A use of a silica composite for inhibiting bacterial, fungal or viral infections and for producing products having antibacterial, antifungal and / or antiviral properties

Abstract

The invention relates to the use of a porous silica composite material containing copper for preventing bacterial infections, inhibiting or reducing the growth of bacteria on surfaces, wherein the bacteria are selected from the genera Enterococcus, Staphylococcus, Streptococcus, or Klebsiella, and / or inhibiting or reducing the growth of fungi, wherein the fungi are selected from the genus Candida, and / or reducing the development of viruses. The invention also relates to the use of above-mentioned composite for the manufacture of coatings for preventing bacterial infections, wherein the bacteria are selected from the genera Enterococcus, Staphylococcus, Streptococcus, or Klebsiella, and / or fungal infections, wherein the fungi are selected from the genus Candida, and / or viral infections.

Description

[0001] A use of a Silica Composite for Inhibiting Bacterial, Fungal or Viral Infections and for Producing Products Having Antibacterial, Antifungal and / or Antiviral Properties

[0002] The subject of the invention is the use of a porous silica composite material with copper for counteracting bacterial infections, wherein the bacteria are selected from the genera Enterococcus, Staphylococcus, Streptococcus, or Klebsiella, and / or fungal infections, wherein the fungi are selected from the genus Candida, or viral infections. The invention also comprises the use of the aforementioned composite for the production of coatings counteracting bacterial infections, wherein the bacteria are selected from the genera Enterococcus, Staphylococcus, Streptococcus, or Klebsiella, and / or fungal infections, wherein the fungi are selected from the genus Candida, or viral infections. Another subject of the invention is the use of a coating made of the composite material for inhibiting bacterial, fungal and / or viral infections.

[0003] The presence in the environment surrounding humans of many bacteria, fungi and viruses, and in particular their uncontrolled growth, may lead to the emergence of a threat of various infectious diseases. Therefore, research is continuously being carried out on the creation of cheap, efficient, and easy to obtain and use antimicrobial materials for applications in such areas as medicine, pharmacy, the food industry, the furniture industry, and this is only a part of the application potential of such materials. Among antimicrobial materials, nanomaterials have recently begun to dominate. Nanoparticles containing metals such as gold [10.1016 / j.colcom.2024.100804], silver [DOI: 10.3390 / ijms22137202] or copper [DOI: 10.3389 / fsurg.2022.905892] are often used. Although such solutions are commonly used, the conducted studies indicate an adverse effect of migrating nanoparticles on the natural environment as well as significant toxicity of nanoparticles agglomerating in mammalian organisms [DOI: 10.3390 / ijms21072375, 10.1016 / j. etap.2019.103220], In addition to the risk associated with the use of metal nanoparticles, there are also other premises for seeking new solutions. Among them, the economic factor dominates, because metals such as gold or silver are among the most expensive on the market. Therefore, development works are constantly being carried out in order to find new cheaper and safer solutions.The achievements of contemporary molecular engineering indicate the enormous possibilities resulting from its application. An important aspect of molecular engineering is the synthesis of large molecules or molecular assemblies "directed toward properties" [DOI: 10.1007 / 978-94-015-8575-0], In the case of the presented invention, molecular engineering techniques were applied to create a nanocomposite with predetermined properties. The nanocomposite in question, owing to the precise arrangement of copper phosphonate groups in the mesoporous silica matrix, constitutes a single-ion catalyst of reactive oxygen species (ROS), and therefore constitutes a specific factory of an antimicrobial agent, which has been demonstrated in the studies presented below.

[0004] Reactive oxygen species (ROS) is a collective term used to describe molecules and reactive intermediates with a highly positive redox potential. This group may include, among others, the superoxide anion radical (O2*“), the hydroperoxyl radical (HO2*), peroxide (O2 ), the hydroxyl radical (OH*), or singlet oxygen (1O2) [DOI: 10.1111 / 1574-6976.12026], Ozone (O3) as well as hydrogen peroxide (H2O2) are also included in this group [DOI: 10.3390 / ijms20102407], The last two examples are commonly used as disinfecting and antiseptic agents. However, they have not found wide application as anti-infective agents due to their lack of specificity toward microbial cells in comparison with host mammalian cells, which means that they may cause unacceptable damage to normal tissue. However, the latest discoveries concerning the methods of generating reactive oxygen species suggest the possibility of the therapeutic use of ROS in the case of actual infections, both systemic and local [DOI: 10.1111 / 1574-6976.12026],

[0005] The antimicrobial solutions used so far in copper-containing surfaces have consisted in the use of an ion-release mechanism, in which the ions affect the integrity of the membrane and / or the cell wall, generate intracellular oxidative stress, and are genotoxic, causing the death of microorganisms [DOI: 10.1186 / sl3756-018-0456-4],

[0006] This method of combating pathogenic microorganisms is associated with many disadvantages, such as the toxicity of copper ions entering the human body and the environment, as well as the need to use large amounts of copper, because the durability of the antimicrobial properties declines as the copper is depleted.An ideal solution would be to design a nanomaterial in which the specific environment of an isolated copper ion is used to generate reactive oxygen species, which in turn eliminate microorganisms.

[0007] The scientific publication entitled "New Class of Antimicrobial Agents: SBA-15 Silica Containing Anchored Copper Ions" (Laskowski L. et al., Journal of Nanomaterials, Volume 2017, Article ID 1287698) discloses a compound based on porous silica matrices containing anchored copper ions. The antibacterial activity of the material was demonstrated against Escherichia coli. Antimicrobial tests were performed both for the pure material and for its application as a modifying agent in plastics.

[0008] In the U.S. patent application US2019 / 0223445A1, an antimicrobial geopolymer composition was disclosed, consisting of porous aluminosilicate aggregates containing various metals, including copper. The key mechanism of action is the rapid release of metal ions from the porous structure of the material, which provides a broad spectrum of biocidal activity against bacteria (including MRSA), fungi, and a wide range of viruses, with explicit emphasis on human coronaviruses and the SARS virus. The invention is intended for various applications, such as paints, plastics, and medical products.

