Anchor peptides: A green and versatile method for polypropylene functionalization

Polymer 116 (2017) 124e132

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Anchor peptides: A green and versatile method for polypropylene functionalization € ker a, c, Felix Jakob a, Kristin Rübsam a, Benjamin Stomps a, c, Alexander Bo Ulrich Schwaneberg a, b, * a

DWI - Leibniz-Institute for Interactive Materials, Forckenbeckstrasse 50, D-52074 Aachen, Germany RWTH Aachen University, Lehrstuhl für Biotechnologie, Worringerweg 3, D-52074 Aachen, Germany €t Potsdam, Fraunhofer-Institut für Angewandte Polymerforschung (IAP), Lehrstuhl für Polymermaterialien und Polymertechnologien, Universita Geiselbergstraße 69, 14476 Potsdam-Golm, Germany

b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 December 2016 Received in revised form 24 March 2017 Accepted 26 March 2017 Available online 27 March 2017

Polypropylene is one of the widest spread commodity polymers in plastic industry with an estimated global consumption of 62.4 million tons in 2020. Surface modification of polypropylene is required for its application as textile fibers, packaging material or filtration membranes. Modification of polypropylene is challenging due to absent functional surface groups. An anchor-peptide-based toolbox for green and versatile polypropylene functionalization was developed. Fusion proteins composed of enhanced green fluorescent protein (EGFP) and anchor peptides (e.g. cecropin A or LCI) were designed and applied to polypropylene surfaces. Resulting protein coatings of EGFP-LCI were characterized by fluorescence and scanning force microscopy. The fusion protein EGFP-LCI formed densely packed monolayers of 4.1 ± 0.2 nm thickness. A microtiter plate-based fluorescence assay was developed to analyze the coating in presence of surfactants. Washing of EGFP-LCI coated polypropylene with 10 mM non-ionic surfactant (Triton X-100) did not detach the protein film, whereas EGFP was removed completely. Anchor peptides promote binding to polypropylene by simple dip-coating at room temperature in water. The high coating density (0.8 pmol/cm2) as well as the number and diversity of provided functional groups offer a viable alternative to conventional modification strategies of functionalizing polypropylene. LCI's role as broadly applicable adhesion promoter was demonstrated by equipping polypropylene with the fluorescent dye ThioGlo-1 via the anchor peptide LCI. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Material binding peptides Anchor peptides Surface modification Immobilization

1. Introduction With an estimated worldwide consumption of 62.4 million tons by 2020, polypropylene is an ubiquitous commodity polymers in the many sectors of industry [1,2], (e.g. packaging [3], textiles [1], medicine [4], and automotive industry [5]). Polypropylene has an excellent chemical resistance, high melting temperature and can be processed for instance by injection molding or extrusion [6]. Synthetic polymers for textile and automotive applications are usually functionalized to improve adhesion and dyeing behavior [5,7]. Yet,

* Corresponding author. DWI - Leibniz-Institut für Interaktive Materialien e.V., Forckenbeckstr. 50, D-52056 Aachen, Germany. E-mail addresses: [email protected] (K. Rübsam), benjamin. [email protected] (B. Stomps), [email protected] €ker), [email protected] (F. Jakob), [email protected]. (A. Bo de (U. Schwaneberg). http://dx.doi.org/10.1016/j.polymer.2017.03.070 0032-3861/© 2017 Elsevier Ltd. All rights reserved.

its unreactive hydrocarbon composition makes its efficient functionalization a challenging task [6]. Functionalized polypropylene is applied in biomaterials such as surgical implants [8], blood filtration membranes as well as filtration membranes for waste water treatment or desalination of seawater [9]. A large variety of processing steps are applied to reduce polypropylene's hydrophobicity to reduce unspecific protein adsorption, biofouling or blood coagulation surface [10]. Surface modification techniques such as plasma or flame treatments, chemical modification or graft polymerization are applied to increase wetting ability and to introduce (hydrophilic) functional groups for further modifications (e.g. adhesion of inks or covalent enzyme immobilization) [11,12]. Surface modification techniques for polypropylene are summarized in reviews by Chan et al. [13] and Penn et al. [14]. Surface binding anchor peptides promise a huge potential to become a widely applied surface functionalization technique which amends to the toolbox of currently used conventional chemical and

