode respectively during the high-amplitude peak. figure 3 102x102mm (300 x 300 DPI)

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Figure 4. PCA results of positive ToF-SIMS spectra of Ti6Al4V electrodes after AC-EPD of BSA. (a) Scores plot on PC1 and PC2 with corresponding (c) PC1 loadings plot for positive mass spectra of Ti6Al4V electrodes after 30 min of immersion (BSA-dip 30’) or AC-EPD, serving as anode resp. cathode during the highamplitude peak (BSA-AC/DC anode 30’ resp. cathode 30’), in 1 mg/ml BSA suspensions. (b) Scores plot on PC1 and PC2 with corresponding loadings plots on (d) PC1 and (e) PC2 for positive mass spectra of BSAAC/DC anode 30’ resp. cathode 30’ compared to a Ti6Al4V electrode serving as AC-EPD anode during the high-amplitude peak for 2 min (BSA-AC/DC anode 2’) in a 1 mg/ml BSA suspension. AC-EPD was carried out in a current-controlled mode in an asymmetric 20 mA AC field superimposed with a 0.1 mA DC offset. figure 4 384x681mm (300 x 300 DPI)

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Figure 5. SEM micrographs of Ti electrodes after CASP deposition. (a) The functionalized surface of a Ti electrode after silanization and isocyanate activation is shown. Other samples were coated for 10 min in a 1 mg/ml CASP suspension by means of (b) immersion or (c, d) EPD in an asymmetric 10 mA AC-field with a superimposed 0.1 mA DC offset as anode and cathode respectively during the high-amplitude peak. (e) Scratched surface of a CASP-AC/DC cathode 10’ sample and corresponding surface profile, showing the coating thickness. figure 5 102x138mm (300 x 300 DPI)

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Figure 6. PCA results of positive ToF-SIMS spectra of Ti electrodes after the various processing steps of CASP coating. (a) Scores plot on PC1 and PC2 with corresponding (b) PC1 and (c) PC2 loadings plots for positive mass spectra of pristine Ti electrodes (Ti), after silanization (Ti + APTES), subsequent isocyanate activation (Ti + APTES + HMDI), and followed by 10 min of immersion (CASP-dip 10’) or AC-EPD, serving as cathode during the high-amplitude peak (CASP-AC/DC cathode 10’), in 1 mg/ml CASP suspensions. AC-EPD was carried out in a current-controlled mode in an asymmetric 10 mA AC field superimposed with a 0.1 mA DC offset. An adsorbed CASP reference on pristine Ti obtained by dropcoating was included (CASP reference). figure 6 384x515mm (300 x 300 DPI)

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Figure 7. PCA results of positive ToF-SIMS spectra of functionalized Ti electrodes after AC-EPD of CASP. (a) Scores plot on PC1 and PC2 with corresponding (c) PC1 and (d) PC2 loadings plots for positive mass spectra of functionalized Ti electrodes (i.e. pretreated using APTES and HMDI) after 10 min of immersion (CASP-dip 10’) or AC-EPD, serving as anode resp. cathode during the high-amplitude peak (CASP-AC/DC anode 10’ resp. cathode 10’), in 1 mg/ml CASP suspensions. (b) Scores plot on PC1 and PC2 with corresponding (e) PC1 loadings plot for positive mass spectra of Ti electrodes serving as AC-EPD cathode during the highamplitude peak for 10 min and that were either not functionalized prior to AC-EPD (CASP-AC/DC no APTES + HMDI), only functionalized using APTES (CASP-AC/DC no HMDI) or with both APTES and HMDI (CASPAC/DC cathode 10’). AC-EPD was carried out in a current-controlled mode in an asymmetric 10 mA AC field superimposed with a 0.1 mA DC offset. figure 7 384x679mm (300 x 300 DPI)

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Figure 8. Scratch adhesion test of AC-EPD CASP coatings on Ti. (a) Overview and (b) detailed SEM micrographs of the scratch tracks on CASP-AC/DC cathode 10’. Scratch tracks were 5 mm in length and representative images were taken at position 0 mm (left), 2.5 mm (middle) and 5 mm (right). Dashed lines indicate the damage zone. figure 8 154x102mm (300 x 300 DPI)

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Figure 9. In vitro antibiofilm activity of CASP-coated Ti substrates. (a) Quantification of the number of C. albicans biofilm cells on CASP-AC/DC cathode 10’ and CASP-dip 10’, relative to control Ti, as measured by cfu counting. Data represent means ± standard errors from 3 independent experiments, each consisting of at least 3 technical repeats, and statistically significant (*p < 0.05) differences are indicated. (b-d) SEM visualization of biofilms developed after 24 h incubation at 37°C. figure 9 102x102mm (300 x 300 DPI)

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