Copper Complex: A Key Role in the Synthesis of Biocidal Polymer Coatings
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A specific copper complex is used here for the development of new antibacterial coatings against two different bacteria strains i.e. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Remarkably, the cationic polymerization of a glycerol-derived monomer was performed using an efficient Cu complex as a photoinitiator. The synthesized coatings which were successfully developed under UV–visible light exposure and under a solvent-free cationic photopolymerization process show excellent adhesion on stainless steel plate according to nanoindentation and scratch tests. The resulting glycerol-derived coatings have been conducted to assess the antibacterial activities against E. coli and S. aureus: a tremendous decrease of 99.9% and 96% of the adhered Escherichia coli and Staphylococcus aureus on the surface of copper containing coatings has been demonstrated in comparison with bare stainless steel supports. Finally, our investigation suggests from live/dead assays data that the synthesized polymer films show biocidal properties due to the release of Cu2+ ions, which are responsible for inhibiting bacterial proliferation.
KeywordsAnti-adherence surfaces Copper complex Cationic photopolymerization Biocide coatings
Hospital-acquired bacterial infections [1, 2, 3], also called nosocomial infections, have become an expanding public health issue since decades and are responsible to several hundreds of thousands deaths every year around the world along with a tremendous increase of the healthcare costs. Despite the development of new antimicrobial drugs for treating infections, multi-drug resistant bacteria have emerged, thereby leading to inefficient treatments. In order to prevent such bacterial infections, antimicrobial polymer coatings have been intensively explored. Polymer coatings (biocidal or otherwise) have been particularly investigated as a means to prevent bacterial adhesion and proliferation, according to either an “active” or a “passive” process. Passive coatings rely on the antifouling properties to inhibit bacterial adhesion [4, 5, 6] (e.g. PEG-containing or perfluorinated coatings) whereas “active” coatings lead to the biochemical death of bacteria simply by contact [7, 8, 9, 10, 11, 12, 13] (like polymers bearing quaternary ammonium functions, guanidine-containing polymers, natural peptides derivative polymers, phospho and sulfo derived polymers, phenol and benzoic acid derivative polymers, organometallic polymers, and polyelectrolyte multilayers), by the use of reactive oxygen species [14, 15, 16, 17, 18] or by the release of inorganic antimicrobial compounds [19, 20, 21, 22, 23] (e.g. zinc oxide, TiO2, montmorillonite-derivative clays, and silver nanoparticles).
Amongst the multitude of metal complexes proposed, copper complexes CuCs are receiving an increasing consideration, especially due to their low cost synthesis and their high potential in polymer chemistry [24, 25]. The design of copper complexes with relatively high lifetime excited states allows its use into various fields such as organic light emitting diodes (OLEDs), photoredox catalysts or photoinitiators for polymer synthesis [26, 27, 28, 29, 30, 31, 32, 33] but few investigations rely on the use of such copper complexes in biomedical field. In light of this observation, our study demonstrates that a chosen type of copper complexes could be used as antimicrobial agents. As is well known, copper ions have been used for centuries to purify and disinfect liquids, solids and tissues [34, 35] either alone or in complexes. Behind to its consideration as being safe to humans [36, 37], and in contrast to its low sensitivity to the body tissues (skin, etc..) , copper is a powerful agent toward micro-organisms . Following this research line, an interesting copper complex is used for the synthesis of new solvent-free materials with interesting antimicrobial properties.
The rise of interest toward renewable resources derivative monomers [40, 41] prompt us to use glycerol as building blocks. Indeed, glycerol is one of nature’s wonders which appears as a component present in all living cells. It is naturally found in wines, beers, bread, and other fermentation products of grains and sugars. Industrially, glycerol is a product of fats and oils and its global market size is likely to exceed USD 3 billion by 2022 with a growing rate at over 8%.
The originality of this investigation is based on the versatility of a copper complex with dual activities: first as a photoinitiator when added to iodonium salt (Iod) for the cationic polymerization of a glycerol epoxidized monomer, and second as a biocidal agent against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). To the best of our knowledge, this study is the first example of copper complex use with such dual performing properties. The photoinitiation ability of the (copper complex/Iod) system and the resulting generation of photoacids able to perform the synthesis of the glycerol-derived coatings under light exposure, are investigated by Real Time Fourier Transform Infrared spectroscopy (RT-FTIR) and UV–visible spectrometry. In addition, the resulting polymer surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and the release of copper ions was evidenced by inductively coupled plasma (ICP). The resulting glycerol-derived coating adhesion on stainless steel plate was demonstrated by nanoindentation and scratch tests, and their antibacterial properties were tested against two bacteria strains, i.e. E. coli and S. aureus.