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physical surface modifications. Even at room temperature, surface binding peptides bind to numerous materials (e.g. platinum [15], gold [16], zinc oxide [17], silicon nitride [18], polystyrene [19e21] and polypropylene [22]), which was described in comprehensive reviews by Care et al. [23] and Seker et al. [24]. Surface binding peptides were applied in the directed immobilization of enzymes on material surfaces. A glutathione S-transferase was immobilized on polystyrene using the binding peptides PS19 and PS23. The PS19- and PS23-GST fusions proteins displayed a directed orientation onto polystyrene substrates (polystyrene petri dishes and polystyrene latex beads) [20]. The immobilization of Cytochrome P450 BM3 and an alkaline phosphatase on gold and/or indium was achieved by tin oxide, gold binding peptide 1 and HighSP-binding peptide [25], [26]. Further polymer binding peptides were reported for polyetherimide (PEI), poly(methyl methacrylate) (PMMA), and Poly(L-lactide) (PLLA) [27]. Polypropylene binding peptides were identified by phage display and patented [22]. Interactions between peptides and surfaces cover a broad spectrum of intermolecular forces [28]. Peptides that bind to polystyrene were reported to be enriched in aromatic amino acids, carrying motifs such as WXXW (X represents any amino acid and W represents tryptophan) [29] and/or contain phenylalanine, tyrosine or tryptophan at the N-terminus of the peptide [19]. The binding mechanism of these aromatic amino acids and the aromatic moieties of polystyrene is primarily based upon the stacking of aromatic ring systems [19,27]. In contrast to this, PMMA binding peptide c2 was found to rely on the C-terminal proline residue resulting in a conformation capable of forming hydrogen bonding to PMMA ester groups [27]. In surface binding peptides, the total number of amino acids limit the number of interactions with the surface [30]. Peptides identified via phage display are fused to capsid proteins of phages (e.g. M13, T7, or l). Thus they are restricted in their size to 6e20 amino acids [24], limiting potential interactions. Previous studies also observed that the binding affinity to surface material can be increased with increasing peptide length [27]. In comparison to single gold binding peptide (GBP)
1 sequences, tandem repeats of GBP-1 in fusion with alkaline phosphatase (5GBP1-AP and 6GBP1AP) showed increased binding to gold particles [26]. Peptides evolved to interact with biological polymers such as membranes of phospholipids over millions of years. One class of polypeptides that ranges among the water-soluble peptides, that interact and/or insert into such biological membranes are antimicrobial peptides (AMPs). AMPs show a detergent-like behavior or form barrel-stave or toroidal pores by self-assembly via a variety of modes of action [31e33]. Initial binding studies of the antimicrobial peptide cecropin-A (CecA, 37 aa, Hyalophora cecropia, a-helical structure) to a synthetic ABA block copolymer (PIB1000ePEG6000ePIB1000) and PMOXA-PDMS-PMOXA polymersomes were conducted by Noor et al. [34] and Klermund et al. [35]. AMPs vary in their length (usually from 10 to 100 amino acids), differ in net charge, amino acid composition, hydrophobicity, and secondary structure [36]. In this study we selected five representative antimicrobial peptides to investigate their potential as polypropylene-binding peptides. Four peptides with a cecropin A-like a-helical structure, one peptide with b-sheet structure and one peptide with a circular structure were selected from the antimicrobial database (APD) [36]. The selected peptides adenoregulin (Ade) [37], cathelicidin-BF (Cat) [38], and cecropin A (CecA) [39] are classified as alpha helical peptides whereas the selected Reutericin (Reu) [40] is classified as circular. The selected helical and circular peptides are unstructured in water and form their secondary structure exclusively when they are in contact with a surface (biological membrane) [33]. The peptide LCI (47 aa, originating from Bacillus subtilis) has a b-sheet

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structure which is reported to be stable in aqueous solution [41]. We investigated the applicability of the selected antimicrobial peptides as polypropylene binding anchor peptides. The functionalization toolbox is based on the anchor peptide fusion proteins EGFP-Ade, EGFP-Cat, EGFP-CecA, EGFP-Reu, and EGFP-LCI (EGFP: enhanced green fluorescent protein). The binding of the fusion proteins EGFP-AP to polypropylene was characterized by fluorescence microscopy. The binding of identified polypropylene anchor peptide LCI was further characterized by scanning force microscopy, and binding assays with and without surfactants. We demonstrated the general applicability of LCI as adhesion promotor by conjugation of the fluorescent dye ThioGlo-1 with LCI and immobilization of the bio conjugate on polypropylene. 2. Experimental 2.1. Materials All used chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, USA), AppliChem GmbH (Darmstadt, Germany) as well as Carl Roth GmbH (Karlsruhe, Germany) and had analytical-reagent grade or higher purity. Synthetic genes were obtained from GeneArt AG (Regensburg, Germany) and oligonucleotides were acquired from Eurofins Scientific SE (Ebersberg, Germany) in saltfree form. Enzymes were obtained from New England Biolabs GmbH (Frankfurt am Main, Germany). Plasmid extraction and PCR purification kits were purchased from Macherey-Nagel GmbH & Co. KG (Düren, Germany) and Qiagen GmbH (Hilden, Germany). The BCA Protein Assay kit was obtained from Novagen EMD Chemicals Inc. (San Diego, USA). Polypropylene fiber material was kindly provided by Coats GmbH (Kenzingen, Germany), polypropylene plates were extruded in house from material obtained from LyondellBasell Industries N. V. (Rotterdam, Netherlands). Black polypropylene microtiter plates (MTP) were obtained from Greiner BioOne GmbH (Frickenhausen, Germany). The solvent toluene (>99%) was purchased by VWR International LLC (Radnor, USA), the alkylsilanes triethoxy(ethyl)silane (96%) and trichloro(octyl)silane (97%) were acquired from Sigma-Aldrich Corp. All chemicals were used without further purification. 2.2. Plasmids and strains Plasmid pET28a(þ) from Novagen (Darmstadt, Germany) was used as expression vector. The plasmid pEGFP was obtained from Clontech Laboratories Inc. (Madison, USA). E. coli strains DH5a and BL21-Gold (DE3) were purchased from Agilent Technologies Inc. (Santa Clara, USA). E. coli DH5a was used as cloning hosts and E. coli BL21-Gold (DE3) was used as protein expression system. 3. Methods 3.1. Generation of EGFP-anchor peptide fusion constructs Fusion proteins of anchor peptides with EGFP was performed to simplify detection and quantification of bound anchor peptides and to provide a visual proof of the general the anchor peptide toolbox applicability. A spacer sequence including 10  alanine and a TEVprotease cleavage site sterically separates EGFP from the anchor peptide. The TEV-protease cleavage site enables the separation and purification of the anchor peptides (without EGFP) to investigate surface binding. The plasmid pET28a::EGFP served as vector backbone for the fusion construct. A detailed description of its generation, as well as a list with all primers (Table S1) can be found in the supplementary material. The synthetic genes of Adenoregulin (Ade; UniProt ID:

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P31107), Cathelicidin-BF (Cat; UniProt ID: B6D434), Cecropin-A (CecA; UniProt ID: P01507), Reutericin (Reu; UniProt ID: O24790), and LCI (UniProt ID: P82243) were codon optimized for E. coli and synthesized by GeneArt (Regensburg, Germany). All synthetic genes contained a spacer sequence consisting of a TEV protease cleavage site sequence followed upstream by a sequence encoding for ten alanine residues (see Table S2) at the 5’-end of the anchor peptide gene. A universal cloning platform was established using phosphorothioate-based ligase independent cloning (PLICing) to generate the EGFP-anchor peptide fusion proteins [42]. For each construct, the corresponding synthetic gene was amplified using primers F-Insert and R-Insert containing phosphorothioated nucleotides (Sequences shown in Table S1). The vector backbone pET28a::EGFP was amplified using primers F-Vector and R-Vector. Iodine cleavage and hybridization was performed according to Blanusa et al. with 0.08 mM insert and 0.04 mM vector. The generated constructs (see Fig. 1A) were used for transformation of E. coli DH5a. The successful construction was verified by sequencing. The negative control consists of the fusion protein EGFP-10xAlaTEV (EGFP) and was generated by inserting a stop codon (TAA) between the gene sequence encoding for the tobacco etch virus (TEV) - protease recognition site and the gene sequence encoding for CecA. Site-directed mutagenesis was performed as previously reported [43], using the pET28a::EGFP-cecropin A template and SDM-primers F-EGFP-neg and R-EGFP-neg. One LCI variant that contains a cysteine-residue between the TEV-protease recognition site and the sequence of LCI was generated to verify the attachment of anchor peptides lacking a fusion tag. Site-directed mutagenesis was used as previously reported [43] with pET28::EGFP-LCI as template and SDM-primers F-Cys-LCI and R-Cys-LCI. The PCR product was digested (20 U DpnI; overnight, 37  C) and purified using PCR clean-up kit (Macherey-Nagel), transformed and subsequently verified by sequencing as described in the previous paragraph.

3.2. Production of EGFP-anchor peptide fusion proteins Proteins of the generated fusion proteins (EGFP-AP and the negative control EGFP) were expressed in E. coli BL21 (DE3) gold cells. Precultures of E. coli BL21 (DE3) gold cells in 10 mL LB-media (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 0.1 mM kanamycin)

were incubated overnight (16 h, 37  C, 250 rpm, 70% humidity in 50 mL Erlenmeyer flasks; Multitron Pro, Infors AG, Bottmingen, Switzerland) and used to inoculate the main cultures. The main cultures (100 mL; TB-media: 12 g/L peptone, 24 g/L yeast extract, 4 mL/L glycerol, 2.31 g/L KH2PO4, 12.54 g/L K2HPO4, 0.1 mM kanamycin) were mixed with the inoculum until an optical density of 0.02 was reached. Main cultures were cultivated until an OD600nm of 0.6 (4 h, 37  C, 250 rpm, 70% humidity; 500 mL Erlenmeyer flasks; Multitron Pro, Infors AG) could be observed. Protein overexpression was induced by supplementing isopropyl b-D-1thiogalactopyranoside (IPTG; 0.1 mM final concentration). Upon induction, the cultivation temperature was reduced to 20  C. Cells were harvested after 40 h by centrifugation (3200 g, 10 min, 4  C; Eppendorf centrifuge 5810 R, Eppendorf AG, Hamburg, Germany) and the cell pellets were stored (
20  C). The obtained cell pellets were suspended in tris(hydroxymethyl)-aminomethan (Tris/HCl) buffer (20 mL; pH 8.0, 50 mM) and disrupted by sonication on ice (3  30 s, interval 30 s, 70% amplitude). Soluble proteins were separated from cell fragments and insoluble proteins by centrifugation (3200 g, 15 min, 4  C; Eppendorf centrifuge 5810 R). The supernatant was filtered through a 0.45 mm cellulose-acetate filter (GE Healthcare, Little Chalfont, UK) and subsequently used for coating experiments or further purification.

3.3. MTP-expression of EGFP-anchor peptides Precultures (150 mL/well; LB-media, 0.1 mM kanamycin) were inoculated from a glycerol stock in 96-well plate format and incubated overnight (37  C, 900 rpm, 70% humidity; Multitron Pro, Infors AG). The main cultures (125 mL; Terrific broth, 0.1 mM kanamycin), were inoculated with 10 mL preculture. After incubation (4 h, 37  C, 900 rpm, 70% humidity; Multitron Pro, Infors AG) protein expression was induced by supplementing IPTG (0.1 mM final concentration), followed by a reduction of the cultivation temperature to 20  C. Cells were harvested by centrifugation (3200 g, 15 min, 4  C; Eppendorf centrifuge 5810 R) after 40 h of incubation (20  C, 900 rpm, 70% humidity; Multitron Pro, Infors AG). Cell pellets were stored at
20  C until further use. To disrupt the E. coli BL21 (DE3) gold cells, the pellets were suspended in lysozyme solution (150 mL; 1.5 mg/mL; Tris/HCl buffer, pH 8.0, 50 mM) and incubated (1 h, 37  C, 900 rpm, 70% humidity;

Fig. 1. (A) Vector map of pET28a(þ)::EGFP-anchor peptide. Grey arrows show the direction of transcription. PLICing primers are represented in italic. (B) Schematic representation of the fusion protein EGFP-LCI consisting of the fluorescent reporter protein EGFP, a spacer sequence including 10x alanine and a TEV-protease cleavage site, which separates EGFP sterically from the anchor peptide LCI. Arrows next to EGFP indicate the dimensions of EGFP (determined by YASARA (Yet Another Scientific Artificial Reality Application; v16.3.5) using the 2Y0G crystal structure of EGFP).