[0009] In another U.S. patent application, US2015 / 0030532A1, an antimicrobial material was described, which is obtained by loading a synthetic zeolite (a porous silica material) with metal ions, such as copper, silver, or zinc, in an ion-exchange process. The main application of this material is to impart long-lasting antimicrobial properties to building materials, including paints, plasters, cement, and plastics. It was shown that the material is effective against a wide range of bacteria (including Staphylococcus and Enterococcus) and fungi (including Candida and Aspergillus); however, the document does not disclose any antiviral activity.

[0010] In a scientific publication „Green sol - gel synthesis of novel nanoporous copper aluminosilicate for the eradication of pathogenic microbes in drinking water and wastewater treatment" (Bahaa Ahmed Hemdan et al., Environ Sci Pollut Res 26, 9508-9523 (2019). https: / / doi.org / 10.1007 / sll356-019-04431-8)

[0011] In the description of the Polish patent application PL433328A1, the use of a specific nanocomposite was disclosed, consisting of an SBA-15 mesoporous silica matrix in whichcopper ions are chemically anchored by means of propylphosphonate groups. The biocidal mechanism is expressly described as the localized, catalytic generation of Reactive Oxygen Species (ROS), while the document emphasizes that the release of copper ions is practically zero. The material is proposed for protective applications, such as hygienic masks, for protection against microorganisms and viruses, including coronaviruses, and the experimental data confirm its activity against E. coli bacteria.

[0012] The problem addressed by the invention is to provide a substance for use in counteracting bacterial infections caused by bacteria of the genera Enterococcus, Staphylococcus, Streptococcus, or Klebsiella, particularly antibiotic-resistant strains, as well as fungal or viral infections. Such a substance should be effective against fungi of the genus Candida and against viruses. Additionally, the aforementioned substance should combine a broad spectrum of antimicrobial activity with a high safety profile (low cytotoxicity) by employing a mechanism that is not based on the release of toxic metal ions. Moreover, in the aforementioned applications, it should be neutral toward animal tissues, especially human tissues. Another problem addressed by the invention is the limitation of the growth or the elimination of pathogenic microorganisms such as bacteria, fungi, or viruses on surface layers while maintaining neutrality toward healthy mammalian cells and tissues.

[0013] The first subject of the invention is a porous silica composite material with copper (MS-Cu5), in which copper ions are chemically anchored in a mesoporous silica matrix by means of phosphonate groups, for use in counteracting bacterial infections, wherein the bacteria are selected from the genera Enterococcus, Staphylococcus, Streptococcus, or Klebsiella, and / or fungal infections, wherein the fungi are selected from the genus Candida, or viral infections. In a preferred embodiment of the invention, the bacteria are selected from the group comprising Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae or Klebsiella pneumoniae.

[0014] In a further preferred embodiment of the invention, the fungi of the genus Candida are selected from the group comprising: Candida glabrata, Candida parapsilosis, Candida albicans, Candida tropicalis, Candida krusei.

[0015] In a further preferred embodiment of the invention, the bacteria are antibiotic-resistant.In another preferred embodiment of the invention, the viruses are selected from the group comprising herpes simplex virus type 1, vaccinia virus, influenza A virus, human adenovirus type 5, and human coronavirus strain 229E.

[0016] The second subject of the invention is the use of the porous silica composite material with copper (MS-Cu5), as defined in the first subject of the invention, for producing a coating for use in counteracting bacterial infections, wherein the bacteria are selected from the genera Enterococcus, Staphylococcus, Streptococcus, or Klebsiella, and / or fungal infections, wherein the fungi are selected from the genus Candida, or viral infections.

[0017] Another subject of the invention is a coating comprising the composite material with copper (MS-Cu5), as defined in the first subject of the invention, for use in inhibiting the growth of microorganisms such as bacteria, wherein the bacteria are selected from the genera Enterococcus, Staphylococcus, Streptococcus, or Klebsiella, fungi, wherein the fungi are selected from the genus Candida, and / or viruses.

[0018] The solution proposed in the present application, namely the use, for antimicrobial purposes, of the catalytic generation of reactive oxygen species by surfaces comprising the composite material, in which the catalytic centers are copper phosphonate groups, has hitherto been known only for Escherichia coli.

[0019] The structure of the nanocomposite, the procedure for its preparation, and the mechanism of its biocidal activity were described in the scientific publication "New Class of Antimicrobial Agents: SBA-15 Silica Containing Anchored Copper Ions" (Laskowski L. et al., Journal of Nanomaterials, Volume 2017, Article ID 1287698, DOI: 10.1155 / 2017 / 1287698). The composite material for combating Escherichia coli bacteria presented therein was obtained in powder form, which may be used for producing the surface layers presented in the present invention by embedding the powder grains into the surface of a polymer. The surface layer thus formed is characterized not only by antimicrobial resistance, in the form of a significant reduction in the formation of bacterial and fungal biofilm, but also by a reduction in viral growth. It should be noted that the level of reduction in the growth of microorganisms on the above-mentioned surfaces may be controlled by changing the concentration of copper ions in the composite [DOI: 10.1155 / 2017 / 1287698],The same antimicrobial mechanism is exhibited by surfaces coated with the mesoporous silica composite containing copper phosphonate groups by a solution-growth method (the so-called modified Stober method), by the spin-coating method, or by the dip-coating method. These methods differ only in the manner of depositing the composite layer on the surfaces, whereas their antimicrobial properties, resulting from reactive oxygen species catalyzed in the pores of the silica, are identical and depend solely on the amount of copper ions in the composite. The obtained results showed that there is a specific molar concentration (relative to the SiO2 groups of silica) of active groups (5% — material MS-Cu5, wherein the active group consists here of copper phosphonate molecules) for which the antimicrobial activity is the strongest. At the same time, the intensity of the antimicrobial activity decreases both when the concentration of the active groups is increased and when it is decreased. The material having any concentration of functional groups was designated as MS-Cu.The possibility of controlling the intensity of the biocidal properties by controlling the concentration of active groups is significant, as it allows the activity of the material to be adjusted to specific needs. There are applications in which a strong activity of the material (biocidal) is desired, such as medical applications, where complete eradication of microorganisms is necessary, but there are also applications in which it is desirable only to inhibit the growth of specific microorganisms, such as applications in the dairy industry, where stabilization of the proliferation of bacterial flora, rather than its complete elimination, is required.

