METHOD FOR CONFERRING ANTIMICROBIAL PROPERTIES TO A SYNTHETIC SUBSTRATE, SYNTHETIC SUBSTRATE AND MEDICAL DEVICE

Abstract

The present invention discloses a method for the treatment of synthetic substrates to provide antimicrobial properties.

Description

1 / 37 METHOD FOR CONFERRING ANTIMICROBIAL PROPERTIES TO A SYNTHETIC SUBSTRATE, SYNTHETIC SUBSTRATE AND MEDICAL DEVICE DESCRIPTION

[001] The present invention finds application in the biomedical field and is particularly related to the treatment of synthetic substrates to provide antimicrobial properties. Background of the invention

[002] As medicine and medical practice have evolved, so has the production of medical devices. The most widely used material to date is plastic, thanks to its unique characteristics and desirable attributes that have encouraged its increasing use over time. Among the many advantages of using plastic are, undoubtedly, its low weight compared to all other materials commonly used in medicine, the fact that it can be easily machined to effectively create even very small and complex components, and above all, its flexibility. Furthermore, it is essential to remember its resistance to chemicals, lipids, sterilization methods, disinfectants, and finally, its biocompatibility.Currently, different types of plastic materials are used in the construction of medical devices, from polyethylene to polyamide via nylon, depending on their intrinsic characteristics and the intended use of the device, which may be a suture thread, a catheter for deep venous access or use in urology, or even a filter for embolic protection or hemodialysis. As the use of plastics in clinical practice has progressed, their properties have naturally been refined by researching mixtures of chemical agents, or directly new compounds, capable of responding more effectively to the problems arising from their daily use, until more recently when actual coatings for these materials began to be studied. Petition 870260022917, dated 12 / 03 / 2026, page 13 / 63 2 / 37 functionalize these materials. These coatings can incorporate metals, such as silver or copper, or antimicrobial agents, such as antibiotics or antifungals, to further improve biocompatibility characteristics. However, despite numerous efforts, it has not yet been possible to obtain a plastic material, coated or uncoated, that fully incorporates the desired characteristics. The most representative categories of plastic medical devices will be described below, with particular attention to their limitations and the approaches currently available on the market to overcome them. CATHETERS

[003] Catheters are made from different materials, including polytetrafluoroethylene, polyurethane, polyethylene, and silicones. However, polyurethane remains the best choice, as it ensures a high level of biocompatibility and the optimization between the outer diameter and wall thickness guarantees optimized flow relative to the invasiveness of the device. VASCULAR CATHETERS

[004] The three most common causes of vascular catheter dysfunction and failure are biofilm formation, which leads to the development of a fibrin sheath, infection, and thrombus formation. Biofilm formation is a major factor in early and late catheter failure and in infection-associated catheter failure. A biofilm is a complex structure formed by bacteria that have attached themselves to an artificial surface. Bacterial attachment to the catheter surface and biofilm formation begin soon after catheter placement: electron microscopy has shown bacterial attachment to the surfaces of permanent vascular catheters as early as 24 hours after insertion. The bacteria proliferate and secrete a polysaccharide matrix that provides a medium for the attachment of additional organisms. Catheters are infected by two Petition 870260022917, dated 12 / 03 / 2026, p. 14 / 63 3 / 37 main pathways, depending on the time elapsed after the catheter placement procedure. In the first 30 days after placement, catheters are infected by external pathways, mainly from the patient's skin microflora and the hands of the medical team. After the first 30 days, catheters are infected by internal pathways, including contamination of the catheter hub, leading to hematogenous dissemination and bacteremia. Typical organisms involved in these infections include coagulase-negative staphylococci, Staphylococcus aureus, Pseudomonas, enterococci, and Candida. A clinical biofilm infection is typically resistant to antimicrobial treatment due to the failure of antibiotics to penetrate all layers of the biofilm and the slow-growing nature of the organisms involved, rendering antibiotics ineffective. A biofilm evolves over weeks or months into a more complex structure: a fibrin sheath.A fibrin sheath can surround the catheter surface, starting at the venipuncture site and progressively extending along the catheter until it eventually covers the catheter tip, thus obstructing the catheter lumen. The rate of catheter malfunction due to fibrin sheath formation has been reported to be as high as 50%. Finally, catheter failure resulting from thrombosis is a common problem in hemodialysis patients. Hemodialysis patients have a unique blood physiology that makes them more susceptible to thrombosis formation. These factors include platelet and plasma abnormalities. URINARY CATHETERS

[005] Urinary tract infection (UTI) is one of the most common healthcare-associated infections reported to the CDC's National Healthcare Safety Network, with >560,000 cases occurring and more than 13,000 attributable deaths (2.3% mortality rate) each year. UTI is also the Petition 870260022917, dated 12 / 03 / 2026, p. 15 / 63 4 / 37 is the leading cause of secondary nosocomial bloodstream infections, with approximately 17% of hospital-acquired bacteremia of urinary origin attributed to a mortality rate of up to 10%. Among hospital-acquired UTIs, 75% of cases are associated with urinary catheters, termed catheter-associated urinary tract infections (CAUTIs). In short-term catheterization (<3 days), most episodes of catheter-associated bacteriuria are asymptomatic, with a single isolated organism, and <5% of catheter-associated bacteriuric patients are identified with bacteremia. However, in long-term catheterization (>28 days), almost all patients develop bacteriuria. Two phenomena are observed during catheterization: the incidence of new episodes of bacteriuria by a wide variety of Gram-negative and Gram-positive uropathogenic bacterial species and the presence of persistent strains in the catheterized urinary tract.Since prolonged use of urinary catheters leads to a higher risk of acquiring urinary tract infections (UTIs), it is recommended to minimize catheter use during hospitalization. Furthermore, it is well recognized that the use of urinary catheters favors the adhesion and colonization of microorganisms, leading to infections, which are always associated with the occurrence of microbial biofilms. Biofilms are communities of microorganisms adherent to surfaces, integrated into a self-generated extracellular matrix. Bacteria sequestered within the biofilm are key agents in the pathogenesis of UTIs because they are protected from host immune responses and antimicrobial agents. Within the biofilm, bacteria can transfer genes encoding antimicrobial resistance, and cell-to-cell communication can occur through a process called quorum sensing.

