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DE GRUYTER Current Directions in Biomedical Engineering 2020;6(3):20203122 Leyla Djanklich*, Tonya Andreeva, Xin Xiong, Rumen Krastev and Ralf Kemkemer Evaluation of antibacterial properties of polyelectrolyte multilayer coatings by norm tests Abstract: Medical implants play a central role in modern patients. Both naturally derived and synthetic materials have medicine and both, naturally derived and synthetic materials been successfully used as biomaterials in the recent decades. have been explored as biomaterials for such devices. However, However, when in long-term contact with living tissue most when implanted into living tissue, most materials initiate a materials initiate a host response such as a foreign body host response. In addition, implants often cause bacterial response. Polyelectrolyte multilayer (PEM) coatings have infections leading to complications. Polyelectrolyte multilayer been used to functionalize the surfaces of medical devices in (PEM) coatings can be used for functionalization of medical order to improve the implant integration. Such PEM coatings implants improving the implant integration and reducing are only a few nanometers thick, but may dramatically foreign body reactions. Some PEMs are also known to show modulate the interaction between biomaterial surface and antibacterial properties. We developed a PEM coating biological systems. In addition, PEM productions methods are suggesting that it can decrease the risk of bacterial infections in principle simple. occurring after implantation while being highly Next to issues of the biocompatibility of the implant materials, biocompatible. We applied two different standard tests for bacterial infections and inflammatory processes caused by evaluating the PEM’s antibacterial properties, the ISO norm contaminations and subsequent local or systemic defence (ISO 22196) and one ASTM norm (ASTM E2180) test. We reactions of the body are a major challenge for using implants, found a reduction of bacterial growth on the PEM but to a often requiring clinical treatment or the revision of the implant. different degree depending on the testing method. This result Modern sterilization procedures and the use of antibiotics are demonstrates the need for defining proper method to evaluate common strategies to reduce infections caused by implants. antibacterial properties of surface coatings. However, not only the increasing resistance to antibiotics requires the development of novel material surface Keywords: polyelectrolyte multilayer coatings, implants, modifications and coatings with antibacterial properties. PEM antibacterial coating, norm tests coatings have also been suggested to have antibacterial properties depending on their composition and properties [1, https://doi.org/10.1515/cdbme-2020-3122 2]. For evaluation of the potential antibacterial effect of such modified implant surfaces appropriate testing is required, 1 Introduction either on the lab scale for early research or standardized for R&D close to applications or transfer to market. Two Medical implants are crucial devices allowing modern protocols commonly used in industry are the ISO 22196 test therapeutics or diagnostics and improving the lives of many “Plastics–Measurement of antibacterial activity on plastics surfaces”  and the ASTM E2180 test “Determining the activity of incorporated antimicrobial agent(s) in polymeric or ______ hydrophobic materials” . In these protocols, surfaces are *Leyla Djanklich: Reutlingen University, Reutlingen, Germany tested using a liquid bacterial culture, covered with a plastic e-mail: Leyla.Djanklich@Reutlingen-University.DE film (ISO) or bacteria are added to an agar slurry (ASTM), and Tonya Andreeva: Reutlingen University, Reutlingen, Germany grown for a certain time in contact with the test samples. After Xin Xiong: NMI Natural and Medical Sciences Institute at the incubation, surfaces are rinsed and after further incubation, University of Tübingen, Reutlingen, Germany Rumen Krastev: Reutlingen University, Reutlingen, Germany and bacteria colonies are counted. Although such standard tests are NMI Natural and Medical Sciences Institute at the University of widely used, it is suggested that they are limited in reflecting Tübingen, Reutlingen, Germany the complex practical condition and are thus limited in their Ralf Kemkemer: Reutlingen University, Reutlingen, Germany Open Access. © 2019 Leyla Djanklich et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 License. Leyla Djanklich et al.: Evaluation of antibacterial properties of polyelectrolyte multilayer coatings by norm tests — 2 usability . In this work we developed a PEM coating analysis system (OCA15EC DataPhysics Instruments, suitable for implants and tested its antibacterial activity Germany) equipped with video capture. 3 μL of ultrapure applying the ISO 22196 and ASTM E2180 standard methods. water were placed on thе lеvеlеd surfacе of thе samplе by With both tests, the PEM coating was found to generally microsyringе forming a singlе sеssilе drop. An image was reduce the bacterial growth compared to the positive control taken 5 seconds post droplet formation. The left and right (an uncoated sample). However, the two tests result in angles wеrе determined at 5 different spots for еach spеcimеn different quantitative degrees of bacterial reduction. (prepared in triplicatе) applying thе Young-Laplacе-fitting of thе profilе. 