[0020] The invention is characterized by a number of advantages. First of all, its local mode of action should be mentioned. Only microorganisms that come into contact with the composite are eliminated. The material does not spread in the body or in the environment (it is insoluble, and the active substance is not released). Studies have shown that it is safe for human tissues and cells and is characterized by a broad spectrum of activity. The elimination of bacteria (including drug-resistant strains) and fungi, as well as a noticeable reduction in viral proliferation on coatings produced from MS-Cu5, has been experimentally confirmed. Moreover, owing to the use of a silica matrix, the material exhibits ease of forming coatings and can be mixed with two-component polymers.

[0021] Embodiments of the invention are illustrated in the figures, in which:Figs. 1 and 2 show bar charts presenting the antibacterial activity of a coating made of the porous silica composite material with copper (MS-Cu5), as compared with the control in the form of a well without the material, against various bacterial strains in Fig. 1 and fungal strains in Fig. 2 (X-axis). The studies were conducted at an incubation temperature of 37°C, which applies to all further studies on bacteria, fungi, and viruses. This is the standard temperature for testing antimicrobial activity in relation to the human body. The Y-axis shows the antimicrobial activity, the measure of which is the ability to form a biofilm. The determinations were carried out separately on three microtiter plates, and therefore the OD value (absorbance) of the negative control and the cut-off value derived therefrom (ODc), which allowed the categorization of the strength of biofilm formation, were different in each case (separate for each plate). The obtained OD values for each isolate in the tested plate were averaged (due to triple repetition). The cut-off value, i.e. the cut-off OD (ODc), for each plate was calculated according to the formula: ODc = mean OD value of the negative control + (3 x standard deviation, SD, of the negative control). The biofilm-forming ability was then determined by classifying the tested isolate into one of four groups (i.e. non-biofilm producer / weak / moderate / strong biofilm producer) on the basis of the calculated OD values: - no biofilm formation: isolate OD < ODc (the microorganisms are completely eliminated); - weak biofilm formation: ODc < isolate OD < 2 x ODc (the microorganisms are eliminated to a large extent);

[0022] - moderate biofilm formation: 2 x ODc < isolate OD < 4 x ODc (the microorganisms are partially eliminated and their reproduction is hindered);

[0023] - strong biofilm formation: 4 x ODc < isolate OD (the microorganisms are not eliminated and proliferate or replicate without hindrance).

[0024] Fig. 3 shows the effect of the coating made of the MS-Cu5 composite on the proliferation of normal cells (fibroblasts), as compared with the control in the form of a well without the material, after 72 hours. The study was carried out in three independent experiments, with eight determinations for a given concentration in each of them (n = 24), using the MTT assay. The MTT assay consists in the spectrophotometric measurement of the colored — purple — product, formazan, which is formed during metabolism by mitochondrial cellular enzymes. The intensity of the color, i.e. the amount of the colored product formed, is directly proportional to the number of living cells.A suspension of fibroblasts in DMEM / F12 culture medium, in an amount of 0.5 x 105per well, was poured onto 96-well plates coated with a coating made of the MS-Cu5 composite and onto control plates without the material. After 72 h, 10 pl of MTT solution (5 mg / ml in PBS with Ca+2 / Mg+2ions) was added to each well. After 3 hours, the formed formazan crystals were dissolved in SDS solution (10% SDS in 0.01 N HCI). After 24 hours, the optical density of the product was measured at a wavelength of A. = 570 nm using an Infinite M200 Pro microplate reader (Tecan, Mannedorf, Switzerland). Taking the control samples as 100%, this analysis made it possible to determine the degree of survival / proliferation of normal cells cultured on the tested substrates.

[0025] The test results indicate only a slight decrease in the survival of fibroblast cells, confirming the absence of cytotoxicity of the material toward the cells.

[0026] Another test used to assess the cytotoxicity of the investigated composite is the LDH enzyme release assay. This is a method used in cell viability analysis, based on the determination of the activity of lactate dehydrogenase (LDH) released from damaged or dead cells into the culture medium. Lactate dehydrogenase is a cytoplasmic enzyme present in all cells. When cells are exposed to a toxic effect, the integrity of their cell membrane is disrupted, and the LDH enzyme leaks out of the cells and passes into the medium. Thus, the assessment of damage is carried out by measuring the activity of the LDH enzyme after exposure. A commercially available cytotoxicity detection kit (LDH) was used in the experiment (https: / / www.thermofisher.com / order / catalog / product / C20301).

[0027] A suspension of fibroblasts in DMEM / F12 culture medium, in an amount of 0.5 x 10A5 per well, was poured onto 96-well plates coated with a coating made of the MS-Cu5 composite and with a coating made of mesoporous silica. After 72 h, the medium / culture medium without cells, in an amount of 50 pl per well, was transferred to a new optically clear flatbottom 96-well plate, and 50 pl of the reaction mixture was added to each well, after which the plate was incubated for 30 minutes at room temperature in the dark. After the 30-minute incubation, the reaction was stopped by adding a stop solution, and the absorbance was measured at a wavelength of X = 490 nm using a microplate reader (Tecan, Mannedorf, Switzerland).The negative control (ctr) and the positive control (ctr+) consisted of the medium of fibroblasts seeded into wells not coated with the tested substances, with the difference that the medium of ctr+ was supplemented with the LDH enzyme reagent.

[0028] The maximum LDH enzyme activity (Max) was determined by collecting the medium from fibroblasts seeded into wells without the tested carriers, while simultaneously lysing the cells with the buffer included in the kit, which released the enzyme into the medium and provided a comparison with the control, in which no disruption of cell membrane integrity and no leakage of the enzyme into the medium had occurred.

[0029] Fig. 4 shows the results of LDH enzyme release in the presence of the MS-Cu5 composite and pure mesoporous silica. The low values of released LDH enzymes for both samples indicate the low cytotoxicity of the MS-Cu5 composite, which is comparable to the cytotoxicity of pure mesoporous silica. This means that the introduction of copper phosphonate functional groups into the structure of mesoporous silica does not increase its cytotoxicity toward healthy skin cells, namely fibroblasts.

