Bioactive peptides grafted silicone dressings: A simple and specific method

Materials Today Chemistry 4 (2017) 73e83

Contents lists available at ScienceDirect

Materials Today Chemistry journal homepage: www.journals.elsevier.com/materials-today-chemistry/

Bioactive peptides grafted silicone dressings: A simple and specific method Coline Pinese a, Said Jebors a, Pierre Emmanuel Stoebner a, Vincent Humblot b,
a Causse a, Xavier Garric a, Hubert Taillades c, Jean Martinez a,
a, Le Pascal Verdie d, ** , Gilles Subra a, * Ahmad Mehdi Institut des Biomol
ecules Max Mousseron (IBMM), UMR5247 CNRS, ENSCM, Universit
e de Montpellier, France Laboratoire de R
eactivit
e de Surface (LRS), UMR 7197 Sorbonne Universit
es, UPMC Universit
e de Paris 06, France Laboratory of Experimental Surgery, University of Montpellier, France d Institut Charles Gerhardt de Montpellier (ICGM), UMR5253 CNRS, ENSCM, Universit
e de Montpellier, France a

b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 November 2016 Accepted 28 February 2017

The need for bioactive dressings increases with the population aging and the prevalence of chronic diseases. In contrast, there are very few dressings on the market which are designed to display a chosen bioactivity. In this context, we investigated the surface-functionalization of silicone wound dressing with bioactive peptides. One of the challenges was to avoid multistep grafting reactions involving catalysts, solvents or toxic reagents, which are not suitable for the fabrication of medical devices at an industrial scale. In the other hand, a covalent bonding was necessary to avoid the loss of the biological effect by progressive removal of the peptide in biological fluids generated by the wound. To solve these limitations, we developed a strategy allowing an easy and direct functionalization of silicone. This strategy relies on hybrid silylated bioactive peptides, which chemoselectively react with plasma-activated silicone surfaces. We synthesized three hybrid peptides with wound healing properties, which were grafted on commercially available silicone dressings Cerederm® and Mepitel®. Grafted dressings were evaluated in vitro and enabled a quicker scare recovery and extracellular matrix deposition with human dermal fibroblasts. These results were confirmed by in vivo studies showing an enhanced wound-healing of the pig skin. By this simple method, we transformed inert dressing into bioactive dressing which showed properties of wound healing. © 2017 Published by Elsevier Ltd.

Keywords: Hybrid peptides Silicone Active dressings Surface modification Wound-healing

1. Introduction After an injury or following a surgical intervention, wound care is of particular significance to speed up the recovery of the patient and to minimize his stay in hospital environment. Besides acute wounds (burns, frostbite, abrasion and cuts) so called ‘chronic wounds’ represent a crucial public health issue [1]. These wounds are associated with population ageing and the dramatic increase of chronic diseases (i.e. diabetes, cancer), which negatively affect a complete and durable skin healing. As an example, the prevalence of foot ulcers is estimated at 15% of the diabetic population in the

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (A. Mehdi), gilles.subra@ umontpellier.fr (G. Subra). http://dx.doi.org/10.1016/j.mtchem.2017.02.007 2468-5194/© 2017 Published by Elsevier Ltd.

US [2] whereas 2% of the European population suffers from legs ulcers [3]. To efficiently struggle against such health problems, the patient's management has to be considered in a global way, associating drug-based therapy with the wound care itself. In this context, the design of specialized and efficient dressing is a key towards reduction of wound care associated expenses and improvement of patient comfort. The primary function of a dressing is to provide a physical barrier between the wound and the outside, essentially to avoid over damage and pathogen entrance through the skin. A “smart dressing” would combine this physical function with other bioactivities such as anti-inflammatory [4], antimicrobial properties [5] wound healing properties. These bioactivities can be obtained from released and grafted bioactive molecules that finally stimulate and trigger target cell responses crucial to the wound-healing process [6].

74

C. Pinese et al. / Materials Today Chemistry 4 (2017) 73e83

With very low immunogenicity, very good biocompatibility and displaying a wide range of activities, peptides represent the class of compound of choice for the design of bioactive dressings well suited for each type of wound. In this context, we have established a general grafting strategy enabling easy and modular introduction of desired bioactive peptides, in a very specific way, on a dressing surface. Advantageously, the strategy should be suitable to obtain a single activity or combined activities when several peptides are introduced at the same time. To favor a long-term biological effect and to avoid the release of the active moiety, the linkage between the peptides and the surface of the dressing should be covalent. While the simple coating of drugs or bioactive molecules on a device can be quite straightforward, their covalent anchoring is much more challenging. Peptides have already been grafted on many surfaces [7], including silicone [8]. Traditionally, a multistep reaction pathway is required. It starts with the functionalization of the surface (e.g. introduction of a primary amine with a 3aminopropylsilyl derivative), then a reaction with a spacer and finally followed by the grafting of the molecule of interest. Moreover, to guarantee the desired orientation and subsequent biological activity, the grafted peptide should have a single reactive function in its sequence constituting the anchorage point on the material (e.g. the N-terminus of the peptide reacting with an activated carboxylic acid ester for instance) [9]. To avoid unspecific reaction, it is also possible to protect all the reactive functional groups of the peptide during the grafting reaction. Alternatively, chemoselective ligation reaction could be used, involving two mutually reactive functions (e.g. maleimide thiol [10], thiol-ene [11],alcyne-azide [12]) one situated on the surface and the other displayed at the suitable position on the peptide. Whatever the method chosen, it requires organic solvents, catalysts or toxic reagents. These issues are unacceptable when the final material is applicable in the field of healthcare. This prompted us to propose a general strategy to immobilize bioactive peptides in a safe, simple, controlled and specific way. It relies on the use of hybrid bioorganic/inorganic building blocks bearing a hydroxysilyl group [13], which is able to react directly with surfaces in mild conditions (i.e. water, ethanol) compatible with the stability of biomolecules. Those hybrid peptides could be prepared in solution or on solid support [14] and were used to craft hybrid materials by direct synthesis and by one-step modification of silica [15,16] and glass [17] surfaces. In this paper, we first applied this methodology to prepare bioactive silicone dressings using peptide sequences favoring scare recovery. The key step of the process is the dip coating of the silicone device in an ethanol/water solution containing hybrid peptides that were grafted on catheter via Si-O-Si bonds. Three peptides sequences, found in proteins of the extracellular matrix (ECM) were chosen to demonstrate the efficiency of the approach. These three peptides were grafted on silicone dressings and wound-healing properties of the grafted dressings were determined both in vitro and in vivo. 2. Materials and methods 2.1. Chemicals All solvents and reagents were used as supplied. Solvents used for LC/MS were of HPLC grade. Dichloromethane (DCM), N,Ndimethylformamide (DMF) were obtained from Carlo Erba. Fmoc amino acid derivatives, [benzotriazol-1-yloxy(dimethylamino) methylidene]-dimethylazanium hexafluorophosphate (HBTU), Fmoc-Rink amide CM resin, 2-chloro chlorotrityl PS resin, were purchased from Iris Biotech (Marktredwitz, Germany). Diisopropylethylamine (DIEA), trifluoroacetic acid (TFA) were obtained from