[006] Urinary tract infections that develop with biofilm formation are 1000 times more resistant to antibiotics compared to their Petition 870260022917, dated 12 / 03 / 2026, page 16 / 63 5 / 37 planktonic equivalents, resulting in a very challenging treatment. Furthermore, it is known that urease-producing agents, such as Proteus mirabilis, lead to the breakdown of elements such as calcium, magnesium, and phosphate in urine during short-term catheterizations. Obstruction occurs when encrustation develops to the point where the lumen becomes occluded. In patients with long-term catheterization, 48% developed catheter blockage and 37% developed shunting (where urine continuously flows around the outside of the catheter). These complications can be painful and result in urinary incontinence (which is distressing for the patient).

[007] Fouling results from ionic components in urine crystallizing on the surface of the biomaterial and becoming incorporated into a bacterial biofilm layer. The bacterium most commonly detected in association with fouling is Proteus mirabilis. Proteus produces an enzyme called urease, which cleaves urea to form ammonia and carbon dioxide. The carbon dioxide dissolves to form carbonic acid. However, since more ammonia is formed than carbonic acid, this process results in a net decrease in the concentration of H+ ions, making the urine more alkaline. This change in pH has a profound effect on the solubility of struvite and calcium phosphate. Embolic Protection Filters

[008] Manipulation of atherosclerotic lesions with wires, catheters, balloons, stents, and other intravascular devices during invasive procedures releases atherosclerotic plaque, resulting in distal embolization. This plaque debris leads to absent or slowed flow due to a multitude of factors, including mechanical obstruction of macrovascular and microvascular channels, local platelet adhesion, platelet activation, and thrombosis. Petition 870260022917, dated 12 / 03 / 2026, page 17 / 63 6 / 37 attributable to the release of tissue factors and microvascular spasm by thromboxane release.

[009] Embolic protection devices prevent or reduce plaque debris from reaching the distal bed and thus have the potential to reduce adverse clinical events. The Embrella Deflector Device (Edwards Lifesciences) is an umbrella-like device with two heparin-coated polyurethane membranes mounted on an oval-shaped nitinol frame; the Sentinel device (Claret Medical) consists of 2 polyurethane filters placed in a flexible radiopaque nitinol frame attached to a 100 cm long delivery catheter; the TriGuard embolic protection device (Keystone Heart) features a nitinol mesh coated with chemical and physical substances (Applause™ Heparin Coating SurModics, Inc., Eden Prairie, MN, USA), thus reducing the possibility of thrombus formation; the Embol-X EPD (Edwards Life Sciences) consists of a heparin-coated polyester mesh in a flexible nitinol frame.Generally, problems with these filters are due not only to platelet activation and the consequent formation of thrombi, but also to potential clogging due to clusters of serum proteins that can alter their porosity. Mesh for Abdominal Wall Repair

[010] Until 1958, treatment for abdominal wall hernias was based on sutures, and the biggest problem faced by surgeons at the time was the increased recurrence of the hernia. To overcome this, the concept of using a mesh was introduced by Usher. Synthetic meshes can be permanent or absorbable. Permanent materials are generally composed of polypropylene, polyester, or expanded polytetrafluoroethylene (ePTFE). Each of these materials has benefits and limitations. They are often combined or added to create composite meshes designed to draw out the hernia. Petition 870260022917, dated 12 / 03 / 2026, page 18 / 63 7 / 37 advantage of their strength while combating their deficiencies. A wide variety of these composite meshes have been approved for clinical use. Permanent synthetic meshes are susceptible to infection, limiting their use in contaminated fields. A recent meta-analysis showed that the overall infection rate was 5%. Mesh removal was performed in 70% overall and in 100% of ePTFE grafts. Polypropylene

[011] Polypropylene has been widely used in a wide variety of surgical procedures and is relatively inexpensive. Experimental studies have shown that polypropylene mesh is well incorporated into the anterior abdominal wall within 2 weeks of implantation. However, the inflammatory reaction may predispose to adhesion formation and result in contraction of the mesh and surrounding tissues.

[012] While the inflammatory response generated by polypropylene contributes to limiting its durability, it also increases adhesion formation when the mesh is used adjacent to the intestine. As a result, polypropylene is rarely used alone in the peritoneal cavity. Polypropylene can be combined with a temporary or permanent material to reduce adhesion formation or isolate it from contact with the intestine (i.e., polyglecaprone, carboxymethylcellulose, and omega-3 fatty acid). The inflammatory response to polypropylene also causes the material to shrink by 30 to 50%. In addition to causing separation from native tissue, the shrinkage can lead to coiling of the composite meshes, exposing the polypropylene component to the intestinal surface. Polyester

[013] Polyester is a carbon-based polymer frequently used in fabrics. Initial studies raised concerns about rates of Petition 870260022917, dated 12 / 03 / 2026, page 19 / 63 8 / 37 infection, small bowel obstruction, recurrence, and fistula are higher compared to other synthetic materials. Polyester meshes remain clinically available, with the caveat that they must be separated from the intestinal surface. Polyester may offer some advantages over polypropylene. In an animal model of ventral hernia repair, a polyester mesh coated with a collagen hydrogel matrix (Parietex) showed superior tissue incorporation than a composite mesh of polypropylene and sodium hyaluronate / carboxymethylcellulose (Sepramesh, Bard, Davol, Inc., Warwick, RI). Expanded Polytetrafluoroethylene (ePTFE)

[014] ePTFE is a microporous woven mesh that was originally used in vascular grafts. The material used in abdominal cases generally has two sides: one side is smooth with small pores, and the other has larger pores with grooves and ridges. The material is designed to place the smooth side towards the intestine to minimize adhesions, and the rough side towards the fascia to allow for inward tissue growth. However, experimental studies have shown limited inward fiber growth and minimal inflammatory changes surrounding ePTFE grafts. This may be a result of the small pore size, hydrophobicity, or the electronegative charge of the mesh. In an animal model of ventral hernias, grafts constructed with ePTFE were compared with those of polypropylene. Although the ePTFE grafts showed less evidence of adhesions, there was no inward growth of fibro-collagenous tissue in the ePTFE graft.The polypropylene mesh was fully incorporated. Furthermore, hernia recurrence was 60% in the ePTFE group, compared to 0% in the polypropylene group. All recurrent hernias were at the mesh junction. Petition 870260022917, dated 12 / 03 / 2026, p. 20 / 63 9 / 37 and native tissue, suggesting that the lack of internal growth in the ePTFE resulted in insufficient anchoring of the mesh to the fascia.