[0030] Subsequently, the toxicity of the composite toward tissues was analyzed. Table 1 shows the effect of the coating made of MS-Cu5 on body tissues. This was illustrated by examining the degree of lipid peroxidation in cardiac muscle tissue after treatment with the tested compound in comparison with tissue not subjected to such treatment. The degree of peroxidation was determined spectrophotometrically using the TBARS (thiobarbituric acid reactive substances) assay. Absorbance was measured at 532 nm. The TBARS concentration was calculated from a standard curve for malondialdehyde. On the basis of the phenotypic analysis of tissue sections collected from the cardiac muscle of a pig (Sus scrofa) after treatment with the specified compound MS-Cu5, and after genotypic analysis consisting in the assessment of oxidative damage to genomic DNA following additional digestion with the Fpg protein as a marker of oxidative stress in the lipid peroxidation analysis, no damage was found. The results indicate the absence of any damaging effect of the tested material on the examined tissues.

[0031] Table 1. Degree of lipid peroxidation in cardiac muscle tissue after treatment or no treatment with the tested compound MS-Cu5, as determined spectrophotometrically.Lp. Control without Control after Test sample Test sample after treatment of the treatment of the without treatment of the cardiac muscle cardiac muscle treatment of the cardiac muscle tissue and tissue with MS- cardiac muscle tissue with MS- without digestion Cu5 and tissue with MS- Cu5 and with Fpg protein digestion with Cu5 and without digestion with Fpg protein digestion with Fpg protein Fpg protein

[0032] % damage to Not detected Not detected Not detected Not detected cardiac muscle

[0033] tissue

[0034]

[0035] The subject of the invention is described below in examples of its embodiments covering all its categories and not limiting its scope, as well as in examples of its use.

[0036] Example 1. Antimicrobial coating made of a porous silica composite with copper (MS-Cu5)

[0037] 1.1 The coating was obtained by acetone embedding of the MS-Cu5 nanocomposite into a plastic material, for example polystyrene. In order to prepare such a coating, a suspension of MS-Cu5 is prepared in a solvent that reacts with the coated plastic material. In the case of polystyrene, this is acetone. For this purpose, MS-Cu5 is suspended in the solvent in a proportion of 1 part by weight of the material to 10 to 100 parts by weight of the solvent, depending on the application method described below. The suspension may be applied to the plastic material using a manual tool, for example a brush (in this case a higher concentration of the material in the solvent is preferred), or by a spray method, for example using an airbrush or a spray gun (in this case a lower concentration of the material and / or the use of a suitably wide spray nozzle is preferred).

[0038] 1.2 The coating was obtained by forming the MS-Cu5 composite in the form of a solid, thin layer directly on the object to be coated (e.g. glass, metal, silicon, or made of certain plastics), by growth using the Stober method. In this manner, urological catheters, NiTi implants, and other medical components placed in the patient's body may be coated. It is also possible to coat all kinds of equipment elements of medical facilities that are made of plastics, glass, ormetals. For this purpose, (1) 0.18 mmol of a stoichiometric mixture of tetraethyl orthosilicate (TEOS, CAS: 78-10-4, Sigma-Aldrich) and methyl 3-(trimethoxysilyl)propyl methylphosphonate (PPTES, CAS: 67812-17-3, Sigma-Aldrich) is dissolved (for example by stirring with a magnetic stirrer or in an ultrasonic bath) in 15 ml of distilled water and 35 ml of ethanol. In order to obtain a concentration of 5%, the molar proportions between TEOS and PPTES should be 19:1, that is, the solution should contain 0.171 mmol of TEOS and 0.009 mmol of PPTES. Thus, in order to prepare about 50 ml of the Stober solution, 0.036 g of TEOS and 0.001 g of PPTES should be added. To obtain other concentrations, the proportions between the two silicon sources should be modified accordingly, while maintaining the total number of moles of the sum of TEOS and PPTES. To this solution, 0.08 g of cetyltrimethylammonium bromide (CTAB, CAS: 57-09-0, Sigma-Aldrich) should be added. When it has dissolved, 0.005 ml of 25% aqueous ammonia should be added. The solution should be stirred (for example on a magnetic stirrer) at room temperature for two hours in a closed container. The procedure may be scaled in order to obtain an appropriate amount of solution by multiplying the amounts of the reagents by an appropriate factor.

[0039] Next, (2) the solution should be poured into a Teflon-lined hydrothermal autoclave of suitable capacity. It may also be another container capable of being tightly sealed. The object to be coated with the material layer should be placed in the solution. The tightly sealed autoclave (container) is placed at a temperature of 60°C for a period of 24 to 72 hours, depending on the desired layer thickness. After this time, the object should be removed and rinsed with water. In order to strengthen the layer, the object should be kept at a temperature of 100-120°Cfor l2 hours. After this time, the CTAB should be removed by soaking the object in ethyl alcohol for 20 minutes in an ultrasonic bath and pouring it off (the alcohol), repeated three times.

[0040] The obtained thin films containing phosphonic acid ester groups should be subjected to silanization in order to avoid undesired side reactions between surface hydroxyl units and phosphonic acid groups (3). For this purpose, the obtained thin films are immersed in a solution of chlorotrimethylsilane (CAS: 75-77-4, Pol-Aura) in toluene (2% solution) for 12 hours. After this time, the coated substrates should be rinsed with toluene three times byimmersion and treatment in an ultrasonic cleaner, and then dried thoroughly. A vacuum dryer set to 90°C and 0.2 Pa may be used for this purpose.

