RO134947A2 - Antimicrobial multilayer nanoassembled films and process for manufacturing the same - Google Patents

Abstract

The invention relates to a process for manufacturing nanoassembled multilayer antimicrobial films with applications in the treatment of wastewater infested with gram-negative bacteria. According to the invention, the process consists in coating a glass support with a first layer of organo-silane compound, using the sol-gel method, over which a second layer is added, said layer being a polymer molecularly imprinted with the O antigen of lipo-polysaccharide, resulting in a surface as films, having the capacity of selectively retaining the O antigen, with a decrease of the E-coli indicator from 2 CFU/50 ml to 0 and the coliform indicator from 41 CFU/50 ml to 1.

Description

ANTIMICROBIAL NANOASSEMBLED MULTILAYER FILMS AND METHOD OF OBTAINING THEM

The invention relates to nanoassembled multilayer antimicrobial films with applications in wastewater treatment and to a process for obtaining them. Lipopolysaccharide (LPZ) is a constituent of the outer membrane of gram-negative bacteria. It consists of polysaccharides (antigen O and nucleus oligosaccharide) and lipid A, and the structure of these substances differs depending on the type of gram-negative bacterium. The present invention is based on its own research carried out to immobilize by molecular imprinting the antigen O of lipolysaccharide from the cell membrane of bacteria and in this way to destroy the respective bacterium. Molecular imprinting with proteins is a known method for their identification or for their extraction from biological matrices.

A surface printing process of a basic protein: liposome is presented in Preparation of surface molecularly imprinted polymeric microspheres and their recognition property for basic protein lysozyme, Journal of Chromatography B, 878 (2010) 1731-1738 authors Baojiao Gao, Hongyan Fu , Yanbin Li, Ruikui Du, in which a substrate was first prepared by copolymerizing Nvinylpyrrolidone with 2-hydroxyethylmethacrylate in reverse suspension to give crosslinked copolymer beads, then an esterification reaction of the hydroxyl groups on the surface of the microspheres took place. of methacryloyl, thus introducing on their surface a large number of polymerizable double bonds. In the presence of the liposome temper, by initiation with a redox system, the crosslinking polymerization of the methacrylic acid was performed. After removing the liposome from the surface of the microspheres, the molecularly imprinted particles were obtained with liposime. The process has the disadvantage that for the synthesis of the support particles it includes many phases and the activation of the support surface is very laborious. The procedure was not used for the preparation of antibacterial surfaces.

Another procedure is presented in Surface-Grafted Molecularly Imprinted Polymers for Protein Recognition, Analytical Chemistry, 73 (2001) 5281-5286 authors Alessandra Bossi, Sergey A. Piletsky, Elena V. Piletska, Pier Giorgio Righetti, Anthony PF Turner, and consists from oxidative polymerization, initiated with ammonium persulfate of 3-aminophenylboronic acid in the presence of the following proteins as a template: microperoxidase, horseradish peroxidase, lactoperoxidase and hemoglobin. The layers were deposited on polystyrene microplates. The process has the disadvantage of working with a special monomer, so very expensive. Also, the method is not used to obtain antimicrobial surfaces.

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A process of imprinting a protein by the sol-gel method is described in Molecularly imprinted polymer grafted on polysaccharide microsphere surface by the sol-gel process for protein recognition, Talanta 74 (2008) 1247-1255 authors Feng Li, Jing Li, Shu Sheng Zhang, in which first or prepared spherical chitosan particles strongly crosslinked with formaldehyde and then with epichlorohydrin to introduce epoxy groups. The modified polysaccharide particles were partially treated to recover some amino groups of chitosan and then reacted with glutardialdehyde to be used as a core support for surface printing. Then, the temperate molecule - bovine serum albumin - was covalently immobilized in borate buffer on the surface of the particles by forming immune bonds with the amino groups of chitosan. Next, 2 siloxane monomers: 3-aminopropyltrimethoxysiloxane: APTMS and tetraethoxysiloxane: TEOS self-assembled around the template protein and were subsequently polymerized to the surface of the protein-containing polysaccharide by sol-gel reaction in aqueous solution at room temperature. Finally, the template was removed by oxalic acid treatment, leaving the cavities molecularly imprinted with the protein. The process has the disadvantage that it is very laborious, contains many critical phases, such as the preparation of support particles and immobilization of the enzyme on them, it is a method of covalent immobilization, which involves breaking strong bonds to remove the temper. The process was not used to obtain antibacterial surfaces.