[015] To overcome the limitations of the various devices illustrated, new coatings, materials, and designs have been thoroughly investigated. Handling approaches fall into three main categories: (i) antimicrobial; (ii) antifouling; and (iii) antithrombotic. Antimicrobial strategies include passive antimicrobial release (typically by impregnation into the plastic surface) and non-release contact elimination (by antimicrobial compounds covalently anchored to the plastic material or by incorporation into a hydrogel coating). Antifouling materials or designs prevent bacterial adhesion using non-antimicrobial approaches, such as mechanical methods or unfavorable surface topography or chemistry.Finally, heparinized coatings have been widely applied due to heparin's ability to bind and induce an allosteric conformational change in antithrombin, thereby dramatically accelerating its ability to inhibit FXa, thrombin, and other proteases that contribute to thrombus formation. i) Antimicrobial strategies Silver coating

[016] Silver is one of the most popular antimicrobials for coating medical devices and one of the few antimicrobials approved by the FDA for application in urinary catheters. Silver coatings can be applied to both internal and external surfaces and slowly release silver ionic particles during the first 5 days, providing consistent elution over time. Silver-coated synthetic materials reduce pathogen colonization by releasing silver ions into the surrounding environment, which target bacteria through three mechanisms: (1) function of Petition 870260022917, dated 12 / 03 / 2026, page 21 / 63 10 / 37 membrane impaired by loss of membrane potential, (2) protein dysfunction by destruction of the Fe-S cluster and (3) oxidative stress by depletion of antioxidants. Antibiotic coatings

[017] Antibiotics selectively inhibit the biological activities of microorganisms at low concentrations, which is fundamental for the prevention and control of infectious diseases. Antibiotic coatings are perhaps the most direct method to prevent bacterial infections, designed to inhibit or delay the onset of biofilm formation by a controlled release of high local concentrations of antibiotics at the potential colonization site. Compared to silver coatings, antibiotic coatings have a high activity for targeting the pathogen. To date, many antibiotics have been impregnated into plastic medical devices, including nitrofurazone, gentamicin, norfloxacin, sparfloxacin, vancomycin, and rifampicin.

[018] It is noteworthy that nitrofurazone can cause mammary and ovarian tumors in animals subjected to it and, therefore, has been listed as a Group I banned drug for production animals by the FDA. This side effect of nitrofurazone has led to a slowdown in research in this area. Catheters coated with antimicrobial substances are beneficial for preventing catheter-related urinary tract infections; however, the cost, patient convenience, and complications associated with these catheters must also be considered. The greatest concern with all antimicrobial-treated medical devices is the development of resistance against activated / coated substances. Thus, their use may be losing ground due to limited efficacy and the potential for resistance development. Bactericidal enzymatic coatings Petition 870260022917, dated 12 / 03 / 2026, page 22 / 63 11 / 37

[019] Bactericidal enzymes inhibit bacteria by producing antimicrobial substances (i.e., oxidative enzymes). Specifically, hydrogen peroxide (H2O2) produced by peroxidases is used to attack bacterial cells or oxidize halides into more potent antimicrobials. Recently, bactericidal enzymes have been used as highly active antibacterial materials for experimental catheter coatings. Bactericidal enzymes have several advantages over other conventional antimicrobials as catheter coatings. (1) They are more specific to selected pathogens without disturbing other benign microorganisms in the host, (2) It is very difficult for pathogens to develop resistance to bactericidal enzymes, (3) Bactericidal enzymes are considered natural, non-reactive, and non-toxic to the host.However, the production and purification of bactericidal enzymes are much more expensive compared to conventional antimicrobials, such as silver and antibiotics. Furthermore, bactericidal enzymes are prone to denaturation under extreme conditions during sterilization, storage, and transport of the device. Antimicrobial peptide coatings (AMPs)

[020] AMPs, or so-called host defense peptides, are broad-spectrum antimicrobials that are effective against both Gram-negative and Gram-positive strains, viruses, and fungi. AMPs target pathogens through multiple pathways: (1) cytoplasmic membrane switching; (2) membrane permeabilization; (3) autolysin activation; (4) inhibition of DNA, RNA, and protein synthesis; (5) inhibition of certain enzymes; (6) enhancement of immunomodulation. These AMP-coated catheters have shown excellent antimicrobial and antibiofilm activities against pathogens and have not exhibited toxicity to mammalian cells. ii) Anti-fouling strategies Petition 870260022917, dated 12 / 03 / 2026, page 23 / 63 12 / 37

[021] Antifouling materials or designs are inherently resistant to bacterial attachment and subsequent biofilm formation due solely to their structure, without the need for antimicrobial additives. Antifouling mechanisms include hydration forces, steric repulsion, electrostatic repulsion, and low surface energy. The field of antifouling polymers is growing rapidly and shows promise as an exciting new approach to combat bacterial infections, without the potential to exacerbate antibiotic resistance. PEG coating

[022] PEG is a hydration layer with a large energy barrier and steric repulsion to non-specific protein adsorption. PEG coating on substrates is designed to prevent bacterial adhesion. This coating has demonstrated excellent antibacterial and antifouling activities against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. While PEG-based coating has historically been considered the gold standard for protein-resistant surfaces, there are still disadvantages to using PEG. Recent studies have indicated that PEG elicits an immune response in ~25% of the population. Furthermore, the long-term stability of the PEG coating is compromised by oxidative degradation of the polyether backbone. Hydrogel coatings

[023] The formation of a hydrogel layer is another strategy to achieve protein resistance. Hydrogels are networks of loosely cross-linked polymers that can swell and retain large amounts of water. Similar to PEG-grafted surfaces, the hydrogel forms a hydration layer, which increases the hydrophilicity of the surface and establishes a barrier to inhibit the adsorption of non-specific proteins. Often, the approach of Petition 870260022917, dated 12 / 03 / 2026, page 24 / 63 13 / 37 hydrogel coating is coupled to silver-based treatment. However, it has been reported that the hydrogel layer increases the aggregation of planktonic cells and newly nucleated crystals, leading to even faster catheter blockage than in the case of uncoated silicone. Polyzwitterion coatings