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Multitron Pro, Infors AG) followed by a centrifugation step (3200 g, 30 min, 4  C; Eppendorf centrifuge 5810 R). 3.4. Purification of EGFP-anchor peptide fusion proteins The EGFP-anchor peptide fusion proteins were purified via chromatography and dialysis (Supplementary material). Protein concentrations were determined with the BCA protein assay kit (Novagen, Merck KGaA) and protein homogeneity was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) using a 5% stacking gel and a 12% separating gel [44]. 3.5. Fusion construct cleavage and purification of anchor peptides To generate anchor peptides without fused EGFP, the fusion constructs were cleaved with the tobacco etch virus (TEV) protease, which cleaves the amino acid sequence ENLYFQG/S site-specifically [45,46]. Fusion protein EGFP-LCI is specifically cleaved by the TEVprotease (Sigma-Aldrich Inc.) into LCI and EGFP. The optimum cleavage efficiency (>99%) occurred at a ratio 30:1 (EGFP-LCI:TEV) in a cleavage buffer (pH 8.0; 25 mM Tris/HCl, 500 mM NaCl, 0.5 mM EDTA, 1 mM DTT; 30  C, 24 h). The cleavage mixture was transferred onto a Protino® Ni-NTA Column (GE Healthcare) to purify the cleaved anchor peptide. Both EGFP and TEV contain a His6-Tag and attached to the column, while pure anchor peptide was collected in the flow through. 3.6. Binding of EGFP-anchor peptide fusion proteins (EGFP-AP) to polypropylene surfaces Synthetic polypropylene fibers (Coats GmbH, Kenzingen, Germany) were coated with EGFP-AP. Fluorescence intensity of EGFPAP cell lysates was measured with a 96-well FLUOstar Omega MTP reader (BMG Labtech GmbH, Ortenberg, Germany; excitation (exc.) 485 nm, emission (em.) 520 nm, gain 750). The fluorescence of EGFP-AP cell lysates was normalized to 14.000 relative fluorescence units (RFU) by addition of Tris/HCl buffer (pH 8.0, 50 mM), which corresponds to 1.07 mM protein. EGFP-LCI fluorescence standard curve data are shown in supporting information (Fig. S1). The volume of 1 mL bovine serum albumin BSA (1 mg/mL) was supplemented to 1 mL of the fluorescence normalized EGFP-AP samples. The selected polymer fibers (length 2 cm) were incubated for 10 min at room temperature in 2 mL of the normalized EGFP-AP/BSA solutions, followed by two subsequent washing steps with 10 mL Tris/HCl buffer (pH 8.0, 50 mM). The coated polymer fibers were protected from light. Fluorescence microscopy was performed with the confocal microscope TCS SP8 (Leica Microsystems CMS GmbH, Mannheim, Germany). The standard GFP mode of the TCS SP8 software was used (exc. 488 nm, argon laser 20% intensity, em. 500e600 nm, gain 800). Transmission pictures were taken with the PMT Trans detector (gain 250). Subsequently overlay pictures of transmission and fluorescence microscopic measurements were generated. 3.7. Determination of film thickness by scanning force microscopy Modification of silica wafers used in this experiment is described in the supplementary material. The modified silicon surfaces were incubated with purified proteins EGFP and EGFP-LCI (5.5 mL, 75 mg/mL; Tris/HCl buffer pH 8.0, 50 mM; 160 min, room temperature). The coated wafers were rinsed with Tris/HCl buffer (pH 8.0, 50 mM) or ddH2O and dried under atmospheric conditions. Layer thickness and homogeneity were determined with scanning force microscopy (SFM) measurements in intermittent mode using a Bruker Dimension Icon with Nanoscope 8.10 software and OTESPA

