Procedure to obtain an antimicrobial composite material, material thus obtained and use thereof

Abstract

The present invention refers to an antimicrobial composite material based on a semiconductor (WO3), a metal (Ag), and a carbon support. The present invention also relates to the procedure of its preparation from chitosan and soluble salts of Ag and W. The antimicrobial material can be used as a surface coating or additive in many different areas, such as automotive and construction industry.

Description

[0001] DESCRIPTION

[0002] PROCEDURE TO OBTAIN AN ANTIMICROBIAL COMPOSITE MATERIAL, MATERIAL THUS OBTAINED AND USE THEREOF

[0003] The invention relates to a procedure to obtain a semiconductor / metal / carbon composite material with antimicrobial properties. Accordingly, the instant application further relates to said material and use thereof, preferably, as a surface coating or additive in automotive and construction industry.

[0004] STATE OF ART

[0005] Until now, the continuing challenge of infectious diseases caused by pathogenic microorganisms (microbes such as fungi, bacteria, and virus) provoking a huge burden on global public health and economies means that infection diseases still remain a serious problem around the world. These pathogens are usually able to inhabit the surfaces of a multitude of objects and devices. One significant route for the spread of diseases is by the transmission of the pathogens through contact with contaminated surfaces. Surface treatments can help to reduce or even avoid these hazards.

[0006] The design of advanced materials by engineering antibacterial surfaces is a main application area of novel materials. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are one of the major bacterial pathogens that causes a variety of infections.

[0007] In view of this, the development of new antimicrobial technologies, not based on traditional antibiotics and free of pollutants or heavy metals, is of great interest.

[0008] Thus, some materials have been disclosed in the state of the art, such as the one described in the document: Michal Jakubczak et al. Materials Chemistry and Physics, 297, 2023, 127333, wherein activated carbon (C) was used as a substrate for in situ surface decoration with graphene oxide (GO) and bioactive TiO2 / Ag nanocomposite particles. The nanocomposite described in said document needs to be photoactivated on order to have an antimicrobial effect. This drawback is also present in many other antimicrobial compounds known in the state of the art. The document: H.l. Hamouda et al. Journal of Environmental Chemical Engineering, 9(2), 2021 ,105034 describes multi-walled carbon nanotubes decorated with silver nanoparticles for antimicrobial applications. In this case, we can observe a reduction in microbial concentration, rather than complete inhibition. Other problems affecting some prior art composites are their cytotoxic effect, low stability, and poor antibacterial effect. To solve the mentioned drawbacks of the antimicrobial materials of the prior art, the present invention provides a new composite material with outstanding stability and antibacterial activity against pathogens such as S. aureus and E. coli, low cytotoxicity and, advantageously, does not need to be photoactivated.

[0009] In order to solve the mentioned drawbacks of the antimicrobial materials of the prior art, the present invention provides a new one that presents excellent stability and antibacterial activity against pathogens such as S. aureus and E. coli, low cytotoxicity and, advantageously, does not need to be photoactivated.

[0010] DESCRIPTION OF THE INVENTION

[0011] The inventors have demonstrated that a composite material based on a semiconductor (WO3), a metal (Ag) and a carbon support (graphite) possesses excellent antimicrobial properties, stability and low cytotoxicity, so that it can be used as an antimicrobial additive or coating in a multitude of fields, such as in automotive and construction industry.

[0012] Also, a procedure to prepare said composite material has been developed.

[0013] Then, in a first aspect, the present invention relates to a procedure to obtain a composite material comprising: a) preparing a dispersion of chitosan in ethanol, b) preparing a dispersion of a-Ag2WC>4 in ethanol, c) adding the dispersion of a-Ag2WC>4 to the chitosan dispersion, wherein the amount of a-Ag2WC>4 is between 0.1 and 20% by weight in relation to the amount of chitosan, d) drying the resulting material at a temperature between 50 and 100 °C for 1 to 48h, e) heating the dried material obtained in step d) at a temperature between at 200- 700°C for 10-360 min, generating the final composite material.

[0014] The above-defined procedure leads to the generation of a composite material comprising particles of silver (Ag) and wolframium trioxide (WO3) both supported on carbon, in particular graphite (Ag / WCh on graphite).

[0015] In a preferred embodiment, the dispersion of step a) is prepared under ultrasounds, more preferably at a frequency between 20-50 kHz, with a preferred power range of 300-800W for preferably 5-60 minutes.

