Layered double hydroxide (LDH) based bacterial adhesion / biofilm inhibitive polymeric composite material

Abstract

The invention relates to a magnesium- aluminium layered double hydroxide (Mg-Al LDH) based filler product comprising boron and functionalised with phosphocholine chloride calcium salt tetrahydrate (PC), the synthesis method of this filler product, and obtaining a new material by preparing a composite material of this filler product, which has antibacterial activity, with a thermoplastic polymer or thermosetting polymer. In the production of the composite material that is the subject of the invention, as the polymer, preferably unsaturated polyester resin (DPes) is used. The composite material comprising the filler product of the invention has the feature of preventing bacterial adhesion / biofilm formation. Said material has the potential to be used for various applications, including medical devices, industrial processes, food production, storage and sales areas, food contact surfaces, areas requiring cleaning and hygiene, and different surface materials.

Description

[0001] LAYERED DOUBLE HYDROXIDE (LDH) BASED BACTERIAL ADHESION / BIOFILM INHIBITIVE POLYMERIC COMPOSITE MATERIAL

[0002] Technical Field of the Invention

[0003] The invention relates to a magnesium- aluminium layered double hydroxide (Mg-AI LDH) based filler product comprising boron and functionalised with phosphocholine chloride calcium salt tetrahydrate (PC), the synthesis method of this filler product, and obtaining a new material by preparing a composite material of this filler product, which has antibacterial activity, with a thermoplastic polymer or thermosetting polymer. In the production of the composite material that is the subject of the invention, as the polymer, preferably unsaturated polyester resin (DPes) is used. The composite material comprising the filler product of the invention has the feature of preventing bacterial adhesion / biofilm formation. Said material has the potential to be used for various applications, including medical devices, industrial processes, food production, storage and sales areas, food contact surfaces, areas requiring cleaning and hygiene, and different surface materials.

[0004] The State of the Art

[0005] Layered double hydroxide (LDH) is a nanostructured anionic synthetic clay that is called two-dimensional layered double because it contains cationic layers and anions within hydrotalcite-like layers. The anions inside the LDH layers can be replaced with other anionic molecules

[0001] . LDHs attract great attention in many technologically important fields such as catalysis, separation and biomedicine due to their interesting properties such as compositional / structural flexibility, anion exchangeability and biocompatibility. In recent years, studies on the development of antimicrobial surfaces for medical devices, household products, and food packaging have increased due to the increasing impact of disease-causing microbes on public health. Inorganic coatings that exhibit good mechanical strength, chemical stability, and physicochemical compatibility with ceramics and metallic objects are of particular interest for anti- microbial applications [2], LDHs are synthesised by different methods, categorised as direct or indirect methods. Direct synthesis methods, such as coprecipitation, are also known as single-step methods, while indirect synthesis methods involve multiple steps, such as anion exchange, anion exchange with regeneration of calcined material, and exfoliation-recombination [3].

[0006] Phosphocholines / Phosphorylcholines (PC) constitute the main (head) group of phospholipid-derived chemical materials consisting of choline and phosphate groups, which have functions called biomembrane imitation or biomimetic. Since the choline group is positively charged and the phosphate group is negatively charged, the entire molecule is in the zwitterionic state, and the net electrical charge of the head group is zero and it is hydrophilic. PC has been used as a building block for a wide variety of polymeric biomaterials due to its good biocompatibility and excellent resistance to nonspecific protein adsorption [4], PC polymers have the ability to inhibit adverse biological reactions by means of their ability to suppress cellular adhesion resulting from their fundamental ability to interact with proteins without causing changes in their structure. This feature of PCs stems from the fact that the chemical and electronic configuration of the zwitterionic head group structures provides reversible interaction with biological macromolecules approaching the surface in association with water molecules in the environment and can also control the adsorption of these macromolecules and the formation of any possible intermediate layer that bacteria may form. Due to their properties, PC polymers and their derivatives are today preferred particularly in innovative material groups such as cardiovascular system apparatus in which bacterial adhesion and biofilm formation are not desired in any way (coronary stents, vascular grafts), ophthalmic applications (intra-ocular lenses, contact lenses), and other anti-infective applications (urological systems and orthopaedic applications) [5]-

[0007] Boron can quickly form complexes by interacting with the hydroxyl groups of aromatic and aliphatic compounds containing -OH. By incorporating boron into the synthesis of petroleum-based polymers with a linear structure, the thermal, mechanical, flameretardant properties and biocidal activities of the polymers can be improved [6]. Boron is mostly found as naturally occurring boric acid. However, other borate compounds can be easily converted to boric acid. The functionality of boron-containing compounds such as boric acid (H3BO3) and its alkyl or aryl substituents (boronic acid) (R-BOH-R; R: alkyl, aryl), borax, diazaborine and potassium tetraborate as antibacterial agents has been widely investigated and said compounds are widely used as buffers in fertilisers, insecticides, detergent additives and pharmaceutical compounds [7], Boric acid (BA) and its salts are well-known antibacterial and antifungal agents. Boric acid is a small, nonpolar molecule that can cross through the biological membranes, which are permeable barriers that separate the cell from its environment. The non-ionised form of boric acid can interact with or pass through the membrane of the microbial cell and interfere with many enzymatic processes in the cell. Boron compounds can also react with hydroxyl-rich compounds such as phospholipids, lipopolysaccharides, and glycoproteins found in microbial membranes. Such interactions can result in changes in membrane functional activity, including that of membrane-bound enzymes. The mode of action of boric acid can be attributed to two possible main processes: inhibition of membrane proteins and / or inhibition of enzymes and co-enzymes inside cells [8].