[0041] The hydrolysis of phosphonic acid ester groups to phosphonic acid groups (4) can be carried out in two ways. The first is a selective two-step method (4a). For this purpose, the coated materials are immersed in a solution of bromotrimethylsilane (CAS: 2857-97-8, Sigma-Aldrich) in dichloromethane (1% solution) for 12 hours. After this time, the coated items should be rinsed with toluene three times by immersion and treatment in an ultrasonic cleaner, and then dried thoroughly. A vacuum dryer set to 90°C and 0.2 Pa may be used for this purpose. To complete the procedure, the dried items are immersed in a 1:1 mixture of water and methanol for 2 hours. After this time, the coated items should be dried thoroughly. A vacuum dryer set to 90°C and 0.2 Pa may be used for this purpose. An alternative method is acid hydrolysis (4b). For this purpose, the coated materials are immersed for 12 hours in a mixture of 35-38% hydrochloric acid (40%) and ethyl alcohol (60%). After this time, the coated items should be rinsed with toluene three times by immersion and treatment in an ultrasonic cleaner, and then dried thoroughly. A vacuum dryer set to 90°C and 0.2 Pa may be used for this purpose.

[0042] The final step is the functionalization of the obtained layers (5), which is carried out by immersing them in a 1% solution of copper acetylacetonate (CAS: 13395-16-9, Pol-Aura) in tetrahydrofuran (THF) for 12 hours. The solution may be reused multiple times. Alternatively, another weak copper salt dissolved in an appropriate solvent may be used. After this time, the coated items should be rinsed with tetrahydrofuran three times by immersion and placement in an ultrasonic cleaner for at least 1 hour in order to remove any residual copper salt, and then dried thoroughly. A vacuum dryer set to 90°C and 0.2 Pa may be used for this purpose.

[0043] 1.3 Coating obtained by forming the material directly on the substrate by dip-coating or spincoating.

[0044] Using this method, objects made, for example, of glass, metals, silicon, or certain plastics may be coated. For this purpose, a solution (1) should be prepared containing the following molar ratios of reagents: 0.9 TEOS : 0.1 PPTES : 75 EtOH : 20 H2O : 0.02 HCI : 0.012 P123, where TEOS denotes tetraethyl orthosilicate, PPTES denotes methyl 3-(trimethoxysilyl)propyl methylphosphonate, and P123 denotes the surfactant commercially known as Pluronic P123(CAS: 9003-11-6, Sigma-Aldrich). The solution is prepared in two stages. For example, to prepare approximately 60 mL of solution for use in dip-coating or spin-coating (the procedure may be scaled), in the first stage 2.0 g of ethanol and 2.5 g of water (H2O) with hydrochloric acid (HCI) are used in proportions such that the pH is 1.25. Then, TEOS and PPTES are added to the prepared solution in the appropriate molar ratios. To obtain a 5% concentration of functional groups, these amounts are 3.328 g and 0.195 g, respectively. To obtain other proportions, the amounts of TEOS and PPTES should be recalculated accordingly. The second stage involves preparing, in a separate vessel, an ethanolic solution of the surfactant Pluronic P123 by adding 1.2 g of P123 to 54.4 g of ethanol. Both solutions should be stirred at room temperature for 2 hours. After this time, the two solutions should be combined and stirred again for a further 3 hours at room temperature. Finally, 4 g of an aqueous hydrochloric acid solution at pH = 1.25 should be added. The solution should then be aged for approximately 2 hours before beginning the deposition of thin films. Deposition of the films on the coated objects may be carried out by the dip-coating method (2a). For this purpose, the object to be coated should be immersed in solution (1) and then slowly withdrawn under conditions of at least 70% humidity, which ensures the formation of a smooth and uniform layer. Under the same humidity conditions, the coated object should be left until the layer has dried. The films may also be obtained by the spin-coating method (2b), which is mainly applicable to coating flat surfaces. For this purpose, the object should be set in rotational motion, and the rotation speed should be selected depending on the desired film thickness, with higher speed resulting in a thinner film. Then, under conditions of at least 70% humidity, solution (1) should be applied near the axis of rotation in an amount sufficient to cover the entire surface. Under the same humidity conditions, the coated object should be left until the layer has dried. Further processing of objects coated according to procedures (2a) and (2b) is identical to that used in the method described in section 1.2, namely stages (3) to (5).

[0045] The porous silica coating preparation methods described above lead to the formation of a composite with antimicrobial properties, the use of which in preventing the growth of bacteria, fungi, and / or viruses is the subject of the present invention. It should be emphasized that these coatings are fully safe for healthy cells of living organisms, as confirmed by studies of the survival / proliferation of normal cells (fibroblasts) and cytotoxicity.Example 2. The antibacterial activity of the coating made from the MS-Cu5 composite against bacteria of the genus Enterococcus, using Enterococcus faecalis ATCC 51299— a strain exhibiting high-level aminoglycoside resistance (HLAR) and vancomycin resistance (with the vanB gene present), i.e. VRE (vancomycin-resistant Enterococcus)— as well as Enterococcus faecium ATCC 700221 (VRE), results in inhibition of bacterial growth and reduction of bacterial biofilm formation. Incubation was carried out at 36.5°C, Fig. 1. E. faecalis and E. faecium strains may cause very serious infections at various sites, for example urinary tract infections. The effect of inhibiting biofilm formation on surfaces coated with the MS-Cu5 composite may be used, for example, for coating the surfaces of medical equipment such as hospital bed frames and medical carts, as well as medical accessories having direct contact with the patient's body, such as catheters or intubation tubes. The conducted studies also indicate that coatings made from MS-Cu5 are of particular importance in applications related to their biocidal activity against drug-resistant hospital strains. In light of the experience of the 2019-2022 pandemic and awareness of the threats associated with the spread of pathogenic microorganisms, the use of MS-Cu5 coatings to cover all kinds of high-touch surfaces in public spaces is also justified.