[0016] In a preferred embodiment, the dispersion of chitosan in step a) is prepared by adding chitosan to ethanol in proportion between 0.001g to 1.000g per 20 mL of ethanol.

[0017] Preferably, chitosan of medium molecular weight is used. The term "medium molecular weight" refers to the average molecular weight of chitosan, which is related to the average size of chitosan polymer chains. In numerical terms, the molecular weight of medium molecular weight chitosan is the range of 103to 104g / mol. Chitosan of medium molecular weight is commercially available.

[0018] In a preferred embodiment, the dispersion of a-Ag2WC>4 in step b) is prepared by adding a- Ag2WC>4 to ethanol in proportion between 0.001g to 1.000g per 20 mL of ethanol.

[0019] In a preferred embodiment, in step c), the amount of a-Ag2WC>4 is between 0.5 and 10% by weight in relation to the amount of chitosan, more preferably, between 1 and 5% by weight in relation to the amount of chitosan.

[0020] In a preferred embodiment, steps d) and e) are carried out in an oven.

[0021] In a preferred embodiment, a-Ag2WC>4 used in step b) is obtained through the method of coprecipitation in aqueous medium that comprises the next steps: i) preparing a solution of Na2WC>4.2H2O in water, ii) preparing a solution of AgNCh in water, iii) heating the solutions prepared in steps i) and ii) d at a temperature between 50 and 100°C, and then adding the AgNCh solution to the Na2WC>4.2H2O solution, thus generating a white precipitate, iv) stirring the solution containing the white precipitate obtained in step iii) for IQ- 360 min constantly, and then centrifuging and washing it with distilled water until pH between 5-7 is obtained in the wash water, v) drying the precipitate obtained in step iv) at a temperature between 50-100°C for 6-48h.

[0022] In step iv), when washing with water, the pH decreases, as the ions not used in the formation of a-Ag2WC>4 are removed.

[0023] In a preferred embodiment, the concentration of Na2WO4.2H2O in the solution of step i) is between 0.2x10-6and 2.0 mol / L. More preferably, between 1x10-5and 1x10-3mol / L.

[0024] In a preferred embodiment, the concentration of AgNCh in the solution of step ii) is between 0.4x1 O'6and 4.0 mol / L. More preferably, between 2x1 O'5and 2x1 O'3mol / L.

[0025] Preferred embodiments of temperature and time used were 20-100 °C and 0.1-6 hours.

[0026] A second aspect of the invention relates to the composite material obtainable by the procedure described in the first aspect of the invention (composite material of the present invention). Said composite material comprises silver (Ag) and WO3 particles deposited on graphite.

[0027] The particles of Ag present cubic crystalline structure with space group Fm-3m; the particles of WO3 monoclinic crystalline structure with space group P21 / c and the graphite has hexagonal crystalline structure and P63mc space group. These particles were obtained from the thermal decomposition of chitosan with a-Ag2WC>4, resulting in micrometric porous carbon particles (graphite) ranging from 10-80 pm of average diameter, as measured by Scanning electron microscopy (SEM), with WO3 and Ag particles on their surface both ranging in size from 80 to 2000 nm of average diameter, as measured by Scanning electron microscopy (SEM).

[0028] The above-described composite material can be used as a coating on a substrate to provide it with antimicrobial properties, preferably, with antibacterial properties. Preferably, the substrate is of stainless steel. This surface coatings can be used automotive and construction industries, for example, in the fabrication of handrails, handles, furniture, switches, among others.

[0029] In a preferred embodiment, the coating is fabricated by the spin coating method. This method preferably comprises the next steps:

[0030] -the substrate, preferably a stainless-steel substrate, is cut and sanded with sandpaper, and then the coating was performed using the spin coating method comprising:

[0031] - 0.01-1.00g g of the composite material of the present invention, 0.010-1.00g g of polyethylene glycol and 1-400 pL of non-ionic surfactant (preferably composed of polyoxyethylenes, which are polyglycol ether derived from polyethylene) are added per 1- 100 mL of water,

[0032] -the water solution formed in the previous step is left under stirring for 1-48 h for better system homogeneity,

[0033] - the homogenised water solution obtained in the previous step is added to the substrate under rotation (preferably at 300-5000 rpm) for 30-360 s and then the substrate is heating at 100-700 °C for 10-360 min for better adhesion of the coating. In a preferred embodiment, between 10 and 500pL of the homogenised water solution are added per 40 mm2of substrate surface. Preferably, the homogenised water solution is added to the substrate for 30-360 s.