[0008] Biofilm is a complex microbial aggregate that forms on a wide variety of surfaces, often encapsulated by an extracellular protective matrix containing various types of biopolymers. Some important pathogenic or potentially pathogenic microorganisms maintain their viability on surfaces for a long time by forming biofilms in the presence of suitable environments and conditions. Meanwhile, pathogenic bacteria continue to survive in the biofilm layer they create, which is resistant to detergents and disinfectants and even mechanical effects, and this point can be a source of contamination for bacteria. Food infections and intoxications caused by biofilm formation pose a great risk in the food industry, especially in important food sectors such as the dairy industry, meat industry and ready-made food industry. Biofilmforming pathogenic and potentially pathogenic microorganisms pose a great risk to the food industry as well as to domestic, health, water treatment and other industrial processes. For many types of bacteria, adhesion to a surface is a prerequisite for growth. Since the main factor that enables this is the polysaccharide-based compounds they secrete, preventing adhesion is of primary importance in order to reduce the risk of bacterial infection. It is known that surfaces / materials containing phosphocholine (PC) have the ability to reduce the adhesion of many bacterial strains by preventing the formation of any conditioning layer that could allow bacteria to adhere to the surface. This feature of PCs stems from the fact that the chemical and electronic configuration of the zwitterionic head group structures provides reversible interaction with biological macromolecules approaching the surface in association with water molecules in the environment and can also control the adsorption of these macromolecules and the formation of a possible intermediate layer that bacteria may form.

[0009] In the state of the art, there are various studies in which biofilm formation is prevented by adding antimicrobial products to surface materials. Apart from this, there are also studies stating that bacterial adhesion is prevented by pre-conditioning the surfaces with a surfactant. In polymer-based bioadhesion inhibiting material types (composite / nanocomposite), modified polyurethane, silicone rubber, and surface- coated polypropylene are mostly used as the polymer main matrix.

[0010] In the state of the art, de Castro et al. (2018) and de Castro et al. (2020) used the same LDH synthesis in their two studies. Borate-intercalated magnesium-aluminium layered double hydroxide was synthesised by co-precipitation method at constant pH. The aim of this study was to evaluate the availability and leaching of boron from Mg-AI bilayer hydroxides intercalated with borate anions (Mg2AI-B-LDH) and H3BO3 (boric acid) in a sandy soil and the boron bioavailability of these two sources in sequential plantings of sunflower [9, 10].

[0011] In another document in the state of the art, Hirota et al. (2005) compared the adhesion of four species (Staphylococcus aureus, Streptococcus mutans, Pseudomonas aeruginosa and Candida albicans) isolated in association with biomedical devicerelated infections to three different surfaces: 2-methacryloyloxyethyl phosphorylcholine (MPC) coated, uncoated and hydrophobic coverslips. All four bacterial species were found to bind significantly less to MPC-coated surfaces than to uncoated surfaces. However, although it is known that hydrophobic surfaces reduce bacterial adhesion and thus prevent an important step in the formation of bacterial biofilms that lead to biomedical device-related infections and complications, they attributed this effect to the superhydrophilicity of MPC-coated surfaces

[0011] .

[0012] In the state of the art, the single syntheses of boric acid and phosphocholine chloride calcium salt tetrahydrate with LDH (LDH with boron and LDH with phosphocholine) show limited antibacterial properties and are effective against a single group of bacteria.

[0013] Due to the limitations and inadequacies of the solutions in the state of the art, it has been necessary to make a development in this field in order to both increase the antibacterial activity of boron in the synthesis of layered double hydroxides in order to prevent biofilm formation and to synthesise a new generation polymer-based material with cationic surface properties.

[0014] Brief Description and Aims of the Invention

[0015] The invention relates to a magnesium- aluminium layered double hydroxide Mg-AI (LDH) based filler product comprising boron and functionalised with phosphocholine, the synthesis method of this filler product, and obtaining a new material by preparing a composite material of this filler product, which has antibacterial activity, with a thermoplastic polymer or thermosetting polymer. The composite material comprising the filler product of the invention has the feature of preventing bacterial adhesion / biofilm formation.

[0016] An aim of the invention is to develop a new filler product with antibacterial effect and a composite material containing this filler product. The subject of the invention is the synthesis of a new filler product based on Mg-AI LDH containing boron functionalised with phosphocholine, and a new material is obtained by preparing the composite material of this antibacterial filler product with a thermoplastic polymer or thermosetting polymer, preferably DPes, both the filler product and said composite material have an antibacterial effect. In the invention, the B-LDH-PC filler product is effective against many bacteria due to the structure formed according to the formation order of the filler product in the synthesis conditions, that is, the "phosphorus-boron clusters" structure is distributed in the exfoliated LDH matrix and eventually, the composite material created with DPes containing this filler product also has properties that prevent bacterial adhesion / biofilm formation.

[0017] Another aim of the invention is to develop a material that prevents microorganisms that may cause infection on frequently contacted surfaces from forming bioadhesion / biofilm. In the preparation of the polymeric composite material of the invention, an inorganic (layered double hydroxide compound-LDH, a synthetic clay) compound was chosen as the reagent to prevent bioadhesion / biofilm formation, and this compound was functionalised with phosphocholine and boron. The phosphocholine (PC) contained in the filler product of the invention has the feature of reducing the adhesion of many bacterial strains by preventing the formation of any conditioning layer that could allow bacteria to adhere to the surface. This feature of phosphocholines stems from the fact that the chemical and electronic configuration of the zwitterionic head group structures provides reversible interaction with biological macromolecules approaching the surface in association with water molecules in the environment and can also control the adsorption of these macromolecules and the formation of a possible intermediate layer that bacteria may form.