[024] Polyzwitterions possess both cationic and anionic groups along the polymer backbone, which are generally charge-neutral. Polyzwitterions are highly hydrophilic, forming a hydration layer on the surface. Polyzwitterions also repel the uptake of non-specific proteins through electrostatic and steric repulsion. Polyzwitterionic surfaces have been shown to be a potent alternative to conventional PEGylated antifouling surfaces. The three most common zwitterionic polymers are phosphorylcholine (PC), sulfobetaine, and carboxybetaine. Unlike permanently charged polymers, zwitterionic polymers can switch between anionic and cationic forms through a controlled hydrolysis process, where the release of dead bacteria and the antifouling agent occurs on the same substrate. However, the long-term stability of zwitterionic surfaces remains a concern.The surface hydration layer of polizwitterion can break down and lose its antibacterial activity, leading to bacterial adhesion to the surface of the modified coating. Nitric oxide releasing coating

[025] Nitric oxide (NO) is a chemically unstable lipophilic gas and one of the smallest endogenously produced molecules against infections. NO has exhibited local / biofilm bactericidal dispersal effects through amino- and sulfhydryl nitration, lipid peroxidation, tyrosine nitration, DNA cleavage, and stimulation of motility and dispersal regulation. Once generated by Petition 870260022917, dated 12 / 03 / 2026, page 25 / 63 14 / 37 activated immune cells, NO can diffuse through bacterial cell membranes to destroy the microorganism by exerting nitrosative and oxidative stress. Common NO donors are S-nitrosothiols, such as S-Nitroso-N-acetyl-DL-penicillamine (SNAP) and S-Nitrosoglutathione (GSNO), which can be blended into polymeric materials for slow NO release. NO-releasing polymeric coatings have been widely applied to prevent biofilm-related infections in implanted biomedical devices. However, very few studies have been conducted with urinary catheters impregnated with NO or NO donor. Long-term storage stability, NO donor diffusion in the physiological environment, and material toxicity remain important concerns requiring further investigation. iii) Antithrombotic coatings

[026] There are two antithrombotic coatings available on tunneled catheters for hemodialysis: Carmeda BioActive Surface (CBAS) in catheter products from Spire Biomedical and Trillium Biosurface developed by BioInteractions Ltd. (Reading, Berks, UK) available in Tyco-Kendall products. Both surfaces use catheter-bound heparin as an anticoagulant. Heparin is not only a strong anticoagulant, but many studies have shown that it reduces thrombin-activated factors and smooth muscle cell proliferation. However, the initial clinical benefit observed for blood-contact devices with a heparinized coating is generally not maintained, presumably due to the inability to sustain heparin surface activity as a consequence of enzymatic or chemical degradation with reduced availability of high-affinity antithrombin binding sites.In particular, heparin and heparan sulfates are depolymerized and degraded by heparanase, an endo-D enzyme. Petition 870260022917, dated 12 / 03 / 2026, page 26 / 63 15 / 37 glucuronidase is produced by a variety of cells and tissues, including fibroblasts, endothelial cells, platelets, activated immune cells, hepatocytes, and cancer cells. Plasma heparanase activity is significantly elevated among patients with atherosclerosis, renal failure, and type 2 diabetes, as well as after surgery, which may further contribute to the early reduction of the clinical benefit of heparin-linked prostheses. Significantly, unfractionated and low molecular weight heparin is susceptible to cleavage by heparanase, with a reduction in the local concentration of high-affinity antithrombin binding sites and neutralization of anticoagulant properties.

[027] Polyphenols are widely found in many plant products, such as green tea, red wine, cocoa, and fruits. They are readily available and inexpensive and are generally recognized as safe by the U.S. Food and Drug Administration.

[028] Yang L and colleagues [6] describe the procedure for obtaining a coating on a quartz or silicon wafer using tannic acid coordinated by metal bonds with Fe3+. The Fe-TA film proves effective in preventing platelet adhesion, however, the procedure is long and complex and involves a pretreatment with a very aggressive boiling solution (98% H2SO4: 30% H2O2 = 3:1) and a subsequent passage in trimethylchlorosilane (4% dichloromethane) for 4 hours.

[029] The approach described above uses complex procedures and critical processing conditions and is specific only to certain types of plastic substrates.

[030] The inventors of the present application have disclosed the use of polyphenols for the functionalization of tissues of animal origin for the manufacture of biological prostheses [1-6]. Petition 870260022917, dated 12 / 03 / 2026, page 27 / 63 16 / 37 Summary of the invention

[031] The inventors of the present patent application have surprisingly discovered that synthetic substrates can be treated in order to impart antimicrobial properties to them, so that these substrates can be used for the manufacture of medical devices. Brief Description of the Figures

[032] Figure 1: Scanning electron microscopy evaluation of the internal and external surface of original (untreated) polyurethane samples and after treatment with two variants of the caffeic acid-based solution (treated with CA 1 and treated with CA 2).

[033] Figure 2: Scanning electron microscopy evaluation of original (untreated) polyamide mesh samples and after treatment with two variants of the caffeic acid-based solution (treated with CA 1 and treated with CA 2). At the bottom of the figure, the EDX evaluation result comparing the untreated sample with the one subjected to the variant treated with CA 1 is reported. It can be observed how the amount of C or O atoms increases significantly in the treated sample, confirming the stable interaction with the polyphenol mixture (rich in C and O).

[034] Figure 3: Scanning electron microscopy evaluation of original (untreated) silicone samples and after treatment with two variants of the caffeic acid-based solution (treated with CA and treated with CA 2).

[035] Figure 4: Proton nuclear magnetic resonance analysis of polyurethane samples after treatment with two variants of the caffeic acid-based solution (P1 and P2). Petition 870260022917, dated 12 / 03 / 2026, page 28 / 63 17 / 37

[036] Figure 5: Carbon-13 nuclear magnetic resonance analysis of untreated (NT) polyurethane samples and after treatment with a caffeic acid-based solution (P2).

[037] Figure 6: Carbon-13 nuclear magnetic resonance analysis of untreated polyamide samples (CTRL) and after treatment with two variants of the caffeic acid-based solution (P1 and P2).

[038] Figure 7: Proton nuclear magnetic resonance analysis of silicon samples before (CTRL) and after treatment with two variants of the caffeic acid-based solution (Sample 1 and Sample 2).

[039] Figure 8: Carbon-13 nuclear magnetic resonance analysis of silicon samples before (CTRL) and after treatment with a caffeic acid-based solution (Sample 2).

[040] Figure 9: Percentage reduction in protein adhesion to different plastic supports evaluated in bovine serum albumin and bovine thyroglobulin.

[041] Figure 10: Percentage reduction in the adhesiveness of different bacterial strains on different types of plastic supports.

