WO2017191555A1

WO2017191555A1 - Use of a mesoporous oxide film in obtaining a transparent and ultra-thin ceramic coating which inhibits the formation of bacterial biofilms on the coated surface

Info

Publication number: WO2017191555A1
Application number: PCT/IB2017/052537
Authority: WO
Inventors: Paolo Nicolás CATALANO; Martin Gonzalo BELLINO; Galo Juan de Ávila Arturo SOLER ILLIA; Cristina Susana COSTA; Ramón Augusto PIZARRO; Magdalena PEZZONI
Original assignee: Consejo Nacional De Investigaciones Científicas Y Técnicas (Conicet); Comisión Nacional De Energía Atómica (Cnea)
Priority date: 2016-05-03
Filing date: 2017-05-02
Publication date: 2017-11-09
Also published as: AR104508A1

Abstract

The invention relates to the use of a mesoporous oxide film, synthesized by the sol-gel technique, self-assembly of surfactants and removal of the structuring agent by calcination, having a pore size of between approximately 4 nm and approximately 9 nm, characterized in that it is used in obtaining a transparent and ultra-thin ceramic coating which inhibits the formation of bacterial biofilms on the coated surface, wherein the film is deposited on the surface by the dip coating or spin coating technique.

Description

USE OF A MESOPOROUS OXIDE FILM, IN OBTAINING A TRANSPARENT AND ULTRADELGED CERAMIC COATING THAT INHIBITS THE FORMATION OF BACTERIAL BIOFILMS ON THE COVERED SURFACE

The present invention relates to the use of a mesoporous oxide film, synthesized by the technique of soldering, self-assembly of surfactants and removal of the structuring agent by calcination, which has a pore diameter of between about 4 nm and about 9 nm, characterized in that it is used in obtaining a transparent and ultra-thin ceramic coating that inhibits the formation of bacterial biofilms on the coated surface, where the film is deposited on the surface by means of the dip coating or spin coating technique.

BACKGROUND OF THE INVENTION

The field of mesoporous materials has evolved in recent decades, since the discovery of the first materials based on silica and molded through the incorporation of micelles as templates (Kresge et al., Nature, 1992, 359: 710; Beck et al., J Am Chem Soc, 1992, 114: 10834; Yanagisa a et al., Bull Chem Soc Jpn, 1990, 63: 988). The possibility of combining mild chemical synthesis methods with molecular self-assembly has opened a wide field of research with extremely interesting applications (Soler-Illia et al. Chem. Rev. 2002, 102: 4093-4138; Brinker et al., Adv Mater. 1999, 11: 579-585). In this way, a wide range of inorganic materials and organic-inorganic hybrid matrices with a variety of composition, pore diameter, organic or biological functions located in the inorganic skeleton, in the pores or on the inner surface of said pores. The processing of mesoporous materials such as thin films offers the opportunity to create novel coatings that combine the advantages of controlling the shape, symmetry and pore diameter together with their possible surface modifications, with outstanding optical properties and the possibility of being applied on different substrates metallic or ceramic. In recent years, the production of accessible and functionalizable thin mesoporous films, whether the surface of pores or the volume of pores can be modified by different functional groups, biological molecules or nanoparticles, has been extensively explored (Sánchez, et al Chem. Mater. 2011, 20: 682-737 .; Innocenzi et al., Chem. Soc. Rev. 2013, 42: 4198-4216).