2 Materials and Methods 2.3 Bacteria cultivation and tests A Gram-negative bacterial strain was used for both of the 2.1 Preparation of surface coatings testing methods (Escherichia coli, DSMZ 10199). The liquid culture was prepared in Tryptic Soy Broth (TSB) (Sigma- Materials. Polyethylenimine (PEI) (MW 750 kDa) and Aldrich), the cultivation occurred on Tryptic Soy Agar plates poly(acrylic acid) (PAA) (MW 100 kDa), both from Sigma- (TSA) (Sigma-Aldrich). Aldrich (Steinheim, Germany), and poly(allylamine hydrochloride) (PAH) (MW 120-200 kDa) from Alfa Aesar International Organization for Standardization (ISO) (Massachusetts, USA) were all used as received. PAA and 22196: Measurement of Antibacterial Activity on Plastics and PAH were dissolved in 0.5 M NaCl solution to a concentration other non-porous Surfaces. of 2 mg/ml, pH 7.0. PEI (2 mg/ml, pH 7.0) was dissolved in A modified protocol, according to ISO 22196 was applied. ultrapure water and deposited as a first layer, acting as an Before inoculation, the E. coli were incubated in TSB for 24 h anchoring layer for the adsorption of consecutive layers. The at 37°C and bacteria number was determined with a whole coating procedure was conducted in a sterile photometer. Thereafter a cell suspension of E. coli was environment under a laminar flow bench. prepared and a 150 µL drop was placed onto the surfaces of Preparation of PEM coatings. PEI(PAA/PAH) coatings 5 the coated samples and the controls. The bacterial inoculum were prepared by layer-by-layer (LbL) technique using the on the test samples was covered with a plastic film (Bemis™ hand dipping method  on microscope slides 2×2.5 cm Curwood Parafilm™). The samples were then incubated in the (Thermo Scientific™ Polysine Adhäsionsobjektträger). The dark for 24 h at 37°C. After incubation, the E. coli on the film build-up was pursued at 25°C by alternating dipping of surface were removed by 5 mL of sterile washing solution the glass slides into PAA and PAH solutions (for 10 min) (0.85% Saline, 0.2% Tween 80). Then serial dilutions were followed by three washing steps (2 min each) in water. After made from the washing solution and afterwards incubated on the last deposition step, the coatings were dried in a nitrogen TSA plates for 24 h at 37°C. The number of colony forming stream. The same microscope glass slides, but uncoated were units (CFU) was counted. Thereafter the number of viable used in the antibacterial tests as a negative control, and thin bacteria was determined according to the ISO norm . copper foil 2×2.5 cm (Kupferfolie, unbeschichtet, 99.8 %, Alfa Numbers re given in log reduction relative to the negative Aesar™) was used as a positive control. control sample. 1. Preparation of a liquid culture 3. Recovery of the bacteria - Determination of the CFU 2.2 Characterization of surface coatings Ellipsometry. The thickness and the refractive index of the 2. Generation of a bacterial colonized surface PEM films were monitored at five different locations on each sample (prepared in triplicate) by spectroscopic ellipsometer (a) ISO (b) ASTM Sentech SE800 (Sentech Instruments GmbH, Germany) with wavelength range from 280 to 850 nm, at an angle of incidence 70 deg. The raw data were fitted by four-layer model Figure 1: General procedure of the standard test methods and considering the contribution from air, PЕM, SiO , and Si. main difference: (a) a cover film protects the inoculum according to ISO 2296; (b) a agar-slurry contains the inoculum Static water contact angles were measured by the sessile drop according to ASTM E2180. method using an optical contact angle measuring and contour Leyla Djanklich et al.: Evaluation of antibacterial properties of polyelectrolyte multilayer coatings by norm tests — 3 American Society for Testing and Materials (ASTM) slightly antibacterial by showing a 1.4 Log-reduction in E2180: Determining the Activity of Incorporated comparison to the positive control (copper foil). The measured Antimicrobial Agent(s) In Polymeric or Hydrophobic Log-reduction means that the reduction of living bacterial cells Materials. as result of the incubation on the coating is nearly over 90%. An adapted protocol was used. The bacterial culture was The results of the measured antibacterial activity applying the prepared in TSB and incubated for 24 h at 37°C, then bacteria protocol according to ASTM E2180 is shown in Figure 2 (C). were counted with a spectrometer. The culture was then The antibacterial activity was calculated in percentage diluted to an end concentration of 10 cells/mL into a semi- reduction in compliance with the norm. With this test method solid agar-slurry cooled down to 45°C. 500 µL of the prepared the PEM coating shows only a moderate antibacterial property suspension was spread on the samples. Subsequent the with a reduction of approximately 20.6%. The positive control samples were incubated 24 h at 37°C in the dark. Afterwards (Cu-foil) led to a reduction of nearly 100% as expected. the samples and controls were placed in a neutralizing To compare the results of the two tests methods we calculated medium, first treated with ultrasound, then vortexed for the log -reduction according to the ISO norm for the transferring the inoculum into the solution. The solutions were experimental results, the CFU, for the