Use of esters from vegetable oils for production of polymers having antimicrobial activity, polymeric materials and articles

Abstract

The present invention relates to the use of esters from vegetable oils for the production of polymers having antimicrobial activity, polymeric materials and articles. The present invention provides the use of plasticizers based on fatty acid esters of glyceroformal for the production of polymers having antimicrobial properties. The invention allows the production of articles with PVC, polyurethane, epoxy resins, SBR and NBR rubbers and other polymers, which articles have the ability to disrupt viruses and bacteria deposited on their surfaces.

Description

Technical Field

[0001] The present invention relates to the use of esters from vegetable oils in formulations for producing polymeric materials having antimicrobial properties on their surfaces. The invention enables the production of articles from PVC, polyurethane, epoxy resins, SBR and NBR rubbers and other polymers that have the ability to destroy viruses and bacteria that settle on their surfaces. Background Art

[0002] Floors, tables, chairs, counters, etc. are typically sanitized by distributing a disinfectant, usually consisting of an alcohol solution such as ethanol, onto their surfaces. However, after treatment, the alcohol or disinfectant is removed mechanically or by evaporation. As a result, the surface is once again exposed to microorganisms and pathogens.

[0003] It is therefore desirable to have a material that is capable of inactivating viruses and bacteria when they settle on the surface of the material to avoid the accumulation of pathogens on the surface, thereby making the disinfection effect permanent.

[0004] Furthermore, polymeric materials made from materials with antimicrobial properties (typically floor coverings, wall coverings, clothing, disposable gloves and items) do not require sterilization. The use of these materials in buildings and institutions open to the public, such as hospitals, schools, workplaces, etc., significantly improves the hygiene conditions of these environments.

[0005] Especially in hospitals, where patients inevitably come and go frequently and are therefore particularly susceptible to infection, the need to prevent people from coming into contact with potentially contaminated objects or materials is crucial to preventing the spread of infectious diseases among hospital patients.

[0006] Articles such as containers or floor coverings are now available on the market and are claimed by the manufacturers to have antimicrobial properties. Typical examples are coatings containing silver nanoparticles (AgNPs) or glass based on silver phosphate.

[0007] About the antimicrobial activity of AgNP, known silver nanoparticles can continuously release Ag+ silver ions, and due to electrostatic attraction and affinity for sulfur proteins, silver ions can adhere to cell walls and cytoplasmic membranes. After passing through the lipid protective membrane (this membrane is semi-permeable to cations such as K+ and Na+) and penetrating into the cytoplasm, silver ions can inhibit protein synthesis by denaturing the ribosomes in the cytoplasm. In addition, Ag+ ions can cause deoxyribonucleic acid (DNA) to change. Since sulfur and phosphorus are important components of DNA, the interaction of silver ions with DNA sulfur and phosphorus can cause problems with DNA replication, cell reproduction, or even cause the destruction of microorganisms.

[0008] However, for these mechanisms to occur, direct contact between the silver particles and the microorganisms is necessary. However, when silver nanoparticles are dispersed in a polymer matrix, the Ag+ silver ions of the particles, trapped in the tangle of polymer chains, tend to bind to the atoms of the negatively charged chains, forming strong ionic bonds, making it difficult for them to interact with the functional groups of binding proteins located on the microbial cell membrane or to diffuse across the membrane.

[0009] Therefore, due to the immobility of the silver ions fixed on the particles and the strong ionic bonds with the polar atoms of the polymer chains, even a surface completely covered with Ag+ would have difficulty in breaking these bonds to form others by interacting with the functional groups of binding proteins located on the cell membrane of microorganisms or by diffusion across the membrane.

[0010] To limit interactions with polymer chain anions, specific polymers must be made with certain structural features to enable the silver nanoparticles to be effectively activated ("Antibacterial waterborne polyurethane coatings impregnated with in-situ formed and capped silver nanoparticles viap-sulfonatocalix[4]arene", Progress in Organic Coating, 2023).

[0011] Therefore, due to the inevitably low efficacy of silver nanoparticles dispersed in standard polymer materials, their use requires that the Ag+ ions be supported by specific polymer mixtures that must be deposited on a substrate and subjected to a crosslinking process. However, with the increasing complexity of the coating process, especially for shaped articles, there are challenges due to the anchoring of the protective coating to the substrate.

[0012] Finally, there's the thorny issue of silver ion toxicity. If silver cations can interact with functional groups such as cysteine ​​sulfur in viral and bacterial proteins, disrupting their function, the same thing could happen with sulfur in human cell proteins. In safety data sheets, soluble silver salts carrying Ag+ cations are classified as reproductive toxic (Category 1B), with a risk indication of H360D: They may harm the unborn child.

[0013] In particular, AgNPs are known to act by interacting with free viral particles or with cell-bound viral particles in the intercellular space. In the first case, AgNPs cause changes in virion morphology; in the second case, they can inhibit the early stages of viral replication, such as binding to host cells or penetration of the virus itself.

[0014] The publication "Protective hybrid coating against envelope containing silver, copper and zinc cations effective against HIV and other enveloped viruses (BMC Microbiology, 2016)" reports a study on the antiviral efficiency of a specific polymer composed of tetraethyl orthosilicate, (trimethoxysilyl)propyl methacrylate, methyl methacrylate, and silver, zinc, and copper nanoparticles dispersed in them. The study reported good virucidal activity against herpes simplex virus, but low or no activity against three other tested viruses, including influenza virus.

[0015] Therefore, regardless of the antimicrobial efficacy or toxicity of silver ions, the fact remains that the ability of silver nanoparticles to be dispersed in common polymer matrices such as PVC, PU, ​​etc. is severely hampered, as Ag+ ions are only active when dispersed in a specific coating. To substantiate what has been written, there is no evidence on the market of articles made from polymeric materials whose manufacturers explicitly claim that they can protect against viruses.