Sigma-Aldrich (St. Louis, MO, USA). Triisopropylsilane (TIS) was obtained from Acros. 3- isocyanatopropyldimethylchlorosilane was purchased from Fluorochem(UK) (France). The following abbreviations were used: ICPDMCS e isocyanatopropyldimethylchlorosilane. Other abbreviations used are recommended by the IUPAC-IUB Commission. 2.2. Hybrid peptide synthesis The peptides were prepared by microwave-assisted peptide synthesis using the Fmoc/tBu SPPS strategy on a LibertyTM Microwave Peptide Synthesizer (CEM Corporation, Matthews, NC) providing MW irradiation at 2450 MHz. Temperature was set up at 70
C as previously described [18]. [19] Compound 3 was prepared on Fmoc-Rink amide ChemMatrix resin (1.2 mmol g1) and compounds 1 and 2 were synthesized on 2-chloro, chloro trityl polystyrene (0.8 mmol g1). 2.2.1. Anchoring step After swelling resins in DCM for 15 min, anchoring on the Fmoc Rink amide linker was performed by a standard deprotection/ coupling cycle (see above). Anchoring on 2-chloro, chloro trityl linker was performed by using a solution of Fmoc-Pro-OH and Fmoc-Gly-OH, for compounds 1 and 2 respectively, (1.175 mL, 17.6 mmol, 2 eq) in DMF (40 mL) containing DIEA (3.060 mL, 17.6 mmol, 2 eq). The resin was stirred overnight and washed with DMF (2), DCM/MeOH 1/1 v/v (2), DMF (2), DCM (2) and dried overnight in vacuo. The experimental loading of the resin was determined by measuring the optical density of the dibenzofulvene-piperidine adduct formed during Fmoc deprotection with a piperidine/DMF 2/8 v/v solution at 299 nm and the loading was calculated according to a previously reported method [18]. 2.2.2. Coupling step On a 0.1 mmol resin scale (either H-Rink amide ChemMatrix resin 1.2 mmol/g or 2Cl-chlorotrityl-PS resin 0.8 mmol/g), coupling reactions were performed using 5 eq. of amino acid (0.5 mmol, 2.5 mL of 0.2 M solution in DMF), 5 eq. of HBTU (0.5 mmol, 2.5 mL of 0.2 M solution in DMF), and 10 eq. of DIEA (1 mmol, 0.5 mL of 2 M NMP solution). Resin was stirred and heated at 70
C under microwave irradiation (50 W) for 10 min. The resin was filtered and then washed with DMF (2) and DCM (2). 2.2.3. Deprotection step Fmoc removal was carried out in a DMF/pip 8/2 v/v solution under MW irradiation (50 W, 70
C) using two cycles of 30 s and 3 min. The resin was filtered and then washed with DMF (2) and DCM (2). 2.2.4. Acetylation step Acetylation of the N-terminus of peptide 3 was performed by treating the resin with acetic anhydride/DCM (1:1) for 5 min (twice). The resin was washed three times with DCM and dried under vacuum. A TNBS test was carried out on a few beads to check that acetylation was complete. 2.2.5. Cleavage of peptides The peptides were cleaved from resins in TFA/TIS/H2O, 95/2.5/ 2.5, v/v/v. The resin was stirred in the cleavage mixture for 2  2 h. The resin was filtered and the solution was concentrated under vacuum. The unprotected peptide was precipitated by addition of diethyl ether. Peptides were purified by preparative RP-HPLC and analyzed by analytical HPLC and LC/MS.

C. Pinese et al. / Materials Today Chemistry 4 (2017) 73e83

2.2.6. Introduction of the silyl moiety To a solution of peptide (0.1 mmol) in 100 mL of DMF was added DIEA (2.1 eq) and 3-isocyanatopropyldimethylchlorosilane (1.2 eq). The reaction mixture was allowed to stir for 2 h at room temperature. The reaction was monitored by analytical HPLC. Then the reaction mixture was poured in diethylether (30 mL). The precipitate was filtered and washed three times to remove excess of ICPDCS and DIEA. All crude compounds were analyzed by analytical HPLC and LC/MS and used without further purification (Supporting information Table S1). 2.3. Grafting method 2.3.1. Surface activation Two commercially available silicone dressings were used for this €lnycke study: Cerederm® from Cereplast and Mepitel® from Mo Healthcare. The flat and plain topography of Cerderem® was particularly adapted for cell proliferation in vitro studies. Mepitel® was chosen for in vivo assays. This dressing is widely used after dermabrasion to take profit of its macroporous topography, allowing exudates to evacuate. An activation of the silicone surface was performed to generate silanol groups, which are the anchoring points for hybrid peptides covalent grafting. Silicone parts were submitted to 90s oxygen plasma treatment with a low-pressure chamber plasma apparatus from Diener Electronics. The settings were: 13.56 MHz radiofrequency, 0.6 mbar, 60 W. 2.3.2. Dip-coating Immediately after plasma activation, silicone pieces were dipped into a 10 mM solution of the hybrid peptide water (1/1000 TFA, v/v)/ethanol, (15/85, v/v), using a homemade dip-coater at a constant withdrawal velocity of 5 cm min1. Coated silicone pieces were placed 24 h at 50
C for aging and washed with a water/ethanol 3/7 solution for 5 min. 2.4. Disinfection Disinfection was performed in two steps: 1- washing of the grafted dressings with three successive ethanol/water 70/30 v/v baths for 15 min for Cerderem® and 5 min for Mepitel®; 2- placing the dressings under UV irradiation in a laminar flow hood for 3 h. 2.5. Cells adhesion Primary human dermal fibroblasts (Promocell, C12352) were cultured in fibroblast growth medium (Promocell, C23010) supplemented with 5000 U mL1 penicillin, 5000 mg mL1 streptomycin and 2.5 mg mL1 amphotericin B. Sample disks (2 cm2) of Cerederm grafted with hybrid peptides 1, 2 and 3 noted respectively Cer-RGD, Cer-Ten-X and Cer-Coll and non-grafted Cerderm® (noted non-grafted-Cer) were placed in non-adhesive TCPS 24-well culture plates. Seventh-passage human dermal fibroblasts were seeded at 100 000 cells per well in triplicate. The same seed was performed on cell culture treated polystyrene wells as a control experiment. Samples were incubated at 37
C and 5% CO2. Cell quantification was obtained using PrestoBlue assay (Invitrogen, A132661), a cell-permeable resazurin-based viability reagent, which reflects the number of living cells present at a given time point. After one day, the proliferative medium was removed and replaced by 1 mL of fresh medium containing 10% of PrestoBlue. After 1 h of incubation at 37
C, 200 mL of supernatant was analyzed in fluorescence (excitation 560 nm, emission 590 nm) with a Victor3 multilabel counter (Perkin Elmer, USA). The mean values for three samples were reported and expressed as arbitrary fluorescence unit (a.f.u.).