2 Materials and methods
Chemical structure of the compounds used in this study
Glycerol diglycidyl ether (GDE)
Iodonium 4-(2-methylpropyl)phenyl-hexafluorophosphate (Iod)
Copper complex (CuC)
Rhodamine B (RhB)
2.2.1 Photopolymerization Method
For the cationic photopolymerization, 23.4 mg of Iod (3 wt % with respect to the glycerol-derivative monomer), 5 mg of CuC (0.9 wt % with respect to the glycerol-derivative monomer) were introduced and dissolved into the glycerol-derivative formulation (GDE, 535 mg). The kinetics of photopolymerization were followed by RT-FTIR using a Thermo-Nicolet 6700 instrument. The liquid samples were applied to a BaF2 chips by means of a hundred μm-calibrated wire-wound applicator. The RT-FTIR experiments were performed under air and at room temperature, by means of a Lightningcure LC8-03 lamp from Hamamatsu, equipped with a xenon lamp (200 W, 300–700 nm range). The end of the flexible light guide was placed at a distance of 4 cm. The maximum UV light intensity at the sample position is fixed at 70 mW/cm2. The decreases of epoxy function of the GDE monomer at 3050 cm−1 and the increase of polyether at 1080–1100 cm−1 were followed by RT-FTIR and demonstrated the efficiency of the cationic photopolymerization. Equation 1 allows to follow the conversion rate during the irradiation
2.2.2 UV–Visible Absorption Measurements
The UV–Vis absorption spectra were carried out using a Cary 60 UV–Vis spectrometer from 200 to 800 nm.
2.2.3 X-Ray Photoelectron Spectroscopy (XPS)
XPS measurements were performed using a K-alpha Thermo Scientific photoelectron spectrometer with monochromatized Al Kα excitation (hν = 1486.6 eV). The compositions of the surface film were determined by measuring integrated photo-peaks intensities after a Shirley background subtraction. The peaks areas were corrected from sensitivity factors. The X-ray spot diameter was 400 microns. Survey spectra were acquired with an energy step of 1.0 eV over a binding energy range of 0–1200 eV, using a pass energy of 200 eV. The high resolution spectra for the C1 s, O1 s and Cu2p were obtained with a pass energy of 50 eV with a 0.05 eV energy step. The dwell time was 100 ms for all acquired spectra. The analysis chamber base pressure was by 8.0 × 10−10 torr. For the charge compensation, electron flood gun combined with an argon ion gun were used.
2.2.4 Preparation of the Coatings
Square stainless steel substrate plates (1.5 × 1.5 cm) were washed with toluene and ethanol before being dried under vacuum at 40 °C for 4 h. Glycerol-derivative solution with CuC was deposited on stainless steel substrate and layed down by means of a 100 μm calibrated wire-wound applicator. Samples have been first irradiated under a Lightningcure LC8-03 lamp for 600 s (light intensity = 70 mW/cm2). Both sides of the stainless steel substrate were irradiated under the same conditions. For Glycerol-derivative solution without CuC, cationic photopolymerization was performed with the Iod photoinitiating system and the resulting coatings on stainless steel substrates have been likewise done.
2.2.5 Hardness of Coatings by Pencil Test
The measurement of pencil hardness begins with the lowest pencil and continues up the scale to determine the maximum hardness able to scratch the surface of the glycerol-derived coatings (Method: ASTMD3363-74, 2000). This method which has been recommended by National Coil Coating Association, provides inexpensive means of evaluating the film hardness and adhesion of the polymer coating on stainless steel substrate. The hardness of the coating was determined relative to a standard set of pencil leads. The surface hardness is determined by scratching the leads across the coating at a controlled angle of 45º and the values is given to the pencil able to induce a scratch on the surface of the coating. The pencil hardness was measured using a No. 553 pencil hardness tester (Yasuda Seiki Seisakusho LTD.). Pencils were supplied by Staedtler Mars Lumograph 100 (Germany).
2.2.6 Nanoindentation and Scratch Resistance Tests
Nanoindentation and scratch tests were carried out on the coatings deposited on steel substrates with a Nano Indenter (Agilent Technologies G200) using a Berkovich tip (Micro Star Technologies). Twenty-five Nanoindentation tests per sample were performed. Experiments parameters are the same as those in reference.