[0016] The mechanism of action of silver nanoparticles has been shown to highlight the extreme difficulty of producing polymeric materials with antimicrobial activity by inserting substances with intrinsic biocidal properties between polymer components. This difficulty stems from the fact that, as is well known, chemical processes require efficient collisions between the reacting molecules, i.e., they have a certain kinetic energy and the correct orientation, and therefore the reaction environment must necessarily be liquid or gaseous.

[0017] Silver nanoparticle coatings have shown their effectiveness against bacteria, but there are no studies in which AgNP-coated materials have shown antiviral properties. This is evidenced by the fact that products currently on the market are advertised as having antibacterial properties.

[0018] Thus, as things stand, ceramic-based materials with coatings of metal nanoparticles are available on the market, which have the ability to protect against bacteria, but no common materials have the property of destroying viruses deposited on them.

[0019] Furthermore, respiratory viruses can remain contagious for days, posing a considerable risk of infection to anyone who comes into contact with contaminated surfaces. For bacterial infections to occur, either the skin barrier must be compromised or the protective tissues of the bronchi, lungs, or mucous membranes must be damaged by the virus or lesions. These viruses are responsible for more deaths worldwide than any other infectious agent. SARS-CoV-2, in particular, causes respiratory infections and has been responsible for nearly 7 million deaths since its emergence in 2020. This tragedy has significantly highlighted the importance of disinfecting the environment, particularly schools, hospitals, and general places open to the public, not only against bacteria but, most importantly, against viruses.

[0020] WO 2023 / 067492 A1 mentions fatty acid esters of glycerol formal as components of alcoholic solutions containing antimicrobial lipids. The experimental study examined the antimicrobial properties of alcoholic solutions of soybean fatty acids, including those with epoxidized unsaturated double bonds. Table 2 lists the samples tested, and Sample 4 is an ethanol-based gel containing 2.5% epoxidized soybean fatty acids and 2.5% epoxidized fatty acid esters of glycerol formal, exhibiting disinfectant properties. The addition of epoxidized fatty acid esters of glycerol formal was intended to test whether their presence could enhance the antimicrobial effects of the fatty acids. Specifically, the claimed disinfectant composition is intended to be used as a virucidal agent and to maintain its ability to destroy pathogens such as viruses and bacteria for extended periods after application to a surface. Therefore, this patent application relates to alcoholic mixtures of antimicrobial lipids, rather than solid materials with antimicrobial properties. After application of the disinfectant composition and evaporation of the solvent, a very thin layer of lipid molecules remains on the surface. These molecules adhere to the surface like a lubricating film, but they are not an integral part of the material. Furthermore, the effect of the lipid layer is limited in time, since due to the natural evaporation of all liquid compounds (including high-boiling compounds such as fatty acid esters) even at room temperature, after a certain period of time the disinfecting effect ceases, since the esters have disappeared by evaporation. Clearly, the document in question relates to alcohol mixtures of antimicrobial lipids, not to solid materials having antimicrobial properties. Summary of the Invention

[0021] After subjecting a PVC polymer material plasticized with a fatty acid ester of glycerol formal (GFE) in which the double bonds of the hydrocarbon chain had been partially epoxidized, it was surprisingly discovered that the polymer possessed antimicrobial properties. These tests are essential for its approval as a material for use in medical devices. In fact, the test results showed that the polymer was cytotoxic, and upon further investigation, it was found to also possess antimicrobial properties, particularly antiviral.

[0022] Subsequently, following exploratory tests, the Department of Public Health and Pediatric Sciences of Torino University (Italy) conducted an in-depth study based on the evaluation of viral and bacterial replication on PVC and polyurethanes, formulations into which increasing amounts of GFE had been introduced.

[0023] As shown in the following examples, the materials of the present invention, which consist of polymers mixed with epoxidized esters of glycerol formal, exhibit antimicrobial properties if the concentration of the epoxidized ester is greater than 10% by weight. The polymers may be PVC, polyurethane, polyester, and all other polymers compatible with the epoxidized esters. To date, no polymeric materials with antimicrobial, and in particular antiviral, properties have been invented.

[0024] The results of the experimental studies show that the polymer material has interesting antimicrobial properties at a concentration of more than 10% of GFE in the formulation. Most importantly, the inactivation activity against enveloped viruses (i.e., viruses with a protective membrane, such as the SARS-CoV-2 coronavirus or hepatitis viruses) is excellent.

[0025] This result is considered remarkable because, as explained above, currently available materials with antimicrobial properties are made from specialized materials with certain structural features suitable for supporting coatings of silver nanoparticles or other transition metals. These materials are primarily of ceramic origin. Furthermore, they have little or no effectiveness against viruses, particularly respiratory viruses.

[0026] The polymeric materials of the present invention constitute an innovation that can improve health from the perspective of preventing the spread of diseases in the environments in which people live. In fact, the polymers can be used to make floor coverings, wall coverings, disposable gloves, tablecloths, shoe soles and many other items with antimicrobial properties, not only against bacteria but also, and most importantly, against viruses, especially respiratory viruses, which are not transmitted through blood, and therefore wounds, but rather by simple inhalation.

[0027] Fatty acid epoxy esters of glycerol formal have been patented as a plasticizer (U.S. Patent No. 9,969,862). Adding plasticizers to polymers changes their flexibility properties because it reduces the elastic modulus, and materials with these characteristics are called plasticizers. More generally, according to the IUPAC definition, a plasticizer is a substance or material that is incorporated into a material to increase its flexibility, processability, or extensibility. Therefore, by adding plasticizers to thermoplastic polymers such as PVC or polyurethanes and elastomers such as NBR or SBR rubbers, the required flexibility of the final product (PVC) can be obtained, its workability (polyurethanes are therefore also referred to as viscosity reducers), or its elasticity (rubbers are therefore also referred to as extensions).