[0018] Another aim of the invention is to synthesise a material with antimicrobial and cationic surface properties. The filler product of the invention, which is a synthetic anionic clay and comprises boron, has been functionalised with zwitterionic phosphocholine (phosphocholine chloride calcium salt tetrahydrate) in order to gain cationic properties. The purpose of imparting cationic properties to the filler product of the invention is that cationic materials are the best-known antimicrobial substances, they are widely used in disinfection applications, and they show a high affinity for microbial interfaces due to the negative charge of microbial interfaces, especially when applied at pH levels from alkaline to neutral. By functionalising this boron-containing Mg-AI LDH with phosphocholine, antibacterial filler product is synthesised, and this synthesised product is used to obtain a composite material with an unsaturated polyester. This composite material has the feature of preventing bacterial adhesion / biofilm formation. It has been observed in the invention that, due to the structure formed between boron, LDH and phosphocholine in the order of formation under synthesis conditions, that is, as a result of the distribution of the "phosphorus-boron clusters" structure in the exfoliated LDH matrix, both the B-LDH-PC filler product and the polymeric composite material obtained with this filler product have antibacterial properties and prevent biofilm formation. Description of Drawings

[0019] Figure 1. Biofilm formation levels of various bacteria on material surfaces

[0020] (A: E. faecium P22-2e, B: S. Epidermidis, C: E. faecium P53-2c, D: S. aureus ATCC 25923, M1 : DPes + B-LDH-PC (at a ratio of 2%), M2: DPes + B-LDH-PC (at a ratio of 5%), M3: DPes + B-LDH-PC (at a ratio of 7%))

[0021] Detailed Description of the Invention

[0022] The invention relates to a magnesium- aluminium layered double hydroxide Mg-AI (LDH) based filler product comprising boron and functionalised with phosphocholine, the synthesis method of this filler product, and obtaining a new material by preparing a composite material of this filler product, which has antibacterial activity, with a thermosetting polymer or thermoplastic polymer.

[0023] In the invention, the LDH compound, which is an inorganic synthetic clay, is synthesised and functionalised with phosphocholine. By blending this functionalised LDH-phosphocholine product with DPes, a composite material based on unsaturated polyester resins is prepared. The material of the invention has the feature of preventing bacterial adhesion / biofilm formation. The filler product, which is a synthetic anionic clay and contains boron, has been modified with zwitterionic phosphocholine (phosphocholine chloride calcium salt tetrahydrate) in order to gain cationic properties. Said material has the potential to be used for various applications, including medical devices, industrial processes, food production, storage and sales areas, food contact surfaces, areas requiring cleaning and hygiene, and different surface materials. The phosphocholine (PC) contained in the filler product of the invention has the feature of reducing the adhesion of many bacterial strains by preventing the formation of any conditioning layer that could allow bacteria to adhere to the surface. In addition, the final structure of the filler product is the exfoliated (layers dispersed) LDH structure containing triborate Ba’1, Ba’2, tetraborate B2and monoborate [B(OH)4]’1clusters whose end groups are modified with PC. By means of the distribution of this "phosphorus-boron clusters" structure in the exfoliated LDH matrix, the expected and desired effect on bacteria was realised. Likewise, with the homogeneous distribution of the B-LDH-PC filler product within the composite structure, the final composite product also showed an inhibitory effect on biofilm formation.

[0024] Synthesis method of the boron-containing filler product functionalised with phosphocholine and showing antibacterial activity comprises the process steps of: i. dissolving magnesium chloride hexahydrate (MgCl2.6H2O) and aluminium chloride hexahydrate (AICI3.6H2O) salts in deionized water, ii. dissolving boric acid (H3BO3) in deionized water, iii. adding the salt solution into the HsBOs-aqueous solution dropwise under vigorous stirring, by co-precipitation method under nitrogen atmosphere, iv. adding sodium hydroxide (NaOH) to increase the pH of the mixture to 10, v. closing tightly and subjecting to reaction at room temperature after feeding nitrogen gas, vi. precipitating and washing with deionized water at the end of the reaction, vii. dispersing the precipitated material after washing in deionized water, viii. raising the temperature of the material by placing it in an autoclave, ix. adding phosphocholine chloride calcium salt tetrahydrate (PC) dissolved in water as much as the boric acid used in the synthesis, to the liquid in the autoclave, x. subjecting to the reaction after closing the autoclave lid and feeding nitrogen gas, and xi. at the end of the reaction, lyophilising (freeze drying) the existing liquid to obtain a powder white filler product.

[0025] In an embodiment of the invention, synthesis method of the filler product showing antibacterial activity comprises process steps of: i. preparing the salt solution containing 1.5 mol / L magnesium chloride hexahydrate (MgCl2.6H2O) and 0.5 mol / L aluminium chloride hexahydrate (AICI3.6H2O), ii. preparing 1 .25 mol / L boric acid (H3BO3) solution, iii. adding 1 unit of salt solution into 2 units of HsBOs-aqueous solution dropwise under vigorous stirring, by co-precipitation method under nitrogen atmosphere, iv. adding 2 M sodium hydroxide (NaOH) to increase the pH of the mixture to 10, v. closing tightly and subjecting to reaction at room temperature for 24 hours after feeding nitrogen gas, vi. precipitating in a centrifuge at 4500 min-1and washing with deionized water at the end of the reaction, vii. dispersing the precipitated material after washing in deionized water, viii. raising the temperature of the material by placing it in an autoclave, ix. adding phosphocholine chloride calcium salt tetrahydrate (PC) (0.005 mol) dissolved in water as much as the boric acid used in the synthesis, to the liquid in the autoclave, x. closing the autoclave lid, feeding nitrogen gas, and reacting at 250 rpm at 80 °C for 24 hours, and xi. at the end of the reaction, lyophilising (freeze drying) the existing liquid to obtain a powder white filler product.