2.2.7 Copper Release Experiments
The amount of copper ions release from the glycerol-derived films containing Cu ions was determined by inductively coupled plasma spectrophotometer (ICP, Thermo Jarrell Ash IRIS-AP). For this, the samples (square samples with a 1.5 × 1.5 cm length) were immersed in 10 mL of phosphate buffer saline (pH 7.0) for different time periods (4 days).
2.2.8 Antibacterial Property of the Coatings
Initial adhesion assays on glycerol-derived coatings with and without CuC and stainless steel substrates were performed using E. coli ATCC25922 and S. aureus ATCC6538. The bacteria were incubated aerobically in Luria–Bertani broth overnight at 37 °C, under stirring. The resulting cultures were diluted to an optical density (OD 600 nm) of 0.05 in sterile LB broth. Then, the glycerol-derived coatings deposited on stainless steel substrates (1.5 × 1.5 cm) were immersed in the bacterial formulation. The vials containing the substrates were placed on a slantwise rotating wheel in order to avoid the sedimentation of bacteria. The solutions with the coatings were incubated for 3 h, and shaken at 150 rpm to allow for initial adhesion (INFORS AG-CH 4103, Bottmingen-Basel, Switzerland). After the incubation time, the coatings were rinsed seven times with sterile saline solution (NaCl, 0.9% w/v), to remove any bacterial cell not adhering to the coating.
Each sample was then transferred to 2 mL of sterile saline solution and vortexed for 30 s. The samples were then plunged in a second 2 mL sterile saline solution and sonicated in a Branson 2200 sonicator for 3 min. Samples were once more transferred to another 2 mL of sterile saline solution and vortexed for 30 s. Suspensions obtained by the treatment of vortex and sonication were assembled, serially diluted and plated on plate count agar (PCA) medium for viable CFU counting. The bacteria removed during the three steps made up the loosely attached biofilm population. A hundred µL of this solution was spread on a PCA plate. The process was repeated successively for 24 pre-dried substrates. The plates were incubated overnight at 37 °C. Counting colony forming units (CFUs) allowed determining in fine the total bacterial adhesion. Levels of adhesion were reported as numbers of cells per square centimeter.
2.2.9 Statistical analysis
All values corresponding to the anti-adherence property of the coatings towards E. coli and S. aureus are reported as mean ± standard deviation. Student’s t test was used to make statistics analyses on the antibacterial tests and for the calculation of significance level of our antibacterial data. Differences were considered statistically significant for p < 0.05. Ten samples were evaluated for each group.
22.214.171.124 Cell Viability
A well-known live/dead assay method allows the evaluation of the cell viability after E. coli and S. aureus were culturing on the native stainless steel substrates and the glycerol-derived coatings for 3 h based on the previously reported method . Briefly, the bacteria adhered were harvested using an aqueous solution (1000 mL) containing Tween 80 surfactant (2 g) and sodium chloride (5 g). Subsequently, 15 µL of a cell suspension was mixed with a 9 µL assay solution [3 µL SYTO 9 solution (1.67 mM in dimethyl sulfoxide (DMSO)) and 6 µL propidium iodide solution (1.67 mM in DMSO)] and incubated for 15 min at 25 °C. The stained cell solution was moved to slide cover glass and then fluorescence images of the cells were visualized with a confocal laser scanning microscope (CLSM 700, Carl Zeiss, Germany).
3 Results and Discussion
3.1 Cationic Photopolymerization and Photochemical Mechanisms
3.2 Properties of the Photosynthesized Coatings
3.3 Antibacterial Properties of the Copper Based Coatings
This study dealt with the use of an efficient CuC/Iod photoinitiating system for the synthesis of anti-adherent and biocidal glycerol-derived coatings. The resulting coatings show a perfect elastomeric behavior and indicate a good adherence to the stainless steel substrate as well as a good resistance to fracture. Moreover, anti-adhesion property was shown to be efficient even at a relatively low concentration in CuC (0.9 wt%): an average decrease of respectively 99.9% and 96% of the adhered E. coli and S. aureus on the surface of CuC-containing coatings was demonstrated in comparison with bare stainless steel substrates, after only 3 h. Interestingly, the live/dead assays evidenced that the polymer films containing CuC show biocidal properties due to the release of Cu2+ ions. The success of the biocidal properties of the CuC coatings prompts us to go further in the antibacterial experiments. The synthesis of new Ag or Cu derivative complexes with visible absorption and photoinitiating properties are under progress and we expect to increase the biocidal activities of the resulting coatings for a longer incubation time. Such coatings could be used for hand-care utensils like pliers, for example.
The authors would like to thank CNRS, UPEC and French National Agency (sPECTRAL project) for financial support.
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