[0034] The present invention also refers to a coated substrate comprising the composite material describe in the first aspect of the invention deposited on at least a portion of the substrate, preferably, on a substrate of stainless steel.

[0035] Also, the composite material described in the second aspect of the invention can be used as an antimicrobial (preferably, antibacterial) additive. More preferably, it can be used as an antimicrobial (preferably, antibacterial) additive of polymeric matrices, with preferably application in the automotive and construction industries. This additive can be directly incorporated in the manufacture of injectable and mouldable polymeric products.

[0036] In summary, the present invention provides a composite material based on a semiconductor (WO3), a metal (Ag) and graphite with antimicrobial properties and having notable advantages over composite materials already known in the prior art, as mentioned below: -displayed excellent stability

[0037] -it does not need to be photoactivated to exhibit its antimicrobial effect

[0038] -low cytotoxicity

[0039] -enhances antimicrobial activity (obtaining a semiconductor / metal nanoparticle heterojunction, that is, a material formed by two or more types of different materials, causes the electron flow between the semiconductor and the nanoparticle to increase due to the plasmonic effect, making it more efficient to generate reactive oxygen species (ROS), even in the dark)

[0040] -versatile application as an antimicrobial agent on different surfaces and as an antimicrobial additive in various polymeric bases. Also, the procedure used for the preparation of the material involves advantages: -one pot synthesis

[0041] -mild synthesis conditions (low temperatures and times)

[0042] -no generation of synthetic secondary residues.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 : (A) XRD patterns and (B) Raman spectra of the samples synthesized with without a-Ag2WO4(QT) and with 1 (QT1A), 2 (QT2A) and 4% (QT4A) wt of a-Ag2WO4.

[0046] FIG. 2: SEM images of the samples (A)QT; (B) QT1A; (C) QT2A and; (D) QT4A.

[0047] FIG. 3: (A) E.coli and (B) S. aureus growth as a function of different concentrations of the samples QT, QT1A, QT2A, and QT4A.

[0048] FIG. 4: Cell viability in % by the MTT method after 24 hours of exposure. Quantitative data analysis. (A) Treatment with QT, (B) QT1A, (C) QT2A, and (D) QT4A. * vs CTRL: * p<0.5; ** p<0.01 ; *** p<0.001 ; **** p<0.0001. Light microscopy images of 3T3 cells after 24 hours of exposure to (E) QT, (F) QT1A, (G) QT2A, and (H) QT4A. Final resolution of 100X.

[0049] FIG. 5: (A) SEM image of SSQT4A sample; (B) contact angle of the stainless steel (SS) and stainless steel coated with the QT4A (SSQT4A) sample; Time kill tests using SS and SSQT4A samples against (C) E. coli and (D) S. aureus.

[0050] EXAMPLES

[0051] To illustrate the invention, the following are examples of the invention that have been carried out by the inventors. Example 1 : Synthesis of a-Ag2WO4

[0052] In two separate beakers, 50.0 mL of distilled water was added, and 1x1 O'3mol of Na2WC>4.2H2O and 2x1 O'3mol of AgNCh, respectively, were added, its complete dissolution, the solutions were heated to 70°C, and then the AgNCh solution was added to the Na2WC>4.2H2O solution, generating a white precipitate. This solution was left under constant stirring for 20 min, and then it was centrifuged and washed with distilled water until pH = 7. After that, the precipitate was dried at 60°C for 12h.

[0053] Example 2: Synthesis of Ag / WOs Supported on Carbon

[0054] In a beaker, 1.00 g of chitosan (CAS: 9012-76-4, medium molecular weight, Sigma-Aldrich) was added to 20.0 mL of ethanol and dispersed under ultrasound for 5 min. In another beaker, 0.01 , 0.02 and 0.04g of a-Ag2WC>4 were also dispersed in 20.0 mL of ethanol for 5 min. After this time, the a-Ag2WC>4 dispersion was added to the chitosan dispersion for 10 min. The resulting material was oven dried for 5 h at 60 °C. After drying, the material was taken to a conventional oven at 400°C for 30 min, generating the final material. The samples without and with 1 , 2 and 4% wt of a-Ag2WC>4 were called QT, QT1 A, QT2A and QT4A.