[0028] A first object of the present invention is therefore the use of esters derived from vegetable oils for the production of polymeric materials having antimicrobial activity.

[0029] A second object is the use of glycerol formal fatty acid esters, in which the double bonds of the hydrocarbon chain are partially or completely epoxidized, for producing polymeric materials having antimicrobial activity.

[0030] Another object is the use of these esters for producing polymeric materials of various types (thermoplastics and thermosets, elastomers or biopolymers).

[0031] Yet another object is materials and articles made of polymeric materials comprising said esters. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Further objects and advantages of the present invention will become apparent from the following description of non-limiting examples with reference to the accompanying drawings, in which the antiviral capabilities of the polymeric materials of the present invention are demonstrated:

[0033] - Figure 1 is a histogram showing the results of a standard plaque formation assay used to determine the antiviral activity of different PVC matrices;

[0034] - Figure 2 Shown are histograms of a standard plaque formation assay used to determine the antiviral activity of different PVC matrices at different incubation times;

[0035] - Figure 3 is a histogram showing the results (PFU / mL) of replicate standard plaque forming assay experiments used to determine the antiviral activity of different polyurethane tissue samples after 5 hours of incubation. DETAILED DESCRIPTION

[0036] The invention consists in the use of esters (GFE) obtained from fatty acids in which the double bonds of the hydrocarbon chain may be partially or completely epoxidized and glycerol formal in formulations for making polymeric materials having antimicrobial properties.

[0037] The invention also relates to the production of polymeric materials with acquired antimicrobial activity by incorporating into the composition an ester obtained from a fatty acid, wherein the double bonds of the hydrocarbon chain are partially or completely epoxidized.

[0038] In order for the antimicrobial activity of the material to be effective, the concentration of GFE must be greater than 10% wt.

[0039] Fatty acids are all carboxylic acids with a carbon number of 6 to 22 that are derived from animal fats or vegetable oils, including those obtained by the oxidative cleavage process of vegetable oils.

[0040] Epoxidized esters of fatty acids with glycerol formal are disclosed in EP 3068829 B1 and US Pat. No. 9,969,862 B. The structures of two isomers of epoxidized esters of linoleic acid are shown in the figure below, in which only one double bond is epoxidized: (the 6-atom linoleic acid-epoxide, 1,3-dioxan-5-yl ester isomer) and the 5-atom isomer (the linoleic acid-epoxide, (1,3-dioxolan-4-yl)methyl ester):

[0041]

[0042] Table 1 below shows the fatty acid composition of some vegetable oils:

[0043] Table 1

[0044]

[0045] These esters can be obtained by direct esterification of fatty acids with glycerol formal and subsequent epoxidation of the double bonds with a mixture of hydrogen peroxide and formic acid (performic acid), or alternatively, by transesterification of fatty acid methyl esters with glycerol formal and subsequent epoxidation of the unsaturated moieties.

[0046] The polymer material consists of any polymer: PVC, polyurethane, polyester, epoxy, polyamide, elastomer, mixed with GFE together with other additives with their specific activity (heat stabilizers, flame retardants, etc.), and combinations thereof.

[0047] Epoxidation of the double bonds contained in the oleic, linoleic and linolenic acid chains is necessary to increase the compatibility of the plasticizer in polar polymers (such as PVC and PU) and thus reduce its exudation. In fact, polymer materials in which there is a significant exudation of plasticizer cannot be placed on the market.

[0048] Esters of short chain saturated fatty acids up to 14 atoms, such as caprylic acid, pelargonic acid, lauric acid, myristic acid, have sufficient polarity to allow them to be well compatible with polar polymers and are also high enough in molecular weight to have acceptable volatility in many applications.

[0049] In the case of GFE used for non-polar or low-polarity polymers such as SBR and NBR rubbers, epoxidation of the double bonds of the hydrocarbon chain of the fatty acid is not necessary because there are no compatibility problems.

[0050] Furthermore, since testing of fatty acid esters whose double bonds are not epoxy groups has shown a better ability to destroy pathogens, it is preferred to control the epoxidation of the double bonds to a level that limits the migration of the plasticizer from the polymer to the level allowed by the acceptance specifications without further epoxidation.

[0051] A great advantage of the present invention is that finished products made from polymeric materials containing GFE, such as flooring, wall coverings, packaging, medical gloves, clothing, blankets, etc., have the ability to destroy both bacteria and enveloped viruses.

[0052] In many PVC applications, plasticizers are added to increase flexibility. In applications such as disposable gloves, the amount of plasticizer can even exceed 50% by weight of the total. Therefore, if a certain degree of flexibility, as measured by Shore A hardness, is desired in a PVC polymer, if GFE is used as a plasticizer at a concentration above 10%, such a polymer will, in addition to the desired flexibility, also be endowed with properties that will destroy microorganisms that settle thereon. In order for the polymer to be effective, the amount of GFE added to the polymer (in % by weight of the total) must be at least 10%, preferably at least 20%, more preferably at least 30%, 36%, 40%, or 50%.

[0053] Other polymers, such as polyurethanes, epoxies, SBR and NBR rubbers, are already inherently flexible, and plasticizers are often added as solvents during processing to reduce their viscosity. This is particularly true when the molecular weight of the polymer is too high. Therefore, antimicrobial activity can be imparted to polymeric materials by mixing them with GFE and increasing the molecular weight of the polymer to obtain a product with the desired degree of flexibility combined with antimicrobial properties.

[0054] The fatty acids constituting GFE preferably have carbon numbers of C6-C 22 More preferably, the fatty acids include lauric acid, myristic acid, oleic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, and eruric acid. 18 –C 20 The double bonds present in the chain are at least partially epoxidized in order to increase its compatibility with the polymer.

