Development of innovative anti-adhesive materials in diagnosis tools

Universidade do Minho Escola de Engenharia Tugce Caykara October 2022 Tugce Caykara UMinho|2022 Development of Innovative Anti-adhesive Materials in Diagnosis Tools Development of Innovative Anti-adhesive Materials in Diagnosis Tools October 2022 Universidade do Minho Escola de Engenharia Tugce Caykara Work developed under supervision of Doctor Carla Joana Santos Marinho Silva and Professor Doctor Lígia Raquel Marona Rodrigues Universidade do Minho Escola de Engenharia Development of Innovative Anti-adhesive Materials in Diagnosis Tools PhD thesis Ph.D. in Chemical and Biological Engineering ii Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-SemDerivações https://creativecommons.org/licenses/by-nc-nd/4.0/ ) iii Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools ACKNOWLEDGMENTS I would first like to thank the ViBrANT Consortium for giving me this wonderful opportunity to be a part of this Marie-Curie International Training Program. It was a great experience to study and learn bacterial adhesion related topics with the leaders in their field. I am so grateful to all the members of the consortium for their support through my education and help in overcoming numerous obstacles. I would like to thank my supervisors Dr. Carla Silva and Prof. Ligia Rodrigues. I would not be able to complete my thesis without help of my supervisors. Their guidance meant a lot to me through the project. I would also like to thank my colleagues from CENTI (Centre for Nanotechnology and Smart Materials) and CEB (Centre of Biological Engineering) at University of Minho, and also HydruMedicals for their support and use of their facility. This project has received funding from the European Union`s Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie Grant Agreement No. 765042. Therefore, I would also like to acknowledge the European Union for their support. Last but not the least, I would like to thank my family and my friends for their lifetime support and encouragement through my years of study and my life in general. This accomplishment would not have been possible without them. Thanks for all your encouragement! iv Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. v Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Development of Innovative Anti-adhesive Materials in Diagnosis Tools ABSTRACT Spread of infections is a growing concern in the healthcare sector, leading to high medical expenses, antibiotic resistance and even death. One recently explored alternative to reduce the spread of infections is by using anti-adhesive surfaces. Different strategies can be used to develop such antiadhesive surfaces, as for example manipulating the surface wettability. Polyethylene terephthalate (PET) is one of the most widely used polymers in the healthcare sector. Although it provides advantages including excellent chemical, physical and mechanical properties and biocompatibility, its anti-adhesive properties still need to be improved. In this thesis, PET surface was modified to improve its antiadhesive properties using three different approaches, namely (1) rendering the surface hydrophilic by chemically grafting a natural polysaccharide (gum Arabic); (2) rendering the surface hydrophilic by UV polymerization of acrylates (sodium acrylate and 3-sulfopropyl acrylate potassium salt); and (3) rendering the surface superhydrophobic by in situ growth of nanoparticles and low surface energy molecules. Determination of contact angle, AFM, FTIR, XPS and UV-VIS spectrophotometer analysis on treated surfaces clearly showed that the abovementioned modifications were successfully accomplished. The hydrophilic modified PET surfaces were found to be anti-adhesive, while the superhydrophobic one was contrarily promoting bacterial adhesion which was thought to be due to bacterial orientation on the ununiformly structured surface, when evaluated for their anti-adhesiveness against an Escherichia coli expressing YadA ( Yersinia enterocolitica adhesin) herein used as a model adhesive microorganism. Finally, the performance of the modified materials was assessed regarding crystal formation using artificial urine. Although, the modified materials did not perform better than the unmodified PET regarding crystal formation, the gum Arabic modified PET and the acrylate modified PET could still be useful for intermittent catheterization due to their anti-adhesive properties, besides exhibiting a great potential also for other applications, such as diagnosis tools. Keywords: anti-adhesive surfaces; bacterial adhesion; grafting; polyethylene terephthalate; surface modification. vi Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Desenvolvimento de materiais anti-adesivos inovadores para dispositivos médicos RESUMO A propagação de infeções é uma preocupação crescente no setor da saúde, pois acarreta elevados custos para o setor hospitalar, bem como potencia o aumento da resistência a antibióticos e pode mesmo levar à morte da pessoa infetada. Uma alternativa recentemente explorada para reduzir a propagação de infeções é o uso de superfícies anti-adesivas. Para desenvolver estas superfícies podem ser usadas diferentes estratégias, como por exemplo manipular a molhabilidade da superfície. O polietileno tereftalato (PET) é um dos polímeros mais utilizados no setor de saúde. Embora apresente vantagens como excelentes propriedades químicas, físicas, mecânicas e biocompatibilidade, as suas propriedades anti-adesivas ainda precisam ser melhoradas. Nesta tese, a superfície do PET foi modificada para melhorar as suas propriedades anti-adesivas, usando três abordagens diferentes, nomeadamente (1) tornando a superfície hidrofílica através do enxerto químico de um polissacarídeo natural (goma arábica); (2) tornando a superfície hidrofílica através da fotopolimerização de acrilatos (acrilato de sódio e sal de potássio de acrilato de 3-sulfopropilo); e (3) tornando a superfície superhidrofóbica pelo crescimento in situ de nanopartículas e moléculas de baixa energia superficial. As superfícies tratadas foram caracterizadas por ângulo de contato, AFM, FTIR, XPS e análise por espectrofotometria UV-VIS, evidenciando claramente que as modificações acima mencionadas foram realizadas com sucesso. As superfícies hidrofílicas do PET modificado demonstraram propriedades antiadesivas, contrariamente à superfície super-hidrofóbica, que promoveu a adesão bacteriana, pensandose ser devido à orientação das bactérias na superfície irregular do PET, quando foi efetuado o teste de adesividade usando uma Escherichia coli expressando YadA (adesina Yersinia enterocolitica ), como um microrganismo adesivo modelo. Por fim, avaliou-se o desempenho dos materiais modificados quanto à formação de cristais, utilizando urina artificial. Embora os materiais modificados não tenham tido um desempenho significativamente melhor que o PET virgem quanto à formação de cristais, o PET modificado com goma arábica e o PET modificado com acrilatos pode ser extremamente útil para o cateterismo intermitente devido às suas propriedades anti-adesivas, para além de apresentar um enorme potencial para outras aplicações, como a utilização em kits de diagnóstico. Palavras-chave: superfícies anti-adesivas; adesão bacteriana; enxerto; tereftalato de polietileno; modificação da superfície. vii Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools OUTPUTS Articles in peer reviewed journals Caykara T, Sande MG, Azoia N, Rodrigues LR, Silva C (2020). Exploring the potential of polyethylene terephatalate in the design of antibacterial surfaces. Medical Immunology and Microbiology, 209, 363372. ( doi: 10.1007/s00430-020-00660-8) Caykara T, Silva J, Fernandes S, Braga A, Rodrigues J, Rodrigues LR, Silva C (2021). Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach. Materials Today Communications, 28, 102684. (doi: 10.1016/j.mtcomm.2021.102684) Caykara T, Rodrigues LR, Silva C (2022). Biomaterial surface engineering towards anti-adhesive urinary catheters. ( to be submitted ) Caykara T, Fernandes S, Braga A, Rodrigues J, Rodrigues LR, Silva C (2022). Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephatalate surfaces. ( to be submitted ) Caykara T, Fernandes S, Braga A, Rodrigues J, Rodrigues LR, Silva C (2022). Can superhydrophobic surfaces prevent bacterial adhesion? ( to be submitted ) Other related articles in peer reviewed journals Sande MG, Caykara T, Silva C, Rodrigues, LR (2020). New solutions to capture and enrich bacteria from complex samples. Medical Immunology and Microbiology, 209, 335-341. ( doi: 10.1007/s00430020-00659-1) xiv Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools LIST OF FIGURES CHAPTER 1 Figure 1.1. Factors affecting bacterial adhesion onto material’s surfaces. ......................................... 9 Figure 1.2. Stages of biofilm formation on catheter surfaces [adapted from 9, 19, 67]. ................... 14 Figure 1.3. Antimicrobial surfaces obtained by the incorporation and release of antimicrobial agent or through binding of an antimicrobial agent onto the surfaces. ............................................................. 17 Figure 1.4. Anti-adhesive surfaces via hydrophilic and superhydrophobic approaches...................... 18 CHAPTER 2 Figure 2.1. Surface action on anti-adhesive and biocidal surfaces. .................................................. 36 Figure 2.2. Surface modification methodologies used to develop PET anti-bacterial surfaces. .......... 37 Figure 2.3. Chemical structure of some molecules used for surface modification of PET using “grafting to”, “grafting from” and coating methodologies. .................................................................. 40 CHAPTER 3 Figure 3.1. Schematic representation of PET modification with gum Arabic. .................................... 60 Figure 3.2. FTIR spectrum of unmodified PET (PET control) and diamine treated PET (PET amine).. 61 Figure 3.3. Acid orange colorimetric assay; a) unmodified PET sample (PET control), b) aminolysed PET sample (PET-amine), c) glutaraldehyde modified aminolysed PET sample (PET-GTA), d) absorption values from UV–vis spectroscopy for PET control, PET-amine and PET GTA. ...................................... 61 Figure 3.4. 3D graphical visualization of the initial WCA with factors A (curing temperature) and B (curing time). Factors C (gum Arabic concentration) and D (contact time) were kept at the central point. ........................................................................................................................................................ 66 Figure 3.5. Contour plots for factors A (curing temperature) and B (curing time), for the models attained for a) the initial WCA, b) WCA after 5 washing cycles and c) WCA after 10 washing cycles. Factors C (gum Arabic concentration) and D (contact time) were kept at the central point. ................. 68 Figure 3.6. Overlay plots highlighting the area were responses fulfil the set criteria (initial WCA of 35 ° and WCA 5X and 10X of 42 °). a) Factor B (curing time) versus A (temperature), keeping C = 3 % and D = 8 h (central point); b) Factor C (gum Arabic concentration) versus A (temperature), keeping B = 3 h and D = 8 h. ..................................................................................................................................... 69 xv Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Figure 3.7. SEM images for samples a–c) PET control, b–d) PET gum Arabic treated samples at two different magnifications. .................................................................................................................... 71 Figure 3.8. AFM images for the samples a, b) PET control, c, d) PET gum Arabic samples. ............. 72 Figure 3.9. Fluorescence microscopy images of Escherichia coli BL21 cells harboring pRSFduet_GFP and pASK_IBA2_YadA plasmids a) PET control and b) PET gum Arabic. Scale bar, 10 µm................. 73 CHAPTER 4 Figure 4.1. Water contact angle values for the PET samples after plasma treatment. ....................... 87 Figure 4.2. Overlay plot of factor Power (B) versus Speed (A). ......................................................... 89 Figure 4.3. Surface topography images from AFM: a) PET Untreated, Plasma treated samples with b) 50 % power and 4 m/min speed, c) 100 % power 4 m/min speed, d) 50 % power 12 m/min speed, e) 100 % power 12 m/min speed, f) 75 % power 8 m/min speed. ......................................................... 91 Figure 4.4. Proposed mechanism for atmospheric plasma treatment and UV grafting polymerisation of acrylates on PET surface. ................................................................................................................. 92 Figure 4.5. Surface topography images obtained by AFM of a) Plasma treated PET with 100 % power, 4 m/min speed, b) NaAc grafted PET, c) KAc grafted PET. ................................................................ 94 Figure 4.6. Fluorescence microscopy images of Escherichia coli BL21 cells harboring pRSFduet_GFP and pASK_IBA2_YadA plasmids a) untreated PET sample b) NaAc treated PET Sample and c) KAc treated PET sample. ......................................................................................................................... 95 CHAPTER 5 Figure 5.1. Reaction mechanism proposed for the in-situ growth of fluorinated silica NPs on hydrolysed PET. .............................................................................................................................. 104 Figure 5.2. AFM images of PET surfaces– a) PET untreated, b) PET hydrolysed, c) PET NP. .......... 106 Figure 5.3. SEM images of untreated (a), b), c)), hydrolysed (d), e), f)) and NP modified (g), h), i)) PET samples, at different magnifications (1000 X, 5000 X and 50000 X respectively). ............................ 107 Figure 5.4. SEM images of PET NP modified surface a) top view b) cross-section view at a magnification of 50000 X. .............................................................................................................. 108 Figure 5.5. SEM images of PET NP modified surface after 10 cycles of water and ethanol washing: a) top view - 1000 X; b) top view - 50000 X and c) cross-section view - 50000 X. ................................. 109 xvi Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Figure 5.6. Fluorescence microscopy images of PET samples after incubation with bacteria: a) PET untreated after 1 h; d) PET NP after 1 h; b) untreated PET after 4 h; e) PET NP after 4 h; c) untreated PET after 6 h; f) PET NP after 6h. Scale bar, 10 µm. ....................................................................... 110 Figure 5.7. SEM visualization of NPs treated samples with different magnifications a) 5 000 X b) 10 000 X and c) 50 000 X, after the bacterial adhesion test (4 h). ........................................................ 111 CHAPTER 6 Figure 6.1. Experimental setup for the dynamic urinary assessment. ............................................ 121 Figure 6.2. SEM analysis of PET samples before exposure to artificial urine using the dynamic urine testing setup; a) untreated PET, b) PET - GA, e) PETNaAc d) PETKAc. .......................................... 122 Figure 6.3. SEM analysis of PET samples before exposure to the artificial urine using the dynamic urine testing set up; a) untreated PET and b) nanoparticle treated PET. ........................................... 123 Figure 6.4. SEM analysis of PET samples after 30 days exposure to artificial urine using the dynamic urine testing setup; a) and b) untreated PET, c) and d) gum Arabic treated PET, e) and f) NaAc treated PET and g) and h) KAc treated PET. ................................................................................................ 123 Figure 6.5. SEM analysis of PET samples after 30 days exposure to the artificial urine using the dynamic urine testing set up; a) and b) untreated PET and c) and d) nanoparticle treated PET. ........ 124 xvii Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools LIST OF TABLES CHAPTER 1 Table 1.1. Limitations of urinary systems used in in vitro studies. .................................................... 15 CHAPTER 2 Table 2.1. Polyethylene terephthalate surface modification methodologies in relation to its antibacterial effectiveness, durability, and cost. ..................................................................................................... 37 CHAPTER 3 Table 3.1. Coded and actual levels for the variables studied in the central composite design. .......... 56 Table 3.2. Values for WCA after functionalization (OX), after 5 washing cycles (5X) and after 10 washing cycles (10X), according to the 24 factorial design (block 1) and to the central composite design (block 2), ordered by standard order. ................................................................................................ 63 Table 3.3. Analysis of variance (ANOVA) for the linear model obtained for the initial WCA. ............... 65 Table 3.4. Analysis of variance (ANOVA) for the linear model obtained for WCA after 5 and 10 washing cycles. .............................................................................................................................................. 67 Table 3.5. Surface roughness and energy of PET control and PET gum Arabic (optimized gum Arabic treated sample). ............................................................................................................................... 70 CHAPTER 4 Table 4.1. Coded and actual levels for the Design of Experiment Variables. ..................................... 83 Table 4.2. Water contact angles, surface energy, roughness and chemistry values found for the PET plasma treated samples.................................................................................................................... 86 Table 4.3. Analysis of Variance (ANOVA) for the linear model obtained for the initial WCA. ............... 88 Table 4.4. Relative composition (in percentage) of the different chemical groups detected with the deconvolution of C 1s and O 1s spectra for plasma treated samples. ................................................. 90 Table 4.5. WCA, surface energy and roughness values of untreated, NaAc treated and KAc treated PET surfaces. ................................................................................................................................... 93 xviii Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools CHAPTER 5 Table 5.1. Contact angle values determined with water and diiodomethane, and surface free energy (SFE) of PET untreated, PET Hydrolysed and PET NP. ..................................................................... 105 Table 5.2. Surface roughness values (Ra) of treated and untreated PET samples determined by AFM. ...................................................................................................................................................... 106 Table 5.3. SEM-EDS results for atomic concentrations of untreated and treated PET samples. ....... 109 CHAPTER 6 Table 6.1. SEM-EDS analysis of material surfaces after being tested with artificial urine. ................ 