75

2.6. In vitro wound-healing capacity Scare recovery was evaluated with an in vitro scare model. Third-passage human dermal fibroblasts were seeded at 400 000 cells per well on 2 cm2 pieces of Cer-RGD, Cer-Ten-X and Cer-Coll and on non-grafted-Cer, in quadruplicate. After 24 h of incubation in culture medium at 37
C to ensure the cell's layer confluence, a scare was created by scratching the surface with a 200 mL tip. Pictures were taken every hour for 38 h by a phasecontrast inverted microscope (Inverse1 Zeiss Axiovert 200 M). The acquired images were analyzed using ImageJ software to determine the percentage of the scare surface covering into the scratched area by measuring and comparing the width of the scratch using the following equation: 100-(wt  100)/w0) where w0 and wt were the scratch width at the start of the experiment and at time t. A scare covering value of 100% indicated a full closure of the scratched region. 2.7. Extra cellular matrix deposition Seventh-passage human dermal fibroblasts were seeded at 900 000 cells per well on 2 cm2 pieces of Cer-RGD, Cer-Ten-X and Cer-Coll and on non-grafted Cerderem®, in triplicate. Samples were incubated at 37
C and 5% CO2. After 24 h, ascorbic acid (150 mg ml1) and insulin (5 mg ml1) were added. After five days, cell mediums were collected and the protein content was analyzed. The production of fibronectin and collagen was quantified respectively using an Elisa kit (Biomatick - EK U04199) and a kit SirCol soluble collagen assay (S1000, bicolor, UK) following the manufacturer protocol. 2.8. In vivo study Animals and experimental procedures complied with the article R-214-89 of french rural code providing guidelines for the care and use of laboratory Animals and were approved by the local Ethical committee and the french ministry (2015092216479994). One male white pig weighing approximately 25 kg was obtained from a local farm. Four types of Mepitel® dressings (2  3 cm pieces) were used for this study: three were grafted with hybrid peptides 1, 2 and 3 (noted respectively Mep-RGD, Mep-Ten-X and Mep-Coll) and one was non-grafted (noted non-grafted-Mep). Each type of dressing was used to treat three wounds. Fifteen partial-thickness wounds were created on the back of the pig. Wounds were separated by ~2 cm and the arrangement of dressings was randomized before each surgery to minimize crossreactivity and interference between dressings. Full-thickness skin samples were excised after euthanasia of the pig (after 5 days) and evaluated histologically for re-epithelialization. 2.8.1. Surgery and wound care The day of surgery, pig was administered a fentanyl patch (50 mg/h), which was replaced after 72 h. Before anaesthesia, a premedication was given to the pig by an intramuscular injection of ketamine (20 mg kg1), Rompun 2% (2 mg kg1) and atropine (0.06 mg kg1). After intubation, anaesthesia was induced by intravenous injection of Nesdonal (7.5 mg kg1) and maintained by inhalation of 1.5% isoflurane. During intervention, NaCl was given by perfusion. The back of the pig was shaved and cleaned with Betadine. Templates of wound areas (1  2 cm) were delimited with a skincompliant marker. Fifteen partial-thickness wounds were created using a scalpel at a depth of 350 mm. The skin was discarded, and hemostasis from the donor sites was achieved with saline-soaked gauze and pressure. Dressings were cut into 2  3 cm pieces,

76

C. Pinese et al. / Materials Today Chemistry 4 (2017) 73e83

applied to the randomly assigned wounds, and secured in place using a sterile gauze, polyurethane film, tape, and a dog tube (Wanimo) for extra protection and to limit the risks of dressings displacement. The animal was observed regularly for wound care, pain management, and evidence of infection. Every dressing and protection was replaced at postoperative day 3. At the day 5, pig was euthanized after wounds examination. Full-thickness samples (including 0.5 cm margin of unwounded skin surrounding each wound) were excised and processed for histology. Photographs were taken at days 3 and 5 and pictures were processed using ImageJ software. The total wound area was measured for each wound and at each time point. The percentage of scare recovery was calculated using the following equation: 100(wt  100)/w0) where w0 and wt represent the wound area at the start of the experiment, at t ¼ 3 and at t ¼ 5 days. 2.8.2. Histological study Specimens were fixed, dehydrated, and embedded within paraffin blocks. Histological sections were prepared using a microtome, and subsequently deparaffinized with xylene, dehydrated using decreasing concentrations of ethanol, and then stained with hematoxylin and eosin (HE). All the histological planks were digitalized and read with NDP view software. The measurement of granulation tissue thickness was performed using Image J software. The thickness of the center of the wound was analyzed; three images were randomly selected and five measurements of each image were performed. 2.9. Statistical analyses Results corresponded to separate experiments performed in triplicate and values are given as standard deviation mean (SD). Statistical analyses were performed using the SigmaStat software. Comparison between several groups was performed using a nonparametric Kruskal-Wallis method (a level of p < 0.05 was considered statistically significant). For analyses of hybrid peptides, XPS calculations and other in vitro and in vivo detailed procedures, see supporting information. 3. Theory/calculation 3.1. Peptide density calculation Assuming a homogeneous peptide layer, the thickness of the layer (d) was estimated from the N1s over Si2p intensity ratio using the following equation: Equ.1: Calculation of the thickness of the layer of peptide grafted on silicone

!! pep

IN1s ¼ ISi2p

14 rpep MSi sN1s TN1s lN1s 1  exp

rSi Mpep sSi2p TSi2p lSi Si2p exp



 lpepdsinq N1s



!!