[0055] Another object of the present invention is a polymeric material having antimicrobial activity, comprising the aforementioned fatty acid esters, generally referred to as GFE, in which the double bonds of the hydrocarbon chain may be epoxidized. These materials comprise GFE as a component of the polymer formulation in an amount suitable for advantageously balancing the properties of the plasticizer with those of the antimicrobial properties, preferably in the amounts indicated above in percentages.

[0056] The polymeric materials of the present invention may include, but are not limited to, hygiene articles, hospital articles, work or kitchen utensils, stationery items such as pens and folders for collecting documents, floor coverings, wall coverings, packaging, medical gloves, clothing, blankets.

[0057] Yet another object of the present invention is an article produced from the above material for use in health and public facilities such as hospitals, sports centers, stadiums, workplaces, banks, post offices, restaurants.

[0058] In the following, experimental data obtained using the esters of the invention will be presented.

[0059] In particular, the results of tests to determine the ability to inactivate microorganisms deposited on the surfaces of polymer samples plasticized with varying amounts of GFE demonstrated the ability of the surfaces of these materials to inactivate enveloped viruses and bacteria.

[0060] The inactivation efficiency increases with increasing concentration of GFE in the polymer. Experimental tests have shown that it must be mixed with the polymer in a concentration not less than 10% by weight of the total weight of the mixture in order to effectively detect the antimicrobial activity.

[0061] Virus inactivation only works against viruses with a lipid protective envelope, and this is demonstrated by the fact that tests on naked viruses showed no inactivation.

[0062] The object of the present invention relates to the creation of a solid polymer material in which the antimicrobial component consisting of esters of fatty acids with glycerol formal is part of the material and not a coating or surface deposit, as contrary to that according to the above patent application WO 2023 / 067492 A1.

[0063] It is noteworthy that substances with intrinsic antimicrobial characteristics can give the solid polymer material uniformly mixed with it the ability to inactivate or destroy microorganisms settled thereon without the need for specific treatment of its surface, such as the addition of a specific coating of metal nanoparticles, which is even surprising to technicians in the science of microbiology.

[0064] In particular, the surprising results of the present invention have been subsequently verified by microbiologists who conducted experimental studies on the esters according to the present invention to explain these results. These experimental studies suggest the following: Chemical reactions necessarily occur in liquid or gaseous environments where molecules can move freely to interact. This is particularly true for biochemical reactions, which require macromolecules such as proteins and lipids to be oriented in a certain manner so that chemical interactions can occur between them and with chemical compounds introduced into the aqueous environment.

[0065] In addition to suitable liquid environmental conditions, the biochemical processes that lead to the destruction of pathogens by biocidal substances also require a minimum concentration of biocidal compounds and the absence of factors that could interfere with the action of the biocidal product. The correct orientation of the reactive molecules (steric factors) is very important, and therefore, even in an aqueous environment, the action of antimicrobial agents can be hampered by conditions that limit the mobility and the possibility of effective interaction between the antimicrobial agents and the functional groups contained in the protein macromolecules of the microorganisms.

[0066] All tests on the antimicrobial activity of substances are always carried out in a suitable aqueous environment with physiological pH, in which molecules can move freely without hindrance or interference, and therefore, if a chemical compound is antimicrobial in an aqueous environment, it cannot be inferred that it will retain its antimicrobial activity even when dispersed in a solid matrix that greatly restricts its movement within the interior and prevents its release from leaking outwards.

[0067] In the specific case of PVC plasticized with epoxidized ester GFE, the mobility of these molecules is very low due to the high molecular weight (340 g / mol) and intermolecular interactions with the polymer. Furthermore, despite the moderate mobility of the molecules occurring within the polymer material, and even when the molecules migrate and appear on the surface of the material, they remain strongly anchored to the polymer chains because of the strong compatibility between the polymer and the macromolecules of the plasticizer. The compatibility is due to the formation of strong dipole-dipole intermolecular bonds between the chlorinated polymer and the polar groups of the GFE. The higher the molecular weight and polarity of the plasticizer, the greater the attraction to the polymer.

[0068] It is therefore truly surprising that a biocide substance dispersed within a solid polymer material can impart antimicrobial properties to its surface without requiring specific treatment of the material, despite the fact that the biocide molecules are trapped within the polymer. As explained in the introduction, it is necessary to create a composite coating composed of silver nanoparticles containing Ag+ surface ions, which is then applied to the stone to create a specific type of composite material with antimicrobial properties on the surface. In this case, the free Ag+ ions on the surface can be absorbed by the particles containing the biological fluid in which the microorganisms are immersed.

[0069] Experts speculate that when microorganisms settle on the surface of the polymer material in question, their protective lipid membrane comes into contact with GFE molecules dispersed within the polymer and therefore present on its surface. GFE molecules can migrate within the lipid membrane, disrupting its integrity and, therefore, its functionality. In extreme cases, the membrane can completely disintegrate following microbial attack. It should be remembered that the components of polymeric solids have minimal mobility, as they are held together not by covalent or ionic bonds but by dipole-dipole or dispersion bonds. These considerations support the fact that antimicrobial efficacy is related to the ester concentration in the polymer: the higher the ester concentration, the higher the antimicrobial efficacy.

[0070] The fatty acid esters of glycerol formal have structural characteristics that make the polymers in which they dissolve antimicrobial, since, in addition to their cell membrane-dissolving properties, they are also compatible with polymers in the sense that they can be dispersed homogeneously in polymers at concentrations up to over 50% by weight. In the case of polar polymers such as PVC or PU, their epoxy compatibility must be improved by adequately matching the double bonds of the hydrocarbon chain of the fatty acid.