A biofilm is a community of microorganisms attached to a surface. In it, bacteria or other microorganisms are embedded in a matrix (composed of water and polymeric substances) that provides structural stability and protection against different stressors, such as lack of nutrients, desiccation, the presence of antibiotics or biocides, etc. (Mah, et al Nature. 2003 426: 306-310; Flemming, et al Nat. Rev. Microbiol. 2010 8: 623-6332010). The phenomenon of bacterial adhesion on surfaces constitutes only the first stage in the formation of a biofilm. Cell proliferation and the formation of a complex matrix make up the second stage and distinguish biofilms from the simple adhesion of bacteria on surfaces. Bacterial biofilms can be formed by one or multiple bacterial species, presenting between 10 and 1000 times greater resistance to antimicrobial agents such as antibiotics and other agents with respect to free or planktonic bacteria (Mah and O 'Toóle, Trends in Microbiology 2001, vol 9 ). They can develop on a wide variety of surfaces, being responsible for about 65% of hospital infections. Biofilms can be formed in medical implants such as catheters, artificial hips, heart valves and contact lenses, and their impact on medicine is relevant throughout the world for its harmful effects in health and economic terms (Nickel, et al Eur. J Clin. Microbiol 1985 4: 213-218; Willcox, et al Biomaterials 2001. 22: 3235-3247). On the other hand, the development of microorganisms on industrial-type surfaces, such as water or hydrocarbon pipe systems, can have a detrimental effect on them (Walker, JT, Mackerness, CW, Rogers, J. and Keevil, CW Microbial Biofilms. 1995. HM Lappin-Scott and JW Costerton (eds.). Cambridge University Press, Great Britain, 196-204; Lenhart TR, et al, Biofouling 2014. 30: 823-835). In addition to generating "bio-fouling", biofilms can promote physical-chemical reactions producing corrosion of the material that composes said pipes. The provision of biocides represents a non-negligible part of the costs of certain industries; However, the action of these compounds is usually inefficient due to the high resistance of biofilms to eradication treatments. From all the above it is clear that to combat biofilms it is more convenient to avoid their formation than to use physical or chemical treatments once they have been installed. New alternatives are therefore required that provide industrial feasibility, low cost and high effectiveness at the time of eradicating or avoiding their formation. In this framework, from the nanotechnology, the regulation of the topography of a surface is a promising proposal in the development of strategies to avoid the formation of biofilms. With this objective, the possibility of using mesoporous materials from titanium substrates (Varióla et al., International Journal of Nanomedicine, 2014, 9: 2319-2325) and chemically treated microparticulate powders (Izquierdo-Barba et al. , Acta Biomaterialia, 2011, 7: 2977-2985; Kinnari et al., Journal of Biomedical Materials Research Part A, 2009, 89A: 215-223). However, these studies demonstrated only effects on bacterial adhesion without demonstrating effects on the formation of bacterial biofilms. In addition, thin mesoporous coatings that can inhibit the formation of bacterial biofilms and that can be applied on various surfaces without altering their optical quality have not been described so far. It is therefore that there is still a need to develop thin coatings that inhibit the formation of bacterial biofilms and retain their optical transparency to be applied on surfaces that require it.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to the use of a mesoporous oxide film, synthesized by the soldering technique, self-assembly of surfactants and removal of the structuring agent by calcination, which has a pore diameter of between about 4 nm and about 9 nm, in obtaining a transparent and ultra-thin ceramic coating that inhibits the formation of bacterial biofilms on the coated surface, where the film is deposited on the surface by means of the dip coating or spin coating technique.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Distribution of pore diameters of the silica mesoporous films used in the study, obtained by water adsorption isotherms. MS-4 and MS-9 correspond to films synthesized with Si (OEt) ₄ (TEOS) / CTAB and Si (OEt) ₄ (TEOS) / F127, respectively. The inserts represent transmission microscopy images of said films.

Figure 2. Representative patterns of low-angle X-ray scattering (2D SAXS) of the silica mesoporous films used in the study. The 3D hexagonal structure is shown in the case of MS-4 (pore diameter of 4 nm) and the cubic structure in the case of MS-9 (pore diameter of 9 nm).

Figure 3. Anti-biofilm effect of silica mesoporous films, in submerged biofilms and in biofilms formed in the air-liquid interface (ALI). a) Average number of viable cells (colony forming units or CFUs) adhered to the NMS (non-mesoporous), MS-4 and MS-9 films after 4, 8 and 24 hours of incubation in culture medium, b) Staining with violet crystal of biofilms grown on NMS, MS-4 and MS-9 surfaces for 24 hours. The images of the biofilms stained with violet crystal, which was eluted and quantified by measuring absorbance at 575 nm. c) Quantification of extracellular polymeric substances (polysaccharides, proteins and DNA) present in the 24-hour biofilm matrix grown on NMS, MS-4 and MS-9 surfaces. Asterisks demonstrate significant differences between MS-9 and NMS (Student's t-test): * p <0.05; ** p <0.01; *** p <0.003.

Figure 4. Scanning electron microscopy images of 24-hour biofilms grown on NMS, MS-4 and MS-9 films. The bar represents 5 μπι.

DETAILED DESCRIPTION OF THE INVENTION

Although there are previous reports describing the effects of mesoporous materials on bacterial adhesion, these refer to the use of titanium substrates and microparticulate powders, without showing conclusive results in this regard. On the contrary, according to the present invention, the ceramic coating disclosed herein can be applied on various surfaces producing not only the inhibition of bacterial adhesion but also the inhibition of the formation of bacterial biofilms. Likewise, the ceramic coating of the invention can be in the form of an ultra-thin, transparent, inert, low-cost, scalable and robust sheet without modifying the original appearance of the surface on which it is deposited, not altering the optical properties of the material.

The present invention relates to the use of a mesoporous oxide film, synthesized by the technique of soldering, self-assembly of surfactants and removal of the structuring agent by calcination, which has a pore diameter between about 4 nm and about 9 nm, in obtaining a transparent and ultra-thin ceramic coating that inhibits the formation of bacterial biofilms on the coated surface, where the sheet is deposited on the surface by the technique of dip coating or spin coating. In accordance with the present invention, the dip coating technique is understood as the immersion coating process where the substrate is introduced into a precursor solution and removed at controlled speed. Likewise, the spin coating technique is understood as the process where an amount of precursor solution is deposited on the substrate and then it is rotated at high speed to achieve a uniform distribution of the solution by centrifugal force.