d lpep Si2p sinq

where, q is the photoelectron collection angle (45
), TN1s and TSi2p are the relative sensitivity factors of N and Si, respectively, provided by the spectrometer manufacturer. The Scofield photoionization cross sections s are equal to 1.8 for N 1s and 0.817 for Si2p [20]. lyx is the inelastic mean free paths of electrons x in the matrix y. They were calculated using the Quases program based on the TPP2M formula [21].

rpep and rSi are the density of peptide and silicium respectively. Mpep and MSi are the molecular weight of peptide and silicone, respectively. The number of peptide per square centimeter npep could be estimated by using the following equation: Equ.2: Calculation of the number of peptide grafted on the silicone dressing   r dN A pep npep molecules:cm2 ¼ Mpep where is the Avogadro number. 4. Results and discussion 4.1. Hybrid peptide design and syntheses Three different peptides were selected (Table 1). Taken together, their sequence displayed all types of functionalities found in peptides (carboxylic acid, guanidine, alcohol, aliphatic chain …). This diversity offered a significant proof of the versatility of the grafting method, which could be generalized to any peptide sequence. The hybrid peptide 1 is based on the tripeptide RGD sequence contained in vitronectin and other proteins of the extra-cellular matrix, which binds efficiently integrin [22]. The linear sequence GRGDSP was already use to promote cell adhesion and proliferation on silicone [9] and titane [23]. Compound 2 is a short hybrid silylated derivative of the elastokine peptide family, presenting the consensus sequence xGxxPG [24]. These sequences, often repeated as oligomers, are described to promote fibroblast proliferation [25] and matrix production and have been used in combination with collagen to improve neoskin flexibility in burns healing [26]. The EGLEGP sequence is found in tenascin X, a glycoprotein of the ECM and has been used in combination with other short sequences from ECM glycoprotein (fibrillin-1) to obtain peptide promoting woundhealing and vascularization [27,28]. The hybrid decapeptide 3 is designed on the ProHypGly tripeptide motif of collagen, repeated three times. The use of natural collagen proteins in biomimetic scaffolds enhance cell adhesion, cell migration, growth factor and extra cellular protein secretion [29,30] while short synthetic sequences derived from collagen have been also designed and used for cell culture [31]. Noteworthy, modifications of ‘Ten-X’ and ‘RGD’ peptides reported in the literature always concerned the N-terminus, keeping the C-terminus free for bioactivity. Thus, the reaction with ICPDMCS was performed at the N-terminus of hybrid peptides 1 and 2. Beta alanine spacers were added between the bioactive sequence and the silyl moiety to favor interaction with cells. The side chain of an extra C-terminal Lysine residue was chosen as a spacer for the short collagen sequence 3. The synthesis of hybrid peptides was performed using the classical SPPS approach with silylation done in solution, after side chain protecting group removal and concomitant resin cleavage by TFA treatment. The hybrid peptide was obtained as its dimethylhydroxysilyl derivative, generated by hydrolysis of corresponding chlorodimethylsilyl moiety during the reaction workup. Noteworthy, hybrid peptides were purified by preparative reversed phase HPLC. Syntheses and purification were performed using an already described procedure set up in our group [32]. See supporting information (Figs. S1eS6). 4.2. Grafting of the hybrid peptide The principle of grafting relies on the specific condensation of the hybrid dimethylhydroxysilyl peptide with a surface silanol function (Fig. 1).

C. Pinese et al. / Materials Today Chemistry 4 (2017) 73e83

77

Table 1 Hybrid peptides 1e3. Compound

Sequencesa

1

R-(bAla)4-GRGDSP-OH ‘RGD’ 2

R-(bAla)4-EGLEPG-OH ‘Ten-X’ 3

Ac-Lys(R)-[Pro-Hyp-Gly]3-NH2 ‘Coll’ a

R ¼ ClMe2Si-(CH2)3NHCO-.

This type of chemistry was already used to modify the surface of silicone with simple organosilanes. For example, Yeh et al. reported the modification of silicone surfaces by grafting of a zwitterionic silane to lead to a superhydrophilic material presenting interesting antifouling properties [33]. For instance, no complex biomolecules such as functional peptides were introduced so far by this one step procedure. Two types of activation are reported in the literature to generate silane groups on the surface: (i) chemical treatment [34] (strong acids or bases) and (ii) plasma treatment [35]. Chemical treatment was excluded in our case for two main reasons. First, we wanted to avoid biomaterial damage, which could affect either the peptide or the mechanical properties of the silicone. Second, such treatments would have necessitated extensive washing steps to wash out reagents from the catheter. Oxygen plasma treatment was preferred. It preferentially generates oxygen-containing activated species on the silicone surface, yielding hydroxysilane functions. We have previously demonstrated that dimethyl hydroxysilyl peptides do not react together and are stable as monomers at low pH [32]. Consequently, we envisioned that an acid (~pH 1.5) solution of the hybrid peptide constituted a smart way to perform the grafting step. Dip coating is a convenient procedure, which is well suitable for industrial production. Indeed, silicone devices could be immersed for a short time in a hybrid peptide solution and removed. The dip coating solution can be reused for a large number of times, being economically advantageous. To establish the dip coating procedure and before using hybrid peptides, the hybrid fluorescent derivative 5 was synthesized and used as a convenient model to visualize grafting. Non-silylated compound 4 was used as a control (Fig. 2). The synthesis of compounds 4 and 5 have been described previously [36]. Pieces of Mepitel® were activated by plasma oxygen and dip-coated at a

constant withdrawal velocity of 5 cm min1 either in a 10 mM water (1/1000 TFA v/v)/Ethanol 85/15 solution containing the hybrid compound 5 or the non silylated derivative 4 as a control. Coated silicone pieces were placed at 50
C for 24 h for aging. As expected, silicone dressing pieces incubated in the solution containing the non-hybrid fluorescent compound 4 lost their fluorescence during the washing steps (DMF, water, ethanol). Indeed, no covalent attachment was possible as the fluorescent dye did not display the required hydroxylsilane moiety (Fig. 2 A). In contrast, Mepitel® pieces dip-coated in the solution containing the hybrid compound 5 kept their fluorescence after extensive washings in various solvents and even after one week of incubation in PBS buffer (Fig. 2 B). This qualitative experiment demonstrated the covalent nature of the grafting when dimethylsilyl derivatives were used. These observations were comparable to those obtained when compound 5 was grafted on silicone catheter pieces, for which a density of ~0.1 hybrid peptide/nm2 was obtained by XPS measurement [5]. Different samples of peptide-grafted Cerederm® or Mepitel® dressings were prepared in the same conditions, by the dip coating procedure with the hybrid peptides 1, 2 and 3 to yield Cer-RGD and Mep-RGD, Cer-Ten-X and Mep-Ten-X, and Cer-Coll and Mep-Coll. Samples were analyzed by XPS to determine the number of peptides per surface unit and the thickness of the layer. 4.3. Surface characterization XPS analyses were conducted on all Mepitel® silicone dressings, non-grafted and grafted with the three modified peptides samples, after 5 min of washing bath like in in vivo study condition. As an example, XPS spectra of a non-grafted silicone sample (Mep) and of a Mep-RGD, after a 5 min wash in PBS, are reported in