[0071] Examples of antiviral activity

[0072] To quantify the antiviral activity of glycerol formal epoxidized esters, several tests were performed based on the evaluation of viral replication on different polymer materials. Human coronavirus strain OC43 (HCoV-OC43), herpes simplex virus type 1 (HSV-1) and human adenovirus (HAdV) have been used as viral models. HCoV-OC43 is a surrogate closely related to Sars-CoV-2 and is considered an example of an RNA virus. HSV-1 and HAdV are ubiquitous viruses belonging to the Herpesviridae and Adenoviridae families, respectively, with double-stranded (ds) DNA genomes. Both have a protein coat (capsid), but while HSV-1 has an additional outer phospholipid bilayer (pericapsid), HAdV lacks such a layer. They were then used as prototypes of "enveloped" (HSV-1) and "naked" (HAdV) viruses to evaluate viral replication on different PVC-based matrices. Herpes simplex virus type 1 (HSV-1) and human adenovirus (HAdV) were used as viral models.

[0073] 1. Preparation of Virus Broth

[0074] HSV-1 and HAdV stocks used in the experiments were prepared by infecting African green monkey kidney cells (VERO) grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After the full cytopathic effect appeared, cells and supernatants were harvested and lysed by three freeze-thaw cycles (nitrogen / 37°C). Viruses were aliquoted and stored at -80°C.

[0075] Viral titers for HSV-1 and HAdV were calculated as plaque-forming units (PLU), while for HCoV-OC43, infectivity was determined by measuring absorbance at 595 nm using a Victor X4 multi-label plate reader. -4 The dilution values ​​were obtained and expressed for each concentration of compound as a percentage of the mean absorbance of the uninfected control (mock) (set to 100%).

[0076] 2. Experimental samples

[0077] Table 2 below shows the composition of the samples used for the tests. GFE-S stands for epoxy fatty acid ester of glycerol formal, wherein the fatty acid is obtained from soybean oil, and PVC-FA indicates that 32% of GFE-S and 5% of a fatty acid with a shorter chain than lauric acid (C6-C11 ) plasticized PVC, PVC-GFL indicates PVC plasticized with 36% glycerol formal laurate (abbreviated as GFL), and DOTP stands for dioctyl terephthalate, which serves as a typical reference phthalate-based plasticizer. In the GFE-S used in this test, approximately 85% of the double bonds of the fatty acids are epoxidized.

[0078] Table 2

[0079]

[0080]

[0081] 3. Sample Preparation

[0082] The formulation components of the PVC polymers indicated in the table above were turbo-mixed at a temperature of 70-80° C. The mixture was then processed for several minutes on a laboratory calender using rollers heated to 160° C. to produce plasticized PVC sheets. The sheets were then rolled and pressed into a mold (200° C., 100 bar) to achieve a constant thickness. Finally, the sheets were cut into round test pieces with a diameter of 2 cm and a thickness of 2 mm.

[0083] The polyurethane (PU) matrix was produced by coating polyester fabric with a plastisol consisting of PU with increasing concentrations of GFE added.

[0084] The samples were exposed to UV light (20 min per side) to remove contaminants that could interfere with viral analysis. 500 μl of virus inoculum (over 10 10 ELT) was added to the sample, centrifuged at 2000 rpm for 15 min to allow virus adsorption, and then incubated at room temperature for the indicated time. The sample was harvested after gently scraping the sample to remove adsorbed virus particles, and 100 μl of the sample was used to determine the virus titer by standard plaque formation.

[0085] 4. Virus Titration

[0086] The viral titers of HSV-1 and HAdV were calculated as plaque forming units (PFU). The titration of HSV-1 was performed by standard plating method on confluent VERO cells in 96-well plates. The virus suspension was serially diluted (10 -3 to 10 -10) and 100 μl was inoculated per well; infection was carried out at 37°C for 48 hours. After incubation, the plates were fixed and stained with 0.1% crystal violet solution, and the cytopathic effect was evaluated by microscopic observation. The virus titer was calculated as PFU plaque-forming units (PFU / ml):

[0087] Virus titer (PFU / mL) = number of plaques * 0.1 mL / dilution factor

[0088] HAdV titers were determined by a standard plate method on confluent VERO cells in 24-well plates. Samples were serially diluted in DMEM supplemented with 10% FBS and inoculated on cell cultures at 0.3 mL per well; infection occurred at 37°C for 2 hours. After the adsorption step, the culture medium was removed and replaced with 1% methylcellulose (Sigma-Aldrich) and diluted in DMEM 10% FBS. The cells were incubated at 37°C, 5% CO2 for 7 days. The cytopathic effect was assessed by microscopic observation. The viral titer was calculated as PFU / mL:

[0089] Virus titer (PFU / mL) = number of plaques * 0.3mL / dilution factor

[0090] The infectivity of HCoV-OC43 coronavirus was determined by measuring the absorbance at 595 nm using a Victor X4 multilabel plate reader. -4 The dilution values ​​were obtained and expressed for each concentration of compound as a percentage of the mean absorbance of the uninfected control (mock) (set to 100%).

[0091] 5. Antiviral activity against HSV-1 virus on PVC

[0092] To determine the antiviral activity of PVC samples plasticized with increasing concentrations of GFE-S, 100 μl samples containing HSV-1 obtained after 5 h of adsorption on the samples were analyzed by a standard plaque formation assay, performed as described above. Figure 1The results of a standard plaque forming test are shown, which was used to determine the antiviral activity of different PVC samples after 5 hours of incubation. HSV-1 (500 μL) was placed on the indicated PVC samples and an untreated virus (called "HSV-1 control") was used as a positive control, i.e. no polymer material was placed at the bottom of the wells. The infected plaques were counted under a microscope and the average value is expressed as plaque forming units / mL (PFU / mL). Each result is the average of two experiments, each performed in duplicate. The results obtained for PVC samples plasticized with GFE-S after 5 hours of adsorption show that 36% by weight of GFE-S is able to completely inhibit HSV-1 replication. Even samples with concentrations below 23% showed a strong ability to remove HSV-1, in fact, their concentration was reduced by 85%, from 7x10 10 to 1x10 10 PFU / ml( Figure 1 ).