[042] Figure 11: Thrombin generation assay performed on polyurethane (PU), silicone (SI), polyamide (PA) and polyester (PE) samples before (NT) and after treatment with a caffeic acid-based solution. The samples are compared with reference material consisting of Medical Steel (MS, with high propensity to thrombin generation) and low-density polyethylene (LDPE, with low propensity to thrombin generation). Objective of the invention

[043] In a first objective, the present invention discloses a method for imparting antimicrobial properties to a synthetic substrate. Petition 870260022917, dated 12 / 03 / 2026, page 29 / 63 18 / 37

[044] In a particular embodiment, the said synthetic substrate is selected from the group comprising: polyurethane, polyesters, polyamides, silicones, PEEK, polytetrafluoroethylene and expanded polytetrafluoroethylene.

[045] In a particular embodiment, the method of the invention may also provide said synthetic substrate with one or more properties selected from the group comprising: inhibition of surface adhesion to serum proteins, resistance to bacterial adhesion to tissue, inhibition of thrombin generation.

[046] In a second objective, the present invention discloses a synthetic substrate obtained according to the method described and a medical device comprising such substrate.

[047] In a particular embodiment, the said medical device is selected from the group comprising: catheters, such as vascular catheters, urinary catheters, embolic protection filters, mesh for abdominal wall repair. Detailed description of the invention

[048] In accordance with the first objective of the invention, a method for imparting antimicrobial properties to a synthetic substrate is disclosed.

[049] In particular, the aforementioned synthetic substrate is represented by a plastic substrate.

[050] In a preferred embodiment, the said plastic substrate is represented by a material selected from the group comprising: polyurethane, polyesters, polyamides, polyethylene, silicones, PEEK, polyacrylates, acrylic hydrogels, Teflon, polysiloxane, fluorinated polymers.

[051] In particular, polyesters include, for example: polyethylene terephthalate, nylon, Dacron, polyglycolic acid, polylactic acid, polycaprolactone. Petition 870260022917, dated 12 / 03 / 2026, p. 30 / 63 19 / 37

[052] In particular, polyamides include Kevlar.

[053] In particular, polyethylene includes low, high and ultra-high molecular weight polyethylene.

[054] In particular, polyacrylates include polymethyl methacrylate and polymethyl acrylate.

[055] In particular, polysiloxane includes Silastic.

[056] In particular, fluorinated polymers include polytetrafluoroethylene and expanded polytetrafluoroethylene.

[057] According to the present invention, the disclosed method may comprise a preliminary pre-treatment step of said substrate.

[058] In particular, the said pretreatment comprises incubating the substrate in an alcohol pretreatment solution.

[059] More specifically, the said alcohol has a concentration of about 10-100% (v / v) and preferably 100% (v / v).

[060] In one embodiment, the incubation of the pretreatment is continued for a period of time from 2 minutes to 24 hours.

[061] In a preferred embodiment, the incubation of the pretreatment is carried out for approximately 10 minutes.

[062] According to a preferred embodiment of the invention, prior to said pretreatment step, the pretreatment solution is maintained at a temperature of about -25 °C to -15 °C.

[063] In a preferred embodiment, the pretreatment solution is maintained at a temperature of about -20 °C.

[064] In a preferred embodiment, the pretreatment solution is maintained at the disclosed temperature for a period of time from 10 minutes to 5 hours. Petition 870260022917, dated 12 / 03 / 2026, p. 31 / 63 20 / 37

[065] For the purposes of the present invention, the method of the invention comprises a step of bringing said substrate into contact with a caffeic acid-based treatment solution.

[066] In particular, the aforementioned caffeic acid-based treatment solution has a caffeic acid concentration between 0.1-10 mg / mL.

[067] In a preferred embodiment, the said treatment solution has a caffeic acid concentration of about 2-4 mg / mL.

[068] The treatment solution of the invention is prepared by dissolving caffeic acid in 70% (v / v) of the final volume in alcohol.

[069] In particular, for the preparation of the treatment solution, caffeic acid is dissolved in a C1-C4 alcohol.

[070] In a preferred embodiment, the C1-C4 alcohol is selected from the group comprising: methanol, ethanol, isopropanol or butanol.

[071] The pH of the treatment solution is then adjusted to a range between 2.5 and 9.0 and, preferably, between pH 5.5 and pH 8.0.

[072] According to one embodiment of the present invention, the treatment solution comprises a second component.

[073] For the purposes of the present invention, said second component is selected from the group comprising polyphenols and their salts or esters, phenolic compounds and their salts and derivatives, antibiotics or antimicrobial agents, methylated phenols, fatty acids and their esters and metal-based solutions.

[074] In particular, the said polyphenols are selected from the group comprising: resveratrol, aloin, cyanin, epigallocatechin, tannic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, narigenin, gallic acid, hesperidin, quinic acid, eleonolic acid, pinoresinol, luteolin, apigenin, tangeritin, isorhamnetin, kaempferol, myricetin, eriodictiol, Petition 870260022917, dated 12 / 03 / 2026, page 32 / 63 21 / 37 theaflavin, thearubigins, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid, salicylic acid, baicalein, 5,7-dihydroxy-4-phenylcoumarin, rutin hydrate, 5,8-dihydroxy-1,4-naphthoquinone, 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone, ethyl 3,4-dihydroxy-cinnamate, butyl gallate, 4-hydroxyl-4-biphenylcarboxylic acid, oleuropein, garlic acid, magnolol, curcumin, ethyl 3,5-dihydroxy-benzoate.

[075] In particular, the said phenolic compounds are selected from the group comprising: vanillin, cinnamic acids, phenylalanine, coumarins, xanthones, catechins, flavononides, flavones, chalcones, flavanonols, flavanols, leucoanthocyanidin, anthocyanidin, hydroxycinnamic acids, phenylpropanoids.

[076] Derivatives of such phenolic compounds, represented by salts and esters, are also included.

[077] In particular, the said antibiotics or antimicrobial agents are selected from the group comprising: penicillins, aminoglycosides, carbapenems, glycopeptides and lipoglycopeptides, such as vancomycin, monobactams, aztreonam, oxazolidinones, such as linezolid and tedizolid, rifamycins, streptogramins, such as quinupristin and dalfopristin, cephalosporins, tetracyclines, macrolides, fluoroquinolones, sulfonamides.