78

C. Pinese et al. / Materials Today Chemistry 4 (2017) 73e83

Fig. 1. One step specific grafting of bioactive hybrid peptides on silicone dressing.

supplementary information (Fig. S5). Both analyses confirmed the presence of carbon and nitrogen elements, together with the strong signature of silicone. A small contribution of the nitrogen N1s around 400 eV was observed on the control sample, suggesting some contamination of the commercial substrate. The Mep-RGD silicone sample presented a clear and significant increase of the N1s peak, suggesting the grafting of the RGD-peptide after incubation (see Fig. S5, left spectra). The successful grafting was also confirmed by looking at the atomic composition of all silicone surfaces showed in Table 2. The atomic percentage of nitrogen went from <1% for the nongrafted sample, to more than 5% for the Mep-RGD sample, as shown in Table S1, resulting on a N1s/Si2p ratio 6.4 time more important for the Mep-RGD compared to the non-grafted samples. At this point it is very important to emphasis that this phenomenon was observed for all 3 peptides grafted silicones. This clearly demonstrated the successful grafting of the different peptides on the silicone surfaces. Then, the XPS data were used to evaluate the density of grafted peptides. The drastic increase of the N1s signal following the

Table 2 Atomic percentages of the Mepitel dressings grafted with peptides RGD, Coll and Ten-X after 5 min washed in PBS.

Fig. 2. Mepitel® silicone dressing dip-coated in a 10 mM water (1/1000 TFA v/v)/ Ethanol 85/15 solution of compound 4 (picture A) and in a 10 mM solution of the hybrid fluorescein 5 (picture B). Dressings were placed 24 h at 50
C then washed (5 min) with DMF1, H2O1 and EtOH1 before pictures were taken.

Samples

C1s

O1s

Si2p

N1s

N1s/Si2p

Mep-RGD Mep -Coll Mep -Ten-X Mep non-grafted

53,81 51,76 49,83 45,67

21,97 27,12 29,5 34,31

18,8 16,04 16,98 19,16

5,42 5,08 3,69 0,86

0,288 0,317 0,217 0,045

C. Pinese et al. / Materials Today Chemistry 4 (2017) 73e83 Table 3 Equivalent thickness of peptide layers together with the equivalent amount of peptides per unit area (nm2) for grafted Mepitel®. Samples

Thickness (Å)

pep/nm2

Mep-RGD Mep-Coll Mep-Ten-X non-grafted Mep

6,50 6,97 5,84 1,11

0,42 0,45 0,38 0,07

79

silicone surface (see Supporting Information for detailed equations). We estimated the equivalent layer thickness of the grafted peptides, and consequently from this we calculated the thickness the density of grafted peptide on the silicon surfaces, in peptides/ nm2. These data are summarized in Table 3. The grafting was estimated to around 0.4 peptides per nm2 of Mepitel® silicone samples following the PBS washing process.

peptide grafting (Fig. S5), prompted us to choose the N1s element as a marker to quantify the amount of molecules grafted on the

Fig. 3. A: Human dermal fibroblast adhesion on Cerederm® samples grafted with hybrid peptides 1, 2 and 3 (Cer-RGD, Cer-Ten-X and Cer-Coll), on Cer-non-grafted and on TCPS (high control), n ¼ 3. B: Percentage of gap length recovery of an in vitro scare colonized by human dermal fibroblasts, data are means (SD), n ¼ 4, (*)p < 0.05 when compare to Cernon-grafted. C: Illustration of in vitro scare recovery by human dermal fibroblast at t ¼ 0, 12, 23 and 38 h, scale bar ¼ 100 mm.

80

C. Pinese et al. / Materials Today Chemistry 4 (2017) 73e83

4.4. Cell adhesion For cell adhesion assays, Cerederm® silicone dressing has been chosen because its flat, full-thick surface which guaranteed a regular contact surface with cells, thus facilitating the comparison between samples. Dermal human fibroblasts were seeded on the different wound dressings and the viability of cells was evaluated after 24 h. As shown in Fig. 3 A, human dermal fibroblasts adhered on CerRGD, Cer-Ten-X and Cer-Coll with adhesion values of 30 000, 28 000 and 37 000 respectively, while adhesion values (20 500) on non-grafted Cerederm®were lower. Human dermal fibroblasts were significantly more abundant on Cerederm® modified peptides compared to non-grafted Cerederm® (pvalue compared to Cer-nongrafted: pCer-RGD, and p Cer-Coll ¼ 0.029 pCer-Ten-X ¼ 0.034). Interestingly, there were few differences between polystyrene treated for cell culture (TCPS, used as high-control) and Cer-Coll. Cer-RGD and Cer-Ten-X were 15e19% less efficient for cell adhesion than TCPS but were still 20e26% more effective than untreated silicone. These data suggested that peptide-modification provided a suitable surface for cell adhesion. 4.5. In vitro wound-healing capacity Cell proliferation is one of the main indicator of a woundhealing potential. Proliferation was evaluated with an in vitro scare model. A straight defect was created on a confluent layer of cells seeded on grafted and non-grafted Cerederm® dressings. The percentage of scratched area (the scare) colonized by cells was evaluated as a function of time at t ¼ 12, 23 and 38 h after creation of the defect. As shown in Fig. 3-B, cells progressively colonized the gap of the defect. Two parameters were measured by image analysis as a function of time: the reduction of the length of the defect, expressed as the percentage of the initial length, and the reduction of the total area of the defect, also expressed as the percentage of the initial defect area. After 38 h, the length of the defect was reduced by 83, 93 and 84% on Cer-RGD, Cer-Ten-X and Cer-Coll respectively. These percentages were significantly higher than the 66% length reduction observed on non-grafted Cerderm® (pvalue when compared to non-grafted cerederm: p Cer-RGD ¼ 0.006, p CerTen-X ¼ 0.001 and p Cer-Coll ¼ 0.01). The same tendency was observed for the area reduction corresponding to 94, 95 and 88% of scare recovery on Cer-RGD, Cer-Ten-X and Cer-Coll respectively. Only 79% of recovery was measured on Cer-non-grafted. Noteworthy, no significant difference was observed between peptide modified Cerederm® and high control TCPS (Fig. 3-C).