The work Development of lipid A-imprinted polymer hydrogels that selectively recognize lipopolysaccharides, Biosensors and Bioelectronics 38 (2012) 215-219 authors Kei-lchi Ogawa, Masumi Hyuga, Tomoko Okada, Norihiko Minoura describes a process of molecular printing in lipid hydrogel A from LPZ. The process consists in the use as functional monomers of acryloillisine and acryloylphenylalanine, as a matrix monomer of acrylamide and as a crosslinker of methylenebiscrilamide (MBA) and as a template lipid A was used. Sodium hydroxide. The process has the disadvantage that it uses as functional monomers two monomers very difficult to prepare, non-industrial and therefore very expensive and as an acrylamide matrix monomer, known as carcinogenic. In addition, lipid A imprinting ensures LPZ retention but does not provide antimicrobial activity, especially since hydrogel is an environment conducive to bacterial proliferation.

US 9329186 B2 (2016) describes the production of molecularly imprinted polymers with phosphorylated peptides and proteins. The process uses as functional monomer monomers based on aryl derivatives of urea or thiourea. The disadvantage of this process is that it uses monomers that are very difficult to prepare and expensive, in addition, the molecularly imprinted polymers are intended for medical imaging or optical sensors and not for antimicrobial surfaces.

The technical problem solved by the invention consists in the preparation of multilayer antimicrobial films by depositing, by sol-gel, on a glass support, a first layer of organosilane compound, over which a second layer of molecularly imprinted polymer is added by solgel method, the imprinting taking place with the O antigen of the lipopolysaccharide, the surfaces thus obtained having a selective retention capacity of the O antigen, the active part of the outer cell membranes of gram-negative bacteria and thus destroying these bacteria.

The antimicrobial nanoassembled multilayer films according to the invention remove the disadvantages of the aforementioned processes in that a first solgel layer is formed over a glass support by the reaction between 3- Mercaptopropyl trimethoxysilane -MPTES and ammonium hydroxide, in volumetric ratio MPTES: 25% aqueous solution ₄ OH of 1: 3,5 ... 4,5, over which a second layer is formed, of molecularly imprinted sol-gel polymer, obtained by the reaction of 3- (2-trimethoxysilyl) propylmethacrylate -MAPTES with ammonium hydroxide , in volumetric ratio MAPTES: 25% aqueous solution of NH ₄ OH of 1: 5 ... 6, formed in the presence as a template, of antigen O from lipopolysaccharide (LPZ), in a ratio of 50,000 units of endotoxin, equivalent to 50,000 units of antigen O, from LPZ at 0.10-0.15 mmol MAPTES, the temperate being subsequently extracted from the second layer, the films thus obtained showing antibacterial activity against gram-negative bacteria such as E-coli and coliforms, the first layer silane ic deposited on the glass being obtained by dissolving MPTES in absolute ethyl alcohol, in volume ratio MPTES: absolute ethyl alcohol of 1: 5 ... 7, at a temperature of 20 ... 30 ° C, the alcoholic precursor solution of MPTES being gradually added , at 20 ... 30 ° C, for 1.5 .... 2.5 hours, under a stirring of 150 .... 350 rpm over a catalytic solution of aqueous ammonium hydroxide solution 25 % and distilled water, in water ratio: 25% NH4OH solution of 1: 2 ... 3, the ratio between MPTES and 25% ammonium hydroxide solution being 1: 3.5 ... 4.5, the soil as follows obtained being sprayed on the glass support and left for 15 .... 45 minutes at a temperature of 20 .... 30 ° C for gelling, and the second layer, molecularly imprinted, being obtained by dissolving MAPTES in ethyl alcohol absolute, in volumetric ratio MAPTES: absolute ethyl alcohol of 1: 10 ... 12, at a temperature of 20 ... 30 ° C, after which an aqueous solution of 0.8-1.2 g / is added to this solution L of LPZ, and the ratio between the solution of LPZ and MAPTES being 2.5 ... 3: 1, the alcoholic precursor solution of MAPTES with tempering being added gradually, for 1.5 .... 2.5 hours under a stirring of 150 .... 350 rot / min over a catalytic solution consisting of 25% aqueous ammonium hydroxide solution, the ratio between MAPTES and ammonium hydroxide solution being 1: 5 ... 6, the soil thus obtained being sprayed over the first silane layer and left to mature 24. ..48 h at 2O ... 3O ° C and another 24 ... 48 h at 45 ... 55 ° C, after which the laminated films obtained on