[0093] from Figure 1 As can be seen in the figure, in addition to PVC plasticized with 36% GFE obtained from soybean oil, PVC blended with 36% GFE obtained from lauric acid also has the same antiviral properties. The best performance is provided by 32% GFE-S with 5% short-chain fatty acids added.

[0094] In order to determine the antiviral activity of PVC samples plasticized with 36% GFE-S against HSV-1 infection kinetics over time, 100 μl of HSV-1-containing samples obtained after the adsorption time indicated on the samples were analyzed by a standard plaque formation test. Each result (PFU / mL) is the average of two different experiments, each titrated in duplicate. Figure 2 In the Figures, a standard plaque formation assay is shown for determining the antiviral activity of different PVC samples at different incubation times (30 minutes, 1.5 hours, 3 hours, 5 hours). HSV-1 (500 μl) was placed on the indicated PVC samples and an untreated virus (referred to as "HSV-1") was used as a positive control. The infected plaques were counted under a microscope and the average value was expressed as plaque forming units / mL (PFU / mL). Each result is the average of two different experiments, each titrated in duplicate. The data obtained show that after only 30 minutes, PVC with a concentration of 23% by weight or more of GFE had killed more than 94% of the HSV-1 virus. After 5 hours, the virus was completely destroyed for samples with 36% and 50% by weight. Dioctyl terephthalate (DOTP) and 10% wt of GFE-S did not show an antiviral effect.

[0095] 6. Antiviral activity against HcoV-OC43 virus on PVC

[0096] The infectivity of HCoV-OC4 was determined by measuring the absorbance at 595 nm using a Victor X4 multilabel plate reader. -4 The values ​​obtained for the dilutions are expressed for each concentration of compound as a percentage of the mean absorbance of the uninfected control (set to 100%). Table 3 below shows the viral replication in % after 5 hours.

[0097] Table 3

[0098]

[0099] 7. Antiviral activity against HAdV on PVC

[0100] To determine the antiviral activity of 36% GFE-S plasticized PVC samples against HAdV adenovirus, 100 μl of HAdV-containing samples obtained after 5 hours of adsorption on the samples were analyzed by standard plaque formation. Table 4 shows the average results (PFU / ml) of replicate experiments.

[0101] Table 4

[0102] sample Concentration after 5 hours PFU / ml comparison <![CDATA[2.5x 10 5 <!-- 10 -->]]> PVC-36 <![CDATA[1.8x 10 5 ]]>

[0103] Tests on non-enveloped HAdV viruses were unsuccessful, confirming the hypothesis that viral inactivation is caused by a breakdown of the protective lipid membrane.

[0104] 8. Antiviral activity against HSV-1 virus on PU

[0105] To determine the antiviral activity of different polyurethane samples with GFE additives obtained from soybean oil, 100 μl of samples containing HSV-1 obtained after 5 h of adsorption on the samples were analyzed by a standard plaque formation test. Figure 3 Shown are the results (PFU / mL) of a repeated standard plaque forming assay experiment used to determine the antiviral activity of different PU tissue samples after 5 hours of incubation. HSV-1 (500 μL) was placed on the indicated tissue samples, and an untreated virus (designated "HSV-1") was used as a positive control. Infected plaques were counted under a microscope and the average value is expressed as plaque forming units / mL (PFU / mL). As can be seen, after 5 hours of contact, samples with 30% and 40% GFE by weight completely destroyed the viral colonies that had settled on their surface. The effects of samples with concentrations of 10% and 20% showed a more moderate effect.

[0106] 9. Antiviral activity of GFE-L against HSV-1 virus on PVC

[0107] To determine the antiviral activity of PVC samples plasticized with 36% wt of GFE-L against HSV-1 virus, 100 μl of HSV1-containing samples obtained after 5 hours of adsorption on the samples were analyzed by standard plaque formation. Table 5 shows the average results (PFU / ml) of repeated experiments.

[0108] Table 5

[0109] sample Concentration after 5 hours PFU / ml comparison <![CDATA[1.0x 10 9 ]]> PVC-GFL <![CDATA[3.5x 10 3 ]]>

[0110] Examples of antimicrobial activity

[0111] Antimicrobial activity on PVC polymer materials

[0112] To evaluate the antimicrobial activity of GFE, Gram-positive bacteria Staphylococcus aureus (ATCC 29213) and Gram-negative Escherichia coli (ATCC 25922) were grown on different PVC-based matrices.

[0113] 1. Preparation of bacterial strains

[0114] All bacteria used in the project were cultured on specific agar media, inoculated into test tubes and kept at -80°C for long-term storage.

[0115] 2. Antibacterial Assay

[0116] Specifically, bacteria were cultured in Mueller Hinton broth (MHB; Becton Dickinson and Company, USA) at 37° C. overnight. After incubation, the bacteria were centrifuged at 4,000 rpm for 10 min, and the resulting pellet was resuspended in 100 μl of MHB and then diluted in MHB until a concentration of 10 was reached. 4 The final concentration of colony forming units (CFU) / mL was determined by colony counting on Mueller Hinton Agar (MHA; Becton Dickinson and Company, USA). Different PVC samples were transferred to multiwell plates and sterilized under UV light for 20 min, and then washed with 1.1 ml of bacterial suspension (10 4The plate was centrifuged at 2000 rpm for 10 min to allow bacteria to adhere to the PVC matrix and incubated at 37 ° C for 5 hours. Bacterial growth controls (represented by the same concentration of bacteria in the absence of material) were incubated at the same temperature for the same time in MHB. At the end of the incubation, a 40 kHz ultrasonic treatment protocol was used at 22 ° C for 10 minutes in 1.1 ml of sterile 0.9% NaCl (Bieffe Medital SpA, Italy) to separate the bacteria adhered to the PVC sample. The total number of bacteria represented by both bacteria firmly adhered to the matrix and planktonic bacteria was quantified by plating on MHA to obtain CFU / ml. All experiments were performed simultaneously for each material and tested in duplicate.