[0026] The filler product of the invention that exhibits antibacterial activity is synthesised using magnesium chloride hexahydrate (MgCl2.6H2O), aluminium chloride hexahydrate (AICI3.6H2O), boric acid (H3BO3), sodium hydroxide (NaOH) and phosphocholine chloride calcium salt tetrahydrate (PC) compounds.

[0027] The final structure of the filler product of the invention showing antibacterial activity is an exfoliated (layers dispersed) LDH structure containing triborate B31, B32, tetraborate B2and monoborate [B(OH)4]’1clusters with end groups modified by PC.

[0028] The filler product of the invention that exhibits antibacterial activity comprises magnesium chloride hexahydrate (MgCl2.6H2O), aluminium chloride hexahydrate (AICI3.6H2O), boric acid (H3BO3) and phosphocholine chloride calcium salt tetrahydrate (PC) in its exfoliated (layers dispersed) LDH structure comprising triborate Bs’1, Bs’2, tetraborate B2and monoborate [B(OH)4]’1clusters with end groups modified by PC.

[0029] In an embodiment of the invention, the filler product of the invention that exhibits antibacterial activity comprises 35-50% magnesium chloride hexahydrate (MgCl2.6H2O), 10-25% aluminium chloride hexahydrate (AICI3.6H2O), 15-30% boric acid (H3BO3) and 15-30% phosphocholine chloride calcium salt tetrahydrate (PC) in its exfoliated (layers dispersed) LDH structure comprising triborate Ba'1, Ba'2, tetraborate B2and monoborate [B(OH)4]'1clusters with end groups modified by PC.

[0030] The final structure of the layered double hydroxide (B-LDH-PC filler product) comprising modified boron and functionalised with phosphocholine is shown below: Final structure of the modified boron-containing and phosphocholine-functionalised layered double hydroxide (B-LDH-PC filler product) described in formula (1 ) comprises exfoliated LDH layers containing Ba'1(triborate), Ba'2(triborate), B2(tetraborate), [B(OH)4]'1(monoborate), PC (phosphocholine chloride calcium salt tetrahydrate) . In other words, the final structure of the filler product is the exfoliated (layers dispersed) LDH structure containing triborate Ba'1, Ba'2, tetraborate B2and monoborate [B(OH)4]'1clusters whose end groups are modified with PC. By means of the distribution of this "phosphorus-boron clusters" structure in the exfoliated LDH matrix, the expected and desired effect on bacteria was realised.

[0031] Formula (1a)

[0032] Formula (1 a) explains Ba-1(triborate) contained in the layered double hydroxide (B- LDH-PC filler product) comprising modified boron and functionalised with phosphocholine.

[0033] Formula (1 b)

[0034] Formula (1 b) explains Ba’2(triborate) contained in the layered double hydroxide (B- LDH-PC filler product) comprising modified boron and functionalised with phosphocholine.

[0035] Formula (1c)

[0036] Formula (1 c) explains B2(tetraborate) contained in the layered double hydroxide (B- LDH-PC filler product) comprising modified boron and functionalised with phosphocholine.

[0037] Formula (1d) Formula (1d) explains [B(OH)4]’1(monoborate) contained in the layered double hydroxide (B-LDH-PC filler product) comprising modified boron and functionalised with phosphocholine.

[0038] Formula (1e)

[0039] Formula (1 e) explains PC (phosphocholine chloride calcium salt tetrahydrate) contained in the layered double hydroxide (B-LDH-PC filler product) comprising modified boron and functionalised with phosphocholine.

[0040] Formula (1f)

[0041] Formula (1 f) explains LDH exfoliated layers contained in the layered double hydroxide (B-LDH-PC filler product) comprising modified boron and functionalised with phosphocholine. The LDH exfoliated layers described in formula (1 f) represent the backbone of the filler product. The end groups described in Formula (1 a), (1 b), (1 c), (1 d) and (1 e) are connected to the backbone of the LDH exfoliated layers filler product described in Formula (1 f).

[0042] The production of the new generation, thermoplastic polymer or thermosetting polymer based polymeric composite material comprising the B-LDH-PC filler product, having cationic surface properties to prevent bioadhesion / biofilm formation of microorganisms that may cause infection on frequently contacted surfaces comprises the process steps of: i. mixing a thermoplastic polymer or a thermosetting polymer and a synthesis filler product comprising phosphocholine-functionalised boron (B-LDH-PC) in an ultrasonic homogeniser, ii. adding cobalt (accelerator) for curing after a homogeneous mixture is observed, iii. adding peroxide M-60 (MEK-P) (hardener), iv. pouring the solution quickly onto the metal smooth surface after achieving homogenisation, and v. bringing the material removed from the surface within 24 hours into tablet / pellet form and leaving it to dry for biofilm formation analysis.

[0043] In an embodiment of the invention, the production of the new generation, thermoplastic polymer or thermosetting polymer based polymeric composite material comprising the B-LDH-PC filler product, having cationic surface properties to prevent bioadhesion / biofilm formation of microorganisms that may cause infection on frequently contacted surfaces comprises the process steps of: i. mixing a thermoplastic polymer or a thermosetting polymer and a synthesis filler product comprising phosphocholine-functionalised boron (B-LDH-PC) at a rate of 0.5-10% in an ultrasonic homogenizer, at 60% amplitude, with vibration for 10 seconds, with a pause of 5 seconds, for a period of five minutes to 10 minutes in total, ii. after observing a homogeneous mixture, adding at a ratio of 0.2% of 8% cobalt (accelerator) for curing and mixing for 2-3 minutes, iii. adding peroxide M-60 (MEK-P) (hardener) and mixing for 1 -3 minutes, iv. pouring the solution quickly onto the metal smooth surface after achieving homogenisation, and v. bringing the material removed from the surface within 24 hours into tablet / pellet form and leaving it to dry for 10-15 days for biofilm formation analysis.