RO 134947 A ^ glass substrates are washed in 2-3 rounds with distilled water in MAPTES ratio: water of 1:10 ... 20 by ultrasound for 3 ... 5 hours per serving, the films having antimicrobial activity against gram-negative bacteria of the E-coli and coliform type.

The invention has the following advantages:

- A very cheap and industrially produced support is used, namely glass plates;

- Commercial monomers are used, with a relatively low cost price;

- The proposed process is simple compared to other protein printing processes;

- There are no particular problems related to the protection of the environment or personnel;

- The films have a very good adhesion to the support;

- The films do not require special storage conditions and can be used as such for water treatment applications infested with gram negative bacteria;

- Films can be easily regenerated due to their very good mechanical strength.

- The process of obtaining the films takes place at temperatures close to room temperature, which means low energy consumption.

Examples of embodiments of the invention are given below.

Example 1. 1.5 mL of absolute ethyl alcohol and then 0.3 mL MPTES are placed in a 10 mL Berzelius beaker. Mix everything vigorously at room temperature (20 ° C) for a good dissolution, obtaining the precursor solution. _{In parallel, 1.05 mL of 25% NH 4} OH solution and 0.52 mL of distilled water are placed in an Erlenmayer beaker with a 10 mL ground stopper and stirred for homogenization to give the catalytic solution. A Teflon-coated magnetic stirring body is introduced into the Erlenmayer beaker at room temperature (20 ° C) and the Erlenmayer beaker is placed on the plate of an unheated magnetic stirrer and the magnetic stirring is started at 150 rpm. Gradually introduce the precursor solution over the catalytic solution, which lasts 2.5 hours, when soil 1 is obtained. Soil 1 thus made is sprayed onto a glass slide, thoroughly cleaned by washing with soap and water and finally with ethanol. .. For polycondensation, allow the glass slide with the first silane layer to stand at room temperature (20 ° C) for 45 minutes. meanwhile, another 2.4 mL of absolute ethyl alcohol and 0.2 mL of MAPTES are added to another 10 mL Berzelius beaker and mixed vigorously at room temperature (20 ° C) for good dissolution. An additional 0.6 mL of 0.8 g / L aqueous solution of LPZ is added and the mixture is stirred for homogenization, thus obtaining the printing precursor solution at a ratio of 50,000 units of endotoxin (equivalent to 50,000 units of antigen O) from LPZ to 0.15 mmol MAPS. _{Place another 1.2 mL 25% NH 4} OH solution in another 10 mL Erlenmayer beaker with a ground stopper. Place the Erlenmayer glass on the plate of a magnetic stirrer, insert a Teflon magnetic stirrer into it and start stirring.