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tips with a spring constant of 12e103 N/m and a resonant frequency of 278e357 kHz (Bruker Corporation, Billerica, USA) under ambient conditions. After scratching the protein layer with a needle the film thickness was determined at the edge of the film. The trichloro(octyl)silane modified wafers were coated with EGFP and EGFP-LCI respectively and incubated in Triton X-100 (100 mL; 2 mM, 5 min, room temperature) to determine surfactant stability. The Triton X-100 solution was removed from the surface by dipping the wafer into ddH2O. The remaining film thickness was determined at the edge of the film as described above. 3.8. Characterization of anchor peptide binding strength in presence of surfactant To analyze the binding strength of anchor peptide LCI towards polypropylene in presence of the surfactants Triton X-100 and alkyl benzene sulfonate (LAS) a 96-well polypropylene microtiter plate based assay was established. The volume of 5 mL cell lysate of EGFPLCI was supplemented to 95 mL Tris/HCl buffer (pH 8.0, 50 mM) in a black polypropylene MTP (Greiner bio-one) and incubated (10 min, room temperature, 600 rpm; MTP shaker, TiMix5, Edmund Bühler GmbH, Hechingen, Germany). In a subsequent washing step, the MTP wells were washed twice with Tris/HCl buffer (100 mL; pH 8.0, 50 mM, 5 min, room temperature, 600 rpm). In the final desorption step, 100 mL of non-ionic surfactant Triton-X 100 (0e10 mM), or of anionic surfactant LAS (0e5 mM) were supplemented to the wells and incubated (5 min, room temperature, 600 rpm). After removal of excess liquid the residual fluorescence of the bound EGFP-APs was measured directly on the polypropylene surface with the 96well microtiter plate reader FLUOstar Omega (exc. 485 nm, em. 520 nm, gain 1000, 35 reads/well). 3.9. Determination of bound anchor peptide amount The MTP binding assay described above was adapted to determine the amount of bound EGFP-LCI on polypropylene. EGFP-LCI fluorescence standard curve data are shown in the supplementary material (Fig. S2). The wells of a black polypropylene MTP (Greiner bio-one) were incubated with BSA (100 mL; 2 mM, 10 min, room temperature, 600 rpm) to prevent unspecific protein adsorption. Varying concentrations of purified EGFP-LCI (100 mL; 0e1 mM) were supplemented to the pretreated wells and incubated (10 min, room temperature, 600 rpm). Three washing steps with Tris/HCl buffer (pH 8.0, 50 mM) and subsequent fluorescence detection (FLUOstar Omega; exc. 485 nm, em. 520 nm, gain 1000, 35 reads/well) were performed. 3.10. Application and detection of purified anchor peptides on polypropylene surface One hundred mL of Cys-LCI (20 mM; diluted with Tris/HCl buffer pH 8.0, 50 mM) were transferred on clean polypropylene plates (extruded from resin: Hostalen PP W2080, LyondellBasell Industries B. V., Netherlands) and incubated (10 min, room temperature). The peptide solution was removed and the polypropylene plate was rinsed with 10 mL deionized water (ddH2O). A negative control was performed with polypropylene plates that were incubated with Tris/HCl buffer (10 mL, pH 8.0, 50 mM). The surface bound peptides were visualized by a fluorescent staining with a maleimide compound (ThioGlo-1, Berry & Associates Inc., Dexter, USA), which reacts with free thiol-groups yielding fluorescent product. The plates were incubated for 2 h, protected from light, kept on ice with 100 mL of ThioGlo-1 (25 mM; diluted from concentrated stock solution with Tris/HCl buffer, pH 8.0, 50 mM). Subsequently, each plate was rinsed with 10 mL ddH2O and

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analyzed with confocal microscopy (TCS SP8, Leica Microsystems CMS GmbH, Mannheim, Germany). Samples were excited with 405 nm, 15% laser intensity. Detection was performed with a HyD1 detector (emission 500e520 nm, gain 300). Subsequently, transmission and fluorescence microscopy was performed and overlay pictures were generated with Leica Application Suite X software. An additional control was performed with polypropylene plates, which were incubated with 20 mM BSA. BSA contains one freely accessible cysteine residue that converts supplemented ThioGlo-1 into its fluorescent product. The plates incubated with BSA were treated identical to those incubated with Cys-LCI. 3.11. Generation of LCI 3D structures The computationally generated 3D structures are based on deposited protein data bank (PDB) files of LCI (2B9K), and EGFP (2Y0G). Dimensions of EGFP were determined using YASARA (Yet Another Scientific Artificial Reality Application, v16.3.5; Fig. 1B). Surface amino acids of anchor peptide LCI were grouped into two categories (hydrophobic in green/hydrophilic in blue) and visualized using Discovery Studio Client, Release 4.0 (Accelrys Software http://accelrys.com/; Fig. 5). 4. Results and discussion The results and discussion section is divided into five parts to characterize and validate the peptide based functionalization toolbox for isotactic polypropylene. The first part investigates polypropylene binding of selected antimicrobial peptides. In parts two and three the polypropylene binding of antimicrobial peptide LCI is characterized by scanning force microscopy and a MTPbinding assay. Part four shows the determination of bound peptide amount. The general applicability of the anchor peptide as adhesion promotor is demonstrated in part five. 4.1. Fluorescence microscopy of coated polypropylene surfaces Anchor peptide binding was visualized by the fused reporter protein (EGFP) fluorescence. Polypropylene fibers were coated with EGFP, EGFP-Ade, EGFP-Cat, EGFP-CecA, EGFP-Reu, and EGFP-LCI, washed to remove unspecific protein adsorption and subsequently analyzed by fluorescence microscopy (Fig. 2). Polypropylene fibers showed no auto fluorescence with applied settings