[078] In particular, the aforementioned methylated phenols are selected from the group comprising: α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol and tocotrienols.

[079] In particular, the said metal-based solution is selected from the group comprising: acetates, sulfates, phosphates, chlorides, nitrites, nitrates or carbonates. Petition 870260022917, dated 12 / 03 / 2026, page 33 / 63 22 / 37

[080] The metal can be selected from the group comprising: iron, silver, gold, zinc, copper, barium, magnesium and aluminum.

[081] For example, barium carbonate, iron(II) chloride, iron(III) chloride, iron(II) nitrate, iron(III) nitrate, aluminum chloride, calcium chloride, calcium carbonate, calcium nitrate, copper sulfate, copper nitrate can be used.

[082] For the purposes of the present invention, the said second component has a concentration of about 0.1-20 mg / mL.

[083] The treatment solution of the invention is prepared by mixing a solution of caffeic acid with a solution of the second component.

[084] In particular, the caffeic acid solution is preferably represented by an alcoholic solution of caffeic acid.

[085] In a preferred embodiment, the caffeic acid solution is prepared by dissolving caffeic acid in 70% (v / v) of the final volume in alcohol.

[086] In particular, for the preparation of the treatment solution, caffeic acid is dissolved in a C1-C4 alcohol.

[087] In a preferred embodiment, the C1-C4 alcohol is selected from the group comprising: methanol, ethanol, isopropanol or butanol.

[088] In a preferred embodiment, the solution of the second component is prepared by dissolving the second component in an aqueous buffer.

[089] For the purposes of the present invention, a suitable buffer may be selected from the group comprising: PBS (phosphate buffer), bicarbonate buffer, Dulbecco's Phosphate Buffered Saline, TBE (tris / borate / EDTA buffer), TE (Tris / EDTA) buffer, buffered saline Petition 870260022917, dated 12 / 03 / 2026, page 34 / 63 23 / 37 with Tris (TBS), SSC (sodium chloride / sodium citrate) and SSPE (sodium chloride / sodium phosphate / EDTA).

[090] Finally, the two solutions are mixed and the pH is adjusted to a range between 2.5 and 9.0 and preferably between pH 5.5 and pH 8.0.

[091] For the purposes of the present invention, the method of the present invention comprises at least one treatment cycle, comprising the steps in which: i) the aforementioned synthetic substrate is incubated in the aforementioned treatment solution and then ii) the aforementioned incubated synthetic substrate is washed.

[092] In particular, the aforementioned incubation step i) is carried out for a period of time from about 5 to 25 minutes and preferably about 15 minutes.

[093] In particular, the aforementioned washing step ii) is carried out for a period of time from about 2 to 120 minutes and preferably about 20 minutes.

[094] For the purposes of the present invention, treatment step i) comprises at least one cycle performed at pH 5.5.

[095] For the purposes of the present invention, treatment step i) further comprises at least one cycle performed at pH 8.0 carried out after treatment and washing at pH 5.5.

[096] In one embodiment of the present invention, the treatment step i) comprises from 1 to 5 treatment cycles performed at pH 5.5.

[097] Preferably, treatment step i) comprises 3 treatment cycles performed at pH 5.5.

[098] In one embodiment of the present invention, the treatment step i) further comprises from 1 to 5 treatment cycles. Petition 870260022917, dated 12 / 03 / 2026, page 35 / 63 24 / 37 performed at pH 8.0, where each step at pH 8.0 is performed after each step at pH 5.5.

[099] Preferably, treatment step i) comprises 3 treatment cycles performed at pH 8.0, where each step at pH 8.0 is performed after each step at pH 5.5.

[100] For the purposes of the present invention, treatment step i) is carried out in the dark.

[101] As per step ii), washing is performed with a washing solution represented by a buffer solution.

[102] In particular, the buffer solution is selected from the group comprising: PBS (phosphate buffer), bicarbonate buffer, Dulbecco's Phosphate Buffered Saline, TBE (tris / borate / EDTA buffer), TE (Tris / EDTA) buffer, Tris-buffered saline (TBS), SSC (sodium chloride / sodium citrate) and SSPE (sodium chloride / sodium phosphate / EDTA).

[103] For the purposes of the present invention, a drying step is additionally performed after the treatment step.

[104] In particular, the said drying step is carried out at a temperature of about 30-45 °C.

[105] In particular, the said drying stage is carried out for a period of time from about 1 minute to 5 hours and preferably about 30 minutes.

[106] According to the present invention, the method disclosed above provides antimicrobial properties to the treated synthetic substrate.

[107] In particular, the aforementioned antimicrobial properties are effective against: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterococcus faecalis, Listeria monocytogenes, Salmonella enterica typhimurium, Streptococcus viridans, non-tuberculous mycobacteria such as Petition 870260022917, dated 12 / 03 / 2026, p. 36 / 63 25 / 37 Mycobacterium chelonae, yeasts such as Candida albicans, and fungi such as Aspergillus brasiliensis.

[108] In a preferred embodiment, the aforementioned antimicrobial properties are versus: Staphylococcus aureus, Escherichia coli and Proteus mirabilis.

[109] In addition, the method of the invention provides one or more of the following properties: inhibition of surface adhesion to serum proteins, resistance to bacterial adhesion to tissue, inhibition of thrombin generation.

[110] In particular, the aforementioned properties of resistance to bacterial tissue adhesion are versus: Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterococcus faecalis, Listeria monocytogenes, Salmonella enterica typhimurium, Streptococcus viridans, non-tuberculous mycobacteria such as Mycobacterium chelonae, yeast such as Candida albicans and fungi such as Aspergillus brasiliensis.

[111] In a preferred embodiment, the aforementioned properties of resistance to bacterial adhesion to tissue are versus: Staphylococcus aureus, Escherichia coli and Proteus mirabilis.

[112] According to a second objective of the invention, a synthetic substrate obtained according to the method of the invention is disclosed.

[113] In particular, the synthetic substrate is made of a material selected from the group comprising: polyurethane, polyesters, polyamides, polyethylene, silicones, PEEK, polyacrylates, acrylic hydrogels, Teflon, polysiloxane, fluorinated polymers, as disclosed above.

[114] Furthermore, a medical device comprising such a substrate is disclosed. Petition 870260022917, dated 12 / 03 / 2026, page 37 / 63 26 / 37

[115] For the purposes of the present invention, a medical device is selected from the group comprising: catheters, such as vascular catheters, urinary catheters, embolic protection filters, meshes for abdominal wall repair, syringes, kits intended for various types of use, laboratory tubes, blood bags, instruments, gloves, trays, thermometers and sutures.