In a particular embodiment of the invention, a silica mesoporous film synthesized by the sol-gel technique, self-assembly of surfactants and removal of the structuring agent by calcination is used, in order to provide anti-biofilm activity to the surface coated with said mesoporous film. On the one hand, said silica mesoporous film is used to inhibit the formation of bacterial biofilms on the coated surface, when said surface is submerged in the bacterial culture medium. On the other hand, said silica mesoporous film is used to inhibit the formation of bacterial biofilms at the air-liquid interface of the coated surface.

According to the invention, the coating with the silica mesoporous film does not modify the original appearance of the surface on which it is deposited.

On the other hand, it is a particular object of the invention to use a mesoporous silica film to inhibit the formation of bacterial biofilms. In more particular embodiments, it is used to inhibit the formation of bacterial biofilms produced by bacteria belonging to the Pseudomonas aeruginosa species. This microorganism is a relevant opportunistic pathogen in humans characterized by the formation of robust biofilms related to processes of pathogenesis and industrial biocorrosion. It is considered as a model organism for studies with biofilms.

The ceramic coating of the invention can be applied commercially in health centers and other places where bacteria-free surfaces are required, for example, food or biomedical processing facilities, play areas and dining rooms. The films described in the present invention could be applied on ceramic surfaces of sinks, tiles and bathrooms or glass surfaces on doors, windows and kitchen utensils. The films could also be applied to metal surfaces of faucets and doorknobs, and medical equipment and instruments.

Mesoporous films (4 and 9 nm in pore diameter) were obtained by combining the synthesis of oxides by sol-gel chemistry, controlling the hydrolysis and condensation of inorganic precursors, with the self-assembling of surfactants or copolymers, which act as pore molds . The starting compounds for the synthesis were Si (OEt) ₄ (TEOS) and ionic surfactants (CTAB) and polymeric surfactants of the POE-POP-POE type (Pluronics-F127 series). After post-stabilization treatments at controlled humidity and temperature, the structuring agent was removed by calcination to obtain the porous films. The operation of the invention has been demonstrated by biofilm assays of Pseudomonas aeruginosa, using non-porous surfaces as control. Two different biofilm formation systems were tested: submerged and air-liquid interface, and their formation was evaluated at different times. The results obtained demonstrated a significant inhibition of cell adhesion and biofilm formation on the surfaces coated with the mesoporous film compared to non-porous surfaces, whether of non-porous silica or glass.

Thus, by means of the present invention, it is possible to combat biofilms of undesirable bacteria, which can become a dangerous source of infection harmful to health or generate corrosion or bio-fouling when they are deposited on a surface. The transparent ceramic coating of the invention has great mechanical resistance and can be applied to ceramic or metal surfaces, without altering its optical properties.

Among these surfaces we can mention those destined to sanitary or food establishments, such as hospitals, restaurants or refrigerators; including homes, household appliances, including sanitary items and appliances.

EXAMPLES OF REALIZATION

Obtaining mesoporous films

Mesoporous silica films were obtained on glass slides (support), following processes reported in the literature, in which the synthesis of oxides by sol-gel chemistry (control of the hydrolysis and condensation of inorganic precursors), with the self-assembly of surfactants or copolymers (which act as pore molds). The starting compounds for the synthesis were Si (OEt) ₄ (TEOS) and ionic surfactants (CTAB) and polymeric surfactants of the POE-POP-POE type (Pluronics-F127 series). After post treatments ^¬ stabilization controlled temperature and humidity, the structuring agent was removed by calcination obtaining mesoporous films. (The procedure is described, for example, in GJAA Soler-Illia et al., "Mesoporous hybrid and nanocomposite thin films. A sol-gel toolbox to create nanoconfined systems with localized chemical properties", J Sol-Gel Technol, 2011, 57 , 299-312, which is attached hereto, as a reference, in its entirety).

Characterization of mesoporous films

The thorough characterization of mesoporous coatings is essential, since the properties of mesoporous films strongly depend on their pore structure. The results that allow to perfectly characterize the described mesoporous films are summarized in Figure 1, Figure 2 and Table 1. MS-4 and MS-9 correspond to films synthesized with Si (OEt) 4 (TEOS) / CTAB and Si (OEt ) 4 (TEOS) / F127, respectively. The numbers 4 and 9 are indicative of the average pore diameter present (Figure 1) that depends on the type of surfactant used. The hexagonal 3D structure for MS-4 films and the cubic structure for MS-9 films are shown in Figure 2.