[0046] Example 3. The antibacterial activity of the MS-Cu5 composite against Gram-positive bacteria, specifically staphylococci (as exemplified by Staphylococcus aureus NCTC 12493 - a methicillin-resistant MRSA strain, and Staphylococcus aureus ATCC 33592— a methicillin-resistant MRSA strain also resistant to gentamicin) and a representative of the streptococci, Streptococcus pneumoniae ATCC 49619 - with reduced susceptibility to penicillin - leads to effective limitation of bacterial growth and bacterial biofilm development. Incubation was carried out at 36.5°C, Fig. 1. The study used key species of Gram-positive cocci that exhibit antibiotic resistance, which is particularly important for epidemiological reasons. The activity of the tested substances / surface preparations may be applied in the creation of coatings for the surfaces of medical equipment and medical accessories, including disposable materials having direct contact with the patient's body, such as intubation tubes and catheters. The study was conducted using the surfaces of 96-well plates coated with the MS-Cu5 composite and uncoated control surfaces, on which biofilm produced by the above-mentioned bacterial strains was grown. Its growth was visible, as indicated by high OD values, on the control surfacenot coated with the MS-Cu5 composite coating. On the surface coated with the MS-Cu5 composite coating, the growth of bacterial biofilm was inhibited.

[0047] Example 4. The antibacterial activity of the MS-Cu5 composite coating against bacteria of the genus Klebsiella (Klebsiella pneumoniae NCTC 13438), Fig. 1. The K. pneumoniae strain used in the study, which produces KPC-type carbapenemase (Klebsiella pneumoniae carbapenemase), belongs to the epidemiologically important group of Gram-negative bacilli that may cause hospital-acquired infections. The reduction in growth and biofilm formation achieved by the investigated MS-Cu5 coatings makes their use possible in the prevention of infections caused by multidrug-resistant bacilli. This may be applied, for example, to coating the surfaces of medical equipment, local dressings, and in procedures aimed at preventing hospital-acquired infections. The study was conducted using the surfaces of 96-well plates coated with the MS-Cu5 composite coating and uncoated control surfaces, on which biofilm produced by the above-mentioned bacterial strains was grown. Its growth was visible, as indicated by high OD values, on the control surface not coated with MS-Cu5. On the tested surface containing the MS-Cu5 composite coating, the development of the bacterial biofilm was inhibited.

[0048] Example 5. The antifungal activity of MS-Cu5 composite coatings against fungi of the genus Candida (Candida glabrata ATCC MYA - 2950, Candida glabrata ATCC 15126, Candida parapsilosis ATCC 22019, Candida albicans ATCC 14053, Candida albicans ATCC 10231, Candida tropicalis ATCC 13803, Candida krusei ATCC 14243), Fig. 2.

[0049] The obtained results indicate an inhibitory effect on the growth and biofilm formation of five key yeast species of the genus Candida. This creates the possibility of applying the mechanism of action of the investigated composite to coating the surfaces of medical equipment and medical accessories, including disposable materials having direct contact with the patient's body, such as catheters and intubation tubes. The study was conducted using the surfaces of 96-well plates coated with the MS-Cu5 composite coating and uncoated control surfaces, on which biofilm produced by the above-mentioned strains was grown. Its growth was visible, as indicated by high OD values, on the control surface not coated with MS-Cu5. On the surface coated with the MS-Cu5 composite coating, its growth was inhibited.Example 6. The action of the coating made of the MS-Cu5 composite inhibiting the development of viruses with respect to: herpes virus type 1 (HSV-l, McKrae strain, Arhus University, Denmark), vaccinia virus (WR strain, ATCC® VR-1354), influenza virus type A (H1N1, strain A / Virginia / ATCCl / 2009) (ATCC® VR-1736), human adenovirus type 5 (ATCC® VR-5), human coronavirus strain 229e (ATCC® VR-740). The activity study of the coatings was conducted according to standard EN 16777:2018.

[0050] Tables 2-6 present the effect of the active sample (containing the coating made of the MS-Cu5 composite) on the development of viruses, determined according to standard EN 16777:2018. For all examined viruses, approximately 50% reduction in virus titer was obtained.

[0051] Table 2. Reduction of HSV-l titer. CM - control material, AM - active material (MS-Cu5 composite)

[0052] Test variants Reduction in PFU / ml

[0053] CM >5,2 ± 11,22 %

[0054]

[0055] AM >64,23 ± 5.2 %

[0056] Table 3. Reduction of VACV titer. CM - control material, AM - active material (MS-Cu5 composite)

[0057] Test variants Reduction in PFU / ml

[0058] CM >3,9 ± 1,3 %

[0059]

[0060] AM > 58,4 ± 7,8 %

[0061] Table 4. Reduction of H1N1 titer. CM - control material, AM - active material (MS-Cu5 composite).

[0062] Test variants Reduction in PFU / ml

[0063] CM >0,0 ± 0,0 %

[0064]

[0065] AM > 45,8 ± 1,22 %

[0066] Table 5. Reduction of HAdv-5 titer for all exposure variants. CM - control material, AM -active material (MS-Cu5 composite).

[0067] Test variants Reduction in PFU / ml

[0068] CM > 2,4 ± 0,89 %

[0069]

[0070] AM > 69,76 ± 6,9 %Table 6. Reduction of HCoV 229e titer for all exposure variants. CM - control material, AM -active material (MS-Cu5 composite).

[0071] Test variants Reduction in PFU / ml

[0072] CM > 0,99 ± 0,2 %

[0073]

[0074] AM > 51,5 ± 0,99 %

[0075] Example 6.

[0076] The antiviral activity of coatings obtained by the acetone embedding method was examined. The study was conducted according to standard PN-EN 16777:2019-01, "Chemical disinfectants and antiseptics. Quantitative non-porous surface test for the evaluation of virucidal activity of chemical disinfectants used in the medical area without mechanical action -Test method and requirements (phase 2)." The results are presented in Tables 2-6 above.

[0077] Example 7.

[0078] The stability of the MS-Cu5 material in solution was investigated. For this purpose, studies on the release of copper and phosphorus under physiological conditions were conducted. The tests were carried out in saline solution (0.9% NaCI) at 37°C using a single-use vial system (one vial per time point).

[0079] Ten suspensions were prepared, each containing 0.10 g of powdered material in 10 mL of saline solution, placed in 20 mL borosilicate vials sealed with a cap. The samples were stirred on a multi-position magnetic stirrer at 200 rpm in a thermostatically controlled chamber at 37°C.