[0044] The thermoplastic polymer or thermosetting polymer-based new generation polymeric composite material produced in this way contains a filler product containing boron functionalised with phosphocholine (B-LDH-PC).

[0045] In an embodiment of the invention, unsaturated polyester resin (DPes) is used as the thermoplastic polymer or thermosetting polymer described in process step (i). In an embodiment of the invention, the thermoplastic polymer or thermosetting polymer-based new generation polymeric composite material produced in this way contains 0.5-10% of filler product containing boron-functionalised with phosphocholine (B-LDH-PC).

[0046] For the characterization studies of the synthesised filler product, Fourier transform infrared spectrophotometry (FTIR), X-ray diffractometer (XRD), Brunauer-Emmet- Teller (BET) analyses were used, and antimicrobial activity tests were performed. For FTIR analysis, spectra were recorded in the wave number range of 500-4000 cm1by using tablets prepared by diluting the filler product / potassium bromide (KBr) ratio to 1 / 200 mg. XRD patterns of the samples were obtained with a scanning speed of 2° / min in the range of 26=5°-120°. The crystal structures of the products, whose structures were examined by FTIR analysis, were also confirmed by XRD results. For BET surface area analysis of the products, the samples were dried under vacuum at 60 °C for 5 hours before analysis. Sixteen points of the adsorption isotherm (P / Po=O.O5-1 ) were taken into account to calculate the specific surface area according to the BET method. Pore size distributions were determined according to BJH and DFT methods using adsorption / desorption data. Since the structures in powder form must be turned into tablets / pellets under pressure in order to perform the antimicrobial activity test analysis of the filler products, the powder product, placed in a 4 mm diameter seal in an industrial tablet pressing device, was turned into tablets with a pressure between 400-1000 psi. Filler products and polymeric composite materials brought into tablet form were sterilised by dry sterilisation or autoclaving for antibacterial analysis. For dry sterilisation, it was kept in a dry air steriliser for 3 hours at 130 °C in a dry hot air environment. For autoclaving, it was kept in a pressurised water steam steriliser at 121 °C for 20 minutes and then dried.

[0047] For antimicrobial activity testing, 7 ATCC coded reference strains [Enterococcus f aecium (ATCC 8459), Enterococcus faecalis (ATCC 29212), Escherichia coll (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Listeria monocytogenes (ATCC 191 15), Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (AT CC 12228)] kept in a -80°C deep freezer were incubated on T ryptic Soy Agar (TSA; Oxoid CM 131 ) at 35±1 °C for 24 hours. Bacterial colonies growing in TSA were suspended in 0.90% NaCI (Physiological Saline) to 0.5 McFarland (approximately 1 x108cfu / ml) by densitometer. Then, with the help of a sterile swab from tubes containing 5 ml of bacteria and 0.90% NaCI suspension, each reference strain was swabbed separately onto the surface of Mueller-Hinton Agar (MHA; Oxoid CM 337) poured into 4 mm thick and 90 mm diameter sterile petri dishes prepared in accordance with EUCAST (2013) standards to ensure that the MHA surface was completely covered with the bacterial suspension. Then, chemical substances (filler products) were placed in the middle of the MHA petri dishes prepared in a sterile environment in laminar flow and containing a bacterial suspension at 0.5 McFarland concentration. This process was applied to MHA petri dishes containing 7 different reference strains. After completing the process of placing chemical substances in the middle of the MHA petri dishes containing the bacterial suspension, all petri plates were incubated at 35±1 °C for 16-20 hours. At the end of the incubation period, the resistance of chemical substances on bacteria was revealed by measuring the chemical substance environment in the petri dish with a calliper for antimicrobial activity testing.

[0048] In order to detect biofilm formation on material surfaces, results were obtained as a result of spectroscopic analysis (480 nm excitation and 590 nm emission) using fluorescent FilmTracer. Within the scope of this analysis, the biofilm formation rates of Staphylococcus epidermidis and Enterococcus faecium P22-2e, two bacteria that were found to form biofilms, and Staphylococcus aureus and Enterococcus faecium P53-2c, two bacteria that did not form biofilms, were investigated and bioadhesion formation on the surface of the material was examined. In biofilm tests, it was determined that bacteria formed biofilms in the medium containing the highest amount of TSB+1 % sucrose. For this reason, medium containing TSB+1 % sucrose was used as the medium in the experiments using test materials. For this purpose, of the bacteria produced in 24-hour TSA, the concentration of S. epidermidis (biofilm positive), S. aureus (biofilm negative), E. faecium P22-2e (biofilm positive) and E. faecium P53-2c (biofilm negative) in 0.9% NaCI was adjusted to McFarland 0.5. Under aseptic conditions, sterile test materials were taken within the biosafety cabinet using forceps and placed in a 96-well black microplate. 200 pl of medium containing TSB+1 % sucrose was added to the test materials, and then 20 pl of bacterial suspension was added and incubated at 37 °C for 24 hours. At the end of the incubation, the medium on the materials was collected with a pipette and washed with 0.9% sodium chloride (NaCI) to remove non-adherent bacteria. Following the protocol recommended by the manufacturer, the fluorescent dye was prepared in dimethyl sulfoxide (DMSO) and diluted with sterile 0.2 pm filtered ultrapure water to a final concentration of 10 pg / ml. To determine the autofluorescence properties emitted by the materials, fluorescence intensity values were determined at 480 nm excitation and 590 nm emission wavelengths in a multi-mode microplate reader before adding fluorescent dye. 100 pl of fluorescent dye was added to each material and plate. The microplate was covered and incubated in the dark at room temperature for 30 minutes. At the end of the incubation, the fluorescent dye was collected with a pipette and the surfaces were washed with 200 pl sterile water. To detect biofilm formation on black microplate material surfaces, fluorescence intensity values were determined at 480 nm excitation and 590 nm emission wavelengths in a multi-mode microplate reader.