On the other hand, other structural data of MS-4 and MS-9 such as porosity can also be observed in Table 1 and the thickness, as well as the contact angle that is an indicator of hydrophobicity of the films used.

Table 1. Structural data and contact angle measurements (hydrophobicity indicator) of the films used in this study. MS-4 and MS-9 correspond to films with an average pore diameter of 4 or 9 nm, respectively. Non-mesoporous films were also used as control (NMS), synthesized by the same procedure as mesoporous but without the incorporation of the surfactant that allows the formation of mesopores.

Porosity Size Type Thickness Film angle contact pore

(%) (nm)

(nm) (°)

NMS 5 ± 0.5 93 ± 1 23.0 ± 1

MS-4 4.1 ± 0.5 42 ± 2 150 ± 1 16.0 ± 1

MS-9 9.2 ± 0.¡ 37 ± 2 100 ± 1 28.0 ± 2

Inhibition of the formation of bacterial biofilms by mesoporous films according to the invention

Studies of anti-biofilm activity of the films according to the invention were carried out using the prototypical strain of Pseudomonas aeruginosa PAOl. This microorganism is a relevant opportunistic pathogen in humans characterized by the formation of robust biofilms related to pathogenesis and industrial corrosion processes. It is considered as a model organism for studies with biofilms. Quantitative data obtained by counting training units are presented. colonies (UFC). Two biofilm formation systems were used: submerged and ALI. In the first system the coatings were completely submerged in the bacterial culture broth favoring the formation of biofilms over the entire surface, while in the second system, called ALI, the coatings were placed vertically leaving a portion of it outside the surface. Bacterial culture medium favoring the formation of biofilm at the air-liquid interface. For coatings based on mesoporous films, a significant decrease in viable cells was observed after 8 and 24 hours of incubation with inoculums of approximately 10 ⁸ bacteria / mL) (Figure 3a). In addition, the quantification performed by staining with violet crystal

(Figure 3b) clearly demonstrated the inhibition in biofilm formation on mesoporous films. A decrease in the levels of extracellular polymeric substances in the matrix was also observed

(polysaccharides, proteins and DNA) on mesoporous films (Figure 3c). Likewise, scanning electron microscopy images show a smaller number of cells attached to the surface of mesoporous films (Figure 4). In all cases, the inhibitory effect on the formation of bacterial biofilms was greater in the case of MS-9, in both systems studied

(submerged and ALI).

From the exposed results it can be seen that the mesoporous ceramic coatings according to the present invention significantly inhibit the development of biofilms of Pseudomonas aeruginosa, both in the submerged system and ALI.

In the specialized literature, it is described that mesoporous films can be synthesized on various ceramic and metal surfaces (Sánchez, et al. Chem. Mater. 2011, 20: 682-737 .; Innocenzi et al., Chem. Soc. Rev. 2013, 42: 4198-4216). Therefore, any of these ceramic and metallic materials would offer the same inhibitory effect on the formation of bacterial biofilms mentioned here, as they are coated with the mesoporous films revealed in this invention.

Claims

1. Use of a mesoporous oxide film, synthesized by the sol-gel technique, self-assembly of surfactants and elimination of the structuring agent by calcination, which has a pore size of between about 4 nm and about 9 nm, characterized in that it is used in obtaining a transparent and ultra-thin ceramic coating that inhibits the formation of bacterial biofilms on the coated surface, where the film is deposited on the surface by means of the dip coating or spin coating technique.

2. Use of a mesoporous oxide film according to claim 1, characterized in that it is used to coat a surface, in order to provide antibiofilm activity to said surface.

3. Use of a mesoporous oxide film according to claim 1, characterized in that it is used to inhibit the formation of bacterial biofilms on the coated surface, when said surface is submerged.

4. Use of a mesoporous oxide film according to claim 1, characterized in that it is used to inhibit the formation of bacterial biofilms at the air-liquid interface of the coated surface.

5. Use of a mesoporous oxide film, according to claim 1, characterized in that it does not modify the original appearance of the surface on which it is deposited.

6. Use of a mesoporous oxide film according to claim 1, characterized in that it is used to inhibit the formation of bacterial biofilms produced by bacteria belonging to the Pseudomonas aeruginosa species.

7. Use of a film of mesoporous oxide according to claim 1, characterized in that the oxides used are , for example Si0 _2, Ti0 _2, Zr0 ₂ or combinations thereof.

8. Use of a mesoporous oxide film according to claim 1, characterized in that the surfactants used for their synthesis belong to the group of non-ionic polymeric surfactants or ionic surfactants.

9. Use of a mesoporous oxide film, according to the preceding claim, characterized in that the non-ionic polymeric surfactants correspond to the POE-POP-POE type and the ionic surfactants to the quaternary ammonium salt type.