p Cer-Coll ¼ 0.045). Interestingly, collagen secretion on Cer-RGD and Cer-Coll were 30% lower than on TCPS high control and secretion on Cer-Ten-X was 43% higher than high control. Similarly, as shown on Fig. 4 B, fibronectin secretion by human dermal fibroblasts after 5 days was found to be of 3.05 mg/mL on Cer-non-grafted, of 4.92 mg/ mL, 3.42 mg/mL and 4.20 mg/mL on Cer-RGD, Cer-Ten-X and Cer-Coll respectively and of 3.20 mg/mL on TCPS high control. Fibronectin secretion was significantly higher on Cer-RGD and Cer-Coll than on non-grafted Cerderm (pvalue compared to Cer-non-grafted: p CerRGD ¼ 0.004 and p Cer-Coll ¼ 0.022) and on in high control. Taken together, the in vitro results clearly established that dressing modified with hybrid peptides facilitated the adhesion, the colonization of the scare, but also enhanced the production of extracellular matrix protein. The features displayed by modified silicone surfaces suggested that this modified dressings could facilitate the in vivo wound-healing.

4.7. In vivo study: scare recovery on the back of a pig The commercially available silicone wound dressing, Mepitel® was chosen for in vivo studies. Indeed, Mepitel® is commonly used for the treatment of acute and chronic wounds. This dressing is first considered as an inert barrier to protect the wound but also, thanks to its porous structure, is thought to facilitate the exudates evacuation. Therefore, it is relevant to add extra wound-healing properties to this inert silicone dressing. Hybrid-Mepitel® dressings were prepared in the same conditions, by the dip coating procedure with hybrid peptides 1, 2 and 3 yielding Mep-RGD, Mep-Ten-X and MepColl. Pig skin share structural and functional resemblance with human skin. That is why porcine model was chosen to evaluate the

4.6. Extra cellular matrix deposition During the wound-healing process, a phase of cell proliferation follows the inflammatory phase. This colonization phase is important to bring cells in the defect where they will locally synthetize extra cellular matrix (ECM) components, which will compose the neo tissue. The evaluation of extracellular matrix secretion on peptide modified dressings was monitored with the quantification of two important ECM proteins: collagen and fibronectin for which results are reported in Fig. 4A and B respectively. Collagen secretion by human dermal fibroblasts after 5 days on the dressing was found to be of 0.84 mg on Cer-non-grafted, of 2.41 mg, 4.92 mg and 2.24 mg on Cer-RGD, Cer-Ten-X and Cer-Coll respectively and of 3.45 mg on TCPS high control. Collagen secretion was significantly higher on Cer-RGD, Cer-Ten-X and Cer-Coll than on Cer-non-grafted (pvalue compared to Cer-non-grafted p Cer-RGD ¼ 0.03, p Cer-Ten-X ¼ 0.019 and

Fig. 4. A. Synthesis of collagen. B: Synthesis of fibronectin. Proteins were produced by human dermal fibroblasts on grafted, on non-grafted Cerederm silicone dressings and on TCPS (high control). Data are means (SD), n ¼ 3. *p < 0.05 vs non-grafted Cerederm®.

C. Pinese et al. / Materials Today Chemistry 4 (2017) 73e83

81

Fig. 5. A: Pictures of wounds inflicted on the back of a pig at t ¼ 0 and at t ¼ 3 and t ¼ 5 days. B: Percentage of recovery of the scares. Wounds were covered by hybrid peptide treated- and non treated-Mepitel® dressings. Data are means (SD), n ¼ 3. (*)p < 0.05 vs Mep-non-grafted.

in vivo wound-healing potential of silicone-grafted hybrid peptide dressings, compared to the non-grafted dressings. Superficial skin defects (~350 mm depth) were made with a manual dermatome on the dorsal region of a pig. Concerning materialewound interaction, grafted silicone dressings were easily applied to the wounds and showed good adhesion properties. Macroscopic visual inspection of the wounds was carried out at days 3 and 5 and photographs taken were used to investigate the progression of the healing process (Fig. 5). No sign of reject or intolerance (necrosis) was clinically observed supporting the good biocompatibility of the grafted

dressings. The macroscopic scare recovery on three wounds/treated group after 3 days and 5 days of healing is illustrated in Fig. 5A. After 3 days, only 5e15% of all wounds had recovered. In contrast, after 5 days, Mep-RGD, Mep-Ten-X and Mep-Coll allowed a significant increased scare recovery (98%, 92% and 96% respectively) when compared to non-grafted Mepitel® (63%).

82

C. Pinese et al. / Materials Today Chemistry 4 (2017) 73e83

Fig. 6. A: Histological examination (HES stain): (A) wound-healing after treatment with grafted, non-grafted Mepitel® and for native tissues. (B) Calculation of the thickness of the granulation tissue after treatment with grafted, non grafted Mepitel® and for native tissues Data are means (SD), n ¼ 15. (*)p < 0.05 when compared to Cer-non-grafted. Scale bar ¼ 500 mm.

4.8. Histological studies

5. Conclusion

Wounded skin was excised at day 5 and histological analyses were carried out on hematoxylin-eosin-saffron (HES)-stained sections. The histological examination revealed the three layers of the skin: epidermis, dermis and hypodermis. Re-epithelization was complete in all specimens. However corneal stratification was more marked in wounds treated with the grafted dressings (Fig. 6 A) than in wounds treated with the non-grafted dressings suggesting a greater degree of epidermal maturity. More interestingly, the thickness of the granulation tissue varied depending on the wound treatment. Indeed, as presented in Fig. 6 B, the granulation tissue of sites treated with non-grafted Mepitel® had an average of 0.56 mm, which was significantly thicker than for sites treated with grafted peptides (0.29 mm, 0.27 mm and 0.29 mm for Mep-RGD, Mep-Ten-X and Mep-Coll respectively, with p values of p Mep-RGD ¼ 0.04, p Mep-Ten-X ¼ 0.03, p Mep-Coll ¼ 0.03 and p Mep -surg-ctr ¼ 0.03) reflecting the accelerated rate of dermis repair provided by peptide modified dressings. The modification of the dressing with peptides could enhance the macroscopic scare recovery and seemed to accelerate the maturation of epidermis and dermis layers.