[0117] 3. Experimental samples

[0118] Antimicrobial assays were performed on the following matrices reported in Table 6:

[0119] Table 6

[0120]

[0121]

[0122] 4. Antibacterial activity against Staphylococcus aureus

[0123] Table 7 shows the results of an antibacterial assay to evaluate the effects of various treated PVC-based substrates on 5-hour growth of Staphylococcus aureus. Untreated bacteria served as a control for bacterial growth. Colonies were counted, and the average value is expressed as total CFU. To obtain CFU counts, bacterial solutions incubated with the various PVC samples were serially diluted and then plated on agar.

[0124] Table 7

[0125]

[0126] 5. Antibacterial activity against Escherichia coli

[0127] Table 8 shows an antibacterial assay evaluating the effects of various treated PVC-based substrates on 5-hour E. coli growth. Untreated bacteria served as a control for bacterial growth. Colonies were counted and the average value expressed as total CFU. The results showed a significant reduction in E. coli growth, less than 1 Log relative to the control sample, which equates to over 70%.

[0128] Table 8

[0129]

[0130] Medical vinyl gloves with antimicrobial properties

[0131] Flexible PVC sheets suitable for medical glove production were prepared by dipping in a plastisol bath using the following formulation: 100 parts of PVC (polyvinyl chloride) resin obtained by emulsion processing, 100 parts of a plasticizer mixture consisting of 70 parts of PVC-36 and 30 parts of GFL, 1.5 parts of a stabilizer, and 2 parts of a reinforcing filler. The sheets were then cut into circular test pieces with a diameter of 2 cm.

[0132] To determine the antiviral activity of the PVC samples against HSV-1 virus, 100 μl of the samples containing HSV1 were analyzed by standard plaque formation after 5 hours of adsorption on the samples. The table below shows the average results (PFU / ml) of the replicates.

[0133] Table 9

[0134]

[0135] Nitrile elastomer with antimicrobial properties

[0136] Acrylonitrile butadiene rubber (NBR) is an unsaturated statistical copolymer of acrylonitrile and butadiene. Due to its excellent oil and chemical resistance, NBR is widely used in the automotive and petroleum industries for engine oils and fuel transportation equipment, machinery, pumps, and more. Another major use of NBR is in the production of disposable gloves. NBR's applications vary depending on the polymer's acrylonitrile content and molecular weight. The polarity of NBR is provided by the acrylonitrile component. The higher the acrylonitrile fraction, the more polar the polymer.

[0137] Table 10 below shows compositions that can be used to produce nitrile rubber-based elastomers. In this example, GFE-A refers to a glycerol formal fatty acid ester derived from soybean oil, wherein approximately 80% of the double bonds are epoxidized, GFE-B refers to a glycerol formal fatty acid ester whose chain double bonds are not epoxidized, and NBR Control refers to a polymer composition without a plasticizer. Measured in parts per hundred parts of the original polymer (phr), the percentage amounts are identical for both.

[0138] Table 10 Composition of nitrile rubber

[0139]

[0140] The compounds were prepared using a two-stage mixing procedure in a laboratory mixer. First, the polymer was introduced, followed by the addition of process oil, carbon black, stearic acid, and all other components except sulfur and accelerators and CBS, which were added 5 minutes later.

[0141] To determine the antiviral activity of the nitrile elastomer of this example against HSV-1 virus, 100 μl containing HSV1 was analyzed by standard plaque formation after 5 hours of adsorption on the sample. Table 11 below shows the average results (PFU / ml) of repeated experiments.

[0142] Table 11

[0143]

[0144] The data in the table show that bacterial growth was significantly reduced, more significantly for GFE-B.

[0145] Polymer materials for medical gloves

[0146] The use of the polymeric materials having antimicrobial properties of the present invention for the construction of articles, tools, floors and furniture in hospital facilities is of vital importance as it facilitates the spread of viruses and bacteria through infection. One of the most important applications is the production of antimicrobial gloves, particularly antiviral vinyl nitrile and latex gloves.

[0147] Paragraph 8.1 of Annex II to the Medical Devices Directive 93 / 42 / EC states: "The device and its method of manufacture must be designed in such a way as to eliminate or reduce, as far as possible, the risk of infection for the patient, the user and third parties. The design must facilitate handling and, if necessary, minimize the risk of contamination of the device by the patient or vice versa during use." The European legislator therefore requires that medical devices (in our case disposable gloves) not only protect the user from infection in the best possible way, but also the patient and "third parties", i.e. other people present. But this precautionary principle is common to all hospitals worldwide.

[0148] Currently, no specific tests are required for the protection of patients or third parties, as no gloves offer active protection against other people with whom the glove wearer comes into contact. Unfortunately, actual gloves are inert to microorganisms, and microorganisms deposited on the user's hands after contact with an infected patient, or even the bed he lies on or the chair he sits on, can then be transferred to other untouched objects or, even more reprehensible, to another patient, thereby becoming a hidden vector of infection.

[0149] Therefore, the only possible protection at present is for the wearer of the gloves, and as for the standards regarding the classification of gloves as protective against microorganisms, the references are the ISO 374-5:2016 standard: "Requirements for the classification of disposable gloves as providing protection against microbiological risks posed by fungi, bacteria and viruses" and the ISO 16604:2004 standard, which describes the test to be carried out (penetration by bacteriophage Phi-X174).