(Fig. 2A). The applied washing procedure was optimized to avoid unspecific protein adsorption of EGFP (without anchor peptide). Indeed no fluorescent signal was detected for polypropylene fibers coated with EGFP, EGFP-Cat and EGFP-Reu after washing (Fig. 2B). In contrast, polypropylene fibers incubated with EGFP-Ade, EGFPCecA and EGFP-LCI showed a detectable fluorescence after the washing steps, which indicates this fluorescence is not caused by unspecific protein adsorption of EGFP. As a-helical antimicrobial peptides, both Adenoregulin and Cecropin A form amphipathic helices with the hydrophobic side in direct contact to the hydrophobic surface in presence of hydrophobic surfaces [47,48]. In contrast, LCI is a b-sheet peptide with a yet unsolved mechanism of action. EGFP-LCI showed the most intense fluorescence signal on isotactic polypropylene fibers (Fig. 2) and therefore was chosen for further characterization experiments. 4.2. Characterization of EGFP-LCI films by scanning force microscopy The film thickness of EGFP-LCI was determined by scanning force microscopy (SFM) after scratching of an unmodified silicon wafer (SiO2), a triethoxy(ethyl)silane (ethyl-SiO2-) wafer and a trichloro(octyl)silane (octyl-SiO2-) wafer. Both the SiO2-wafer and the two SiO2-functionalized wafers were selected due to their difference in hydrophobicity. The SiO2-wafer, ethyl-SiO2-wafer and the octyl-SiO2-wafer have contact angles of 35.3 ± 0.1, 68.4 ± 0.4 and 108.6 ± 0.3 , respectively. Below a contact angle of ~90 surface properties shift from hydrophobic to hydrophilic. The difference in contact angle of the SiO2-wafer, ethyl-SiO2-wafer and the octylSiO2-wafer gives the opportunity to investigate the influence of hydrophobicity on protein adsorption. The contact angle of the octyl-SiO2-wafer (108.7 ) is comparable to the contact angle of polypropylene (103 ). The octyl-SiO2-wafer served as a model surface for polypropylene to investigate protein adsorption and film thickness. Fig. 3A shows SFM measurements of EGFP-LCI and EGFP films on octyl-SiO2-wafers. A homogenous film of equal height was obtained for the EGFP-LCI by SFM measurement (Fig. 3A). The difference in height between uncoated wafer and EGFP-LCI coated wafer was 4.1 ± 0.2 nm. The film thickness of EGFP-LCI films on SiO2-wafer and on ethyl-SiO2-wafer (Fig. S4 and Fig. S5) was 3.7 ± 1.0 nm and 4.6 ± 1.5 nm. The average film thickness of EGFP-LCI coated on all three tested wafers was 4.0 ± 1.5 nm and was independent of the hydrophobic character of

Fig. 2. Visualization of EGFP-anchor peptide binding on polypropylene fibers by fluorescence microscopy: Uncoated and washed polypropylene fiber; Polypropylene fibers were incubated with EGFP, EGFP-Ade, EGFP-Cat, EGFP-CecA, EGFP-Reu, and EGFP-LCI, followed by two washing steps. Successful coating is visualized by EGFP fluorescence (exc. 488 nm, em. 500e600 nm).

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Fig. 3. Scanning force microscopy measurements of octyl-SiO2-wafers coated with (A) EGFP-LCI and (C) EGFP without anchor peptide. In order to evaluate the binding strength a Triton X-100 solution (2 mM) was applied to wash the surface. Pore formation occurred in (D) EGFP films whereas the (B) EGFP-LCI film remained on the surface but its roughness increased.

the wafers. This height corresponds to the formation of a monolayer of the EGFP-LCI (Fig. 1B). The film that is solely composed of EGFP varies in thickness from 6 to 10 nm (Fig. 3C) and appears to be inhomogeneous in comparison to the EGFP-LCI film (Fig. 3A). The latter indicates that LCI governs the binding of EGFP in the EGFP-LCI fusion construct and prevents further adsorption or formation of aggregates. SFM measurements of EGFP-LCI and EGFP films that were subjected to Triton X-100 (2 mM; 5 min) confirmed that the EGFP-LCI film surface proved less homogenous and thickness was reduced (from 4.1 ± 0.2 nm to 2.5e4 nm; see Fig. 3B), whereas the rough EGFP film thickness was reduced by 66e75% and significant pore formation occurred (see Fig. 3D and Fig. S6). This implies a strong interaction between polypropylene-like wafer and the anchor peptide LCI in contrast to a weak interaction between polypropylene-like wafer and EGFP. 4.3. EGFP-anchor peptide binding in presence of surfactants Binding strength of the fusion protein EGFP-LCI to polypropylene was studied in presence of a nonionic and an anionic surfactant. Interactions between proteins and non-ionic surfactants in aqueous systems are governed by hydrophobic forces [49]. Triton X-100 was selected, as it is frequently used to prevent nonspecific hydrophobic-driven protein adsorption [19]. Interactions between proteins and anionic surfactants are governed by electrostatic and hydrophobic forces [49]. Alkyl benzene sulfonate (LAS) was selected as anionic surfactants to study the binding of EGFP-LCI to polypropylene, as LAS does not denature protein structures even at high concentrations [50]. A prerequisite for the analysis of EGFP and EGFP-LCI binding to polypropylene in presence of surfactants is the stability of EGFP under the applied conditions. Incubation of EGFP in 1 mM Triton X100 and 0.5 mM LAS resulted in a non-significant reduction of EGFP fluorescence (2.8% for 1 mM TritonX-100 and 1.0% for 0.5 mM LAS; Supplementary Fig. S3). The binding of EGFP-LCI to polypropylene was analyzed in a microtiter plate based (MTP) binding assay using cell free lysates and containing EGFP or EGFP-LCI fusion proteins.