The use of silicone wound dressings is common after surgery or to follow an acute or a chronic wound. Besides raising a barrier to protect wounds against infection, the principal advantage of silicone dressings is its ease removal and the improved comfort for the patient. However, silicone dressings do not have any additional benefits on wound-healing in itself. Herein, we report a simple way to bring bioactivity on an inert silicone dressing with peptides. We developed a strategy to immobilize wound-healing peptides in a site-specific way using hybrid dimethylhydroxysilyl peptides. We showed that the silicone dressings grafted with peptides improved in vitro cell adhesion and proliferation in the way that cells recovered completely a created wound on grafted surfaces. We also showed that grafted wound dressings enhanced collagen and fibronectin synthesis by fibroblasts reflecting a capacity to promote extracellular matrix synthesis. In vivo studies provided interesting trends. Indeed, macroscopic scare recovery was almost complete on sites treated with grafted dressings. Moreover, histological studies showed that the granulation tissues on sites treated with peptide grafted dressings was thinner suggesting an interest in the woundhealing process. This straightforward method should be easily

C. Pinese et al. / Materials Today Chemistry 4 (2017) 73e83

applicable to any other bioactive molecule, as far as a contact effect is targeted. The process of covalent grafting is safe and easy to scale-up to an industrial production. The treated silicone is biocompatible and does not contain any organic contaminant that could impair a clinical use, such as residues of coupling reagents (Nhydroxysuccinimide, carbodiimide, copper ions for azide-alcyne CuAAc, etc.), which are commonly used in classical grafting procedures. At last, this methodology opens the way to the generation of silicones exhibiting several biological activities, such as woundhealing and antibacterial capacity. A multi antibacterial silicone can also be foreseen by combining different antibacterial compounds in a single step, in a controlled way, by using a chosen clearly defined mixture of different silylated antibacterial biomolecules including peptides. Acknowledgements Peptide syntheses were performed using the facilities of SynBio3 IBISA platform supported by ITMO cancer. This work was supported by SATT AxLR as part of a maturation program. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.mtchem.2017.02.007. References €rber, Analysis of a survey of 1000 practicing spe[1] J. AcKlode, C. Wax, A. Ko cialists and general practitioners, Phlebologie (2009) 211e218. [2] D.J. Margolis, L. Allen-Taylor, O. Hoffstad, J.A. Berlin, Diabetic neuropathic foot ulcers: the association of wound size, wound duration, and wound grade on healing, Diabetes Care 25 (2002) 1835e1839. [3] Differential diagnosis of chronic leg ulcers, Servier - Phlebolymphology. (n.d.). http://www.phlebolymphology.org/differential-diagnosis-of-chronic-legulcers/(Accessed 17 December 2015). [4] V. Andreu, G. Mendoza, M. Arruebo, S. Irusta, Smart dressings based on nanostructured fibers containing natural origin antimicrobial, antiinflammatory, and regenerative compounds, Materials 8 (2015) 5154e5193, http://dx.doi.org/10.3390/ma8085154. [5] C. Pinese, G. Subra, Simple and specific grafting of antibacterial peptides on silicone catheters, Adv. Healthc. Mater. (n.d.). [6] N. Mayet, Y.E. Choonara, P. Kumar, L.K. Tomar, C. Tyagi, L.C.D. Toit, V. Pillay, A comprehensive review of advanced biopolymeric wound healing systems, J. Pharm. Sci. 103 (2014) 2211e2230, http://dx.doi.org/10.1002/jps.24068. [7] A. Lombana, Z. Raja, S. Casale, C.-M. Pradier, T. Foulon, A. Ladram, V. Humblot, Temporin-SHa peptides grafted on gold surfaces display antibacterial activity, J. Pept. Sci. 20 (2014) 563e569, http://dx.doi.org/10.1002/psc.2654. [8] B. Mishra, A. Basu, R.R.Y. Chua, R. Saravanan, P.A. Tambyah, B. Ho, M.W. Chang, S.S.J. Leong, Site specific immobilization of a potent antimicrobial peptide onto silicone catheters: evaluation against urinary tract infection pathogens, J. Mat. Chem. B 2 (2014) 1706e1716, http://dx.doi.org/10.1039/C3TB21300E. [9] B. Li, J. Chen, J.H.-C. Wang, RGD peptide-conjugated poly(dimethylsiloxane) promotes adhesion, proliferation, and collagen secretion of human fibroblasts, J. Biomed. Mat. Res. A 79A (2006) 989e998, http://dx.doi.org/10.1002/ jbm.a.30847. [10] S.D. Fontaine, R. Reid, L. Robinson, G.W. Ashley, D.V. Santi, Long-term stabilization of maleimideethiol conjugates, Bioconjug. Chem. 26 (2015) 145e152, http://dx.doi.org/10.1021/bc5005262. [11] L. Cheng, X. Zhang, Z. Zhang, H. Chen, S. Zhang, J. Kong, Multifunctional phenylboronic acid-tagged fluorescent silica nanoparticles via thiol-ene click reaction for imaging sialic acid expressed on living cells, Talanta 115 (2013) 823e829, http://dx.doi.org/10.1016/j.talanta.2013.06.060. [12] H. Jiang, S. Qin, H. Dong, Q. Lei, X. Su, R. Zhuo, Z. Zhong, An injectable and fastdegradable poly(ethylene glycol) hydrogel fabricated via bioorthogonal strain-promoted azideealkyne cycloaddition click chemistry, Soft Matter 11 (2015) 6029e6036, http://dx.doi.org/10.1039/C5SM00508F. [13] J. Martinez, G. Subra, A. Mehdi, S. JEBORS, C. ENJALBAL, L. Brunel, F. Fajula, Materiaux Hybrides Peptide-silice, WO2013190148 A1, 2013, http://www. google.com/patents/WO2013190148A1 (Accessed 6 February 2015). [14] S. Jebors, J. Ciccione, S. Al-Halifa, B. Nottelet, C. Enjalbal, C. M'Kadmi, M. Amblard, A. Mehdi, J. Martinez, G. Subra, A new way to silicone-based peptide polymers, Angew. Chem. Int. Ed. (2015), http://dx.doi.org/10.1002/ anie.201411065 n/a-n/a.