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£ î at 350 rpm. Gradually introduce the pre-printing solution over the 25% _{NH 4 OH solution.} The introduction lasts 1.5 hours, resulting in soil 2. Soil 2 is sprayed over the first silane layer on the glass slide. Leave the lamella with the second layer to mature at room temperature (20 ° C) for 48 hours and then in an oven for 24 hours at 55 ° C. Then, the lamella is placed in a crystallizer and 2 mL of distilled water is added over it. Place the crystallizer in an ultrasonic bath, where it is left for 3 hours. Then drain the extraction water and add another 2 mL of distilled water. Ultrasound again for 3 hours. Drain the wash water and add 2 mL of distilled water again. Ultrasound for another 3 hours. Finally, remove the multilayer lamella from the crystallizer and allow to dry in free convection at room temperature (20 ° C). The laminated film slide thus prepared was placed in contact with the wastewater, 50 mL / sample. After 24 hours the E-coli indicator decreased from 2CFU / 50 mL to zero and the coliform indicator from 41CFU / 50mL to 1.

Example 2 Place 2.1 mL of absolute ethyl alcohol and then 0.3 mL MPTES in a 10 mL Berzelius beaker. Mix everything vigorously, on a magnetic hob, at a temperature of 30 ° C, for a good dissolution, obtaining the precursor solution. _{In parallel, 1.35 mL of 25% NH 4} OH solution and 0.45 mL of distilled water are placed in an Erlenmayer beaker with a 10 mL ground stopper and stirred for homogenization to give the catalytic solution. In the Erlenmayer beaker, a Teflon magnetic stirring body is introduced and the Erlenmayer beaker is placed on the plate of a magnetic stirrer at a temperature of 30 ° C and the magnetic stirring is started at 350 rpm. Gradually introduce the precursor solution over the catalytic solution. This gradual introduction lasts 1.5 hours, when soil 1 is obtained. Soil 1 is sprayed on a glass slide, thoroughly cleaned by washing with soap and water and finally with ethanol pa. Allow the glass slide with the first silane layer to stand at 30 ° C on the magnetic stirrer hob for polycondensation for 15 minutes. meanwhile, in another 10 mL Berzelius beaker on a magnetic stirrer, place 2 mL of absolute ethyl alcohol and 0.2 mL of MAPTES and mix vigorously at 30 ° for a good dissolution. Add another 0.5 mL of 1.2 g / L aqueous solution of LPZ and stir for homogenization. The printing precursor solution is thus obtained at a ratio of 50,000 units of endotoxin (equivalent to 50,000 units of antigen O) from LPZ to 0.10 mmol MAPTES. In another 10 mL Erlenmayer beaker, with ground stopper, place 1 mL of 25% NH ₄ OH solution. Place the Erlenmayer beaker on the plate of a magnetic stirrer heated to 30 ° C, insert a Teflon magnetic stirring body into it and start stirring at 150 rpm. Gradually introduce the pre-printing solution over the 25% _{NH 4 OH solution.} Introduction takes 2.5 hours. The soil 2 thus obtained is sprayed over the first silane layer on the glass slide. Allow the lamella with the second layer to stand at maturity at 30 ° C on the shaker plate.