The fluorescence of bound EGFP-anchor was measured directly on the polypropylene microtiter plate both prior to and after surfactant treatments. Detected relative fluorescence units (RFU) correlate directly to the amount of bound EGFP-anchor fusion proteins. The negative control EGFP showed fluorescence values of 80 ± 4.3 RFU after buffer washing (Tris/HCl, pH 8.0, 50 mM). The fluorescence decreased to 50.33 ± 3.2 RFU with increasing TritonX100 concentrations in the washing buffer (0.1 mMe10,000 mM). Untreated wells (without incubation step with EGFP) showed comparable values of 52.8 ± 4.4 RFU, which indicates a complete removal of EGFP from the polypropylene surface when the washing buffer contains TritonX-100 in concentration higher than 0.1 mM. The coating of polypropylene with the fusion protein EGFP-LCI resulted in a fluorescence of 297.6 ± 29.9 RFU when washed with buffer (Tris/HCl, pH 8.0, 50 mM). Application of low Triton X-100 concentrations (0.1e10 mM) did not result in a detachment of EGFPLCI from polypropylene. Increasing the applied Triton X-100 concentrations (20e500 mM), resulted in a decreased fluorescence signal (132.3 RFU corresponding to 44% of initial fluorescence; see Fig. 4A). The detachment of LCI from the polypropylene surface starts in presence of 20 mM Triton X-100, which is below the critical micellar concentration (CMC: 260e270 mM) of Triton X-100 [51]. Detachment of EGFP-LCI below the CMC of Triton X-100 indicates that EGFP-LCI interacts with the hydrophobic Triton X-100 monomers. Increasing the Triton X-100 concentration above CMC, did not lead to further detachment of EGFP-LCI from polypropylene, showing that the detachment of LCI from polypropylene is not driven by micelle formation. The addition of the anionic surfactant LAS to the washing solutions (5e5000 mM) resulted in a sigmoidal decrease of the residual fluorescence signal of EGFP-LCI (Fig. 4B). The CMC of LAS is 280e300 mM. In presence of 200 mM LAS EGFP-LCI retained 106.8 RFU (33% of its initial fluorescence; see also Fig. 4B). A complete removal of EGFP-LCI is detected, when LAS is added in concentrations higher than 400 mM (detected fluorescence of EGFP-LCI equals the detected fluorescence of EGFP negative control). LAS has a stronger effect on LCI binding to polypropylene than Triton X-100, as a complete removal of EGFP-LCI is achieved by LAS addition but

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polypropylene surface. A similar estimation conducted with P450 BM3 and ITO binding peptides showed a surface coverage of 20 fmol peptide/well [25]. LCI contains a total of 22 hydrophobic amino acids, 20 polar or charged amino acids which carry 25 functional groups in the side chains. Fig. 5B shows the structure of LCI in folded state (LCI: 2B9K). Hydrophobic regions (shown in green) are potential interaction sites with polypropylene, hydrophilic regions (shown in blue) are important to maintain the solubility of LCI in water and can provide functional groups like hydroxyl and carboxyl groups for further surface functionalization. Assuming that the coated surface area of one well correlates to its flat bottom (34 mm2; product data sheet, 96 well plate PP, Greiner Bio-one), EGFP-LCI has an estimated surface coverage of 0.8 pmol/cm2 or 4.81  1011 molecules/cm2. This amount of LCI molecules contains 1.92  1012 carboxyl, 1.44  1012 carboxamide, 4.81  1012 hydroxyl and 4.81  1012 amino groups available for further functionalization. 4.5. Coating of polypropylene surface by LCI peptide

Fig. 4. Binding of EGFP-LCI in presence of nonionic and an anionic surfactant. Residual fluorescence of EGFP-LCI (black bars) and EGFP (grey bars) on polypropylene in presence of (A) Triton X-100 and (B) alkyl benzene sulfonate (LAS).

addition of Triton X-100 (even at the highest tested concentration) did not result in a removal of EGFP-LCI from polypropylene. Interactions of LAS with LCI may also occur via electrostatic interactions of the anionic head group of LAS with the positively charged residues arginine (R46) and lysine (K3, K22, K28, K34 and K47) of LCI. A detachment of LCI from polypropylene surface could therefore be caused by the negatively charged micelle surface. Intensive washing with dH2O or buffer did not lead to any leaching of EGFP-LCI from polypropylene and binding studies in presence of Triton X-100 and LAS proved a strong binding of EGFPLCI to isotactic polypropylene. LCI is therefore suitable for further functionalization and tailoring of surface properties. Scanning force microscopy experiments and microtiter plate based binding assay also showed that EGFP-LCI coatings of polypropylene surface can withstand up to 10 mM Triton X-100, whereas EGFP films (without anchor peptide) were removed and showed pore formations as reported for many proteins [52e54]. The monolayer of EGFP-LCI on polypropylene surface can be most likely attributed to a high number of hydrophobic interactions between the LCI and isotactic polypropylene. 4.4. Quantification of protein amount on polypropylene surface Protein coverage of a polypropylene well was determined with an adapted MTP binding assay. A saturation of the fluorescence signal was reached when 0.25 mM EGFP-LCI were used for incubation (167.33 ± 10.01 RFU; Fig. 5A). The detected fluorescence correlates to 0.27 pmol EGFP-LCI/well with a flat bottom (area of 34 mm2; fluorescence standard curve see Fig. S2). This provides a good estimation of how much protein is bound to the