[0055] Example 3: Preparation of a coating on Stainless steel substrate

[0056] The stainless-steel substrates were cut into sizes of 20mmx20mmx1 mm and sanded with 200, 400, 1000 and 1600 sandpaper. The coatings were performed using the spin coating method. 0.100 g of the sample QT4A, 0.040 g of Polyethylene glycol and 10 pL of non-ionic surfactant (Triton X-100) were added to 1 mL of distilled water. This solution was left under stirring for 12 h for better system homogeneity. 30 pL were added to the stainless-steel substrates under rotation of 3000 rpm for 30 s. After this process, the substrates were taken to a conventional muffle furnace at 200 °C for 30 min for better adhesion of the material.

[0057] Example 4: Characterization of the composites obtained in example 2

[0058] To analyze the structural properties of the samples after thermal treatment, X-ray diffraction (XRD) analysis was performed (Figure 1A). In fact, it is observed that the QT sample completely converts to graphite after thermal treatment, with hexagonal structure and P63mc space group. As for the samples with 1% (QT1A), 2% (QT2A) and 4% (QT4A) of a- Ag2WO4, besides the formation of graphite, the total decomposition of a-Ag2WO4 into WO3 (monoclinic with space group P21 / c) and Ag (cubic with space group Fm-3m), showing that the atmosphere generated in the combustion of chitosan favors redox processes for the formation of WO3 and Ag. In this way, there is a decrease in the temperature of thermal decomposition of a-Ag2WC>4 by the atmosphere generated in the combustion of chitosan. This decomposition process is inherent and different from the works published until now. In Figure 1 B it is possible to observe the D (-1350 cm-1) and G (-1570 cm-1) bands of carbonbased materials, referring to defects in the graphite layers and sp2 hybridized carbon bonds, respectively.

[0059] Scanning electron microscopy (SEM) images are shown in Figure 2. It is observed that chitosan after heat treatment has an extremely porous micrometric structure (Figure 2A). When WO3 / Ag is formed in the surface of the carbonic material, irregular structures of these materials (high contrast particles) fill these pores and are deposited on the surface of this carbonic support (Figure 2B-C). As expected, as the concentration of WO3 / Ag increases, the surface becomes increasingly filled with these structures.

[0060] Minimum inhibitory concentration (MIC) tests using E. coli (ATCC 25922) and S. aureus (ATCC 29213) bacteria were performed and are shown in Figure 3. As a negative control in both tests, the antibiotic gentamicin was used. It is observed that the QT sample does not show any significant antimicrobial inhibition at any of the concentrations tested with both microorganisms. For the gram-negative bacterium E. coli, samples QT1A, QT2A and QT4A showed MIC values of 125, 62.5 and 31.25 pg / mL, respectively. For the gram-positive bacterium S. aureus, samples QT1A, QT2A and QT4A presented MIC values of 250, 62.5 and 31.25 pg / mL, respectively. Thus, it is possible to observe that the antimicrobial activity is proportional to the amount of WO3 / Ag formed on the surface of the carbon support. The minimum bactericidal concentration (MBC) tests were performed with the well suspensions without turbidity, inoculated in agar and incubated for 24h. No bacterial growth was observed, confirming that the MBC is equal to the MIC.

[0061] In order to corroborate the bactericidal results, the cytotoxicity of the samples was analyzed using murine 3T3 cells. This strain is often applied in studies with materials before the step with in vivo assays. The cytotoxicity of the materials was analyzed through the % of cell viability, and the data are shown in Figure 4A-D. The viability of the QT sample showed a significant decrease at the lowest concentrations (3.90 and 7.81 pg / ml). The QT 1 A sample showed a decrease in cell viability at concentrations of 31.25 and 62.5 pg / ml. The QT2A had a significant decrease from the concentration of 15.62 pg / ml and the material QT4A from 7.81 pg / ml. In ascending order of decreasing cell viability and increasing cytotoxicity is: QT= QT1A < QT2A< QT4A. The effect of cytotoxicity on cell morphology is shown in Figure 4E-H. It is observed that at concentrations lower than 3.90 and 7.81 pg / ml the number of cells is as large as the control and the morphology is preserved. At concentrations of 15.62, 31 .25 and 62.5 pg / ml, the number of cells begins to decrease and the morphology changes from the original one, present in the control group, since the cell membrane has less definition, irregular cytoplasmic content and low adherence.