124 xix Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools LIST OF ABBREVIATIONS AF Acrylate polymer blend AFM Atomic Force Microscopy ANOVA Analysis of variance aTc Anhydrotetracycline ATR-FTIR Attenuated total reflectance - Fourier Transform Infrared ATRP Atom transfer radical polymerization BP-QAS Benzophenone group terminated cationic quaternary ammonium salts CAE Constant Analyser Energy CHI Chitosan CMC Carboxymethylcellulose Cs Cationic chitosan DBD Dielectric Barrier Discharge DCC N,N′-dicyclohexylcarbodiimide DEDA N,N-diethylethylenediamine DLS Dynamic Light Scattering DLVO Derjaguin-Landau-Verwey Overbeek DNA Deoxyribonucleic Acid DoE Design of experiments DS Dermatan sulfate ECDC The European Centre for Disease Prevention and Control eDNA Extracellular deoxyribonucleic acid EDS Energy dispersive spectroscopy EF Epoxy polymer ePET Expanded polyethylene terephthalate EPS Extracellular polymeric substances ePTFE Expanded polytetrafluoroethylene FimA Adhesin from fimbriae type 1 GMA Glycidyl methacrylate HA Hyaluronic acid xx Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools HAI Hospital Associated Infections HTCC Hydroxypropyltrimethyl ammonium chloride chitosan IC Intermitted catheters IPTG β-D-1-thiogalactopyranoside LAMA 2-lactobionamidoethyl methacrylate LB Luria Broth KAc 3-Sulfopropyl Acrylate Potassium Salts MPEO Methoxy polyethylene oxide NAc Sodium acrylate NP Nanoparticle OFAT One-factor-at-a-time OWRK Owens-Wendt-Rabel&Kaelble Model PANI Polyaniline PapC Adhesin type from pili PBS Phosphate buffer saline PC Polycarboxybetaine PCCs Phosphorylcholine–chitosan PDMAEMA Poly(2-(dimethylamino)ethyl methacrylate) PEG Polyethylene glycol PEI Polyethylenimine PET Polyethylene terephthalate PG Peptidoglycan PHA Polyhexylene adipate PS Polysulfobetaine PTFE Polytetrafluoroethylene PU Polyurethane RAM Rhizome Atractylodes macrocephala RIF Rifampicin ROS Reactive oxygen species SD Sulfadimehoxine SEM Scanning electron microscopy SfAS Adhesin type from fimbriae S xxi Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools TFPDM 2,3,5,6-tetrafluoro-p-phenylenedimethanol TRI Triclosan UV-VIS Ultraviolet – visible spectrophotometer UV Ultraviolet VAN N-(4-hydroxy-3-methoxybenzyl)-acrylamide WCA Water contact angle XPS X-Ray Photoelectron Spectroscopy YadA Yersinia enterocolitica adhesin 1 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools GENERAL INTRODUCTION Context and motivation Research aims Outline of the thesis 2 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools CONTEXT AND MOTIVATION Spread of infections is a growing health problem and the number of infected people is increasing constantly every year in healthcare facilities [1]. Antibiotics are used to treat infections, however, the increase in antibiotic resistance is a great concern. Indeed, 30% of the bacteria found in healthcare facilities have been found to be antibiotic resistant [2]. Biofilms are ideal for the development of such resistance as they protect bacteria conferring them resistance [3]. Thus, for material-associated infections, which are common in the medical scenario, it is important to prevent the initial bacterial adhesion, that ultimately leads to biofilm formation and infection. Inhibition of planktonic bacteria adhesion [4] can be achieved by modifying materials properties that affect adhesion. Some of the strategies that have been reported to prevent bacterial adhesion include the development of:  resistant hydrophilic surfaces, where a hydration layer on the top of surface is formed that prevents bacteria from approaching to the surface [1,4,5,8];  repellent superhydrophobic surfaces, where air pockets are created on surfaces to prevent bacteria from approaching and to enable their easy cleaning [5];  bactericidal surfaces, where bacteria approaching the surface are killed by antimicrobials which are either bonded to the surface or released from it [6];  resistant and bactericidal patterned surfaces, where surface patterns can be designed in a way that either reduces bacterial adhesion or kills the adhered bacteria [7,8]. Since the bacteria deterrence strategies with hydrophilic surfaces and superhydrophobic surfaces are based on anti-adhesion, the spread of infections in these surfaces would be reduced, and consequently also the unnecessary use of antimicrobials, limiting the occurrence of antimicrobial resistance. PET is one of the most used polymeric materials in the healthcare sector due to its desirable properties such as biocompatibility, high uniformity, mechanical strength and resistance against chemicals and/or abrasion. However, PET surfaces are prone to bacterial adhesion [4]. Therefore, the main goal of this thesis was to modify PET surfaces to improve its surface wetting properties towards bacterial adhesion prevention. Furthermore, to assure a durable surface modification, several methods including “grafting to”, “grafting from” and “ in situ nanoparticle growth” for covalent bonding the different studied molecules to the surface were implemented. All modified surfaces were characterized by contact angle measurement, FTIR, AFM, XPS and UV-Vis spectrophotometer analysis and further bacterial adhesion tests were conducted with a modified Escherichia coli . CHAPTER 1 - Biomaterials surface engineering towards anti-adhesive urinary catheters 9 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools thermodynamic approach [16]. However, all these theories do not completely describe adhesion since the bacterial surface is complex, as well as structurally and chemically heterogeneous. Furthermore, extracellular polymeric substances (EPS) and proteinaceous cell appendages bridge between bacteria and the substrate, directly affecting bacterial adhesion and the theories are unable to model it. While EPS is important for biofilm formation, the presence of proteinaceous cell appendages are often essential for the initial bacterial adhesion [17]. Thus, experimental bacterial adhesion studies and design of new anti-adhesive materials remain of critical importance [16]. 1.3. FACTORS AFFECTING BACTERIAL ADHESION Adhesion of bacteria onto surfaces depends on three main factors (Figure 1.1), namely bacteria itself (so-called biological factors), material surface (so-called physicochemical factors) and the surrounding environment (so-called environmental factors) [10] which must be considered when engineering materials to confer them anti-adhesive properties. Figure 1.1. Factors affecting bacterial adhesion onto material’s surfaces. 1.3.1. Biological factors Urinary infections are caused by both internal microflora and external contamination [9]. The most common bacteria found in catheter-related infections are Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Escherichia coli, Citrobacter freundii, Providentia rettgeri, Candida albicans, Morganella morganii, Burkholderia cepacian, Providencia sp., Providencia stuartii [8,9,18]. From all those bacteria, E. coli, E. faecalis, S. epidermidis are frequent in short-term device applications, while P. mirabilis, P. aeruginosa, P. stuartii, M. morgani and K. pneumoniae usually occur in long term applications [18,19]. In indwelling catheters, P. mirabilis biofilm is the main concern, while in CHAPTER 1 - Biomaterials surface engineering towards anti-adhesive urinary catheters 10 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools intermitted catheters, the main infections that occur are caused by E. coli due to the microflora of meatus being pushed into the bladder [9]. Bacteria can be divided into Gram-negative and Gram-positive depending on the cell wall composition [20]. Although both bacterium types are seen, gram-negative bacteria seem to be the dominant bacteria type in urinary infections [8,9,18]. One of the main differences in these two types of bacteria is the thickness of the peptidoglycan layer in the cell wall. Gram-positive bacteria have a thick peptidoglycan layer above the cell membrane, while gram-negative ones have a thin peptidoglycan layer between the cell membrane and outer membrane layer, and it includes a gel-like region which is called periplasmic space [20]. Thus, gram-negative bacteria have a less rigid surface compared to the grampositive ones and, the more flexible cell wall of gram-negative bacteria can promote a better adhesion onto rough surfaces [21]. Furthermore, the outer cell wall in gram-negative bacteria consists of lipopolysaccharides, whereas in gram-positive bacteria teichoic and lipoteichoic acids in the wall are attached to the peptidoglycan layer [20]. Additionally, bacteria are almost always negatively charged [20]. Bacteria cell surface charge originates from dissociation or protonation of carboxyl, phosphate and amino groups and consequently depends on the environmental pH [22]. The negative bacterial charge is mainly due to phosphate groups in teichoic acids for gram-positive bacteria [23] and phosphoryl and 2-keto-3deoxyoctonate carboxylate groups in gram-negative bacteria [20]. However, the bacterial cell is highly heterogeneous and contains various exposed proteins, lipids and exopolysaccharides, that exhibit different charges and hydrophobicity depending on the growth conditions such as environmental pH and ionic strength, to provide adhesive adaptability [10,24]. Surface charge is also influenced by the bacteria age and surface structure. A high surface charge usually goes together with hydrophilic bacteria [25]. The hydrophobicity of bacteria differs according to the bacterial species, and it is also influenced by growth, medium, bacteria age and surface structure [26]. Microbial cell hydrophobicity can be determined by contact angle measurements and the values for some of the abovementioned bacteria can be found in literature [27]. A great variety of structural surface appendages like fimbriae or flagella can be found on the top of the peptidoglycan layer in gram-positive bacteria and on the top of the outer membrane in gramnegative bacteria [22]. The surface appendages are extracellular structures which are important in bacterial growth and survival in diverse environments [28]. Filamentous protein extensions and surface appendages from the cell surface, including flagella, fimbriae, curli and pili, are involved in the nonspecific initial adhesion to abiotic surfaces [10]. On the other hand, non-fibral adhesins on the surface CHAPTER 1 - Biomaterials surface engineering towards anti-adhesive urinary catheters 11 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools of bacteria can play a role in the close contact between bacterial cell and substrate, as well as in the maturation of interactions and irreversible adhesion [10,24]. It has been shown that adhesins like Fimbriae type 1 (FimA), Fimbriae S (SfaS), PapC (which forms pili) [29] and extracellular polysaccharides [24,30,31] and eDNA (extracellular DNA) [32] also promote adhesion. Moreover, bacterial cells are highly dynamic, and they can adsorb ions and macromolecular components. Charged groups may associate or dissociate upon changes in pH or ionic strength of the surrounding fluid and even when approaching a charged surface. This might induce changes in the conformation of different kinds of surface appendages as fimbriae and flagella [22]. Additionally, bacteria can reach a surface by active and/or passive movement. While some bacteria have swimming motility, some are subjected to physical forces like Brownian motion and gravitational forces to bring them close to the surface. In active movement, bacteria have flagella, which are responsible for the swimming ability by generating a propulsive force. They can also play a role in reversible and irreversible adhesion. Flagella can direct the swimming towards a surface in response to the cues in environment like chemical signals, light, temperature, magnetic fields and oxygen [10,24]. 1.3.2. Physical factors Several physicochemical properties of the surfaces affect bacterial adhesion, namely, wettability, surface energy, surface charge, roughness and others like hardness and film thickness [33,34]. The hydrophobicity effect on bacterial adhesion is mostly governed by the hydrophobicity of the bacterial cell [10]. Hydrophobic bacteria show higher adhesion compared to hydrophilic bacteria, while hydrophilic materials were found to be more resistant to bacterial adhesion than hydrophobic materials. Large numbers of bacteria adhere to hydrophobic surfaces with little or no surface charge, however, this number is smaller for hydrophilic surfaces [26]. The number of bacteria becomes even smaller if the hydrophilic surfaces are negatively charged [25]. A study suggests that bacteria adhere with only few strongly binding macromolecules on hydrophilic surfaces whereas bacteria adhere with many weakly binding macromolecules leading to high adhesion with low variability on hydrophobic surfaces. [35]. Anti-adhesive materials via hydrophilic surface modification will benefit from a hydration layer formed on the material surface which acts as a barrier to the bacteria approaching [36]. Although there is an increased interest in hydrophilic surfaces, the bacterial inhibition observed on some natural surfaces like lotus leaves, dragonfly wings and shark skin has inspired the scientific community to also work on the design of superhydrophobic surfaces. These surfaces, due to their superhydrophobic nature, form air pockets on the surface of the materials, preventing bacteria from approaching and consequently CHAPTER 1 - Biomaterials surface engineering towards anti-adhesive urinary catheters 12 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools adhering [10] and, they are also easier to clean due to a weaker binding at the interface [37]. An additional parameter affecting bacterial adhesion is surface energy [38]. Katsikogianni et al. showed that bacterial adhesion was negatively correlated with the total surface free energy and its polar component according to dispersive-polar approach, where total surface free energy is expressed as the sum of polar and dispersive components [39]. Adhesion free energy becomes more negative resulting in an increased bacterial adhesion strength with the increasing hydrophobicity of a surface [40]. However, some studies have shown that low surface energies (20 – 30 mN/m) have the lowest adhesion [34]. This may be due to the chemical and physical properties of bacteria, substrates and water solution used [38]. However, since surface energy values are usually calculated using contact angle values, which are an indication of hydrophilicity and hydrophobicity, conflicting results can arise from using these wetting properties of the materials [37]. Another factor influencing bacterial adhesion is the material surface charge. As previously mentioned, bacterial cells generally exhibit a net negative charge. As most surfaces are naturally negatively charged, bacteria experience electric double layer repulsion [34]. However, positively charged surfaces can be used to kill bacteria by attracting them and damaging their cell wall [33]. Roughness is another parameter showing conflicting results [41]. The general opinion in the literature reports that irregularities on polymer surfaces promotes bacterial adhesion, while ultra-smooth surfaces show lower bacterial adhesion. This surface behaviour has been explained with the increase of favourable sites on the surface area for bacterial attachment [34]. However, other studies showed that roughness had no effect or even inhibited the adhesion of bacteria [42] in cases of creating nano roughness [42]. This kind of conflicting results may be due to the bacteria used, as bacteria type (gram type) [21] and fibral structures like flagella [43] can make a difference. Moreover, it should also be noticed that the surface roughness can affect its hydrophilicity and hydrophobicity. Surface wetting can either be homogeneous or heterogeneous, which have different impacts on bacterial adhesion. The Wenzel `s phenomenon suggests that both hydrophilicity and hydrophobicity are enhanced by an increasing roughness on homogenously wetted surfaces, meaning that a hydrophilic surface will become more hydrophilic, and a hydrophobic surface will become more hydrophobic [44]. The porous surfaces behave according to another phenomenon, the so-called Cassie-Baxter phenomenon. In this state, the water droplet heterogeneously wets the surface causing air pockets and affecting the wettability [45]. In this way, hydrophobic surfaces can prevent bacterial adhesion. Additionally, micro-texturing has also been found to be beneficial on surfaces where bacteria width is bigger than the gap between micro-textures. This reduces the bacterial adhesion by reducing CHAPTER 1 - Biomaterials surface engineering towards anti-adhesive urinary catheters 13 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools the surface area [34]. Thus, it is important to tune the dimensions and shapes of textures according to the bacterial cell to control bacterial cell adhesion [46,47]. Micro and nano texture of the surfaces can also be arranged in a way to mechanically rupture bacterial cell wall, thus providing antibacterial properties while avoiding antimicrobial resistance. However, this mode of action works better for gramnegative bacteria, due to its flexible cell membrane which allows stretching and tearing upon surface adsorption [48]. In addition to the factors discussed above, other factors like hardness and film thickness have attracted attention. While it had been found that increased hardness reduced bacterial cell adhesion in polymers [26,49], there has been reports that soft materials were more resistant to bacterial adhesion [50]. On the other hand, the film thickness was important for soft materials and thicker films reduced bacterial adhesion, which was thought to be due to the stiff substrate having a higher effect on thin films [50]. 