RO 134947 A magnetic for 24 hours and then in an oven for 48 hours at 45 ° C. Then, the lamella is placed in a crystallizer and 3 mL of distilled water is added over it. Place the crystallizer in an ultrasonic bath, where it is left for 5 hours. Then drain the extraction water and add another 3 mL of distilled water. Ultrasound again for 5 hours. Finally, remove the multilayer lamella from the crystallizer and allow to dry in free convection on a magnetic stirrer hob heated to 30 ° C. The dry lamella, with multilayer films thus prepared, was placed in contact with the wastewater, 100 mL / sample each. After 24 hours the E-coli indicator decreased from 1 CFU / 100 mL to zero and the coliform indicator from 120 CFU / 100 mL to 19. Example 3 2 mL of absolute ethyl alcohol were introduced into a 10 mL Berzelius beaker. and then 0.3 mL MPTES. Mix everything vigorously, on a magnetic hob, at a temperature of 28 ° C, for a good dissolution, obtaining the precursor solution. _{In parallel, 1.25 mL of 25% NH 4} OH solution and 0.65 mL of distilled water are placed in an Erlenmayer beaker with a 10 mL ground stopper and stirred for homogenization to give the catalytic solution. Insert a Teflon magnetic stirring body into the Erlenmayer beaker and place the Erlenmayer beaker on a magnetic stirrer at 28 ° C and start stirring magnetically at 200 rpm. Gradually introduce the precursor solution over the catalytic solution. This gradual introduction lasts 2 hours, when soil 1 is obtained. Soil 1 is sprayed over a glass slide, thoroughly cleaned by washing with soap and water and finally with ethanol pa. Allow the glass slide with the first silane layer to stand at 25 ° C on the magnetic stirrer hob for polycondensation for 30 minutes. Meanwhile, 2.2 mL of absolute ethyl alcohol and 0.2 mL of MAPTES are placed in another 10 mL Berzelius beaker on a magnetic stirrer and mixed vigorously at 25 ° for good dissolution. Add another 0.55 mL of 1 g / L aqueous solution of LPZ and stir for homogenization. The printing precursor solution is thus obtained at a ratio of 50,000 units of endotoxin (equivalent to 50,000 units of antigen O) from LPZ to 0.12 mmol MAPTES. In another 10 mL Erlenmayer beaker, 1.1 mL of 25% NH 4 OH is introduced with a ground stopper. Place the Erlenmayer beaker on the plate of a magnetic stirrer heated to 25 ° C, insert a Teflon magnetic stirring body into it and start stirring at 200 rpm. Gradually introduce the pre-printing solution over the 25% _{NH 4 OH solution.} The introduction takes 2.2 hours. The soil 2 obtained is sprayed over the first silane layer on the glass slide. Allow the lamella with the second layer to mature at 25 ° C on the magnetic stirrer hob for 36 hours and then in an oven for 30 hours at 50 ° C. Then the slide is placed in a crystallizer and 4 mL of distilled water is added over it. Place the crystallizer in an ultrasonic bath, where it is left for 4 hours. Then drain the extraction water and add another 3 mL of distilled water. Ultrasound again for 4 hours. Finally, remove the multilayer lamella from the crystallizer and allow to dry

RO 134947 A2 ^ _χ in free convection on a magnetic stirrer hob heated to 23 ° C. The dry lamella, with multilayer films thus prepared, was placed in contact with the wastewater, 100 mL / sample each. After 24 hours the E-coli indicator decreased from 3 CFU / 100 mL to zero and the coliform indicator from 100 CFU / 100 mL to 12. Example 4 1.8 mL of alcohol is introduced into a 10 mL Berzelius beaker. absolute ethyl and then 0.3 mL MPTES. Mix everything vigorously, on a magnetic hob, at a temperature of 25 ° C, for a good dissolution, obtaining the precursor solution. _{In parallel, 1.2 mL of 25% NH 4} OH and 0.5 mL of distilled water are placed in an Erlenmayer beaker with a 10 mL ground stopper and stirred for homogenization to give the catalytic solution. Insert a Teflon magnetic stirring body into the Erlenmayer beaker and place the Erlenmayer beaker on a magnetic stirrer at 23 ° C and start stirring magnetically at 250 rpm. Gradually introduce the precursor solution over the catalytic solution. This gradual introduction lasts 2.2 hours, when soil 1 is obtained. Soil 1 is sprayed on a glass slide, thoroughly cleaned by washing with soap and water and finally with ethanol pa. Allow the glass slide with the first silane layer to stand at 27 ° C on the magnetic stirrer hob for polycondensation for 35 minutes. meanwhile, 2.1 mL of absolute ethyl alcohol and 0.2 mL of MAPTES are placed in another 10 mL Berzelius beaker on a magnetic stirrer and mixed vigorously at 27 ° for good dissolution. Add another 0.55 mL of 1.1 g / L aqueous solution of LPZ and stir for homogenization. The printing precursor solution is thus obtained at a ratio of 50,000 endotoxin units (equivalent to 50,000 antigen O units) from LPZ to 0.11 mmol MAPTES. In another 10 mL Erlenmayer beaker, with a ground stopper, place 1.15 mL of 25% NH ₄ OH solution. Place the Erlenmayer beaker on the plate of a magnetic stirrer heated to 27 ° C, insert a Teflon magnetic stirring body into it and start stirring at 180 rpm. Gradually introduce the pre-printing solution over the 25% _{NH 4 OH solution.} The introduction takes 1.8 hours. The obtained soil is sprayed over the first silane layer on the glass slide. Allow the lamella with the second layer to stand at 27 ° C on the magnetic stirrer plate for 30 hours and then in an oven for 40 hours at 48 ° C. Then, the lamella is placed in a crystallizer and 3 mL of distilled water is added over it. Place the crystallizer in an ultrasonic bath, where it is left for 3.5 hours. Then drain the extraction water and add 4 mL of distilled water. Ultrasound again for 4 hours. Drain the wash water and add 2 mL of distilled water. Ultrasound for another 3 hours. Finally, remove the multilayer lamella from the crystallizer and allow to dry in free convection at room temperature (20 ° C). The lamella with laminates thus prepared was placed in contact with the wastewater, 50 mL / sample each. After 24 hours the E-coli indicator decreased from 4 CFU / 50 mL to 1 and the coliform indicator from 61CFU / 50mL to 4.