[116] The present invention will be further disclosed in the following experimental section. Experimental section Polyurethane (PU)

[117] In particular, polyurethane samples were incubated for 10 minutes in a 100% (v / v) isopropanol solution. Before use, the alcoholic solution was placed at -20 °C for a time interval ranging from 10 minutes to 5 hours.

[118] The samples were subsequently incubated in two different mixtures of caffeic acid-based polyphenols, specifically: variant 1 (P1 in Figure 4) consisting of caffeic acid at a concentration of 2 mg / mL and tannic acid at a concentration of 4 mg / mL was chosen and variant 2 (P2 in Figure 4) consisting of caffeic acid at a concentration of 2 mg / mL and rutin hydrate at a concentration of 1 mg / mL was chosen.

[119] For both variants, caffeic acid was dissolved in 70% of the final volume in isopropanol.

[120] The second polyphenol was dissolved in 30% of the final volume in PBS (phosphate buffer).

[121] Finally, the two solutions are mixed and the pH is adjusted to a range between pH 5.5 and pH 8.0. Petition 870260022917, dated 12 / 03 / 2026, page 38 / 63 27 / 37

[122] The polyurethane samples are then incubated in these caffeic acid-based solutions for 3 cycles at pH 5.5 and an additional 3 cycles at pH 8.0.

[123] An incubation time in a caffeic acid-based polyphenolic solution of 15 minutes was used for each cycle, and each cycle was followed by a wash in TBE (Tris / borate / EDTA buffer) lasting 20 minutes.

[124] After the treatment cycles, the samples were placed in an incubator at 37 °C for 30 minutes. Polyamide (PA)

[125] Polyamide samples were incubated for 10 minutes in a 100% (v / v) ethanol solution. Incubation time can vary from 2 to 24 minutes.

[126] Before use, the alcoholic solution was placed at -20 °C for a time interval ranging from 10 minutes to 5 hours.

[127] The samples were subsequently incubated in two different mixtures of caffeic acid-based polyphenols, specifically: variant 1 (P1 in Figure 6), which consists of caffeic acid at a concentration of 4 mg / mL and tannic acid at a concentration of 8 mg / mL and variant 2 (P2 in Figure 6), which consists of caffeic acid at a concentration between 4 mg / mL and barium carbonate at a concentration of 1 mg / mL.

[128] For both variants, caffeic acid was dissolved in 70% of the final volume in isopropanol.

[129] The metal-based salt was dissolved in 30% of the final volume in PBS (phosphate buffer).

[130] Finally, the two solutions are mixed and the pH is adjusted to a range between 2.5 and 9.0 (in our case pH 5.5 and pH 8.0). Petition 870260022917, dated 12 / 03 / 2026, page 39 / 63 28 / 37

[131] The polyamide samples are then incubated in these caffeic acid-based solutions for 3 cycles at pH 5.5 and an additional 3 cycles at pH 8.0.

[132] An incubation time in a caffeic acid-based polyphenolic solution of 15 minutes was used for each cycle, and each cycle was followed by a wash in TBE (Tris / borate / EDTA buffer) lasting 20 minutes.

[133] After the treatment cycles, the samples were placed in an incubator at 37 °C for a period of 30 minutes. Silicon

[134] Silicon samples were incubated for 10 minutes in a 100% (v / v) isopropanol solution. Incubation time can vary from 2 to 24 minutes.

[135] Before use, the alcoholic solution was placed at -20 °C for a time interval ranging from 10 minutes to 5 hours.

[136] The samples were subsequently incubated in two different mixtures of caffeic acid-based polyphenols, specifically: variant 1 (Sample 1 in Figure 7), which consists of caffeic acid at a concentration of 2 mg / mL and tannic acid at a concentration of 4 mg / mL, and variant 2 (Sample 2 in Figure 7), which consists of caffeic acid at a concentration of 2 mg / mL and ganoderic acid at a concentration of 5 mg / mL was used).

[137] For both variants, caffeic acid was dissolved in 70% of the final volume in isopropanol. The second polyphenol was dissolved in 30% of the final volume in PBS (phosphate buffer).

[138] Finally, the two solutions are mixed and the pH is adjusted to a range between pH 5.5 and pH 8.0.

[139] The silicon samples are then incubated in these caffeic acid-based solutions for 3 cycles at pH 5.5 and an additional 3 cycles at pH 8.0. Petition 870260022917, dated 12 / 03 / 2026, pp. 40 / 63 29 / 37

[140] An incubation time in a caffeic acid-based polyphenolic solution of 15 minutes was used for each cycle, and each cycle was followed by a wash in TBE (Tris / borate / EDTA buffer) lasting 20 minutes.

[141] After the treatment cycles, the samples were placed in an incubator at 37 °C for a period of 30 minutes. Polyester (PE)

[142] Polyester samples were incubated for 10 minutes in 100% (v / v) isopropanol solution. Incubation time can vary from 2 minutes to 24 hours.

[143] The alcoholic solution was placed at -20 °C for a time interval ranging from 10 minutes to 5 hours.

[144] The samples were subsequently incubated in a caffeic acid-based polyphenol mixture, consisting of caffeic acid at a concentration of 4 mg / mL (was chosen), tannic acid at a concentration of 8 mg / mL and a mixture of penicillin (150 pg / mL) / streptomycin (150 pg / mg) / neomycin (100 pg / mL).

[145] Caffeic acid was dissolved in 70% of the final volume in isopropanol.

[146] The polyphenol and antibiotics were dissolved in 30% of the final volume in PBS (phosphate buffer).

[147] Finally, the two solutions are mixed and the pH is adjusted to a range between 5.5 and pH 8.0).

[148] The polyester samples are then incubated in these caffeic acid-based solutions for 3 cycles at pH 5.5 and an additional 3 cycles at pH 8.0.

[149] An incubation time in a caffeic acid-based polyphenolic solution of 15 minutes was used for each cycle, and each cycle was followed by a wash in TBE (Tris / borate / EDTA buffer) lasting 20 minutes. Petition 870260022917, dated 12 / 03 / 2026, page 41 / 63 30 / 37

[150] After the treatment cycles, the samples were placed in an incubator at 37 °C for a period of 30 minutes.