1.3.3. Environmental factors Environmental effects caused by the presence of proteins, ions, pH and flow rate can also play a role in bacterial adhesion. Human urine consists of 91 – 96 % water and the remaining parts comprise inorganic salts, urea, organic compounds and organic ammonium salts [51]. It was found that pH and the ionic strength of the surrounding buffer affect the cell surface and material surface’s hydrophobicity [52]. The pH of urine is between 4.5 - 8 [19]. A problem that arises due to increased pH in the medium is encrustation. It may cause blockage of the catheter leading to damage of the bladder, ureters and kidneys [9]. Encrustation can occur due to metabolic dysfunction but generally, it is due to bacteria as urease-producing bacteria ( P. mirabilis, P. vulgaris and P. rettgeri ). Urease hydrolyses urea into ammonia and carbon and with increased ammonia, the pH of the urine increases above 8.0, causing calcium and magnesium phosphate to crystallise. However, some urease forming bacteria species do not form crystals due to low levels of urease production. Some of these species include P. aeruginosa, S. aureus, K. pneumoniae, E. coli, M. morganii, and P. stuartii . Nevertheless, other bacteria can still produce mucoid which can also cause catheters blockage. Thus, catheters should be resistant to bacterial adhesion, biofilms formation and encrustation [9]. On the other hand, ionic strength is one of the factors affecting bacterial adhesion. At low ionic strength (≤ 20 mM) adhesion can be driven by electrostatic repulsion, while Van der Waals and hydrophobic interactions can be primary driving forces at higher ionic strengths (50 – 100 mM) [14]. The ionic strength of urine is mainly determined by sodium and chloride due to their abundance [53] and its physiological ionic strength is 150 mmol L-1 CHAPTER 1 - Biomaterials surface engineering towards anti-adhesive urinary catheters 14 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools [54]. Due to the relatively high ionic strength value, it can be said that the adhesion will be driven by Van der Waals and hydrophobic interactions thus, the design of new materials should be more focused on wetting properties of the materials rather than on surface charge. Moreover, other ions like calcium can also enhance bacterial adhesion [14]. While a low amount of iron and manganese weakens bacterial adhesion, phosphate limitations enhance bacterial adhesion [10]. Furthermore, a conditioning film is formed on the surface of the urinary catheter when it enters the body [19]. This conditioning film consisting of proteins, polysaccharides [55] and urine can make the surface more susceptible to bacterial adhesion [8,56]. Tamm-Horsfall glycoprotein and other proteinaceous molecules like serum albumin, fibrinogen, collagen and fibronectin can be found in conditioning films [57]. Tamm-Horsfall, fibrinogen and fibronectin can promote bacterial adhesion [8,58] while albumin inhibits it [59,60] and collagen has been shown to promote and inhibit bacterial adhesion depending on the experimental conditions [61,62]. Urine also includes proteins from urothelial cells, as well as damages at the urethral lining caused during insertion of catheters [19]. Electrolytes, ions, mineral and other organic molecules will also be present on the conditioning film [19,63–65]. Furthermore, initial bacterial adhesion stages in alkaline environments can cause the formation of microcrystals on the catheter surface which are also proven to support bacterial adhesion (Figure 1.2) [8,66]. Figure 1.2. Stages of biofilm formation on catheter surfaces [adapted from 9, 19, 67]. Other important factors to prevent bacteria from reaching and adhering to the surface are flow and shear. If the flow rate is high, it would cause a lower boundary layer, thus bacteria near to the surface are affected by higher shear and at high velocity, limiting the bacteria approach to the surface [10]. Furthermore, antimicrobial substances can be depleted from the surface in a shorter time compared to static conditions. Therefore, catheters should be studied under flow [68]. Unfortunately, the lack of standard testing methods makes the development and study of new materials harder to CHAPTER 1 - Biomaterials surface engineering towards anti-adhesive urinary catheters 15 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools compare, due to the absence of well-documented data on bacterial adhesion onto different materials [19,69]. 1.4. NEW MATERIALS FOR URINARY CATHETERS Catheters are one of the most used medical devices [9] and bacteria with antibiotic resistant genes found in biofilms and in urinary samples of patients, raises even more concerns [7]. Thus, there is a great need to develop better materials that can prevent or reduce bacterial adhesion, and subsequently biofilm formation and related infections [7]. In order to evaluate these newly designed materials, in vitro testing models have been developed [9,70,71]. However, the evaluation is still a challenge as the urinary systems are complex and inconsistent [75] with several difficulties at mimicking listed in Table 1.1. Nevertheless, in vitro models are important to test new materials since they can help to avoid ethical issues and higher cost which comes with in vivo tests [9]; however, there is still a need for standard and realistic models to be developed in order to evaluate the performance of the new materials in equivalent conditions. Table 1.1. Limitations of urinary systems used in in vitro studies. Factors Complexities of urinary systems Bacteria  The variation in bacterium types in urine [8,9,18]  Strain used with reduced abilities to attach in vitro studies [72]  Use of only cultivable strains for in vitro studies [72]  Variation in bacteria colonisation time [73]  Variation in the number of bacteria [19] Urinary fluid  The difference in nutrients and nutrient levels [72]  Lack of Host defence system like antimicrobial proteins and peptides for in vitro models [72]  Presence of proteins, cells and crystals in urine [9]  Oxygen amount for bacterial growth [75]  Other parameters like flow, temperature, osmolarity [68,72] Material  Conditioning film on the material [19,55,63–65]  Crystal formation on the material [8,9,66]  Biocompatibility, stability of the material or modification [72] Strategies to develop new materials to prevent bacterial adhesion in urinary catheters include mainly (1) incorporating antimicrobial agents and (2) developing antifouling surfaces [33]. The most common commercial products use antimicrobial coatings with silver and antibiotics like nitrofural, sparfloxacin, rifampicin, minocycline, and antifouling coatings by combination with hydrophilic coatings and hydrogels [19,74,75]. However, studies are still being conducted to develop more effective CHAPTER 1 - Biomaterials surface engineering towards anti-adhesive urinary catheters 16 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools technologies. 1.4.1. Antimicrobial coatings Antimicrobial studies involve using antimicrobial coatings comprising antibiotics (e.g., rifampicin, triclosan), silver or nanoparticles (carbon nanotube and graphene oxide), enzymes and peptides [8,9,74,76]. Antibiotic coatings are less effective at preventing biofilm formation since urinary tract infections are mostly caused by antibiotic resistant pathogens [9]. Silver is one of the most used antimicrobial agents [74] and it has provided effective results either alone [77] or in combination with other molecules [78]. The combination of silver with antibiotics (amikacin and nitrofurantoin) also showed a synergistic effect by inhibiting bacterial adhesion more effectively compared to the single use of any of them [78]. Although silver plays an important role in the development of antimicrobial catheters, this type of catheter loses its antimicrobial ability in long term uses. The intermittent use can also cause bacterial resistance [74]. Moreover, silver can cause hypersensitive reactions in patients and there is a growing concern about a possible emergence of resistant bacteria [9]. Another bactericide used is nitric oxide which is an endogenously produced bactericidal gas used in urinary catheter studies [79]. Implementation of nitric oxide has shown some better results compared to silver. However, there can be side effects like decrease in blood pressure, inhibition in platelet aggregation, increased bleeding, skin irritation, skin edema/erythema and uncontrolled erection [80]. Thus, other alternatives like antimicrobial enzymes [81] and peptides [82] are being investigated as they are less likely to cause the development of bacterial resistance and they may be less toxic to tissues compared to silver [74]. Nonetheless, their production is more expensive [74,83] and their use might be limited due to enzymatic degradation [84]. Less toxic surfaces with antibacterial properties can also be achieved using natural substances like phenolic compounds, such as vanillic acid which was found to be effective [85], and chitosan which can offer bactericidal and antifouling properties while being cheap and biocompatible [86]. When designing materials, antimicrobials can be incorporated into the surface by using an antimicrobial release approach which affects the material surface and the surrounding or can be bonded to the surface, affecting only the surface of the materials as shown in Figure 1.3. This incorporation strategy is decided according to the type of antimicrobials incorporated [87]. While some leaching antimicrobials may sensitize patients and cause life-threatening anaphylaxis [87], in other cases surface-bond antimicrobials may lose their efficiency [88]. Surfaces modified with antimicrobials can lose their effectiveness after being implanted since film conditioning and dead bacterial cells can CHAPTER 1 - Biomaterials surface engineering towards anti-adhesive urinary catheters 17 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools cover the top of the materials preventing the material surface from functioning [72]. Therefore, it is recommended that antimicrobial strategy is implemented along with another complementary strategy. Figure 1.3. Antimicrobial surfaces obtained by the incorporation and release of antimicrobial agent or through binding of an antimicrobial agent onto the surfaces. 1.4.2. Anti-adhesive surfaces Some other strategies focus on forming anti-adhesive surfaces by modifying the surface physiochemical properties, for example developing hydrophilic or superhydrophobic [74,89] surfaces as shown in Figure 1.4. Among these strategies, catheters with hydrophilic surface properties have already been commercially available [8]. One of the most studied hydrophilic surface modifications has been the formation of polyethylene glycol brushes (PEG) [90], being considered the gold standard; however, these PEG modified surfaces are found to be unstable due to oxidation [91]. Thus, there have been other hydrophilic surface modifications, including poly[N-(2-hydroxypropyl) methacrylamide] (poly(HPMA)) brush [92] or polysaccharides like heparin [93], hyaluronic acid [94] and chitosan [95,96]. Zwitterionic polymers can also be alternatively used to confer hydrophilicity to surfaces. The most common zwitterionic polymers are phosphorylcholine, sulfobetaine and carboxybetaine [74,97,98]. Although they have been extensively studied as the next generation of promising antifouling materials [84], the long-term stability of zwitterionic surface is still a concern [74]. The use of amphiphilic polymeric coatings, by the combination of dodecyl methacrylate, polyethylene glycol methacrylate and acrylic acid, has also proven to prevent bacterial adhesion significantly [56]. An CHAPTER 1 - Biomaterials surface engineering towards anti-adhesive urinary catheters 18 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools amphiphilic coating which was effective against both bacteria and protein adhesion was performed by synthesising a polymer from hydrophobic benzyl methacrylate, hydrophilic polyethylene glycol methacrylate and methacrylic acid [99]. Furthermore, surfactants such as surfactin, rhamnolipids and several other surface-active compounds produced by microorganisms were reported in the literature [100]. Biosurfactants are advantageous due to their low toxicity, biodegradability and biocompatibility. They further show both anti-adhesive and antimicrobial properties thus, offer a great potential to be used in urinary catheters [101]. Figure 1.4. Anti-adhesive surfaces via hydrophilic and superhydrophobic approaches. In general, thin hydrophilic coatings like polymer brushes may not be mechanically stable in the long-term use thus, hydrogel coatings can be promising considering biocompatibility, functional group density and lubricity. However, hydrogel coatings are fragile due to weak interactions between the coating and the substrate. Nevertheless, cross-linking of the coating to the substrate can improve the durability of the hydrogel coating while improving its anti-adhesive performance [102]. Hydrogels can also be used in combination with antimicrobial agents. For instance, Su et al. [103] reported a hydrogel from PEG oligomers with the antimicrobial agent polyhexamethylene guanidine. Although antifouling surfaces are advantageous against antimicrobial resistance, the results obtained using anti-adhesive surface modifications are generally considered to be modest compared to using surfaces modified with antimicrobial agents [56] and combining antifouling with antimicrobial surfaces can increase the life cycle of the surface modification, as dead bacteria cells can be removed from the surface with antifouling functionality [104]. Hence, modifications employing the combination of both strategies are being developed. Polyethylene glycol (PEG) has been a popular choice in studies combining anti-adhesive and antimicrobial functions such as combining PEG for anti-adhesive and CHAPTER 1 - Biomaterials surface engineering towards anti-adhesive urinary catheters 25 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools 37. Q. Zhao, S. Wang, H. Müller-Steinhagen, Tailored surface free energy of membrane diffusers to minimize microbial adhesion. 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Acta Biomater. 2020;114: 117–132. https://doi.org/10.1016/j.actbio.2020.07.025. 34 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools CHAPTER 2 Exploring the potential of polyethylene terephthalate in the design of antibacterial surfaces Polyethylene terephthalate (PET) is one of the most used polymeric materials in the health care sector mainly due to its advantages that include biocompatibility, high uniformity, mechanical strength, and resistance against chemicals and/or abrasion. However, avoiding bacterial contamination on PET is still an unsolved challenge and two main strategies are being explored to overcome this drawback: the anti-adhesive and biocidal modification of PET surface. While bacterial adhesion depends on several surface properties namely surface charge and energy, hydrophilicity and surface roughness, a biocidal effect can be obtained by antimicrobial compounds attached to the surface to inhibit the growth of bacteria (bacteriostatic) or kill bacteria (bactericidal). Therefore, it is well known that granting antibacterial properties to PET surface would be beneficial in the prevention of infectious diseases. Different modification methods have been reported for such purpose. This review addresses some of the strategies that have been attempted to prevent or reduce the bacterial contamination on PET surfaces, including functionalisation, grafting, topographical surface modification and coating. Those strategies, particularly the grafting method seems to be very promising for healthcare applications to prevent infectious diseases and the emergence of bacteria resistance. Keywords: Polyethylene terephetalate; bacterial adhesion; antibacterial properties; surface functionalisation; grafting; topographical modification; coating; water contact angle This chapter is based on the following publication: Caykara T, Sande MG, Azoia N, Rodrigues LR, Silva C. (2020). Exploring the potential of polyethylene terephatalate in the design of antibacterial surfaces. Medical Immunology and Microbiology, 209, 363372. ( doi: 10.1007/s00430-020-00660-8) CHAPTER 2 - Exploring the potential of polyethylene terephthalate in design of anti-adhesive materials 41 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools 2.2.2.1. “Grafting to” method In the past years, there have been different attempts to prevent and/or inhibit bacterial contamination using the “grafting to” method. One of the interesting approaches was the immobilization of enzymes. Lysozyme is known for possessing bactericidal activity due to its capacity to hydrolyse the bacterial cell wall peptidoglycan (PG) and highly cationic conventional lysozyme types can kill the bacteria independently of cell wall PG damage [21]. Meslmani et al. [22] immobilized lysozyme onto woven and knitted crimped PET surfaces. Although the enzyme activity has been reduced to 55– 60 % with the grafting, the resulting samples were able to prevent bacterial adhesion. The anti-adhesive efficiencies of woven and knitted enzyme grafted PET were above 80 % against S. aureus and S. epidermidis , and above 70 % against E. coli , compared to unmodified PET samples. The effectiveness of chitosan, a widely used natural polysaccharide, was investigated within the “grafting to” approach. Due to its positive charge, it has bacteriostatic and bactericidal effects by damaging the bacterial cell wall. Chitosan is broadly used in layer-by-layer methods to provide multifunctional films due to its partially positive charges. However, the layer-by-layer method does not provide stable coatings, because the layers are pH dependent and not resistant to abrasion [8]. Hayder et al. [8] covalently immobilized two separate layers of chitosan and dermatan sulfate (DS), an anionic polysaccharide, on PET using the coupling agent N,N′-dicyclohexylcarbodiimide (DCC). The results showed that chitosan and DS modification increased hydrophilicity. PET–DS–CHI was more hydrophilic exhibiting a contact angle of 71 °, while PET–CHI–DS exhibited a contact angle of 87 °. This was probably due to the higher amount of surface –COOH group on PET–DS–CHI. The tests against bacteria showed that both modified materials were more resistant to biofilm formation compared to the unmodified PET substrate, with PET–DS–CHI coating showing a better inhibition. However, PET–CHI showed a higher reduction of the bacterial adhesion which is thought to be due to the partial positive charges present on chitosan compared to other modified PET samples. The block copolymer of sulfadimehoxine polyhexylene adipate-b-methoxy polyethylene oxide (SD-PHA-b-MPEO) was another multifunctional copolymer studied and it is the combination of hydrophobic PHA to repel bacteria, hydrophilic MPEO to increase the host cell interactions for material integration in vivo and negatively charged SD which is a bacteriostatic antibiotic [23]. A porous structure has been obtained after its grafting onto PET by evaporating the solvent from polymer brush solution. Anti-adhesive efficiency of woven and knitted forms of modified PET samples were between 56–62 % against S. aureus and S. epidermidis , while the efficiency was between 63–64 % against E. coli [24]. Zwitterionic polymer brushes is an attractive approach for surface modification in the biomedical CHAPTER 2 - Exploring the potential of polyethylene terephthalate in design of anti-adhesive materials 42 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools field, since it can provide good biocompatibility and anti-adhesive efficiency due to counteraction of the electrostatic hydration effect [25]. Timma and co-workers [26] developed polyvinylamine polymers that were functionalised with zwitterionic sulfobetaine side chains for PET fabrics. While sulfobetaine provided anti-adhesive properties, protonated amine groups provided bactericidal properties. A high substitution degree of sullfobetaine on the polymer chains might cause pure anti-adhesive properties. Therefore, reducing the substitution degree of sulfobetaine can allow a mixture of anti-adhesive and bactericidal properties due to the existence of uncoupled protonated amine groups. It was shown that the bacterial adhesion decreased with higher amount of substitution degree and 80 % of substitution degree caused the material to lose almost all bactericidal properties. Moreover, the primary action of polymers with 60 % of substitution degree seemed to be dependent of the fibre type. The bactericidal effect against Gram-negative bacteria was noted to be more influenced due to the different structure of the cell wall with the increasing substitution degree. In another study done by Xv et al. [25], a zwitterionic glycidyl methacrylate–phosphorylcholine– chitosan (PCCs–GMA) was photo-immobilized on PET films. Hydroxypropyltrimethyl ammonium chloride chitosan–GMA (HTCC–GMA), cationic chitosan–GMA(Cs–GMA) and pristine PET were used to compare the results. The water contact angle was reduced to 34 °, 36 ° and 47 ° after immobilization of PET– GMA–PCCs, PET–GMA–HTCC, PET–GMA–Cs, respectively. PET–GMA–PCCs has improved surface antibacterial properties and inhibited the adhesion up to 100 % for E. coli and 92 % for S. aureus compared with pristine PET. Although PET–GMA–HTCC and PET–GMA–Cs improved the antibacterial properties, many bacteria were observed on the surface of the tested materials. The live/dead bacteria assessment showed that no live or dead bacteria were observed on PET–GMA–PCCs surface, while there were some dead bacteria on PET–GMA–HTCC and some live bacteria on both pristine PET and PET–GMA–Cs. Both HTCC and chitosan are positively charged; however, the bacterial growth inhibition and contact killing properties of chitosan are limited in neutral conditions thus HTCC showed attached dead bacteria. Due to strong electrostatic hydration effect of zwitterionic PC, the attachment and killing of bacteria was suppressed [25]. 