Claims (2)

AND PROCESS FOR OBTAINING THESE CLAIMS 1. Antimicrobial nano-assembled multilayer films characterized in that a first sol-gel layer is formed over a glass support by the reaction of (3- Mercaptopropyl) trimethoxysilane: MPTES and ammonium hydroxide, in volumetric ratio MPTES: aqueous solution 25% NH ₄ OH of 1: 3,5 ... 4,5, on which a second layer is formed, of molecularly imprinted sol-gel polymer, obtained by the reaction of 3 (2-trimethoxysilyl) -propylmethacrylate: MAPTES, with hydroxide of ammonium, in volumetric ratio MAPTES: 25% aqueous solution of NH ₄ OH of 1: 5 ... 6, formed in the presence as a template, of antigen O from lipopolysaccharide: LPZ, in a ratio of 50,000 units of endotoxin, equivalent to 50,000 O antigen units, from LPZ to 0.100.15 mmol MAPTES, the temperate being subsequently extracted from the second layer, the films thus obtained showing antibacterial activity against gram-negative bacteria such as E-coli and coliforms. Process for obtaining nanoassembled multilayer films according to claim 1, characterized in that the first silane layer deposited on the glass is obtained by dissolving MPTES in absolute ethyl alcohol, by volume ratio MPTES: absolute ethyl alcohol of 1: 5 ... 7 , at a temperature of 20 ... 30 ° C, the alcoholic precursor solution of MPTES being gradually added, at 20 ... 30 ° C, for 1,5 .... 2,5 hours, under a stirring of 150. ... 350 rpm over a catalytic solution consisting of 25% aqueous ammonium hydroxide solution and distilled water, in water ratio: 25% NH ₄ OH solution of 1: 2 ... 3, the ratio between MPTES and the solution of 25% ammonium hydroxide being 1: 3.5 ... 4.5, the soil thus obtained being sprayed on the glass support and left for 15 .... 45 minutes at a temperature of 20 .... 30 ° C for gelling, and the second layer, molecularly imprinted, being obtained by dissolving MAPTES in absolute ethyl alcohol, in volume ratio MAPTES: absolute ethyl alcohol of 1:10 ... 12, at tem perature of 20 ... 30 ° C, after which an aqueous solution of 0.8-1.2 g / L of LPZ is added to this solution, and the ratio between the solution of LPZ and MAPTES is 2.5. .3: 1, the alcoholic MAPTES precursor solution with tempering being added gradually for 1.5 .... 2.5 hours under a stirring of 150 .... 350 rpm over a catalytic solution of aqueous solution of ammonium hydroxide 25%, the ratio between MAPTES and the ammonium hydroxide solution being 1: 5 ... 6, the soil thus obtained being sprayed over the first silane layer and left to mature 24 ... 48 h at 20 ... 30 ° C and another 24 ... 48 h at 45 ... 55 ° C, after which the laminated films obtained on the glass support are washed in 2-3 times with distilled water in MAPTES ratio: water of 1:10 .. .20, by ultrasound, for 3 ... 5 hours per serving.