results of the ASTM then diluted and spread on TSA, followed by incubation for 24 measurements. The Log-reduction for the ASTM experimental h at 37°C. Afterwards the CFU could be counted according to data is significantly smaller than the one determined by the the ASTM norm . Numbers are given in percent reductions ISO standard method. relative to the negative control sample. The moderate antibacterial action of the PAH-finished PEM could be attributed to the positive surface charge and high charge density that may yield strong electrostatic interaction between PAH-chains and bacterial surface. PAH has been 3 Results and Discussion identified to bind to phosphate moieties present in the lipopolysaccharides on the outer membrane of Gram-negative PEM offer a simple, versatile platform for controlling the bacteria. A similar mechanism of antibacterial action was interaction between materials and cells and can have found for other cationic polymers . antibacterial properties. We used PEM as a well-defined model system to compare the both test procedures described in ISO 22196 and ASTM E2180. The coatings comprise five bilayers of the weak synthetic polyelectrolytes PAH and PAA - PEI(PAA/PAH) . PEI was applied as an adhering layer. Ellipsometry measurements showed that PEI(PAA/PAH) PEM have thickness of about 15.0 nm. The contact angle of the coating was 72°. Weak polyelectrolytes are charged only in a small pH range, hence, their polymeric chains conformations, surface and bulk properties can be easily modulated upon changing their net charge by adjusting the pH of the deposition solutions. At our Figure 2: Results for the antibacterial properties of the PEM coating and Cu-foil (positive control) according to (A) ISO22196, (B) deposition conditions the amine groups of PAH are protonated ASTM measurment accoring to ISO calculation yielding a Log- and carry positive charge. The degree of ionization of PAH at reduction value and (C) the ASTM measurment evaluated that conditions is about 85% . The coatings are overlaid according to the ASTM standard in %-reduction, all compared with the positively charged PAH and have in total a net to the uncoated control. positive charge. In summary, the PEM coating induced a reduction in living bacteria incubated in contact with the samples, however the The antibacterial activity of the coated samples against E. coli measured Log-reductions are moderate compared to the evaluated using the two different standard norms is presented positive control of Cu-foil, Nonetheless, the quantitative in Figure 2. The results show that both test methods degree of the antibacterial properties highly depends on the demonstrate a reduction in bacteria number compared to the test method demonstrating the need for careful consideration negative control (uncoated sample). ISO 22196 results are of the test methods even if relying on norm standards. given in log reductions (figure 2 (A)). The positive control (Cu foil) yield in a reduction of 8.0-Log demonstrating the high antibacterial properties. The PEM coating performs Leyla Djanklich et al.: Evaluation of antibacterial properties of polyelectrolyte multilayer coatings by norm tests — 4  Séon L, Lavalle Ph, Schaaf P, Boulmedais F. Polyelectrolyte Multilayers: A Versatile Tool for Preparing Antimicrobial Author Statement Coatings. Langmuir 2015;31:12856-12872. Research funding: The authors acknowledge the funding  International Organization for Standardization. ISO 22196: by the BMBF program Materialinnovationen für gesundes Measurement of Antibacterial Activity on Plastics and other non-porous Surfaces or Hydrophobic Materials. 2011. Leben: ProMatLeben – Polymere, project PolyAntiBak,  American Society for Testing and Materials. ASTM E2180 (FKZ: 13XP5073D) and the Servier Medical Art for providing Determining the Activity of Incorporated Antimicrobial the pictures in Fig. 1. Agent(s) In Polymeric or Hydrophobic Materials. 2018. Conflict of interest: Authors state no conflict of interest.  Matias D. Campos, Paola C. Zucchi, Ann Phung, Steven N. Leonard, Elizabeth B. Hirsch. The Activity of Antimicrobial Informed consent: Informed consent has been obtained from Surfaces Varies by Testing Protocol Utilized. PLos ONE all individuals included in this study. Ethical approval: The 2016;11(8): e0160728 research related to human use complies with all the relevant  Kolasinska M, Krastev R, Gutberlet T, Warszynski P. Layer- national regulations, institutional policies and was performed by-layer deposition of polyelectrolytes. Dipping versus spraying. Langmuir 2009;25(2):1224–1232. in accordance with the tenets of the Helsinki Declaration, and  Choi J, Rubner MF. Influence of the Degree of Ionization on has been approved by the authors' institutional review board or Weak Polyelectrolyte Multilayer Assembly. Macromolecules equivalent committee. 2005;38:116-124.  Timofeeva L, Kleshcheva N. Antimicrobial polymers: Mechanism of action, factors of activity, and applications. Appl Microbiol Biotechnol 2011;89:475-492. References  In Decher G, Schlenoff JB, editors. Multilayer Thin Films Sequential Assembly of Nanocomposite Materials. John Wiley & Sons; 2012.
Current Directions in Biomedical Engineering – de Gruyter
Published: Sep 1, 2020
Keywords: polyelectrolyte multilayer coatings; implants; antibacterial coating; norm tests
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