The general applicability of LCI as adhesion promotor was demonstrated by conjugation of the fluorescent dye ThioGlo-1 by maleimide-coupling with an additionally introduced free cysteine thiol group at the N-terminus of LCI. The conjugation of ThioGlo-1 with a free thiol group results in a fluorescent derivative. The successful immobilization is analyzed by fluorescence microscopy after immobilization. Bovine serum albumin (BSA), a well-studied protein, is known to show unspecific adsorption to polypropylene surface and harbors a thiol group accessible for ThioGlo-1 coupling [55]. The coupling reaction with ThioGlo-1 carried out in solution resulted in a ~3-times higher fluorescence signal for 1 mM BSA (2580 RFU) compared to 1 mM Cys-LCI (802 RFU). Samples of BSA and Cys-LCI (20 mM each) were applied on polypropylene surfaces (1 cm2, extruded from resin: Hostalen PP W2080, LyondellBasell Industries B. V.). Fig. 6 shows the degree of bound protein available for functionalization (A: neg. control, buffer only; B: coated with BSA; C: coated with Cys-LCI) after washing (10 mL dH2O water per plate) via the intensity of the ThioGlo-1 fluorescence. Although BSA showed a high maleimide coupling signal in solution, only few thiol groups are available for maleimide coupling on the polypropylene surface. Cys-LCI shows a comparably high and uniform coverage of the surface with thiol groups, which are available for maleimide coupling. The introduction of accessible thiol groups (through a cysteine) in combination with the maleimide fluorescence label proved to be a straightforward and user-friendly method to functionalize polypropylene fibers and surfaces. 5. Conclusion In this study we identified and characterized PP-anchor peptides, and demonstrated their potential as adhesion promoters. Anchor peptides offer tremendous opportunities as a functionalization toolbox for the introduction of specific numbers of hydroxyl, carboxyl, thiol and amines to improve adhesion of coatings and to modify polypropylene surfaces via the attached functional moieties. The main performance criteria for polypropylene functionalization by anchor peptides is the coating density (0.8 pmol/cm2) as well as number and diversity of provided functional groups for further modifications. SFM measurements (see Fig. 3A) and fluorescence microscopy measurements (see Fig. 2) demonstrate that EGFP-LCI binds on SiO2-wafer, ethyl-SiO2-wafer and octyl-SiO2wafer (polypropylene-like) as a monolayer. Binding assays revealed that 4.81  1011 molecules bind to one cm2, of wafer. Hence, 1 g of LCI (corresponding to 5.23 mmol) is sufficient to cover 654 m2 of a polypropylene surface. Furthermore, the applicability of LCI as

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Fig. 5. A: Binding saturation of EGFP-LCI in polypropylene MTP well (contact area 0.95 cm2). Residual fluorescence of EGFP-LCI was determined in triplicated after 3 cycles of washing with TrisHCl buffer (pH 8.0, 50 mM); B: Three-dimensional structure of LCI (with hydrophobic regions in green and hydrophilic regions in blue. Hydrophobic areas are capable of interacting with polypropylene. Hydrophilic areas both ensure solubility and provide functional groups for further modifications. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Visualization of anchor peptide binding to polypropylene surface via fluorescence microscopy (polypropylene plates incubated 10 min at room temperature with (A) 50 mM Tris/HCl pH 8.0, (B) 20 mM bovine serum albumin, (C) 20 mM Cys-LCI, washed in 10 mL ddH2O and stained with fluorescent dye ThioGlo-1, exc. 405 nm, em. 500e520 nm).

adhesion promotor was demonstrated by successful functionalization of polypropylene with the fluorescent dye ThioGlo-1. Initial investigations on the mechanical stability of the EGFP-LCI monolayer on polypropylene plates were performed applying highpressure steam (60 bar, 60  C, 60 s; Fig. S7). Results support the high application potential of the anchor peptide technology. In conclusion, the green anchor peptide toolbox for functionalization of polypropylene can generally be applied to a large variety of polypropylene based materials (e.g. polypropylene fibers, yarns, flat surfaces and likely other polypropylene based materials (e.g. membranes). The possible methods of application range from standard dip coating and spraying to foulard processing. Its simplicity in handling renders the anchor peptide toolbox especially attractive for polypropylene substrates with complicated 3D structures, e.g. hernia implants, or microfluidic devices.

Abbreviations Ade Adenoregulin; Cat Cathelicidin-BF; CecA Cecropin-A; EGFP enhanced green fluorescent protein; LAS linear alkyl sulfate; Reu Reutericin; RFU relative fluorescence units.

Author contributions All authors have given approval to the final version of the manuscript.

Funding sources Our research was supported by the German Federal Ministry of Education and Research (BMBF) [FKZ: 031A227F]. Acknowledgment The authors thank the Alliance “FuPol” (Funktionalisierung von Polymeren), the German Federal Ministry of Education and Research (BMBF) [FKZ: 031A227F] for financial support. We thank Dr. Marco Bocola for support in 3D structure visualization of LCI. Appendix A. Supplementary data Supplementary material includes: Primer sequences; Description of pET28a::EGFP generation; Nucleotide sequences of ordered synthetic genes Ade, Cat, CecA, Reu, and LCI; Quantification of fluorescence intensity of EGFP-LCI; Scanning force microscopy measurements of SiO2-wafers and ethyl-SiO2-wafers coated with EGFP and EGFP-LCI; Scanning force microscopy measurements of pore formation of EGFP film on octyl-SiO2-wafers, washed with Triton X-100 (2 mM); Preliminary mechanical stability estimation. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.polymer.2017.03.070. References [1] N. Yaman, E. Ozdogan, N. Seventekin, H. Ayhan, Plasma treatment of polypropylene fabric for improved dyeability with soluble textile dyestuff, Appl. Surf. Sci. 255 (15) (2009) 6764e6770.

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