2.2.2.2. “Grafting from” method The “grafting from” is an alternative surface modification technology, being one of its main advantages the controllable molar mass and grafting density. Lepoittevin et al. [20] studied the grafting density by modulating the monomer/free initiator ratio. PET films were pre-treated with polyethylenimine (PEI), followed by the reaction with a surface initiator (bromoisobutyryl bromide). CHAPTER 2 - Exploring the potential of polyethylene terephthalate in design of anti-adhesive materials 43 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Finally, the Atom Transfer Radical Polymerization (ATRP) of 2-lactobionamidoethyl methacrylate (LAMA) was carried out and glycopolymer brushes were grown on the surface of the PET films with different grafting degrees. With the higher grafting degree, the authors obtained a water contact angle of 11 ° and a surface energy of 44.1 mN/m. The study showed the great potential for this type of carbohydrate and further studies should be performed to evaluate its potential for inhibiting bacterial adhesion. Researchers have been showing an increased interest in the use of natural compounds as alternatives to synthetic active agents. Recent studies on some natural compounds like vanillin monomer which has bacteriostatic effect depending on target (more effective towards Gram-positive bacteria) [27] and thyme, which presents bacteriostatic and bactericidal effect, have been reported. In a study conducted by Mani and co-workers [28], the vanillin derived biobased monomer, N-(4-hydroxy-3methoxybenzyl)-acrylamide (VAN), was used to modify the PET surface using the photopolymerization technique and N,N-diethylethylenediamine (DEDA) as a crosslinker. The surface modification with VAN reduced the contact angle from 80 ° to 62 ° and it was found that VAN grafted PET inhibited the adhesion of the Gram-positive bacteria Rhodococcus wratislaviensis and S. aureus by 85 % and 97 %, respectively. However, the inhibition of the Gram-negative bacteria E. coli and Pseudomonas aeruginosa was limited by 50 %. Rhizome Atractylodes macrocephala (RAM), is another herbal product which has declared antibacterial properties. Shu et al. [29] worked with RAM grafting on PET non-woven substrates. They have found that the grafting of RAM was highly improved when pre-grafted polymerization of acrylic acid or plasma treatment was applied. It was further improved when both acrylic acid and plasma treatments were applied prior to grafting of RAM. The effectiveness against S. aureus and E. coli increased with the increased grafting percentage. Bedel et al. [30] performed ATRP polymerization of thymol monomer. The results showed that the water contact angle increased from 81 ° to 99 ° for samples treated with thymyl methacrylate. The total surface energy of PET was reduced from 44.7 to 40.5 mN/m. Furthermore, the thymyl methacrylate treated samples were highly antibacterial exhibiting up to a 99 % decrease in the bacterial attachment against P. aeruginosa , Listeria monocytongenes and S. aureus . In a recent study, Gallarato et al. [31] explored the antibacterial properties of polyaniline (PANI) coating grafted from PET. The authors further microstructured PANI coated surfaces with direct laser interface. They have found that the water contact angle increased from 72 ° to 84 ° for PANI coated PET film and it further increased to 101 ° with additional laser treatment on the PANI coating. PANI film reduced the bacterial adhesion of P. aeruginosa by 74 %. Microstructure on PET–PANI film show to reduce the bacterial adhesion by 97 %. The author also showed that the percentage of live bacteria was CHAPTER 2 - Exploring the potential of polyethylene terephthalate in design of anti-adhesive materials 44 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools lower in modified surfaces and the live bacteria ratio was lower compared to dead bacteria on modified surfaces which proves its bactericidal effect. A multifunctional coating where zwitterionic polymer brushes of polycarboxybetaine (PC) and polysulfobetaine (PS) formed on PDMAEMA (poly(2-(dimethylamino)ethyl methacrylate)) grafted PET sheets were developed by Jin and collaborators [32]. Both cationic killing behaviour from PDMAEMA and zwitterionic repelling behaviour from PS and PC have been obtained. Water contact angles of 30.4 ° and 30.6 ° with a polymerization time of 8 h have been obtained for PS and PC modified samples. A significant reduction of E. coli attachment to PC formed PDMAEMA grafted PET sheets was observed [33]. Further improvements in antibacterial properties can be also performed with additional functionalisation of grafted samples. Arslan et al. [34] studied the antibacterial effects of amine, chlorine, hydrogen peroxide, and triclosan functionalisation of grafted vinyl monomer on PET fibres. They have found that co-polymerisation of vinyl monomers improved antibacterial properties and further functionalisation with triclosan showed the highest growth inhibition zone in all samples. The most promising vinyl grafting type with the bacterial inhibition was found to be the co-polymerisation of 4vinylpyridine which also gave higher inhibition zone compared to its oxidized or chlorine forms. 2.2.3. Surface topography modification As previously mentioned, surface roughness and topography greatly affect bacterial adhesion [2]. In addition, it has been shown that the surface roughness can affect the surface hydrophilicity. Surface wetting can either be homogeneous or heterogeneous, impacting differently the bacterial adhesion. The Wenzel’s phenomenon suggests that both hydrophilicity and hydrophobicity are enhanced by an increasing roughness on homogenously wetted surfaces, meaning that a hydrophilic surface will become more hydrophilic, and a hydrophobic surface will become more hydrophobic [35]. The porous surfaces behave according to another phenomenon so-called the Cassie–Baxter phenomenon. In this state, the water droplet heterogeneously wet the surface and affect the wettability [36]. Gillett and collaborators [37] studied the effect of laser modification on surfaces, creating pit structures with 15 µm in diameter and 20 µm gap between each other on PET surfaces. The laser modification increased the roughness more than 30 times, from Ra = 0.81 µm to 30.1 µm. The surface modification has also affected the water contact angle, increasing it from 76.9 ° to 87.7 °. Moreover, the modification was found to affect the E. coli distribution on the surface. Although more mature bacteria seem to accumulate around pits, there were no bacteria observed inside the pits. The authors CHAPTER 2 - Exploring the potential of polyethylene terephthalate in design of anti-adhesive materials 45 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools suggested that this could be due to the presence of air pockets inside the pits following the Cassie– Baxter state. It should be noted that Gram-negative bacteria have an extra outer membrane which can ease the interaction with nano-irregularities [38]. Lithography is another method that can be used to modify the surface topography as an alternative to the laser surface modification. It has been reported that the size of the micropatterns can be arranged to prevent microorganisms to adhere and create biofilm onto the materials. Arisoy and coworkers [39] combined nanoimprinted shark skin pattern samples with 1.6 and 3 µm height, 1.3 and 2 µm width, 2.7 and 2 µm spacing by lithography with bactericidal effect of TiO2 on PET substrates. When shark skin patterned TiO2 samples were compared to smoother surfaces with the same chemistry, 70 % reduction of the E. coli adhesion was observed. In addition, shark skin patterns led to 80 % reduction in the bacterial adhesion as compared to flat PET surfaces. Moreover, it was shown that if the spacing is bigger than the width of the bacteria, then bacteria tend to adhere onto the surface between patterns rather than being repelled by them. Wang et al. [40] also evaluated the E. coli adhesion on micropatterned PET surfaces obtained by quartz photomask for six different pattern dimensions. The results showed that the shape of the microstructures affect the adhesion of cells and the live/dead cell ratio. In addition, the authors found the minimum adhesion with the smallest micropattern design (i.e., 1 µm). 2.2.4. Coating In the recent years, the layer-by-layer (LbL) methodology has been widely used. Within this method, the cationic and anionic polyelectrolyte layers can be bond through ionic bonds to form a thin coating film. Alvarez et al. [4] showed that the surface of a PET film could be successfully coated by positively charged chitosan and negatively charged hyaluronic acid to create a potentially antifouling surface. Chitosan has contact killing properties, while hyaluronic acid is hydrophilic, and it can repel bacteria due to a steric effect formed by water absorption. This type of coatings can have a nanometre scale thickness. Gallego et al. [5] used a similar method to coat the PET surface with chitosan and hyaluronic acid, obtaining coating thicknesses ranging from 45 to 385 nm depending on the number of bilayers (from 5 to 10). The water contact angle of the PET film was 77 ° and decreased to 54 ° after the deposition of the first bilayer. However, the contact angle has not shown any specific trend and varied between 54 ° and 77 ° with the further deposition of bilayers. The authors also observed a reduction of bacterial adhesion against E. coli with almost complete bacterial inhibition for ten layers of HA/CHI. However, it is important to bear in mind that the coating degradation is a problem when using CHAPTER 2 - Exploring the potential of polyethylene terephthalate in design of anti-adhesive materials 46 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools the layer-by-layer surface modification. Indeed, the authors reported that 50 % of the coating was degraded in the first 24 h and 90 % during the first 6 days when exposed to enzymes. This value was 18 % when enzymes were absent and remained stable for more than a month. Further incorporation of triclosan (TRI) and rifampicin (RIF) antibiotics into HA/CHI layer has been done and further antibacterial improvements have been observed. While the reduction in bacterial adhesion was found to be 80 % for 5 bilayers of HA/CHI layers, it was found to be even higher > 99 % for TRI and RIF incorporated HA/CHI layers [13]. An improvement of the material’s chemical and physical stability was attempted by Park et al. [6] by cross-linking the carboxymethylcellulose (CMC) polysaccharide and chitosan LbLassembled multilayers on PETG (polyethylene terephthalate glycol modified) samples. The authors used a maximum of 20 bilayers of CMC/CHI to coat PETG and a thickness of 1818 nm was obtained, that was lower than 2 µm, which represents the thickness at which there is a risk of peeling. Moreover, the authors observed that when cross-linking was performed on 20 bilayers of CMC/CHI, the surface roughness increased from 20.3 to 57.7 nm. The cross-linking led to a super-hydrophilic surface exhibiting a reduction of the water contact angle from 35.34 ° to 4.86 ° (10 bilayers of CMC/CHI) and this change of the contact angle was thought to be due to surface roughness. Bacterial adhesion against Streptococcus mutans was reduced by 75 % using cross-linked samples as compared to control PETG. Another study showed that an abrasion resistant coating incorporating chitosan can also be developed using a sandblasting method. Wieckiewiz et al. [7] formed a chitosan film layer on PET surfaces using the sandblasting method. Prior to chitosan coating, the silica coated sands formed a tribo-chemically hydrophilic adhesive silicate layer by high impact, to improve the stability. In addition, fluoropolymers have been explored as antibacterial agents. Bao et al. [41] studied the use of 2,3,5,6-tetrafluoro-p-phenylenedimethanol (TFPDM) containing acrylate polymer blend (AF) and TFPDM sandwiched epoxy polymer structure (EF) to coat PET. While AF lowered the PET water contact angle from 60 ° to 41 ° and the roughness from 1.6 nm to 1.4 nm; EF lowered the water contact angle to 51 ° and the roughness to 1.3 nm. Both modified materials prevented the initial bacterial adhesion and biofilm formation by Bacillus subtilis and E. coli . However, AF performed slightly better reducing by 28 % the B. subtilis adhesion and by 69 % the biofilm formation, and by 89 % the E. coli adhesion and by 94 % its biofilm formation. It was also supported that the use of fluoride damages cell membrane by live/dead bacterial viability study. The combination of topographical modification and coatings has been reported to increase its effectiveness. For instance, Yamada et al. [42] coated a PET film with nanoscale moth-eye cone-shaped protrusions from a hydrophilic resin made of urethane acrylate and polyethylene glycol (PEG) derivatives CHAPTER 2 - Exploring the potential of polyethylene terephthalate in design of anti-adhesive materials 47 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools [43] with a size of approximately 200 nm in depth and diameter. Bacteria counts were reduced significantly with the use of moth-eye film compared to uncoated PET substrate due to specific structure of moth-eye film. It was also observed that the flat hydrophilic resin coated film reduced bacterial adhesion compared to uncoated PET film. This was attributed to bactericidal characteristics of PEG derivatives. 2.3. CONCLUSION AND OUTLOOK In this review, recent surface modification approaches for granting antibacterial properties on PET were analysed according to the main methodologies used, namely, functionalisation, grafting, surface topography modification, coating and their combinations. Current developments show that treated PET surface presents a significantly higher antibacterial activity than the pristine form. The most popular methodologies for imparting antibacterial activity to PET surface appear to be the grafting and coating methods. High durability of the surface modification and preservation of PET mechanical properties are particularly important properties for in vivo applications, where material experiences excessive wear and thus must always be considered when modifying PET surface. The studies also showed that coating and grafting methods can be advantageous for providing significantly higher hydrophilic surfaces, being the water contact angle one of the most used characterization methods. Low contact angles were found in literature (around 4 ° with the coating method and around 11 ° with the “grafting from” method) for treated polyethylene terephthalate. As stated by several authors, a surface with a high hydrophilicity behaviour (low contact angle) shows a great potential for developing anti-adhesive PET products for the medical sector. Further topographical surface modifications after grafting methods have been also used to improve the materials antibacterial properties. Furthermore, combining anti-adhesive properties with bactericidal properties is a popular strategy for achieving high antibacterial efficiencies for PET materials. However, one must consider the potential bacterial resistance, the cost–benefit ratio, the durability of the modification, as well as the possible toxic effects of the antibacterial agents on the environment when choosing the most suitable modification approach for the envisaged PET products. Moreover, it is also important to keep in mind that different types of bacteria react differently to the presence of bactericidal agents due to their different shape (spheres, rods or spirals) and outer membrane structure. Finally, the use of natural and biological molecules to improve the antibacterial properties of PET materials is a very promising approach to further develop new strategies against infectious diseases CHAPTER 2 - Exploring the potential of polyethylene terephthalate in design of anti-adhesive materials 48 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools 2.4. REFERENCES 1. C. Adlhart, J. Verran, N.F. Azevedo, H. Olmez, M.M. Keinänen-Toivola, I. 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In vitro antibacterial and cytotoxic activities of plasma- CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 57 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools statistical calculation, the variables were coded according to Eq. (1): 𝑥𝑖= (𝑋𝑖− 𝑋0) ∆𝑋𝑖 ⁄ (Eq. 3.1) where 𝑥𝑖 is the independent variable coded value, 𝑋𝑖 is the independent variable real value, 𝑋0 is the independent variable real value on the center point and ∆𝑋𝑖 is the step change value. The selected variables and their levels are listed in Table 3.1. Due to constraints in the independent variables curing time (B) and reaction time (D), the minimum level tested in the central composite design had to be adjusted. Minimum curing time and reaction time were set at 5 min (0.08 h), so the coded value changed from - √2 (-1.141) to -1.280 for curing time and from - √2 (-1.141) to -1.131 for the reaction time. State-Ease® ‘Design Expert’ (version 12) software was used for regression and graphical analyses of the data obtained. The statistical significance of the regression coefficients was determined by Student’s t-test, the second-order model equation was determined by Fischer’s test and the proportion of variance explained by the model obtained was given by the multiple coefficient of determination, R2. The optimum range of the variables were obtained by the graphical and numerical analysis using the ‘Design expert’ program, based on the criterion of desirability. 3.2.5. Water contact angle (WCA) The hydrophilicity of the modified surfaces was evaluated by static WCA measurement using the Optical Tensiometer Attension Model Theta Basic by Biolin Scientific, at room temperature. MiliQ water with a droplet size of 3 µL was used. The measurements were repeated at least 4 times for each sample at different locations. Contact angle measurements were also performed with diiodomethane. Owens-Wendt-Rabel&Kaelble Model (OWRK) equation was used to calculate polar and disperse components of surface energy by using contact angle values from water and diiodomethane. 