[0150] In the article "Testing for viral penetration of non-latex surgical and examination gloves," European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 322–326, several types of medical gloves were compared. Table 1 of the article summarizes the test results for viral penetration of the different types of gloves tested. The virus tested was bacteriophage FX174.

[0151] Table 12 summarizes the results of the virus penetration test, where Brand A Method B refers to a sterile powdered exam glove made of polyvinyl chloride and Brand B refers to a non-sterile powder-free exam glove made of nitrile (YES indicates the virus has penetrated the material):

[0152] Table 12

[0153]

[0154] The table shows the results for three different glove conditions: Unstressed refers to a new glove, Stressed refers to a glove that has been stretched and rubbed in a certain way, and Punctured simulates the fact that a glove may experience micro-cracking during use.

[0155] The term microperforation describes pores that are too small to be discerned by the user, but large enough to allow microorganisms to pass through. The flexible nature of the glove material allows microperforations to open and close depending on whether the glove is stretched. Microperforations can be caused by manufacturing defects, material fatigue, or extensive wear. Permeation can occur by diffusion, capillary action, or forced movement through the microporous polymer membrane.

[0156] The table shows that in gloves fresh from the packaging (unstressed gloves), vinyl gloves have slightly better permeability than nitrile gloves, while in stressed gloves, permeability is higher: 13% for vinyl and 20% for nitrile. In punctured gloves, 100% vinyl gloves show some viral permeability.

[0157] If the plasticizer for vinyl gloves is a GFE ester, the permeation test results for vinyl will be better than those obtained with ordinary plasticizers because the virus, when passing through the polymer entanglement, will necessarily move in an environment composed mainly of antiviral lipid molecules, which leads to their destruction. Therefore, at least under stressed conditions, we should expect zero permeation and much better performance in punctured gloves.

[0158] Nitrile gloves do not contain plasticizers because the polymer already has the desired flexibility characteristics. However, by introducing even moderate amounts of GFE plasticizers, we can expect a reduction in viral and bacterial permeability. The reduction in permeability will be proportional to the concentration of GFE in the polymer. They can be as little as 10 parts per 100 parts of GFE polymer (10 phr) to achieve satisfactory results.

[0159] in conclusion

[0160] According to the above, the results of the examples show that mixing GFE esters and polymers compatible therewith in fractions greater than 10% wt allows to solve the drawbacks of the prior art and demonstrates that important advantages have been achieved.

[0161] In fact, using the polymers according to the present invention, there is no need to treat objects or surfaces to disinfect them against microorganisms. Thanks to the new material, it is now possible to produce plastic or elastomeric objects that have the ability to kill viruses and bacteria when these viruses settle on their surfaces. In other words, it is possible to achieve significant results in the production of materials that can improve environmental hygiene.

[0162] Furthermore, essentially any polymer material can be produced, and particularly those used in hospitals, as they are important in creating the healthiest possible environment.

[0163] Variations and modifications of the polymers according to the present invention that are within the capabilities of those skilled in the art are intended to be covered by the appended claims.

[0164] For example, the use of these antimicrobial materials is very useful in hospitals. The materials are those that are used in hospitals as they prevent the spread of viruses and bacteria.

[0165] Experimental data clearly demonstrate that the destruction of enveloped viruses is not only effective but also rapid. In fact, the tests presented in the examples, conducted under specific conditions, showed a complete viral destruction time of approximately 30 minutes. However, those conditions involved contacting a suspension of cells infected with a particular virus with the polymer surface, and while they showed a complete viral destruction time of approximately 30 minutes, under realistic conditions, the destruction time would be considered instantaneous, as viruses are contained in small bioparticles expelled by an infected person through breath or sneezing, or are present on their hands or clothing.

[0166] In fact, in this case, the problem is not the large amount of biological fluid, about 50 ml, contained in a specific hole in contact with the surface, but the tiny biological droplets that are deposited on objects touched by the infected person or on materials that come into contact with other infected materials (such as the infected person's clothes). The extremely small amount of these droplets compared to the relatively large surface area on which they are deposited makes the destruction of the virus instantaneous.

Claims

1. Use of plasticizers based on fatty acid esters of glycerol formal for producing polymers having antimicrobial properties.

2. The use of the plasticizer according to claim 1, wherein The double bonds in the hydrocarbon chain of the fatty acid having double bonds may be at least partially epoxidized in order to increase the compatibility of the ester with polar polymers.

3. The use according to claim 1 or 2, wherein The carbon number of the fatty acid is C6 to C22.

4. The use according to any one of claims 1 to 3, wherein The fatty acids include caprylic acid, pelargonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and erucic acid.

5. The use according to any one of claims 1 to 4, wherein The polymer is selected from thermoplastics, thermosets, elastomers or biopolymers, possibly mixed with additives, and combinations thereof.

6. The use according to claim 5, wherein The polymer is selected from polyvinyl chloride, polyurethane, acrylonitrile-butadiene rubber (NBR), styrene-butadiene rubber (SBR), cellulose acetate, polyester resin, and epoxy resin.

7. The use according to any one of claims 1 to 6, wherein The esters are present in a concentration of at least 10% by weight, preferably at least 20% by weight, at least 30% by weight, at least 40% by weight or at least 50% by weight of the total.

8. A polymeric material having antimicrobial activity, comprising a fatty acid ester of glycerol formal, wherein the double bonds of the hydrocarbon chain are partially or completely epoxidized.

9. The polymer material according to claim 8, wherein The ester is present in an amount of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% by weight.

10. The material according to claims 8 and 9 for use in the production of healthcare products, hospital products, work or kitchen utensils, stationery items such as pens and folders for collecting documents, floor coverings, wall coverings, packaging, medical gloves, clothing, coverings.

11. Articles produced from the material according to any one of claims 7 to 10 for use in health and public facilities such as hospitals, sports centres, stadiums, workplaces, banks, post offices, restaurants.

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