[0080] Every 24 hours, one vial was collected, and the suspension was centrifuged at 5°C at 25,000 rpm for 30 minutes. The supernatant was then filtered through a 0.22 pm membrane, after which the filtrate was immediately acidified to approximately 1% HNO3. For ICP analysis, 9 mL of the suspension was collected in order to standardize the measurement and enable decantation of the liquid above the sediment without the risk of disturbing it. The copper and phosphorus contents in the filtrates were determined by ICP-MS.

[0081] Phosphorus was determined in parallel as an indicator of the transfer of solid or colloidal particles from the SBA-POO2Cu material; the presence of phosphorus in the filtrate (<0.22 pm)was interpreted as a signal of residual solid phase and was used to distinguish truly dissolved copper (Cu2+) from copper bound to particles.

[0082] Fig. 5 presents the results of copper and phosphorus release into saline solution (0.9% NaCI, 10 mL), in which 0.10 g of powdered MS-Cu5 material was suspended. The X-axis shows the duration of the experiment, after which a suspension sample was collected, and the Y-axis indicates the concentration of the examined element in the supernatant obtained after filtering the powder from the suspension.

[0083] It was found that the copper concentration in the filtrate increased gradually and monotonically from 0.122 mg / L to 0.381 mg / L over a period of 10 days. The observed release profile was approximately linear and did not exhibit an initial rapid release ("burst release") effect, which indicates controlled, slow release of only a small, labile fraction of copper.

[0084] The total copper release after 10 days corresponded to only approximately 0.030-0.094% of the total Cu content in the sample (0.10 g of material), which in absolute values amounted to approximately 3.81 pg of Cu per vial. Such a low level of release confirms the high chemical stability and the durable binding of copper ions within the structure of the MS-Cu5 material.

[0085] At the same time, the phosphorus concentration in the filtrate remained practically constant within the narrow range of 0.054-0.081 mg / L throughout the entire study period. This corresponded to the release of only approximately 0.025-0.038% of the total phosphorus content. The absence of an upward trend and the low, stable values indicate a lack of hydrolysis of the phosphonate groups responsible for structural anchoring, while the detected amounts of phosphorus are attributed to trace transfer of particles below the filtration threshold or to the background of the solution.

[0086] These results clearly indicate that the MS-Cu5 material is characterized by very high stability under physiological conditions, exhibiting:

[0087] • minimal copper leaching (less than 0.1% after 10 days),

[0088] • no significant release of phosphorus,

[0089] • no deterioration of the anchoring structure.Such properties make the material particularly useful in applications requiring controlled bioavailability of metal ions while maintaining the structural integrity of the carrier.

[0090] Example 8.

[0091] The ability of the MS-Cu5 composite to generate reactive oxygen species (ROS) was investigated. For this purpose, studies were carried out using the fluorogenic probe CellROX™ Green (Invitrogen, Life Technologies, USA), which enables direct detection of ROS in the environment of the tested sample.

[0092] The CellROX™ Green reagent was dissolved in saline solution (0.9% NaCI) at a volume ratio of 1:4. Next, grains of mesoporous silica containing copper (MS-Cu5) were placed on a glass substrate, after which a drop of the prepared probe solution was applied onto their surface. Observations were carried out using a fluorescence microscope (Leica DMi8) equipped with a 40x objective, recording both bright-field images and the fluorescent signal. Microscopic images were collected immediately after addition of the probe.

[0093] Fig. 6 presents fluorescence microscopy images of the MS-Cu5 material showing the formation of reactive oxygen species (ROS). The images show three different locations of the same MS-Cu5 powder sample (a, b, and c). The upper image was obtained in bright-field mode, while the lower image was obtained in fluorescence mode. A distinct green fluorescence signal was observed, localized inside the silica grains and in their immediate surroundings. The intensity and localization of the signal indicate the formation of reactive oxygen species within the mesoporous matrix and in the vicinity of the copper centers.

[0094] The absence of a significant fluorescent signal outside the area of the material particles indicates that ROS generation is surface-based and local in nature, limited to the structure of the composite. This means that the biological effect, in particular the antimicrobial activity, is manifested mainly in the direct contact of microorganisms with the surface of the material. The obtained results are consistent with the spectroscopic data and theoretical modelling and confirm that the MS-Cu5 composite demonstrates the ability to catalytically generate ROS in a physiological environment. This mechanism is associated with the presence of Cu(ll) centers anchored in a phosphonate environment within the mesopores.Based on the available data, it is assumed that different types of reactive oxygen species may coexist within the structure of the material, including: singlet oxygen Ch), superoxide anion radical (Ch*-), hydroxyl radical (*OH), and hydrogen peroxide (H2O2). The exact identification of the individual ROS species has not been unambiguously resolved; however, their combined presence is responsible for the observed catalytic activity of the material.

[0095] These results indicate that the MS-Cu5 composite acts as a heterogeneous catalyst generating ROS under physiological conditions, with this process occurring locally within the material, which ensures high efficiency of action while simultaneously limiting the migration of active species into the external environment.

[0096] Example 9.

[0097] The enzymatic activity of composite coatings made from the MS-Cu5 material, as well as of the MS-Cu5 material in powder form, was investigated. The aim of the experiment was to evaluate the ability of MS-Cu5 coatings and the MS-Cu5 material itself to generate reactive oxygen species (ROS) and to catalyze the oxidation of potassium iodide. This phenomenon was used as a model reaction to confirm the catalytic activity of the material, analogous to enzymatic activity.

[0098] The generation of ROS by materials containing transition metal ions is a well-known phenomenon and is often exploited in studies on materials with catalytic, antibacterial, or biomimetic properties.

[0099] The method is based on the oxidation of iodide ions (I-) to molecular iodine (l2) in the presence of reactive oxygen species generated by the catalytic material. The iodine formed then reacts with starch, producing a characteristic complex with an intense blue color.

[0100] This reaction is a classic chemical test used for the detection of iodine and, indirectly, for the detection of oxidation processes occurring in the system. The appearance of a blue coloration indicates the formation of iodine and, thus, the presence of oxidative processes associated with ROS generation.