3.2.6. Acid orange colorimetric method Acid orange coloration was used for the determination of the presence of amino groups on the substrate`s surface. The method used was adapted from Gallego et al. [21]. Samples with 2.5 X 2.5 cm were immersed in 6 mL of dye solution in acidic conditions (0.028 g/L, pH=3 adjusted with HCl 37 % in MiliQ water) and agitated at 100 rpm, at 40 °C. Following that, samples were removed and rinsed intensively with aqueous acidic solution (pH = 3) to remove unbound dye. The absorbed dye was CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 58 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools subsequently desorbed by immersing the films in an alkaline NaOH solution, at pH = 12 and then the absorbance of the final solution was measured in a Perkin Elmer Lambda 35 UV-VIS Spectrophotometer using an incident wavelength of 484 nm. 3.2.7. ATR-FTIR Fourier Transform Infrared Spectrophotometer (FTIR), Perkin Elmer Spectrum 100, with universal attenuated total reflectance (ATR) accessory with diamond crystal was used. The spectra were collected between the region of 4000 – 600 cm-1 wavenumbers with a 4 cm-1 resolution. 3.2.8. SEM The morphological analyses of the samples were performed using a Scanning Electron Microscope NanoSEM – FEI Nova 200 (FEG/SEM) with a secondary electron detector with 10 keV energy and a working distance between 7 and 8 mm. Before the morphological analysis, the samples were coated with a thin film of Au/Pd (80/20 by weight) 10 nm thick. For each sample, several images were taken by SEM, referring to different magnifications. 3.2.9. AFM A Keysight Technologies 5500 Atomic Force Microscope was used to perform the AFM measurements. The measurements were performed in air in tapping mode (Mac Mode) at room temperature. The silicone material cantilevers with a spring constant of 13-77 N/m and radius < 10 nm were used. The images were analysed by the Keysight PicoView and Gwyddion software. All images were taken at 5 X 5 µm surface area. 3.2.10. Bacterial adhesion 3.2.10.1. Bacterial strains E. coli BL21 (DE3) expressing green fluorescent protein (GFP) and an adhesive protein from Yersinia so-called Yersinia adhesin A (YadA), was used to test bacterial adhesion in the materials. The plasmid pRSFduet_GFP was kindly provided by Maria Sande (unpublished) and was obtained by amplification of GFP from pETduet_GFP(mut3b) [22]. The plasmid pASK_IBA2_YadA was kindly provided by Dirk Linke [23]. CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 59 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools 3.2.10.2. Preparation of bacteria E. coli BL21 DE3 (NZYTech, #MB006) harboring pRSFduet_GFP and pASK_IBA2_YadA, was cultivated at 37 °C, 200 rpm in LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) with the addition of kanamycin (50 µg/mL) and ampicillin (100 µg/mL). For the adhesion experiments, the cultures were grown at 37 °C and 200 rpm in LB (150 mL) up to an optical density at 600 nm (OD600 nm) of 0.6. Isopropyl β-D-thiogalactopyranoside (IPTG) and anhydrotetracycline (aTc) were added (final concentration of 1 mM and 100 ng/mL, respectively) to induce the heterologous protein expression (GFP and YadA). The cultures were then incubated for 5 h at 37 °C and 200 rpm. Next, the cells were harvested by centrifugation at 5000 rpm for 5 min and washed two times with 1 X PBS (phosphate buffer saline). Afterwards, the bacterial concentration was adjusted to an OD600 nm of 0.8 using 1 X PBS. 3.2.10.3. Bacterial adhesion test Prior to the test, modified and unmodified PET film samples were cut into 1 X 1.5 cm size rectangles. To ensure reproducibility of the results, three replicates of each sample were prepared. The samples were immersed into 1 % detergent solution and placed in an ultrasonic bath for 1 min, rinsed with water to remove contaminants and any possible previously attached microorganism. After that, samples were air dried in the flow chamber and UV treated for 30 min. Each sample was then placed into a falcon tube containing 14 mL of bacterial cells already prepared (OD600 nm of 0.8) and were incubated at 4 °C for 4 h in an orbital shaker at 12 rpm. After incubation, PET samples were removed and washed gently with 1 X PBS buffer to remove unattached bacteria. The bacteria adhered on PET surfaces were visualized using a fluorescence microscope (Olympus BX51), at 60X magnification, coupled with an DP71 digital camera. Fluorescence imaging was done with 470-490 nm excitation and a 520 nm filter in the microscope optical path. 3.3. RESULTS AND DISCUSSION 3.3.1. Water contact angle (WCA) analysis of PET control WCA gives an indication about the wettability of the surface. However, some environmental conditions like dirt [24], humidity [25] and temperature [26,27] can affect the WCA. Thus, it is normal to expect some variations among each measurement. An average WCA of 70 ° ± 5 ° was found for PET control, in close agreement with other values found in literature for pristine PET (e.g. 72 ° [28] or 77 ° CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 60 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools [21]). The values follow a normal distribution, showing that all the individual measurements fall within the control limits. A surface with a high hydrophilicity behavior (i.e., low contact angle) exhibits a great potential as an anti-adhesive surface thus highlighting the need to further functionalize PET films and one of the commonly used approaches is grafting method to immobilize natural substances including polysaccharides [29]. 3.3.2. Pre-treatment of PET substrate Aminolysis of PET involves a reaction of primary amines with the ester linkage of PET which results in the formation of an amide linkage and hydroxyl group by chain cleavage [20,30]. By using diamine, free primary amine ending groups on the surface of PET were obtained, that can be available for further reaction for gum Arabic attachment (Figure 3.1). Figure 3.1. Schematic representation of PET modification with gum Arabic. The aminolysed PET samples were evaluated by ATR-FTIR and the acid orange coloration method to characterize the presence of amine groups on the surface. The ATR-FTIR data in Figure 3.2 shows stretching of the amide groups at 1649 cm−1 (amide I, C=O stretching) and 1548 cm−1 (amide II, NH deformation) on PET sample [31,32]. Although there were no medium or strong bands representative of the stretching of amine groups between 3000 and 3600 cm−1, the broad weak peak observed between 3000 and 3600 cm−1 can be due to presence of exchangeable protons from hydroxyls and amines. However, with a penetration depth as low as 0.6 µm [31], ATR-FTIR lacks high sensitivity for the thin surface modifications. Therefore, it can be assumed that the existence of characteristic peaks in the ATR-FTIR spectrum indicates some degree of bulk material modification, which is also supported by the literature [21,33]. CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 61 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Figure 3.2. FTIR spectrum of unmodified PET (PET control) and diamine treated PET (PET amine). Additionally, the presence of free amines was also confirmed with a colorimetric method. Acid orange has affinity to primary amine groups in acidic conditions due to electrostatic interactions occurring between the dye and amine groups [34]. The dye treated PET samples can be seen in Figure 3.3. The aminolysed samples showed a higher amount of dye absorption, meaning that the aminolysed samples obtained orange color after the dye application, indicating the presence of primary amine groups, as found also in previous studies [21,34]. Figure 3.3. Acid orange colorimetric assay; a) unmodified PET sample (PET control), b) aminolysed PET sample (PET-amine), c) glutaraldehyde modified aminolysed PET sample (PET-GTA), d) absorption values from UV–vis spectroscopy for PET control, PET-amine and PET GTA. Further modification of the aminolysed surfaces was performed with glutaraldehyde. Glutaraldehyde is a well-known crosslinker. The possible crosslinking of glutaraldehyde with amine groups and gum Arabic is shown in Figure 3.1. It is expected that glutaraldehyde forms Schiff bases with the amine groups. Although Schiff bases can be considered unstable and the exact mechanism is still unknown [35], several reports have shown that the bonds created by glutaraldehyde can be stable CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 62 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools due to bulky hydrophobic part near to amine part of Schiff bases [36], or further reaction of the Schiff bases with glutaraldehyde [37]. It is also possible that the monomeric cyclic hemiacetal and multimeric cyclic hemiacetal forms of glutaraldehyde react with the amine groups on the surface since this form is more likely to be stable in acidic conditions [35]. In order to evaluate glutaraldehyde modification, glutaraldehyde treated samples were further characterized with acid orange colorimetric assay, whereas the low absorbance of the orange dye indicates the reduced amount of primary amine groups on glutaraldehyde treated samples as shown in Figure 3.1, thus confirming the bonding of glutaraldehyde on aminolysed PET surfaces. It was also observed that there was a slight yellow interference on the glutaraldehyde treated samples which may be due to the formation of Schiff bases [38-40]. Moreover, further characterization of the surfaces using water contact angle led to values of 64 ± 1 ° and 75 ± 6 ° for aminolysed and glutaraldehyde modified samples, respectively. 3.3.3. Statistical analysis of reaction parameters Organic reactions can be highly affected by the reaction parameters such as reaction time, reagents concentration and curing conditions. In a first step, screening experiments were conducted to identify the factors that influence PET grafting efficiency and to verify if any changes to their settings should be made to improve the process. The effects of different experimental variables on PET grafting process were simultaneously investigated, using a full factorial design experiment (Table 3.2, block 1). Four variables (curing temperature, curing time, gum Arabic concentration and contact time) were considered. The pre-treatment methods (glutaraldehyde modification and aminolysis) were performed for all the samples in the same conditions. The grafting process was then performed in aqueous solutions with 0.5 % 0.1 M HCl (pH values ranged between 3.4 and 4) at 40 °C, where the grafting parameters were varied according to the design planning. CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 63 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Table 3.2. Values for WCA after functionalization (OX), after 5 washing cycles (5X) and after 10 washing cycles (10X), according to the 24 factorial design (block 1) and to the central composite design (block 2), ordered by standard order. Assay Variables Responses Block Curing temperature (°C) Curing time (h) Gum Arabic concentration (%) Reaction time (h) Contact angle (0X) Contact angle (5X) Contact angle (10X) 1 1 80 0.5 1 1 49±7 49±6 53±6 2 1 120 0.5 1 1 41±4 41±6 44±4 3 1 80 3.5 1 1 37±6 41±5 45±3 4 1 120 3.5 1 1 27±5 34±9 34±6 5 1 80 0.5 5 1 55±2 52±5 55±5 6 1 120 0.5 5 1 49±4 49±5 50±2 7 1 80 3.5 5 1 58±2 50±8 58±3 8 1 120 3.5 5 1 21±3 29±5 37±1 9 1 80 0.5 1 15 39±3 46±7 48±4 10 1 120 0.5 1 15 42±4 39±3 40±3 11 1 80 3.5 1 15 46±8 58±4 53±6 12 1 120 3.5 1 15 25±4 32±3 33±8 13 1 80 0.5 5 15 57±6 52±3 59±4 14 1 120 0.5 5 15 35±5 54±4 51±2 15 1 80 3.5 5 15 45±6 32±3 29±3 16 1 120 3.5 5 15 24±3 42±3 57±3 17 1 100 2 3 8 46±8 45±6 53±5 18 1 100 2 3 8 42±5 49±4 54±3 19 1 100 2 3 8 43±3 57±4 59±3 20 2 71.7 2 3 8 50±1 61±7 55±4 21 2 128.3 2 3 8 24±3 32±2 30±5 22 2 100 0.08 3 8 43±3 55±4 53±4 23 2 100 4.12 3 8 25±5 31±3 46±5 24 2 100 2 0.17 8 50±5 50±6 46±4 25 2 100 2 5.83 8 31±4 46±8 48±4 26 2 100 2 3 0.08 38±3 39±4 47±2 27 2 100 2 3 17.9 38±6 44±2 48±3 28 2 100 2 3 8 37±5 51±5 44±3 29 2 100 2 3 8 48±8 52±8 47±9 The initial WCA and WCA after 5 and 10 washing cycles (one cycle of washing consisted of immersing the sample in distilled water at 40 °C, 100 rpm agitation for 30 min) were chosen as the response variables. At least four measurements were performed in different locations of the sample, being the average values and standard deviation presented as the final response. The experimental matrix and the results for the factorial design are shown at Table 3.2 (block 1). After the grafting reaction in the conditions set by the planning, the obtained PET grafted films were washed for 5X or 10X successively, to understand the resistance and stability of the produced gum Arabic coatings. After each washing cycle, the samples were removed and placed in a new container for another washing cycle. After 5 or 10 consecutive cycles, the samples were allowed to air dry and WCA was measured in the dried samples. CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 64 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools As can be observed in Table 3.2, the initial WCA after the functionalization process varied considerably (from 21 ° to 58 °), being this variation identical after washing (from 29 ° to 58 ° and to 59 °, after 5 and 10 washing cycles, respectively). However, higher average WCA were observed after washing (the mean WCA increased from 41 ° to 45 ° and 48 °, after 5 and 10 washing cycles, respectively). The increase in the WCA with washing was expected, due to the low stability of the coating in some assay conditions and due to the presence of unreacted components that are being removed during the washing process. However, comparing with PET control samples that have a mean WCA of 70 °, a significant decrease in the WCA was achieved after grafting and washing, being concluded that a stable grafting was achieved. Table 3.2 also shows that, independently of the other variables, increasing curing temperature from 80 °C to 120 °C resulted in a decrease of the WCA, and consequently, in a higher efficiency in the gum Arabic linkage. Finally, it is also possible to observe that lower WCA occurred when higher curing temperatures and times (more intense curing conditions) were used. Indeed, curing temperature (A) and curing time (B) have played a critical role in PET coating. According to the Student’s t-test results, the most important factors affecting the initial WCA were the curing temperature (A) and curing time (B), thus these factors were considered for building the linear model. Factor C, concentration, the first order interactions AB and AC and the fourth order interaction ABCD were also considered in the linear model, due to their t-test value (above t-value limit). Using the selected factors for building the linear model, the Fischer’s test was used to evaluate the model significance. The ANOVA statistical analysis for the initial WCA is summarized in Table 3.3. A model F-value of 28.15 implies the model is significant (p-value < 0.0001), i.e., there is only a 0.01 % chance that an F-value this large could occur due to noise. CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 65 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Table 3.3. Analysis of variance (ANOVA) for the linear model obtained for the initial WCA. Factors WCA (0X) Sum of Squares Df Mean Square Fvalue p-value Model 1996.0 6 332.67 28.15 < 0.0001 A 930.25 1 930.25 78.73 < 0.0001 B 441.00 1 441.00 37.32 < 0.0001 C 90.25 1 90.25 7.64 0.0172 AB 196.00 1 196.00 16.59 0.0015 AC 156.25 1 156.25 13.22 0.0034 ABCD 182.25 1 182.25 15.42 0.0020 Residual 141.79 12 11.82 Lack of fit 133.12 10 13.31 3.07 0.2705 Pure error 8.67 2 4.33 Cor Total 2137.79 18 The curvature and residual lack of fit were found to be insignificant, and the proportion of variance explained by the model, given by the multiple coefficient of determination, R2 was found to be 0.9337, highlighting the model adequacy to predict the WCA in the design space. The obtained final model equation for the initial WCA, in terms of coded factors is: 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑊𝐶𝐴 = 41.11 − 7.63𝐴 − 5.25𝐵 + 2.37𝐶 − 3.50𝐴𝐵 − 3.12𝐴𝐶 + 3.38𝐴𝐵𝐶𝐷 (Eq. 3.2) Figure 3.4 shows the 3D surface for the initial WCA, where a clear interaction effect between curing temperature and curing time is observed, thus suggesting lower WCAs values when these 2 factors are simultaneously maximized. CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 66 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Figure 3.4. 3D graphical visualization of the initial WCA with factors A (curing temperature) and B (curing time). Factors C (gum Arabic concentration) and D (contact time) were kept at the central point. The same statistical analysis was performed for the WCA after 5 and 10 washing cycles, being the Fischer’s test used for evaluating the model significance and the ANOVA statistical analysis summarized in Table 3.4. CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 73 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools exact mechanism of antimicrobial action is unknown, there are some studies suggesting that the presence of antimicrobial enzymes like oxidases, peroxidases and pectinases might be the reason for its antimicrobial activity. Some other studies highlight that the presence of the high amounts of salts may be the reason for the antimicrobial properties of gum Arabic [50,51], since glucuronic acid is found as calcium, magnesium and potassium salts in nature [52]. Furthermore, it was also seen that roughness obtained through modification did not promote the adhesion indicating that surface hydrophilicity can be more important at bacterial adhesion. Figure 3.9. Fluorescence microscopy images of Escherichia coli BL21 cells harboring pRSFduet_GFP and pASK_IBA2_YadA plasmids a) PET control and b) PET gum Arabic. Scale bar, 10 µm. 3.4. CONCLUSION AND OUTLOOK PET surface was modified with gum Arabic through treatments with diamines and glutaraldehyde. The use of WCA as a response for gum Arabic grafting onto PET films was used to find the optimum design parameters within the design of experiment range towards PET bacterial anti-adhesiveness. The parameters chosen were concentration, reaction time, curing temperature and curing time. It was found that curing temperature and curing time were the most significant parameters affecting the WCAs and their simultaneously maximization leads to a decrease of the WCA values. Using optimized conditions (1 % GA concentration, 1 h of reaction time, curing temperature of 120 °C and curing time of 3 h) an initial WCA of 27 ° was found. It was further shown that the treated samples were stable and resistant to several cycles of washings. Furthermore, the total surface energy and polar component of surface energy were found to be higher for treated samples, having increased from 49 mJ/m2 to 70 mJ/m2 and from 7 mJ/m2 to 29 mJ/m2, respectively. This corresponds to the requirements for obtaining a low bacterial adhesion on the surface. Finally, the bacterial adhesion susceptibility of treated and untreated CHAPTER 3 - Modification of PET surfaces with gum Arabic towards its bacterial anti-adhesiveness using an experimental factorial design approach 74 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools materials was evaluated against E. coli expressing YadA, an adhesive protein from Yersinia so-called Yersinia adhesin A. 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Sci. 2013;10: 1270–1279. https://doi.org/10.3844/ajassp.2013.1270.1279. 