[0049] In the boron LDH (B-LDH) spectra, due to the structural formation, a broad band formation with two peaks is observed at 1434 / 1442 and 1305 cm-1, which overlapped. In the 1033 cm1broad shoulder [BO3] structural vibrations, B-0 stretching and in plane B-OH stretching band in the boric acid (BA) structure shifts and appears as broad bands with two peaks at 1039 and 922 cm-1in the B-LDH spectrum in this region, which overlapped. BA caused the formation of a double band in the spectra as [B(OH)4]’ (borate anion). As a result of FTIR spectra examination, it was observed that the peaked bands at 1434 and 1305 cm1in the B-LDH spectra disappeared, and instead, in the B-LDH-PC spectra, a new formation consisting of the overlapping of three new peaks broad, three bands at 1478, 1420 and 1343 cm1, and a new broad shoulder at 1248 cm1appeared. The small shoulder at 1248 cm1(in some cases it can be seen at 1258 cm-1) arises from the vibrations of PO2 groups in which no bridge is formed between the oxygen atoms in the asymmetric phosphate structures, and when the amount of B in the molecule is high, it expands and appears with a weak intensity, and this is a decrease in intensity resulting from the formation of P-O-B bonds. It is reported that the increased intensity of the band is related to the lack / absence of the formation of P-O-B bonds. In addition, the very small peak at 1552 cm’1, which is also observed in PC spectra, appeared from the bending vibrations of H-O-H bonds belonging to crystal water. Asymmetric (asym.) stretching belonging to the P-O3 structure at 1 150 cm-1in the PC spectrum and symmetric (sym.) sharp long intense stretchings belonging to the P-O3 structure at 1089 cm’1and 1092 cm’1and along with these, -O- B stretchings in BA spectra, those in the intensely long band at 1 195 cm’1, which are generally caused by B-O-H structural in-plane bendings and those in the broad long shoulder at 1100 / 1 114 cm-1resulting from B-0 bonds asym. / sym stretchings in the tetrahedral structure BC , appeared as a broad intense band with a peak at 1097 cm’1overlapping with a broad broadshoulder at 1 129 cm-1with shifted peaks, with their intensity being decreased as expected due to PC / BA-LDH-structural interactions. P- O-C stretching bands at 1021 and 1000 cm1and C-H bending bands in the C-O-P bond at 963 cm1in the PC spectra appeared as the formation of a broad absorption with two peaks, with a wide sharp band at 1000 cm’1and a shorter broad sharp band with a peak at 968 cm’1, with decreased intensities, by binding of the phosphate molecule to the B-OH structures in the LDH layers / mainly in the structure formed by the BA-LDH interaction. The band at 918 cm’1(P-0 stretchings) in the PC spectrum and the two-peaked corner shoulder at 925 and 91 1 cm1in the BA spectrum decreased in intensity due to the reaction between PC and B-LDH and appeared as a short, wide new band at 926 cm’1. The intensity of the sharp short band at 873 cm’1in the PC spectrum decreases due to the binding of the phosphate groups to the BA structure, and its peak is shifted, resulting in the formation of a short, wide new band at 872 cm’1. Under the applied synthesis conditions, an associated B-LDH-PC structure was formed through the formation of B-O-P bridges from the reaction with PC, without disrupting the [BCU] network in the B-LDH tetrahedral structure.

[0050] The crystal structures of the filler products of the invention were determined by XRD analysis results. FTIR and XRD results of the synthesised and used products confirm each other. Accordingly, the boric acid used has a fully crystalized structure. The characteristic crystal peaks given for boric acid in the literature are compatible with the peak values of 26=14.5° / 14.7° and 28°

[0012] , As reported by Ojha et al. (2018), according to the XRD analysis results of boron particles, the presence of many peaks in a sequential in the region up to approximately 26=20-45°, especially between 26=20-30°, or the presence of peaks / broad shoulders within broad peaks, is an indication that boron oxide (B2O3) structures have changed from crystalline to amorphous

[0013] . It confirms that while exfoliation (disorder of layers) occurs in the name of LDH during the formation of B-LDH, an amorphous structure will be formed with the distortion of the crystal structure, and at the same time, there will be formations of crystalline B structures in this structure (26=60.987°; d=0.1518) (Table 1 ). Therefore, the structure is in the form of crystals consisting of OHs with B (triborate Ba’1, B32, tetraborate B2and monoborate [B(OH)4]’1) in a fully exfoliated amorphous structure

[0015] . In other words, the B-LDH product has a delaminated structure, and the dispersed layers are bound to the specified B-OH species. The structure in B-LDH maintained its exfoliated state when the B-LDH-PC structure was formed by reaction with PC, and it has been defined in the literature that the resulting structure is predominantly in the form of "cage compounds" or "phosphorus-boron clusters" with PC bonding to the edges of the B-P lattice structure, and in these structures, B atoms are generally located in the centre and the P compound is preferred to be placed at the edges

[0014] , The broad shoulders / peaks at 26=21 .476° and 34.810° in the B-LDH- PC XRD graph results (Table 1 ) appear to be indicative of amorphous B-P lattice structures and the peak at 26=61 .671 ° as an indicator of boron oxide formations in the crystal structure. The findings of the sections where the description "small shoulder at 1248 cm-1(in some cases can be seen at 1258 cm-1)" in the interpreted FTIR spectra of B-LDH-PC confirm this situation. In summary, B-LDH-PC is a fully exfoliated structure, but since it also contains amorphous and crystalline regions belonging to the B-P formation, new peaks have appeared (seen when compared with the B-LDH XRD result (Table 1 )).