83

[15] S. Jebors, S. Cecillon, C. Faye, C. Enjalbal, M. Amblard, A. Mehdi, G. Subra, J. Martinez, From protected trialkoxysilyl-peptide building blocks to bioorganicesilica hybrid materials, J. Mat. Chem. B 1 (2013) 6510e6515, http:// dx.doi.org/10.1039/C3TB21326A. [16] J. Ciccione, T. Jia, J.-L. Coll, K. Parra, M. Amblard, S. Jebors, J. Martinez, A. Mehdi, G. Subra, Unambiguous and controlled one-pot synthesis of multifunctional silica nanoparticles, Chem. Mater 28 (2016) 885e889, http://dx.doi.org/ 10.1021/acs.chemmater.5b04398. [17] S. Jebors, C. Enjalbal, M. Amblard, A. Mehdi, G. Subra, J. Martinez, Bioorganic hybrid OMS by straightforward grafting of trialkoxysilyl peptides, J. Mat. Chem. B 1 (2013) 2921e2925, http://dx.doi.org/10.1039/C3TB20122H. [18] C. Echalier, S. Al-Halifa, A. Kreiter, C. Enjalbal, P. Sanchez, L. Ronga, K. Puget,
, M. Amblard, J. Martinez, G. Subra, Heating and microwave assisted P. Verdie SPPS of C-terminal acid peptides on trityl resin: the truth behind the yield, Amino Acids 45 (2013) 1395e1403, http://dx.doi.org/10.1007/s00726-0131604-z. [19] M. Amblard, J.A. Fehrentz, J. Martinez, G. Subra, Methods and Protocols of modern solid phase peptide synthesis, Mol. Biotechnol. 33 (2006) 239e254. [20] J.H. Scofield, Hartree-Slater subshell photoionization cross-sections at 1254 and 1487 eV, J. Electron Spectrosc. Relat. Phenom. 8 (1976) 129e137, http:// dx.doi.org/10.1016/0368-2048(76)80015-1. [21] S. Tanuma, C.J. Powell, D.R. Penn, Calculations of electron inelastic mean free paths. V. Data for 14 organic compounds over the 50e2000 eV range, Surf. Interface Anal. 21 (1994) 165e176, http://dx.doi.org/10.1002/sia.740210302. [22] E. Ruoslahti, RGD and other recognition sequences for integrins, Annu. Rev. Cell Dev. Biol. 12 (1996) 697e715, http://dx.doi.org/10.1146/ annurev.cellbio.12.1.697. [23] M. Schuler, G.R. Owen, D.W. Hamilton, M. de Wild, M. Textor, D.M. Brunette, S.G.P. Tosatti, Biomimetic modification of titanium dental implant model surfaces using the RGDSP-peptide sequence: a cell morphology study, Biomaterials 27 (2006) 4003e4015, http://dx.doi.org/10.1016/ j.biomaterials.2006.03.009. [24] J. Attia-Vigneau, C. Terryn, S. Lorimier, J. Sandre, F. Antonicelli, W. Hornebeck, Regeneration of human dermis by a multi-headed peptide, J. Invest. Dermatol 134 (2014) 58e67, http://dx.doi.org/10.1038/jid.2013.290. [25] A. Kamoun, J.M. Landeau, G. Godeau, J. Wallach, A. Duchesnay, B. Pellat, W. Hornebeck, Growth stimulation of human skin fibroblasts by elastinderived peptides, Cell Adhes. Commun. 3 (1995) 273e281. [26] W.F. Daamen, J.H. Veerkamp, J.C.M. van Hest, T.H. van Kuppevelt, Elastin as a biomaterial for tissue engineering, Biomaterials 28 (2007) 4378e4398, http:// dx.doi.org/10.1016/j.biomaterials.2007.06.025. [27] T.N. Demidova-Rice, L. Wolf, J. Deckenback, M.R. Hamblin, I.M. Herman, Human platelet-rich plasma- and extracellular matrix-derived peptides promote impaired cutaneous wound healing in vivo, PLoS One 7 (2012) e32146, http:// dx.doi.org/10.1371/journal.pone.0032146. [28] T.N. Demidova-Rice, A. Geevarghese, I.M. Herman, Bioactive peptides derived from vascular endothelial cell extracellular matrices promote microvascular morphogenesis and wound healing in vitro, Wound Repair Regen. 19 (2011) 59e70, http://dx.doi.org/10.1111/j.1524-475X.2010.00642.x. [29] X. Chen, Y.-Y. Qi, L.-L. Wang, Z. Yin, G.-L. Yin, X.-H. Zou, H.-W. Ouyang, Ligament regeneration using a knitted silk scaffold combined with collagen matrix, Biomaterials 29 (2008) 3683e3692, http://dx.doi.org/10.1016/ j.biomaterials.2008.05.017. [30] H. Fan, H. Liu, E.J.W. Wong, S.L. Toh, J.C.H. Goh, In vivo study of anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold, Biomaterials 29 (2008) 3324e3337, http://dx.doi.org/10.1016/ j.biomaterials.2008.04.012. [31] J. Luo, Y.W. Tong, Self-assembly of collagen-mimetic peptide amphiphiles into biofunctional nanofiber, ACS Nano 5 (2011) 7739e7747, http://dx.doi.org/ 10.1021/nn202822f. [32] C. Echalier, A. Kalistratova, J. Ciccione, A. Lebrun, B. Legrand, E. Naydenova, D. Gagne, J.-A. Fehrentz, J. Marie, M. Amblard, A. Mehdi, J. Martinez, G. Subra, Selective homodimerization of unprotected peptides using hybrid hydroxydimethylsilane derivatives, RSC Adv. (2016), http://dx.doi.org/10.1039/ C6RA06075G. [33] Y. Sb, C. Cs, C. Wy, H. Cj, Modification of silicone elastomer with zwitterionic silane for durable antifouling properties, Langmuir ACS J. Surf. Colloids 30 (2014) 11386e11393, http://dx.doi.org/10.1021/la502486e. [34] C.N. Hughes-Chinkhota, M. Banda, J.M. Smolinski, R.A. Thomas, D.M. Petibone, J.D. Tucker, G.W. Auner, Oligonucleotide immobilization using 10-(carbomethoxy)decyl-dimethylchlorosilane for mRNA isolation and cDNA synthesis on a microfluidic chip, Sens. Actuators B Chem. 155 (2011) 437e445, http:// dx.doi.org/10.1016/j.snb.2010.12.043. [35] C. Diaz Blanco, A. Ortner, R. Dimitrov, A. Navarro, E. Mendoza, T. Tzanov, Building an antifouling zwitterionic coating on urinary catheters using an enzymatically triggered bottom-up approach, ACS Appl. Mat. Interfaces 6 (2014) 11385e11393, http://dx.doi.org/10.1021/am501961b. [36] C. Echalier, C. Pinese, X. Garric, H. Van Den Berghe, E. Jumas Bilak, J. Martinez, A. Mehdi, G. Subra, Easy synthesis of tunable hybrid bioactive hydrogels, Chem. Mater 28 (2016) 1261e1265, http://dx.doi.org/10.1021/ acs.chemmater.5b04881.