[0101] The reaction scheme can be presented in simplified form:

[0102] 2I~ I2

[0103] I2+ starch -> starch — iodine complex (blue color)9.1. Description of Experiment 1

[0104] In the experiment, MS-Cu5 material coatings prepared by the method described in Example 1.1 were used. The coatings were immersed in a solution containing potassium iodide (KI) and starch. The reaction system was observed visually for changes in the color of the solution. Two material variants were used in the experiment:

[0105] • MS-Cu5 material coatings,

[0106] • control coatings made from pure SBA-15 silica, containing no copper.

[0107] 9.2. Description of Experiment 2

[0108] In the experiment, MS-Cu5 in powder form was used, and the reference material was pure SBA-15 silica in powder form. The powders were immersed in a solution containing potassium iodide (KI) and starch. In addition, a clean glass test tube also served as a control. The reaction system was observed visually for changes in the color of the solution.

[0109] 9.3. Observation results

[0110] After immersion of copper-containing silica coatings and powders in the KI solution with starch, a gradual appearance of an intense blue coloration of the solution was observed in both cases , as shown in Fig. 7 , presenting the enzymatic activity test performed on the coating made of the MS-Cu5 material (three upper wells) and the control in empty wells (three lower wells). The observations were carried out: (a) immediately after addition of the KI solution with starch, (b) after 1 hour, (c) after 2 hours, and (d) after 4 hours.

[0111] A similar effect was observed for the materials in powder form, as shown in Fig. 8, which presents the enzymatic activity test performed on powdered MS-Cu5 material (tube 1 from the left), pure SBA-15 silica in powder form (tube 2 from the left), and the control test in a clean test tube (tube 3 from the left). The observations were carried out: (a) immediately after addition of the KI solution with starch, (b) after 1 hour, (c) after 2 hours, and (d) after 4 hours. The blue coloration is characteristic of the iodine-starch complex, formed as a result of the oxidation of iodide ions to molecular iodine. This observation indicates that, in the presence of copper-containing coatings, the oxidation of iodide took place, which suggests the generation of reactive oxygen species in the system. In the case of the powders and control layers made of pure SBA-15 silica devoid of copper, no change in the color of the solution was observed. The solution remained colorless throughout the entire duration of the experiment.9.4. Interpretation of the results

[0112] The observed coloration of the solution in the presence of copper-containing coatings and powders indicates that the material exhibits catalytic activity leading to the oxidation of iodide ions. This process may be interpreted as the effect of the generation of reactive oxygen species (ROS), which act as oxidizing agents in the reaction system. The absence of a reaction in the case of powders made of pure SBA-15 silica confirms that the presence of copper in the structure of the material is the key factor responsible for the observed catalytic activity. The obtained results therefore indicate that MS-Cu5 powders and layers may exhibit properties similar to enzymatic activity, consisting in catalyzing oxidation reactions in an aqueous environment.

[0113] 9.5. Conclusions

[0114] The conducted experiment demonstrated that the MS-Cu5 material, both in the form of powders and coatings prepared by the method described in 1.1, is capable of initiating the oxidation of potassium iodide in an aqueous solution, as evidenced by the formation of the characteristic iodine-starch complex with an intense blue coloration. This observation indicates the generation of reactive oxygen species in the presence of copper-containing coatings. At the same time, the absence of a reaction in the case of pure silica confirms that this effect is associated with the presence of copper in the structure of the material. The obtained results suggest that the investigated nanocomposites may exhibit biomimetic catalytic properties resembling the activity of oxidative enzymes.

[0115] Example 10.

[0116] The dependence of the enzymatic activity of composite coatings made from the MS-Cu5 material, as well as of the MS-Cu5 material in powder form, on light was investigated. The aim of the experiment was to evaluate the ability of MS-Cu5 coatings and the MS-Cu5 material itself to generate reactive oxygen species (ROS) and to catalyze the oxidation of potassium iodide in the presence of light, as well as in its absence.

[0117] The study was conducted analogously to Example 9, except that the experiment was carried out in complete darkness. For this purpose, the room was completely darkened, and the culture plates with the layers and the vials containing the suspensions were additionally protected from light with aluminum foil.A gradual change of the KI solution with starch to blue was observed in the wells containing the MS-Cu5 coating, as well as in the vials containing MS-Cu5 powder. No change in the color of the solution was observed in the remaining solutions. This result demonstrates the generation of reactive oxygen species by the MS-Cu5 material in the absence of light. It confirms the thesis that light is not a necessary factor for the catalytic generation of ROS.

Claims

Patent claims1. A porous silica composite material containing copper (MS-Cu5), wherein the copper ions are chemically anchored in a mesoporous silica matrix by means of phosphonate groups, for use in the prevention of bacterial infections, wherein the bacteria are selected from the genera Enterococcus, Staphylococcus, Streptococcus, or Klebsiella, and / or fungal infections, wherein the fungi are selected from the genus Candida, or viral infections.

2. The composite material for use according to claim 1, characterized in that the bacteria are selected from the group comprising Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, or Klebsiella pneumoniae.

3. The composite material for use according to claim 1, characterized in that the fungi of the genus Candida are selected from the group comprising: Candida glabrata, Candida parapsilosis, Candida albicans, Candida tropicalis, Candida krusei.

4. The composite material for use according to claim 1 or 2, characterized in that the bacteria are antibiotic-resistant.

5. The composite material for use according to claim 1, characterized in that the viruses are selected from the group comprising: herpes simplex virus type 1, vaccinia virus, influenza A virus, human adenovirus type 5, human coronavirus strain 229E.

6. A use of the porous silica composite material containing copper (MS-Cu5), as defined in claim 1, for the manufacture of a coating for use in the prevention of bacterial infections, inhibiting bacterial growth or reducing bacterial proliferation, wherein the bacteria are selected from the genera Enterococcus, Staphylococcus, Streptococcus, or Klebsiella, and / or fungal infections, wherein the fungi are selected from the genus Candida, or viral infections.

7. A coating comprising the composite material containing copper (MS-Cu5), as defined in claim 1, for use in inhibiting the growth of microorganisms such as bacteria, wherein the bacteria are selected from the genera Enterococcus, Staphylococcus, Streptococcus, or Klebsiella, fungi, wherein the fungi are selected from the genus Candida, and / or viruses.

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