[0051] Table 1. XRD Analysis Results The structures of the synthesised filler products are in powder form, and as a result of the analysis, it is seen that the surface area and pore diameter of the plain B-LDH structure are larger. It can be seen that the surface area and pore diameter of the B- LDH-PC filler product obtained by functionalisation with PC is smaller (Table 2). Therefore, it can be said that the B-LDH-PC product has a smaller surface area than B-LDH and PC penetrates into the dispersed B-LDH layers. Therefore, it has been structurally confirmed that B-LDH-PC is a good filler product for the composite material to be prepared.

[0052] Table 2. BET + Pore Size Analysis Results

[0053] According to the antibacterial activity test results of a total of 7 reference microorganisms with gram "+" and gram "-" adhesion properties; B-LDH product was tested as a control group and it was observed that 7 bacteria showed resistance to this material, meaning that it was not effective in inhibiting the growth of reference microorganisms. It has been determined that the B-LDH-PC synthesis filler product is effective on all reference bacteria except the Enterococcus faecium reference strain. Accordingly, it was determined that it inhibited the growth of the Listeria monocytogenes reference strain by 13 mm (diameter of the bacterial inhibition ring), the growth of the Staphylococcus epidermidis reference strain by 13 mm, the growth of the Enterococcus faecalis reference strain by 12 mm, and the growth of the Staphylococcus aureus reference strain by 12 mm, the growth of the Escherichia coli reference strain by 14 mm and the growth of the Pseudomonas aeruginosa reference strain by 13 mm. Thus, as a result of the analyses, it was determined that the Mg-AI LDH based filler product of the invention comprising boron and functionalised with phosphocholine showed antibacterial properties against gram (+) reference strains of Staphylococcus aureus, S. epidermidis, L. monocytogenes, Enterococcus feacalis and gram (-) reference strains of Pseudomonas aeruginosa and Escherichia coli. Biofilm formation rates of Staphylococcus epidermidis and Enterococcus faecium P22- 2e, two bacteria that have been found to form biofilms, and Staphylococcus aureus and Enterococcus faecium P53-2c, two bacteria that do not form biofilms, were evaluated on the surface of 3 different materials, namely the plate surface, M1 , M2 and M3, using a fluorescent film monitoring method (Table 3). Results showing biofilm formation amounts (RFU; relative fluorescence unit) for each bacterial strain on material surfaces at different filler ratios are presented in Figure 1 .

[0054] Table 3. Naming of Materials

[0055] Accordingly, various levels of biofilm formation are observed among the bacterial species tested on material surfaces with different filler ratios. Biofilm formation amounts are given as fluorescence measurements to reflect the relative biofilm mass formed by each strain on each material. It has been determined that bacteria with biofilm-forming capacity (E. faecium P22-2e and S. epidermidis) show consistent biofilm formation on all material surfaces. In particular, biofilm formation appears to be higher for S. epidermidis compared to E. faecium P22-2e. Non-biofilm-forming bacteria (E. faecium P53-2c and S. aureus ATCC 25923) showed significantly lower levels of biofilm formation on all surfaces compared to biofilm-forming bacteria. Differences in biofilm formation between non-biofilm-forming strains were detected less clearly. The choice of surface material has been shown to have a clear impact on biofilm formation in all bacterial species. While varying levels of biofilm formation were observed in biofilm-forming strains, some strain remained attached to surfaces in non-biofilm- forming strains. Moderate biofilm formation was detected on the microtiter plate surface in all strains compared to other materials. However, among all materials, the least biofilm formation occurred on the M2 surface (Figure 1 ). It was determined that biofilm-forming strains showed a significant increase in biofilm amounts compared to the plate on other materials (M1 and M3), and especially M3 showed high biofilm formation for all strains. The highest biofilm masses were observed for E. faecium P22- 2e and S. epidermidis. As a result, when the biofilm-forming capacities of different bacterial strains on material surfaces with different filler ratios are examined, the biofilm-forming bacteria E. faecium P22-2e and S. epidermidis form biofilms as expected, although they vary depending on the surface material. E. faecium P53-2c and S. aureus ATCC 25923 strains, which do not form biofilms, showed significantly lower biofilm formation, again as expected. The choice of surface material is emerging as a critical determinant of biofilm formation. The effect of the different amounts of filler used on the biofilm formed by strains on the material surface was also different. However, it was determined that the most effective anti-adhesion material was the M2- encoded material synthesised for the first time in this study, and even biofilm-forming strains showed lower growth compared to the plate (control) (Figure 1 ).

[0056] The findings show that both the synthesised filler product (B-LDH-PC) and the composite material prepared with DPes (DPes + B-LDH-PC) have antibacterial properties, and it has been demonstrated that it has a strong potential to be used for various applications, including medical devices, industrial processes, food production, storage and sales areas, food contact surfaces, areas requiring cleaning and hygiene, and different surface materials.