80 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools CHAPTER 4 Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces Enhancing the hydrophilicity of polymeric materials is an important step to achieve antiadhesiveness. Thus, in this study, atmospheric plasma as a pre-treatment was combined with UV grafting process to obtain durable surface modification on polyethylene terephthalate (PET). The most promising conditions for plasma process were found to be 100 % power and 4 m/s speed, leading to a contact angle reduction from 70 ± 6 ° to around 30 °. However, it was observed that these values increased over time due to ageing and washing, ultimately recovering to its initial value. Therefore, the plasma-pre-treated PET samples were further modified through an UV grafting process, using sodium acrylate (NaAc) and 3-Sulfopropyl Acrylate Potassium Salts (KAc). The grafted acrylate PET samples exhibited contact angles of 8 ± 3 ° and 28 ± 13 ° for NaAc and KAc, respectively, while showing durability to ageing and washing tests. Dry film thicknesses for both samples were found to be 28 ± 2 μm. Finally, the anti-adhesive properties of NaAc and KAc treated surfaces were evaluated using an Escherichia coli expressing YadA, an adhesive protein from Yersinia . The modified PET surfaces were highly effective in reducing bacterial adhesion by more than 90 %. Keywords: Grafting; Hydrophilicity; Surface modification; Wettability; Plasma; UV polymerisation This chapter is based on the following publication: Caykara T, Silva J, Fernandes S, Braga A, Rodrigues J, Rodrigues LR, Silva C (2022). Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces. (to be submitted) CHAPTER 4 - Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces 81 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools 4.1. INTRODUCTION The number of patients with infections increase every year. The European Centre for Disease Prevention and Control estimated that 8.9 million Healthcare-associated Infections (HAI) occur each year in European Hospitals and long-term facilities. Additionally, around 30 % of bacteria responsible for those infections were found to be resistant to antibiotics [1]. More than ever, it is extremely important to reduce the spread of infections. A potential way to reduce the spread of infections and antibiotic resistance is using anti-adhesive materials that can in turn reduce the microbial load on surfaces. A popular approach to obtain such anti-adhesive surfaces is to use hydrophilic surfaces [2], since it has been shown that hydrophilic surfaces can prevent bacterial adhesion by forming a hydration layer [3]. One attractive method to obtain such surfaces is plasma treatment. Plasma surface treatment can be applied on many different materials (metal, wood, paper, glass, polymer, ceramic, nonwoven textile, among others) and the medical sector is one of the main application fields [4]. Plasma systems are well known, and they can be used for a variety of treatments like surface cleaning, etching, functionalisation, activation and polymerisation since they change the chemical and physical properties of the surface [5,6]. The gas type used in the plasma system can determine the hydrophilicity and hydrophobicity of the treated surface [2], and hydrophilic surfaces can be obtained using gases such as air, oxygen, nitrogen, helium and argon [5,7]. Atmospheric air can be a viable option for plasma treatment as it is a natural, ecological, inexpensive and abundant gas mixture [8]. Additionally, since it is a dry surface treatment, using plasma eliminates the need to use harmful chemicals and consequently its effluents, hence leading to more sustainable processes [5]. Furthermore, the use of atmospheric plasma can eliminate the drawbacks from low-pressure plasma systems such as expensive vacuum systems, high maintenance costs, and being limited to batch processes and smaller area treatments. Although atmospheric plasma offers these advantages, it has been seldom studied compared to low-pressure plasma [8]. Moreover, UV-induced photopolymerisation is also an attractive technology for both industry and academy, as it is characterized by having a low environmental impact, a versatile technique, and high efficiency. In most cases, a photoinitiator is needed to create the necessary radicals for the initiation of the polymerisation reaction. However, there are some drawbacks with the use of photoinitiators like storage problems, unwanted odour and colour on final products, migration of remaining photoinitator molecules, health concerns and cost [9]. Thus, UV can be combined with plasma to generate radicals like peroxyl or hydroxyl on the surface [6], increasing the efficiency of the polymerisation reaction and CHAPTER 4 - Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces 82 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools removing the unnecessary use of photoinitiators. In this study, we used polyethylene terephthalate (PET) as the base substrate since it is one of the most popular polymers used in the medical context due to its hardness, stiffness, biocompatibility, biological, chemical and mechanical stability [2]. However, PET surfaces need to be modified to prevent/reduce bacterial adhesion. Charged polymer networks exhibit high uptake of water [10], and sodium acrylate (NaAc) and 3-sulfopropyl acrylate potassium salt (KAc) are both used in the preparation of charged polymers commonly used in hydrogel studies [10,11]. Furthermore, poly(sodium acrylate) is known as superabsorbent [12] material, being a good candidate to improve surface hydrophilicity. Thus, a combined process of plasma and UV photopolymerisation for PET modification was employed in this study, in order to render the PET surface hydrophilic. An initial statistical study for the determination of the optimal experimental parameters of the plasma treatment (speed and power) using air as reactive gas was performed. After that, atmospheric plasma treated samples were further subjected to UV polymerisation with NaAc and KAc. Finally, the bacterial anti-adhesive properties of the PET treated surfaces were assessed. 4.2. MATERIALS AND METHODS 4.2.1. Materials PET film (Mylar® A with thickness of 36 µm) was acquired from Isovolta Group. Sodium Acrylate (97 %) and diodomethane (99 %) were purchased from Sigma-Aldrich; 3-Sulfopropyl Acrylate Potassium Salt (>98 %) was purchased from TCI Chemicals. 4.2.2. Pre-treatment with atmospheric plasma A Dielectric Barrier Discharge (DBD) atmospheric pressure plasma was used as a sustainable pre-treatment for PET modification. The films were treated using the atmospheric pressure plasma equipment (Sigma Technologies International – Atmospheric Plasma Treatment System), at room temperature and relative humidity around 45 – 55 %. Air was used as reactive gas. The PET film samples passed between two metallic electrodes, at a power that varied from 15 kW (100 % of maximum power) to 7.5 kW (50 % of maximum power) and at a conveyor speed that varied from 4 m/min (1.1 Hz) to 12 m/min (3.3 Hz). CHAPTER 4 - Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces 89 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools oxygen increase) were set as 30 %. Figure 4.2 shows the overlay plot of factor Power (B) versus Speed (A) for the responses fulfilling these criteria (yellow area), simultaneously. Observing the yellow shaded area, it is possible to conclude that using combined plasma power above 80 % and treatment speed lower than 6 m/min would enable achieving the desired conditions. As such, the study was continued, setting the plasma pre-treatment conditions at the highest power (100 %) and lowest speed (4 m/min), before the UV grafting process. Figure 4.2. Overlay plot of factor Power (B) versus Speed (A). To reveal the chemical composition of the samples, XRay photoemission was used to reveal the chemical composition of the samples. In Table 4.4, the detailed information on carbon and oxygen bonds are listed. The peaks at 285.00, 286.58, 288.99 and 291.53 eV correspond to bonds typical of a benzene ring (C–C, C=C), methylene carbon singly bound to oxygen (–C–O–) and ester carbon atoms (O–C=O), respectively. The peaks at 531.93 and 533.53 eV indicate the carbonyl oxygen (O=C) and singly bonded oxygen atoms in the ester groups (O–C). The chemical changes in treated samples indicated by XPS analysis mainly show a decrease in C=C/C–C and an increase in –C–O–bonds, which is consistent with the expected replacement of hydrogen of aromatic rings by hydroxyl groups [21]. CHAPTER 4 - Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces 90 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Table 4.4. Relative composition (in percentage) of the different chemical groups detected with the deconvolution of C 1s and O 1s spectra for plasma treated samples. C 1s O 1s O/C ratio Peaks deconvolution C1 C2 C3 C4 O1 O2 Functional groups C-C, C=C -C-OO-C=O -C=C O=C O-C Binding energy (eV) 285,00 286,58 288,99 291,53 531,93 533,53 Untreated PET 44.96 15.18 13.12 2.28 11.08 13.03 0.32 PET with 50% power 4m/min speed plasma treatment 37.33 14.25 14.16 3.17 12.74 17.99 0.45 PET with 50% power 12 m/min speed plasma treatment 39.48 15.94 14.18 2.34 10.69 16.12 0.37 PET with 100% power 4 m/min speed plasma treatment 35.29 15.94 14.3 2.27 15.99 15.52 0.46 PET with 100% power 12 m/min speed plasma treatment 38 16.56 14.06 1.95 12.43 16.49 0.41 PET film 75% power 8 m/min speed plasma treatment 43.46 15.09 12.78 1.59 12.93 14.3 0.38 The chemical composition of PET revealed an increase in the O 1s/C 1s ratio from 0.32 to around 0.40 after plasma treatment (Table 4.4), with the highest ratio of 0.46 found for the PET sample treated at 100 % power and with a speed of 4 m/min. The changes in chemical composition with the introduction of these new chemical groups resulted in an increase of hydrophilicity of the polymer surface, as indicated by the results of WCA measurement, which showed a decrease in WCA from 70 ± 6 ° for PET control to less than 30 ° for PET-plasma treated samples. The plasma treatment can also be used to etch away surface molecules and modify the surface topography by increasing roughness, when enough energy is applied [18]. In this way, the adhesion of a coating can be improved by increasing the contact points between the coating and the polymer surface CHAPTER 4 - Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces 91 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools [6]. The plasma treated, and untreated PET samples were further subjected to AFM analysis to verify if morphological changes occurred in the PET surface topography (Figure 4.3). As can be seen from Figure 4.3, untreated PET surface is slightly smoother (with Ra = 3 nm) when compared with the plasma treated samples (Ra values ranging from 4 to 7 nm). Figure 4.3. Surface topography images from AFM: a) PET Untreated, Plasma treated samples with b) 50 % power and 4 m/min speed, c) 100 % power 4 m/min speed, d) 50 % power 12 m/min speed, e) 100 % power 12 m/min speed, f) 75 % power 8 m/min speed. 4.3.2. Grafting of PET with acrylates Although plasma treatment was effective at generating local hydrophilicity in PET, the treatment was not permanent and there was a recovery of the surface hydrophobicity over time. To obtain a hydrophilic surface with higher stability, the surfaces can be further modified with acrylates through the formation of active agents' species like peroxides and hydroperoxides to initiate the grafting process [18]. A proposed functionalisation mechanism with plasma treatment and grafting by UV photopolymerisation can be seen in Figure 4.4. From the plasma treatment optimisation experiments, it was seen that the assay performed at the highest power and lowest speed provided the most effective modification. Even though it was not possible to identify the peroxide functional groups generated in this study, several authors have reported an increasing similar trend with increased plasma power and time [18,22]. Thus, the lowest speed (4 m/min) and highest power (100 %) were used to treat the samples with plasma prior to the grafting of sodium acrylate (NaAc) and 3-Sulfopropyl Acrylate Potassium Salt (KAc). It is also important to note according to our experimental study, the plasma modification CHAPTER 4 - Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces 92 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools procedure with 3 number of passes was shown to be giving stable results for UV photopolymerisation, thus the procedure with 3 number of passes was adopted for further UV polymerisation. Figure 4.4. Proposed mechanism for atmospheric plasma treatment and UV grafting polymerisation of acrylates on PET surface. The plasma treated samples were further modified with acrylates under UV light which resulted in a significative WCA reduction, namely to 8 ± 3 ° (modification with NaAc) making it highly hydrophilic, and to 28 ± 13 ° (modification with KAc) (Table 4.5). The ageing tests showed that WCA increased to 14 ± 4 °, 17 ± 9 ° and 16 ± 8 ° after 1, 2 and 3 weeks of storage for the NaAc grafted sample, respectively, and it remained around 28–30 ° for the KAc grafted sample. The washing tests for the NaAc grafted sample suggest that the UV grafting procedure provided stability and durability to surface treatment. However, the hydrophilicity of the KAc treated sample after 10 washing cycles showed a strong variation in the WCA result, as can be seen in Table 4.5, indicating that this acrylate might lead to a lower treatment uniformity. Additionally, total surface energy of the samples increased from 49 ± 4 mJ/m2 to 75 ± 1 mJ/m2 for NaAc, and to 66 ± 7 mJ/m2 for KAc grafted samples, due to high increase in the polar component of surface energy (to 38 ± 2 mJ/m2 and 33 ± 7 mJ/m2 for NaAc and KAc modified surfaces, respectively). In order to confirm the effect of plasma pre-treatment in the acrylates grafting enhancement, the same UV grafting process was performed without any prior plasma pre-treatment, No-plasma NaAc grafted PET and No-plasma KAc grafted PET (Table 4.5). Under these conditions, the values found for the WCA were significantly higher for these two samples, thus indicating the positive effect of plasma pre-treatment in the surface modification of PET for preparation to the UV polymerisation procedure with acrylates. CHAPTER 4 - Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces 93 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Table 4.5. WCA, surface energy and roughness values of untreated, NaAc treated and KAc treated PET surfaces. Samples Initial WCA (°) WCA After washing 5 cycles (°) WCA After washing 10 cycles (°) WCA After 1 week (°) WCA After 2 weeks (°) WCA After 3 weeks (°) Diiodomethane contact angle (°) SFE Disperse (mJ/m2) SFE Polar (mJ/m2) SFE Total (mJ/m2) Ra (nm) PET Untreated 70 ± 6 N/A N/A N/A N/A N/A 34 ± 7 42 ± 3 7 ± 3 49 ± 4 2.7 ± 0.9 NaAc grafted PET 8 ± 3 8±3 14 ± 9 14 ± 4 17 ± 9 16 ± 8 44 ± 5 37 ± 3 38 ± 2 75 ± 1 3 ± 1 KAc grafted PET 28 ± 13 33 ± 10 55 ± 27 30 ± 11 30 ± 8 28 ± 10 52 ± 5 33 ± 3 33 ± 7 66 ± 7 2.4 ± 0.3 No plasmaNaAc grafted PET 39 ± 25 54 ± 12 58 ± 10 No plasmaKAc grafted PET 23 ± 18 58 ± 16 77 ± 7 CHAPTER 4 - Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces 94 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Figure 4.5 shows that the surface topography, as visualized by AFM analysis, of the plasma pretreated surface has a rougher surface with Ra of 10 ± 2 nm and the structures resembled nanoparticle modified surfaces. Moreover, the surface topography of NaAc and KAc treated surfaces showed a smoother structure, with roughness values 2.62 nm for NaAc and 2.35 nm for KAc like untreated PET sample. The difference in surface nanostructure also corroborates the successful surface modification via photopolymerisation. The unfocused surface appearance of NaAc and KAc treated surfaces indicates that the AFM tip was not effective at displacing polymer brushes and the polymer brushes concealed the surface topography [23]. Figure 4.5. Surface topography images obtained by AFM of a) Plasma treated PET with 100 % power, 4 m/min speed, b) NaAc grafted PET, c) KAc grafted PET. In addition to the structural study, the thickness of the polymeric brushes was measured by UV spectrophotometer using Swanepool method and was found to be 28 ± 2 μm for both NaAc and Kac grafted PET surfaces. This value is thicker than the conventional grafting thickness reported in the literature, and it can expand the uses of this type of polymer brushes [24,25]. 4.3.3. PET anti-adhesiveness Finally, bacterial anti-adhesion studies were performed using E. coli expressing YadA which is an adhesin from Y. entrocolitica [17]. Bacterial adhesins like YadA are known to promote bacterial adhesion, biofilm formation, and provide resistance against bactericidal compounds [17,26,27]. Adhesins mature the bonding between bacteria and the substrate leading to irreversible adhesion [26] thus, it is important to study bacteria with such highly bonding adhesins. The bacterial adhesion onto the untreated PET and NaAc and KAc treated PET samples can be seen in Figure 4.6. It is evident that the NaAc and KAc treated surfaces successfully prevented bacterial adhesion as compared to untreated PET surface. In order to quantify the effect of these modifications on bacterial adhesion, data gathered in Figure 4.6 was further analysed using the ImageJ® software. The total number of cells attached to CHAPTER 4 - Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces 95 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools the surfaces were counted and used to calculate the reduction in bacterial adhesion. The bacterial adhesion was reduced by 90.8 % in NaAc grafted PET and more than 99.9 % in KAc graft PET surfaces. The anti-adhesive behaviour of these type of surfaces are thought to be due to higher hydrophilicity and surface energy [7,15]. In this way, a hydration layer is formed on the top of the surface, making it hard for bacteria to approach [28]. According to a recent review, this reduction can be considered highly effective compared to other modifications on PET substrate [2]. Figure 4.6. Fluorescence microscopy images of Escherichia coli BL21 cells harboring pRSFduet_GFP and pASK_IBA2_YadA plasmids a) untreated PET sample b) NaAc treated PET Sample and c) KAc treated PET sample. 4.4. CONCLUSION AND OUTLOOK Plasma and UV treatments are cost-effective, sustainable and environmentally friendly processes, and the combination of the two can avoid the use of the photoinitiator needed for UV polymerisation. Furthermore, the use of atmospheric plasma can increase the efficiency of an industrial applications. Statistical analysis on plasma treatment parameters showed that power and speed were significant variables influencing the surface WCA, and the highest power and lower speed should be used to increase the plasmatic effect on PET surface. Using these conditions, plasma pre-treated PET surfaces were successfully modified with NaAc and KAc, creating hydrophilic surfaces with WCA less than 10 ° for the NaAc grafted samples. The studies on samples washing and ageing showed that the NaAc treated samples hold a durable modification under the tested conditions, while KAc treated samples exhibit some surface heterogeneity. Finally, the bacterial anti-adhesive performance of treated surfaces was evaluated with E. coli expressing YadA and both modified surfaces were able to inhibit the bacterial adhesion by more than 90 %. The combination of these two technologies can bring several benefits to the modification of conventionally hydrophobic substrates, besides being easily industrialised. Furthermore, achieving surfaces with grafting thicknesses at the level of micrometres can have some other potential uses such CHAPTER 4 - Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate surfaces 96 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools as implants, reservoirs to release therapeutic agents, higher membrane selectivity and sensor selectivity and reduced friction. 