[0062] Expanding the technological application of this material as an antimicrobial agent, stainless steel coatings were performed using the sample with the highest antimicrobial capacity in order to analyze its surface properties, as well as its antimicrobial activity against S. aureus and E. coli. Figure 5A shows the surface of the stainless steel substrate after deposition of the carbon-supported WOa / Ag particles. It is observed that not the entire surface of the substrate is completely covered by the material, but a large part of the substrate is covered. Surface wettability is a central property that governs interactions between solid and liquid phases in biological systems. The liquid phase wets a solid surface maximizing its area in contact with the surface. This, in turn, increases the interaction between the liquid and the solid surface. Thus, more hydrophobic surfaces may limit bacterial adhesion. Figure 5B shows the contact angles for the pure stainless-steel substrate (SS) and after coating (SSQT4A). The contact angle of the SS sample was -65°, while that of the SSQT4A sample was -93°. Thus, in addition to the ROS production effect of the coating, the antimicrobial activity of this sample can be amplified due to the decrease in wettability of this surface.

[0063] The antimicrobial activity of the surface with and without the coating was analyzed through time kill tests, analyzing the bacterial growth as a function of time of E. coli (Figure 5C) and S. aureus (Figure 5D) after the interaction with the sample surface. For both bacteria, an increase in bacterial growth is observed for the pure substrate SS. However, for both bacteria, an abrupt reduction in bacterial growth was observed using the coating with the sample QT4A, showing that the coating translates its antimicrobial efficiency to the surface on which it is deposited.

Claims

CLAIMS1 . Procedure to obtain a composite material comprising: a) preparing a dispersion of chitosan in ethanol, b) preparing a dispersion of a-Ag2WC>4 in ethanol, c) adding the dispersion of a-Ag2WC>4 to the chitosan dispersion, wherein the amount of a-Ag2WC>4 is between 0.1 and 20% by weight in relation to the amount of chitosan, d) drying the resulting material at a temperature between 50 and 100 °C for 1 to 48h, e) heating the dried material obtained in step d) at a temperature between at 200- 700°C for 10-360 min, generating the final composite material.

2. Procedure according to claim 1 , wherein the dispersion of step a) is prepared under ultrasounds.

3. Procedure according to claim 2, wherein the ultrasounds are at a frequency between 20-50 kHz, with a power range of 300-800W for 5-60 minutes.

4. Procedure according to any of previous claims , wherein the dispersion of chitosan in step a) is prepared by adding chitosan to ethanol in proportion between 0.001g to 1.000g per 20 mL of ethanol.

5. Procedure according to any of previous claims, wherein the dispersion of a-Ag2WC>4 in step b) is prepared by adding a-Ag2WC>4 to ethanol in proportion between 0.001g to 1 ,000g per 20 mL of ethanol.

6. Procedure according to any of previous claims, wherein a-Ag2WC>4 used in step b) is obtained through the method of coprecipitation in aqueous medium that comprises the next steps: i) preparing a solution of Na2WC>4.2H2O in water, ii) preparing a solution of AgNCh in water, iii) heating the solutions prepared in steps i) and ii) at a temperature between 50 and 100°C, and then, adding the AgNCh solution to the Na2WC>4.2H2O solution, thus generating a white precipitate, iv) stirring the solution containing the white precipitate obtained in step iii) for 10-360 min constantly, and then centrifuging and washing it with distilled water until pH between 5and 7 is obtained in the wash water, v) drying the precipitate obtained in step iv) at a temperature between 50-100°C for 6- 48h.

7. Procedure according to claim 6, wherein the concentration of Na2WO4.2H2O in the solution of step i) is between 0.2x1 O'6and 2.0 mol / L.

8. Procedure according to claim 6 or 7, wherein the concentration of AgNCh in the solution of step ii) is between 0.4x1 O'6and 4.0 mol / L.

9. A composite material obtainable by the procedure described in any of claims 1 to 8 comprising: silver (Ag) and WO3 particles deposited on graphite.

10. Non-therapeutic use of the composite material defined in claim 9 as an antimicrobial coating or additive.

11. The use according to claim 10, wherein the antimicrobial is selected from an antibacterial, antiviral and antifungal.

12. The use according to any of any claims 10 or 11 , as an additive of polymeric matrices.

13. A coated substrate comprising the composite material describe in claim 9 deposited on at least a portion of the substrate.

14. A coated substrate, according to claim 13 wherein the substrate is of stainless steel.

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