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Claims

CLAIMS1. Synthesis method of a filler product comprising boron functionalised with phosphocholine, showing antibacterial activity, comprising the process steps of: i. dissolving magnesium chloride hexahydrate (MgCl2.6H2O) and aluminium chloride hexahydrate (AICI3.6H2O) salts in deionized water, ii. dissolving boric acid (H3BO3) in deionized water, iii. adding the salt solution into the HsBOs-aqueous solution dropwise under vigorous stirring, by co-precipitation method under nitrogen atmosphere, iv. adding sodium hydroxide (NaOH) to increase the pH of the mixture to 10, v. closing tightly and subjecting to reaction at room temperature after feeding nitrogen gas, vi. precipitating and washing with deionized water at the end of the reaction, vii. dispersing the precipitated material after washing in deionized water, viii. raising the temperature of the material by placing it in an autoclave, ix. adding phosphocholine chloride calcium salt tetrahydrate (PC) dissolved in water as much as the boric acid used in the synthesis, to the liquid in the autoclave, x. subjecting to the reaction after closing the autoclave lid and feeding nitrogen gas, and xi. at the end of the reaction, lyophilising (freeze drying) the existing liquid to obtain a powder white filler product.

2. A method according to claim 1 , comprising the steps of: i. preparing the salt solution containing 1.5 mol / L magnesium chloride hexahydrate (MgCl2.6H2O) and 0.5 mol / L aluminium chloride hexahydrate (AICI3.6H2O), ii. preparing 1 .25 mol / L boric acid (H3BO3) solution, iii. adding 1 unit of salt solution into 2 units of HsBOs-aqueous solution dropwise under vigorous stirring, by co-precipitation method under nitrogen atmosphere, iv. adding 2 M sodium hydroxide (NaOH) to increase the pH of the mixturev. closing tightly and subjecting to reaction at room temperature for 24 hours after feeding nitrogen gas, vi. precipitating in centrifuge at 4500 min-1and washing with deionized water at the end of the reaction, vii. dispersing the precipitated material after washing in deionized water, viii. raising the temperature of the material by placing it in an autoclave, ix. adding phosphocholine chloride calcium salt tetrahydrate (PC) (0.005 mol) dissolved in water as much as the boric acid used in the synthesis, to the liquid in the autoclave, x. closing the autoclave lid, feeding nitrogen gas, and reacting at 250 rpm at 80 °C for 24 hours, and xi. at the end of the reaction, lyophilising (freeze drying) the existing liquid to obtain a powder white filler product.

3. A filler product comprising boron functionalised with phosphocholine, produced by a method according to claim 1 or 2.

4. A filler product according to claim 3, comprises magnesium chloride hexahydrate (MgCl2.6H2O), aluminum chloride hexahydrate (AICI3.6H2O), boric acid (H3BO3) and phosphocholine chloride calcium salt tetrahydrate (PC) in its exfoliated (layers dispersed) LDH structure comprising triborate B31, B32, tetraborate B2and monoborate [B(OH)4]’1clusters with end groups modified by PC.

5. A filler product according to claim 3, comprises 35-50% magnesium chloride hexahydrate (MgCl2.6H2O), 10-25% aluminum chloride hexahydrate (AICI3.6H2O), 15-30% boric acid (H3BO3) and 15-30% phosphocholine chloride calcium salt tetrahydrate (PC) in its exfoliated (layers dispersed) LDH structure comprising triborate Bs’1, Bs’2, tetraborate B2and monoborate [B(OH)4]’1clusters with end groups modified by PC.

6. A filler product according to claim 3, comprising phosphorus-boron clusters dispersed in the exfoliated LDH matrix.

7. A filler product according to any one of claims 3-6.

8. A new generation, thermoplastic polymer or thermosetting polymer based polymeric composite material comprising a filler product according to any of claims 3-7, having cationic surface properties to prevent bioadhesion / biofilm formation of microorganisms that may cause infection on frequently contacted surfaces.

9. A new generation polymeric composite material according to claim 8, wherein said polymer is unsaturated polyester resin (DPes). 10.A new generation polymeric composite material according to claim 8, comprising 0.5-10% of the filler product comprising boron functionalised with phosphocholine (B-LDH-PC).

11. The production method of the polymeric composite material according to claim 8, comprising the process steps of:i. mixing a thermoplastic polymer or a thermosetting polymer and a synthesis filler product comprising phosphocholine-functionalised boron (B-LDH-PC) in an ultrasonic homogeniser, ii. adding cobalt (accelerator) for curing after a homogeneous mixture is observed, iii. adding peroxide M-60 (MEK-P) (hardener), iv. pouring the solution quickly onto the metal smooth surface after achieving homogenisation, and v. bringing the material removed from the surface within 24 hours into tablet / pellet form and leaving it to dry for biofilm formation analysis.

12. A method according to claim 11 , comprising the process steps of: i. mixing a thermoplastic polymer or a thermosetting polymer and a synthesis filler product comprising phosphocholine-functionalised boron (B-LDH-PC) at a rate of 0.5-10% in an ultrasonic homogenizer, at 60% amplitude, with vibration for 10 seconds, with a pause of 5 seconds, for a period of five minutes to 10 minutes in total, ii. after observing a homogeneous mixture, adding at a ratio of 0.2% of 8% cobalt (accelerator) for curing and mixing for 2-3 minutes, iii. adding peroxide M-60 (MEK-P) (hardener) and mixing for 1 -3 minutes, iv. pouring the solution quickly onto the metal smooth surface after achieving homogenisation, and v. bringing the material removed from the surface within 24 hours into tablet / pellet form and leaving it to dry for 10-15 days for biofilm formation analysis.

13. A method according to claims 1 1 or 12, wherein the polymer mentioned in the process step (i) is unsaturated polyester resin (DPes).

14. A new generation polymeric composite material based on unsaturated polyester resin (DPes) produced by a method according to any of claims 1 1 -13.

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