4.5. REFERENCES 1. C. Suetens, K. Latour, T. Kärki, E. Ricchizzi, P. Kinross, M.L. Moro, B. Jans, S. Hopkins, S. Hansen, O. Lyytikäinen, J. Reilly, A. Deptula, W. Zingg, D. Plachouras, D.L. Monnet, Prevalence of healthcare-associated infections, estimated incidence and composite antimicrobial resistance index in acute care hospitals and long-term care facilities: Results from two european point prevalence surveys, 2016 to 2017, Euro Surveill. 2018;23: 1800516. https://doi.org/10.2807/1560-7917.ES.2018.23.46.1800516. 2. T. 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Mater. 2020;30: 2000936. https://doi.org/10.1002/adfm.202000936. CHAPTER 5 - Can superhydrophobic PET surfaces prevent bacterial adhesion? 105 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools promising approach that can be used to achieve superhydrophobic surfaces. The change in the surface’s energy due to the chemical modification was assessed by measuring samples WCA with water and diiodomethane. The obtained results showed that untreated PET surfaces exhibited a WCA of 69 ± 10 °, while hydrolysed surfaces possessed a WCA of 62 ± 6 °. The WCA value found after hydrolysis agreed with other studies [22,23]. For the NPs treated surfaces (PET NP), the measured WCA was 156 ± 12 °. In addition, this surface property was maintained after some ageing of the sample, when the WCA was found to be 157 ± 10 ° after 2 weeks and 151 ± 14 ° after 3 weeks of storage. Furthermore, the samples were washed with water and ethanol to evaluate the durability of the treatment under these conditions. After 10 cycles of washing with water, where each cycle consisted of immersing the sample in water for 30 min at 40 °C, 100 rpm, the WCA was 160 ± 9 °, and after 10 additional cycles of washing with ethanol the WCA was 157 ± 10 °, thus showing that the functionalized PET NP samples possessed a resistant superhydrophobic character under the tested conditions. Table 5.1. Contact angle values determined with water and diiodomethane, and surface free energy (SFE) of PET untreated, PET Hydrolysed and PET NP. Samples Water Contact Angle (°) Diiodomethane Contact Angle (°) SFE Disperse mJ/m2 SFE Polar mJ/m2 SFE Total mJ/m2 PET untreated 69 ± 10 35 ± 4 42.0 ± 1.8 7.2 ± 4.0 49.2 ± 4.2 PET hydrolysed 62 ± 6 35 ± 3 41.9 ± 1.5 10.8 ± 3.1 52.7 ± 3.7 PET NP 156 ± 12 114 ± 14 1.3 ± 1.2 3.3 ± 5.7 6.7 ± 4.3 5.3.3. Atomic force microscopy (AFM) and surface roughness It is well known that surface roughness is necessary to achieve superhydrophobic surfaces [1]. In Table 5.2, surface roughness values for untreated and treated PET determined by AFM technique are summarized. Untreated PET surfaces presented a smoother surface topography with a Ra value of 4.8 ± 1.0 nm when compared to hydrolysed PET (27.8 ± 2.0 nm) and to NPs treated PET (104 ± 20 nm). This last one was considerably rougher, being this an important aspect for the transitioning of the surfaces to the superhydrophobic state [33]. CHAPTER 5 - Can superhydrophobic PET surfaces prevent bacterial adhesion? 106 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Table 5.2. Surface roughness values (Ra) of treated and untreated PET samples determined by AFM. Samples Roughness, Ra (nm) PET untreated 5 ± 1 PET hydrolysed 28 ± 2 PET NP 104 ± 20 When the surfaces were visualized by AFM, the topography of hydrolysed PET was found to be an irregular granular structure with pits on the film surface, whereas the NPs modified PET presented the typical appearance of NP like structures (Figure 5.2). Interestingly, the hydrolysed surfaces also present small granular like structures on the surface intercalated with pits which are created from the etching process, and these can be better identified by SEM images (Figure 5.3, e)). Therefore, the topography of PET surfaces was further studied by SEM analysis to confirm the presence of these structures. Figure 5.2. AFM images of PET surfaces– a) PET untreated, b) PET hydrolysed, c) PET NP. 5.3.4. Scanning electron microscopy (SEM) / Energy dispersive spectroscopy (EDS) and dynamic light scattering (DLS) measurements SEM/EDS analysis was performed to characterize the surfaces morphology and chemistry. The morphological changes on both hydrolysed and NPs modified samples at different magnifications can be seen in Figure 5.3. Hydrolysed PET shows signs of etching, which was expected due to breakage of ester groups on the surface of the PET film into hydroxyl and carboxyl groups [22]. It is well known that etching of PET surface depends on the material characteristics like crystallinity and thickness. During hydrolysis, amorphous regions erode faster than crystalline regions leaving a non-uniform surface structure after short hydrolysing times [23]. The formed hydroxyl groups on the PET surface are the probable location where the covalent bonds with TEOS will be established, and that will lead to the subsequent formation of the NPs on the surface, which can be seen for instance in Figure 5.3 i). 1 CHAPTER 5 - Can superhydrophobic PET surfaces prevent bacterial adhesion? 107 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Figure 5.3. SEM images of untreated (a), b), c)), hydrolysed (d), e), f)) and NP modified (g), h), i)) PET samples, at different magnifications (1000 X, 5000 X and 50000 X respectively). Furthermore, the cross-section of PET NP (Figure 5.4) shows that polycondensation resulted in a clear layer of silica NPs with a thickness around 711 nm on the top of the PET substrate. The growth of the layer thickness is probably due to continuous polycondensation on the surface. In Figure 5.4, the NPs on the surface presented a heterogeneous dimension, with values that vary from 151 nm to 326 nm and with many smaller ones. CHAPTER 5 - Can superhydrophobic PET surfaces prevent bacterial adhesion? 108 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Figure 5.4. SEM images of PET NP modified surface a) top view b) cross-section view at a magnification of 50000 X. The NPs formed during the surface modification process and that remained in the solution, were also analyzed by DLS to determine their size and zeta potential. The average NP size in the solution was 366 ± 26 nm with a polydispersity index (PdI) of 0.27 ± 0.13. PdI is used to describe the particle size distribution [34] and a value lower than 0.1 is indicative of uniformity [34]. While a PdI between 0.03 and 0.06 indicates a monodisperse solution [35], a PdI higher than 0.5 suggests a wide range of particle size distribution [34]. The differences in the NPs size between the surface and the solution herein observed (Figure 5.4) might be due to growth of the layer on the surface and continuous polycondensation which is embedding some nanoparticles while allowing the formation of new ones. Moreover, the DLS analysis provided a zeta potential of – 34 ± 2 mV, indicating a stable NPs solution since a zeta potential around -30 mV can be considered a moderately stable solution [36]. In Figure 5.5, the PET surface morphology after 10 cycles of washing with water followed by 10 cycles of washing with ethanol can be observed. Although at lower magnification, washed and unwashed samples seem to have similar features, at higher magnifications it is possible to visualize a NPs free area in the NPs layer on the PET surface (Figure 5.5, b) which indicates that parts of the coating layer were removed from the surface due to washing. Additionally, by observing the sample cross -section, it can be observed that the layer thickness is lower after washing (Figure 5.5, c), which can be explained by the loss of the outermost NPs of the coating layer. It is also important to note that the sizes of the NPs seem to be smaller after washing. Although covalent bonding of the NPs coating layer to the PET surface was expected, it is possible that some NPs were only adhered or were weakly bonded to the surface, or even were only trapped in the network of bonds formed during CHAPTER 5 - Can superhydrophobic PET surfaces prevent bacterial adhesion? 109 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools polycondensation. Nevertheless, the removal of these fluoroalkylsilane functionalized NPs layer suggests that the treated surfaces have limited durability, even though they continue to provide superhydrophobic surface properties after the washing cycles performed with water and ethanol, as previously shown with WCA measurements. These results strengthen the importance of conducting additional testing such as abrasion when determining the durability of these of surface treatments. Figure 5.5. SEM images of PET NP modified surface after 10 cycles of water and ethanol washing: a) top view - 1000 X; b) top view - 50000 X and c) cross-section view - 50000 X. The treated and untreated samples were further analyzed by EDS to determine the chemical composition of the surfaces (Table 5.3). The elemental analysis of samples showed that untreated and hydrolysed surfaces were mainly constituted by carbon and oxygen atoms, while the NPs treated surfaces, including washed samples, also possessed silicon and fluorine in its composition, thus confirming the modification of the surfaces with TEOS and FAS13. Table 5.3. SEM-EDS results for atomic concentrations of untreated and treated PET samples. Samples / elements (% atomic) C O Si F PET untreated 86 14 n.d. n.d. PET hydrolysed 85 15 n.d. n.d. PET NP 62 28 8 2 PET NP after 10 cycles of washing with water and ethanol 62 27 7 4 n.d.: not detected CHAPTER 5 - Can superhydrophobic PET surfaces prevent bacterial adhesion? 110 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools 5.3.5. Bacterial adhesion tests Finally, the performance of the developed modified PET films was evaluated regarding its bacterial anti-adhesion properties. For this purpose, a heterologous E. coli expressing the adhesin YadA was used similarly to previous reports [26] and as described in the previous chapters. This protein was cloned into a plasmid containing a reporter protein (GFP) to enable its visualization through fluorescence microscopy. Therefore, it was possible to observe the cells binding to the tested materials. The anti-adhesive mechanism of superhydrophobic surfaces depends on the formation of air pockets on the surface that impair the contact between the bacteria and the surfaces. However, the entrapped air from the air pockets will eventually be replaced over time by the immersion of the solution, potentially leading to an increased adhesion [1,37]. Thus, the duration of the experimental setup to assess adhesion can be an important parameter. For this reason, the untreated and fluorinated-NPs treated samples were incubated with the bacterial solution for 1 h, 4 h and 6 h. Figure 5.6 illustrates the fluorescence microscopy images captured for the different adhesion time periods. Figure 5.6. Fluorescence microscopy images of PET samples after incubation with bacteria: a) PET untreated after 1 h; d) PET NP after 1 h; b) untreated PET after 4 h; e) PET NP after 4 h; c) untreated PET after 6 h; f) PET NP after 6h. Scale bar, 10 µm. CHAPTER 5 - Can superhydrophobic PET surfaces prevent bacterial adhesion? 111 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools From Figure 5.6, it seems that after incubation with bacteria for different time periods, the modified PET samples do not seem to show anti-adhesive properties, when compared to the untreated materials using the same incubation times. This result differs from other studies that reported a decreased bacterial adhesion when testing other superhydrophobic materials [38–40]. In our study, the PET modified materials were found to promote the bacterial adhesion over time. In order to better understand these results, the samples immersed in bacterial solution for 4 h were further characterized by SEM, where adhered bacteria on top of the treated surface can be observed (Figure 5.7). In Figure 5.7, c), bacteria seem to position itself in the irregularities where the surface area available for cell adhesion is high. This behavior has also been reported for other superhydrophobic materials where surface irregularities were also found to play a role in promoting bacterial adhesion [41]. In addition, the bacterial cell structure is known to affect the adhesion of E. coli that holds a thinner peptidoglycan layer, making the bacterial cell more flexible when adhering on rough surfaces [42]. Besides, in this study the E. coli is expressing the YadA adhesin which can be helpful when maturing the adhesion [43]. Furthermore, E. coli can explore the local topography through flagella which can improve its adhesion on surfaces with microtopographies [44,45]. Moreover, although it was not evaluated in this study, it is possible that the surface wetting transition from Cassie-Baxter to Wenzel state have been displacing air pockets and causing an increased adhesion [46]. Thus, altogether these factors that confer the bacterial cell the ability to fit irregularities in the modified surface, can explain the increased adhesion that was herein observed. Also, microtopography and nanotopograhy can be considered crucial factors at governing bacterial adhesion in this study. Figure 5.7. SEM visualization of NPs treated samples with different magnifications a) 5 000 X b) 10 000 X and c) 50 000 X, after the bacterial adhesion test (4 h). CHAPTER 5 - Can superhydrophobic PET surfaces prevent bacterial adhesion? 112 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools 5.4. CONCLUSION AND OUTLOOK Superhydrophobic materials have been shown to possess bacterial repellent properties by several authors. In situ growth of NPs on the PET surfaces was studied to create a durable superhydrophobic surface. The WCA of treated surfaces showed a significant increase (226 %) to a value of 156 ± 12 °, when compared to untreated PET (69 ± 10 °). Surface roughness increased to 104 ± 21 nm from the initial 5 ± 1 nm value. This surface modification was further characterised by SEM/EDS showing that the surfaces were properly modified with NPs. Although the samples washing with water and ethanol removed some of the bounded NPs from the material surface, the WCA was still high after the performed washing cycles with water (160 ± 9 °) and additional washing cycles with ethanol (157 ± 10 °), hence indicating that the superhydrophobicity of the surface was maintained in the tested conditions. These results demonstrated that the functionalized surfaces have some degree of durability. Furthermore, bacterial adhesion onto the treated surfaces was evaluated and it was shown that these surfaces promoted bacterial adhesion, contrarily to other reports. This could be explained by the increased bacterial contact within the surface microtopographies, suggesting that surface topography and roughness are crucial factors at determining the bacterial adhesion outcome of a given material. 5.5. REFERENCES 1. X. Zhang, L. Wang, E. Levänen, Superhydrophobic surfaces for the reduction of bacterial adhesion. RSC Adv. 2013;3: 12003–12020. https://doi.org/10.1039/c3ra40497h. 2. T. Çaykara, M.G. Sande, N. Azoia, L.R. Rodrigues, C.J. Silva, Exploring the potential of polyethylene terephthalate in the design of antibacterial surfaces. Med. Microbiol. Immunol. 2020;209: 363–372. https://doi.org/10.1007/s00430-020-00660-8. 3. L. Liu, H. Shi, H. Yu, S. Yan, S. Luan, The recent advances in surface antibacterial strategies for biomedical catheters. Biomater. Sci. 2020;8: 4095–4108. https://doi.org/10.1039/d0bm00659a. 4. Q. Zhao, S. Wang, H. Müller-Steinhagen, Tailored surface free energy of membrane diffusers to minimize microbial adhesion. Appl. Surf. Sci. 2004;230: 371–378. https://doi.org/10.1016/j.apsusc.2004.02.052. 5. C. Neinhuis, W. Barthlott, Characterization and distribution of water-repellent, self-cleaning plant surfaces. Ann. Bot. 1997;79: 667–677. https://doi.org/10.1006/anbo.1997.0400. CHAPTER 5 - Can superhydrophobic PET surfaces prevent bacterial adhesion? 113 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools 6. L. Feng, S. Li, Y. Li, H. Li, L. Zhang, J. Zhai, Y. Song, B. Liu, L. Jiang, D. Zhu, Superhydrophobic surfaces: From natural to artificial. Adv. Mater. 2002;14: 1857–1860. https://doi.org/10.1002/adma.200290020. 7. G. Wen, Z. Guo, W. Liu, Biomimetic polymeric superhydrophobic surfaces and nanostructures: From fabrication to applications. Nanoscale. 2017;9: 3338–3366. https://doi.org/10.1039/c7nr00096k. 8. M. Liravi, H. Pakzad, A. Moosavi, A. Nouri-Borujerdi, A comprehensive review on recent advances in superhydrophobic surfaces and their applications for drag reduction. Prog. Org. Coatings. 2020;140: 105537. https://doi.org/10.1016/j.porgcoat.2019.105537. 9. S. Czyzyk, A. Dotan, H. Dodiuk, S. Kenig, Processing effects on the kinetics morphology and properties of hybrid solgel superhydrophobic coatings. Prog. Org. Coatings. 2020;140: 105501. https://doi.org/10.1016/j.porgcoat.2019.105501. 10. Y. Zhang, L. Chen, Z. Lin, L. Ding, X. Zhang, R. Dai, Q. Yan, X. Wang, Highly Sensitive Dissolved Oxygen Sensor with a Sustainable Antifouling, Antiabrasion, and Self-Cleaning Superhydrophobic Surface. ACS Omega. 2019;4: 1715–1721. https://doi.org/10.1021/acsomega.8b02464. 11. B. Gayani, A. Dilhari, N. Kottegoda, D.R. Ratnaweera, M.M. Weerasekera, Reduced Crystalline Biofilm Formation on Superhydrophobic Silicone Urinary Catheter Materials. ACS Omega. 2021;6: 11488–11496. https://doi.org/10.1021/acsomega.1c00560. 12. S. Zhang, L. Wang, X. Liang, J. Vorstius, R. Keatch, G. Corner, G. Nabi, F. Davidson, G.M. Gadd, Q. Zhao, Enhanced Antibacterial and Antiadhesive Activities of Silver-PTFE Nanocomposite Coating for Urinary Catheters. ACS Biomater. Sci. Eng. 2019;5: 2804–2814. https://doi.org/10.1021/acsbiomaterials.9b00071. 13. H. Qian, M. Li, Z. Li, Y. Lou, L. Huang, D. Zhang, D. Xu, C. Du, L. Lu, J. Gao, Mussel-inspired superhydrophobic surfaces with enhanced corrosion resistance and dual-action antibacterial properties. Mater. Sci. Eng. C. 2017;80: 566–577. https://doi.org/10.1016/j.msec.2017.07.002. 14. Y. Li, J. John, K.W. Kolewe, J.D. Schiffman, K.R. Carter, Scaling Up Nature: Large Area Flexible Biomimetic Surfaces. ACS Appl. Mater. Interfaces. 2015;7: 23439–23444. https://doi.org/10.1021/acsami.5b04957. CHAPTER 5 - Can superhydrophobic PET surfaces prevent bacterial adhesion? 114 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools 15. Y. Zhao, T. Xu, J.-M. Hu, A robust , room-temperature curable and molecular-level superhydrophobic coating with excellent antibacterial and antifouling properties. Chem. Eng. J. 2022;450: 136557. https://doi.org/10.1016/j.cej.2022.136557. 16. K. Han, T.Y. Park, K. Yong, H.J. Cha, Combinational Biomimicking of Lotus Leaf, Mussel, and Sandcastle Worm for Robust Superhydrophobic Surfaces with Biomedical Multifunctionality: Antithrombotic, Antibiofouling, and Tissue Closure Capabilities. ACS Appl. Mater. Interfaces. 2019;11: 9777–9785. https://doi.org/10.1021/acsami.8b21122. 17. J. Li, H. Ding, H. Zhang, C. Guo, X. Hong, L. Sun, F. Ding, Superhydrophobic Methylated Silica Sol for Effective. Materials (Basel). 2020;13: 842. https://doi.org/doi:10.3390/ma13040842. 18. X. Chen, Y. Chen, T. Jin, L. He, Y. Zeng, Q. Ma, N. Li, Fabrication of superhydrophobic coating from non-fluorine siloxanes via a one-pot sol–gel method. J. Mater. Sci. 2018;53: 11253– 11264. https://doi.org/10.1007/s10853-018-2348-7. 19. F. Zhou, Y. Zhang, D. Zhang, Z. Zhang, F. Fu, X. Zhang, Y. Yang, H. Lin, Y. Chen, Fabrication of robust and self-healing superhydrophobic PET fabrics based on profiled fiber structure. Colloids Surfaces A Physicochem. Eng. Asp. 2021;609: 125686. https://doi.org/10.1016/j.colsurfa.2020.125686. 20. B.J. Sparks, E.F.T. Hoff, L. Xiong, J.T. Goetz, D.L. Patton, Superhydrophobic hybrid inorganicorganic thiol-ene surfaces fabricated via spray-deposition and photopolymerization. ACS Appl. Mater. Interfaces. 2013;5: 1811–1817. https://doi.org/10.1021/am303165e. 21. Y. Hu, X. Ma, H. Bi, J. Sun, Robust superhydrophobic surfaces fabricated by self-growth of TiO2 particles on cured silicone rubber. Colloids Surfaces A Physicochem. Eng. Asp. 2020;603: 125227. https://doi.org/10.1016/j.colsurfa.2020.125227. 22. Y. Liu, T. He, C. Gao, Surface modification of poly(ethylene terephthalate) via hydrolysis and layer-by-layer assembly of chitosan and chondroitin sulfate to construct cytocompatible layer for human endothelial cells. Colloids Surfaces B Biointerfaces. 2005;46: 117–126. https://doi.org/10.1016/j.colsurfb.2005.09.005. 23. L. Pérez-Álvarez, E. Lizundia, S. del Hoyo, A. Sagasti, L.R. Rubio, J.L. Vilas, Polysaccharide polyelectrolyte multilayer coating on poly(ethylene terephthalate). Polym. Int. 2016;65: 915– 920. https://doi.org/10.1002/pi.5116. 24. X. Chen, Y. Liu, H. Lu, H. Yang, X. Zhou, J.H. Xin, In-situ growth of silica nanoparticles on cellulose and application of hierarchical structure in biomimetic hydrophobicity. Cellulose. 2010;17: 1103–1113. https://doi.org/10.1007/s10570-010-9445-3. CHAPTER 6 - Preliminary Application as Urinary Catheters 121 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Figure 6.1. Experimental setup for the dynamic urinary assessment. Finally, the treated samples (5 samples for each different modification) were placed in the tubes and the artificial urine was continuously circulated through the tubes for 30 days to evaluate their performance. The testing was performed in HydruMedicals facilities (Guimaraes, Portugal). 6.2.4. Scanning electron microscopy (SEM) The morphological and chemical analyses of the samples were performed at the Materials Characterization Services Laboratory of the University of Minho (SEMAT-UM), using a Scanning Electron Microscopy (SEM) NanoSEM - FEI Nova 200 (FEG/SEM). Before the morphological analysis, the samples were coated with a thin film of Au/Pd (80/20 by weight) with 10 nm thick. For the morphological analysis, a secondary electron detector with 10 KeV energy and a working distance between 7 and 8 mm, was used. For each sample, several groups of images were taken by SEM, referring to different magnifications. For the chemical analysis, energy dispersive spectroscopy (EDS) technique was performed by an Integrated System EDAX - Pegasus X4M, using 15 KeV energy, at a working distance of approximately 6.5 mm. 6.3. RESULTS AND DISCUSSION The urinary catheters can be used in two different ways, (1) short-term usage, that typically last up to 7 days, (2) long-term usage for 28 days or longer [1]. The studies conducted with the modified PET samples were assessed for the long-term usage, as it would be more advantageous to have a more resistant material. It is also important to note that there are two different untreated PET samples used CHAPTER 6 - Preliminary Application as Urinary Catheters 122 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools in this study, as thicker PET films were needed for the nanoparticles modification process (Chapter 5). Figure 6.2 and Figure 6.3 shows the material surface before the urinary solution performance test. The SEM images before urinary tests revealed that no significant differences were found between the untreated PET sample as compared to the hydrophilic treated PET samples shown in Figure 6.2. However, as it can be seen in Figure 6.3, the nanoparticle treated sample has shown to have a different structure which is discussed in Chapter 5. Further SEM images comparisons after artificial urine performance tests are illustrated in Figure 6.4 and Figure 6.5. All surface modifications were found to promote crystal formation compared to the untreated PET samples. Figure 6.2. SEM analysis of PET samples before exposure to artificial urine using the dynamic urine testing setup; a) untreated PET, b) PET - GA, e) PETNaAc d) PETKAc. CHAPTER 6 - Preliminary Application as Urinary Catheters 123 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Figure 6.3. SEM analysis of PET samples before exposure to the artificial urine using the dynamic urine testing set up; a) untreated PET and b) nanoparticle treated PET. Figure 6.4. SEM analysis of PET samples after 30 days exposure to artificial urine using the dynamic urine testing setup; a) and b) untreated PET, c) and d) gum Arabic treated PET, e) and f) NaAc treated PET and g) and h) KAc treated PET. CHAPTER 6 - Preliminary Application as Urinary Catheters 124 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools Figure 6.5. SEM analysis of PET samples after 30 days exposure to the artificial urine using the dynamic urine testing set up; a) and b) untreated PET and c) and d) nanoparticle treated PET. Table 6.1. SEM-EDS analysis of material surfaces after being tested with artificial urine. Detected Elements (Atomic %) PET - Untreated PET-NP PET-GA PET-NAc PET-KAc C 22,8 54,6 49,1 55,9 56,2 N 2,4 11,1 16,9 15,7 13,4 O 12,2 26,8 22,8 20,3 23,9 F n/a 2,9 n/a n/a n/a Na 25,0 1,8 5,5 4,0 3,5 Si n/a 1,3 0,3 0,1 0,1 P 4,6 0,1 0,8 0,4 0,3 S 3,7 0,1 0,6 0,3 0,3 Cl 25,7 0,9 3,6 3,0 2,0 K 3,7 0,2 0,4 0,3 0,3 Further elemental analysis on the modified materials after urinary testing were done by SEM-EDS as shown in Table 6.1. Carbon and oxygen are expected elements which are present on all materials. However, the third element with the largest detected amount was found to be nitrogen, N, which is not present in the modified materials and untreated samples except PET-GA, thus it must be obtained through artificial urinary solution. Since, ammonium chloride, urea and creatinine consist of N element in their structure, this element indicate that at least one of them was present on the crystals formed. The other elements that attracted the attention are Na and Cl which are expected to be from sodium CHAPTER 6 - Preliminary Application as Urinary Catheters 125 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools chloride, ammonium chloride, di-Sodium Hydrogen Phosphate, and sodium sulfite. Additionally, sodium is also expected in the PET-NaAc composition. Moreover, a low amount of P, S, K was detected, and these elements are thought to be from artificial urine components including di-Sodium Hydrogen Phosphate, Potassium phosphate monobasic and sodium sulfite. Some of the crystal formations look like the NaCl crystals reported in the literature [11], however in our work, it is hard to determine the crystals through their shape as they are embedded. Nevertheless, the appearance of the treated surfaces, particularly for hydrophilic ones, were not expected as the hydrophilic properties should have prevented the deposition of crystals [12] and there are currently commercially available catheters with hydrophilic surface modifications [3]. Regarding the superhydrophobic materials, air bubbles might have been the nucleation sites for crystals to develop. Furthermore, surface topography also plays an important role in nucleation, and it was found that rough materials and materials with pores enhance nucleation for a range of crystals [13] which could explain why nanoparticle modified surfaces promoted crystal formation compared to untreated ones. It is important to note that herein we only used artificial urine and, in vivo applications, crystal formation and biofilm formation promote one another [14]. As explained in Chapter 1, the main reason for the encrustation problem is the presence of urease forming bacteria found in catheters. Thus, preventing the adhesion of this type of bacteria is an important step at reducing the crystal formation [4]. Although modified PET materials do not exhibit good dynamic urinary crystal inhibition, they were previously shown to be anti-adhesive (Chapter 3 and Chapter 4), thus they could still provide some degree of protection for short-term applications. 6.4. CONCLUSION AND OUTLOOK The treated and untreated PET samples were placed under dynamic urinary fluid for 30 days. Samples were observed and analyzed at the final timepoint. The SEM analysis showed that all the modified surfaces promoted crystal formation. Although this is not desired, it is known that adhesion of urease producing bacteria promotes crystal formation. Hence, the strong anti-adhesive properties of the materials herein developed anticipates that these materials can probably reduce the crystal formation by reducing bacterial adhesion. For this purpose, further work is recommended using a mixed bacterial culture (including particularly those that produce urease) with artificial urine. CHAPTER 6 - Preliminary Application as Urinary Catheters 126 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools 6.5. REFERENCES 1. V. Zumstein, P. Betschart, W. Albrich, M. Buhmann, Q. Ren, H. Schmid, D. Abt, Biofilm formation on uretral stents - incidence, clinical impact and prevention. Swiss Med. Wkly. 2017;147: w14408. https://doi.org/doi: 10.4414/smw.2017.14408. 2. Z.K. Zander, M.L. Becker, Antimicrobial and Antifouling Strategies for Polymeric Medical Devices. ACS Macro Lett. 2018;7: 16–25. https://doi.org/10.1021/acsmacrolett.7b00879. 3. H. Pelling, J. Nzakizwanayo, S. Milo, E.L. Denham, W.M. MacFarlane, L.J. Bock, J.M. Sutton, B. V. Jones, Bacterial biofilm formation on indwelling urethral catheters. Lett. Appl. Microbiol. 2019;68: 277–293. https://doi.org/10.1111/lam.13144. 4. Y.J. Cortese, V.E. Wagner, M. Tierney, D. Devine, A. Fogarty, Review of catheter-associated urinary tract infections and in vitro urinary tract models. J. Healthc. Eng. 2018;2018: 2986742. https://doi.org/10.1155/2018/2986742. 5. Z. Zhu, Z. Wang, S. Li, X. Yuan, Antimicrobial strategies for urinary catheters. J. Biomed. Mater. Res. - Part A. 2019;107: 445–467. https://doi.org/10.1002/jbm.a.36561. 6. L. Liu, H. Shi, H. Yu, S. Yan, S. Luan, The recent advances in surface antibacterial strategies for biomedical catheters. Biomater. Sci. 2020;8: 4074–4087. https://doi.org/10.1039/d0bm00659a. 7. N.P. Desai, J.A. Hubbell, Biological responses to polyethylene oxide modified polyethylene terephthalate surfaces. J. Biomed. Mater. Res., 1991;25: 829-843. https://doi.org/10.1002/jbm.820250704 8. Y. Liu, T. He, C. Gao, Surface modification of poly(ethylene terephthalate) via hydrolysis and layerby-layer assembly of chitosan and chondroitin sulfate to construct cytocompatible layer for human endothelial cells. Colloids Surfaces B Biointerfaces. 2005;46: 117–126. https://doi.org/10.1016/j.colsurfb.2005.09.005. 9. L. Pérez-Álvarez, E. Lizundia, S. del Hoyo, A. Sagasti, L.R. Rubio, J.L. Vilas, Polysaccharide polyelectrolyte multilayer coating on poly(ethylene terephthalate). Polym. Int. 2016;65: 915–920. https://doi.org/10.1002/pi.5116. 10. X. Chen, Y. Liu, H. Lu, H. Yang, X. Zhou, J.H. Xin, In-situ growth of silica nanoparticles on cellulose and application of hierarchical structure in biomimetic hydrophobicity. Cellulose. 2010;17: 1103– 1113. https://doi.org/10.1007/s10570-010-9445-3. CHAPTER 6 - Preliminary Application as Urinary Catheters 127 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools 11. M. J. Qazi, & H. Salim, C. A. W. Doorman, E. Jambon-Puillet, N. Shahidzadeh, Salt creeping as a self-amplifying crystallization process. Science Advances. 2019;5: eaax1853. https://doi.org/10.1126/sciadv.aax1853. 12. H. Rebl, J. Renner, W. Kram, A. Springer, N. Fritsch, H. Hansmann, O. W. Hakenberg, J. B. Nebe, Prevention of Encrustation on Ureteral Stents: Which Surface Parameters Provide Guidance for the Development of Novel Stent Materials?. Polymers. 12 2020;12: 558. https://doi.org/10.3390/polym12030558 13. J. M. Campbell, F. C. Meldrum, H. K. Christenson, Observing formation of crystals in active sites. Proceedings of the National Academy of Sciences 2017;114: 810-815. https://doi.org/10.1073/pnas.1617717114. 14. D. Stickler, R. Feneley, The encrustation and blockage of long-term indwelling bladder catheters: a way forward in prevention and control. Spinal Cord 2010;48: 784–790. https://doi.org/10.1038/sc.2010.32. 128 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools CHAPTER 7 General Conclusions & Future Perspectives CHAPTER 7 - General Conclusion & Future Perspectives 129 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools The main goal of this thesis was the development of anti-adhesive materials using different surface modification processes such as “grafting to”, “grafting from” and “ in situ nanoparticles synthesis”. Two different strategies to develop such surfaces were implemented. While the first two strategies included the formation of resistant coatings with hydrophilic characteristics, the third one aimed the formation of repellent superhydrophobic coatings. In the first strategy, a natural polysaccharide, gum Arabic was used to modify the PET surface using glutaraldehyde as a crosslinker for covalent attachment of gum Arabic. Design of experiments was performed to improve the process parameters like concentration, reaction time, curing temperature and curing time. The curing time and curing temperature were found to be important parameters to reach hydrophilic features in this “grafting to” process. After determining the optimal conditions, a PET sample was subjected to the optimized modification process. Its water contact angle was 27 ° after initial treatment (a significant decrease from untreated PET sample, that presents a WCA of 70 °) and the sample maintained a contact angle of 30 ° after 10 washing cycles, indicating a high degree of durability and stability to usage conditions. Other characterization methods such as UV-VIS FTIR, AFM and SEM were further used to demonstrate the surface modification. Finally, the modified materials proved to be highly resistant (more than 99.9 %) to the adhesion of E. coli expressing with YadA. In the second strategy, the surfaces were modified with sodium acrylate (NaAc) and 3-sulfopropyl acrylate potassium salt (KAc) by combining plasma and UV photopolymerization methods. The water contact angle values for the samples decreased from 70 ° to 8 ° and 28 ° for both NaAc and KAc, respectively. The water contact angle after 10 washing cycles was 14 ° for NaAc, thus showing that the modification was durable and stable. However, with KAc, the contact angle after 10 washing cycles showed variations, indicating a non-uniform treatment. The surfaces were further characterized with XPS, AFM, UV-VIS to further assess the surface modification. As the last step, the bacterial resistance against E. coli expressing YadA of the modified materials was evaluated. It was shown that the treated samples possessed more than 90 % reduction for both NaAc and KAc treated samples, proving its potential use to prevent bacterial adhesion in surfaces. In the third strategy, the modification of the PET surface was implemented by in situ growth of nanoparticles using TEOS and fluorocarbon chains (to provide a low surface energy character to the surface). The water contact angle of the modified PET samples was around 156 ° and the contact angle of modified PET samples after 10 cycles of washing with water and 10 cycles of washing with ethanol were 160 ° and 157 °, respectively, proving its durability to usage conditions. However, SEM images showed that some removal of the surface coating occurred after the 10th cycle of water and 10th CHAPTER 7 - General Conclusion & Future Perspectives 130 Tugce Caykara Development of innovative anti-adhesive materials in diagnosis tools cycle of ethanol washing. The modified samples were further evaluated for their anti-adhesive properties against E. coli expressing YadA. However, the modified samples were found to promote adhesion instead of inhibiting it as initially expected. This kind of behavior was thought to be due to surface heterogeneity with non-uniform structures that are reported to be an ideal spot for bacteria to adhere. All the above-mentioned modified PET materials were further evaluated in an artificial bladder model using artificial urine, to assess the modified materials performance as an alternative in the development of urinary catheters. The results showed that the modified PET materials which were immersed in running artificial urine for 30 days, did not show any improvement in the formation of crystals compared to unmodified surfaces. Although this was a preliminary study, it was shown that the modified materials promoted crystallization. This indicates that these type of surface modifications might not be the ideal for materials that are envisaged to be used in the long term in urinary applications. However, it is important to note that most of the crystallization is caused by urease producing bacteria in urinary systems. Therefore, the anti-adhesive nature of the modified PET materials herein developed is as important as determining how resistant a material can be against encrustation. A method combining both the use of bacteria and artificial urine could be better and more realistic at determining how effective a material is. Although this was not tested for the PET materials herein modified, the gum Arabic and acrylate modified samples could still be useful for intermittent catheterization as they can reduce bacterial adhesion very effectively. The results also suggest that the nanoparticle modified PET samples should not be used in urinary catheters as they seem to promote bacterial adhesion instead of preventing it. Instead, other applications where bacterial adhesion is envisaged should be explored for these materials. For instance, the modified nanoparticles may find application in systems where bacteria capture and/or enrichment is envisaged, for example for application in sensors for rapid detection of bacteria. The hydrophilic modified materials exhibit very interesting anti-adhesive properties, which open several potential applications, particularly for the healthcare sector. Furthermore, these surfaces could be used in diagnosis tools to increase the sensitivity and selectivity of the analysis by preventing nonspecific adhesion on sensors and reducing unwanted adhesion on the other parts of diagnostic tools. In this way, they can reduce misdiagnosis by providing faster and more accurate results thus, preventing wrong use of drugs and reducing the cost and effect on antimicrobial resistance. While surfaces that work with antimicrobial agents can be highly effective against initial stages of bacterial adhesion, accumulation of dead bacteria on the surface will change the surface properties, thus once the surface is covered, the expected antimicrobial effect might be highly inhibited. Moreover,