[151] The treated and untreated plastic specimens were subjected to scanning electron microscopy (SEM) for surface evaluation, nuclear magnetic resonance (H- and C-NMR) for characterization of the interaction with the support, and tests for evaluation of the anti-adhesiveness of various bacterial strains and serum proteins and evaluation of thrombogenicity. Results Scanning electron microscopy (SEM) Polyurethane samples

[152] Figure 1 highlights how samples treated with caffeic acid (CA)-based solutions do not differ macroscopically from untreated (NT) samples when compared at low magnification (50X). By increasing the magnification, it is possible to appreciate a uniform coverage which, based on specific variations of the caffeic acid-based solution (CA-1 and CA2), can be modulated in thickness and texture. Polyamide samples

[153] Figure 2 highlights how samples treated with caffeic acid-based solutions (TREATED WITH CA 1) do not differ macroscopically from untreated samples (NT) when compared at low magnification (400X). The presence of the polyphenol-based coating was confirmed by EDX analysis, which highlighted an increase in the presence of C and O atoms compared to the NT samples. Energy-dispersive X-ray spectroscopy (also abbreviated as EDX) is an analytical technique that enables the chemical characterization / elemental analysis of materials. A sample excited by an energy source (such as the electron beam of an electron microscope) dissipates some of the absorbed energy by ejecting an electron from the shell. Petition 870260022917, dated 12 / 03 / 2026, pp. 42 / 63 31 / 37 inner. A higher-energy electron from the outer shell then proceeds to fill its place, releasing the energy difference as an X-ray that has a characteristic spectrum based on its originating atom. This allows for compositional analysis of a given sample volume that has been excited by the energy source. The position of the peaks in the spectrum identifies the element, while the signal intensity corresponds to the element's concentration. By increasing the magnification, however, it is possible to appreciate a uniform coverage that, based on specific variations in the caffeic acid-based solution (CA-1 and CA-2), can be modulated in thickness and texture. Silicone samples

[154] The silicone samples showed different behavior compared to the other types of materials analyzed. As is evident in Figure 3, treatment with the caffeic acid-based solution ensures the formation of a coating that, however, is only visible at high magnifications (starting from 6000X). The coating is visible as a result of the formation of cracks due to the prolonged permanence of the SEM electron beam. This attribute makes the coating very interesting, as it is not detectable macroscopically. Assessment by nuclear magnetic resonance (NMR) Polyurethane samples

[155] 1H NMR investigations show no substantial differences in the peaks of the various treatments with caffeic acid-based solutions in the polyurethane samples. Spectra P1 and P2 are substantially identical (Figure 4), showing no release of caffeic acid and confirming the stability of the treatment. In particular, treatment P2 showed a decrease in the peak to about 68 ppm in 1C-NMR, corresponding to the formation of Petition 870260022917, dated 12 / 03 / 2026, pp. 43 / 63 32 / 37 a covalent bond with the terminal hydroxyl groups of the polyurethane chain (Figure 5). Polyamide samples

[156] 1H NMR investigations (Figure 6) confirm the stability of the interaction between the caffeic acid-based polyphenol solution and the polyamide samples. Furthermore, the disappearance of the peak at 3.7 ppm observable in the control sample (CTRL) is indicative of the formation of a covalent chemical bond between the terminal amino groups of the polyamide and the caffeic acid solution used for coating (the signal highlighted in yellow corresponds to the signals of the protons at position 1 of the chain). Silicone samples

[157] 1H NMR investigations confirm the stability of the interaction between the caffeic acid-based polyphenol solution and the silicone samples. In particular, 1H NMR showed a significant ¹H-mediated interaction, confirmed by the presence of several peaks in the region between 1.4 and 0.6 ppm (Figure 7). Furthermore, the disappearance of the peak at 60 and 185 ppm observable in the control sample (CTRL) (Figure 8) is indicative of the formation of a covalent chemical bond between polyphenols and silicon surfaces. Inhibition of surface adhesion to serum proteins

[158] Samples of different plastic substrates treated and untreated with a caffeic acid-based polyphenolic solution according to the invention were incubated for 24 hours at 37 °C in a phosphate buffer containing 50 pg / mL of bovine serum albumin (66 kDa) or bovine thyroglobulin (330 kDa), with moderate but constant agitation. Subsequently, all samples were subjected to 3 washes in phosphate buffer for 3 minutes each, to remove any protein residues that were not firmly bound to the surface. The protein bound to the surface was measured and, considering the Petition 870260022917, dated 12 / 03 / 2026, pp. 44 / 63 33 / 37 The amount of protein quantified in the untreated samples was set to a value of 100, and the percentage reduction in the variously treated samples was derived. As reported in Figure 9, treatment with the caffeic acid-based solution of the invention can ensure a reduction in protein adhesion greater than 90% overall. Resistance to bacterial adhesion to tissue

[159] Anti-adhesive bacterial activity was evaluated against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Proteus mirabilis (P. mirabilis). Bacteria were cultured overnight in Tryptic Soybean Broth (TSB) at 37 °C. Total bacterial load was assessed by serial dilutions of 10 factors in TSB (10⁻¹ to 10⁻⁷), seeded onto Petri dishes with appropriate selective medium and incubated overnight. After incubation, the CFU was counted to determine the effective concentration of the microorganism. Furthermore, the optical density at 600 nm was determined from each mosaic dilution to verify the linearity between it and the effective microbial load of the broth.

[160] Samples of polyurethane (PU), polyamide (PA), silicone (SI), and polyester (PE), before (NT) and after treatment with a caffeic acid-based solution (CA, n=5 for each treatment type), were prepared using a biopsy puncture (3 mm diameter) to obtain the same effective surface area for bacterial adhesion. To eliminate any bacterial load before the adhesion test, the samples were washed with PBS and incubated overnight at room temperature in PBS supplemented with gentamicin (300 μg / mL) under moderate but constant agitation. After overnight incubation, the samples were washed extensively in PBS to remove any remaining antibiotics that could distort the test results. Subsequently, the treated and untreated samples Petition 870260022917, dated 12 / 03 / 2026, pp. 45 / 63 34 / 37 were individually exposed to bacterial suspensions of S. aureus, E. coli, and P. mirabilis (bacterial load 1x107 CFU / mL) for 90 minutes at room temperature under moderate but constant agitation.

[161] Subsequently, the samples were subjected to three moderate vortex passages to facilitate the detachment of loosely bound bacteria and serial dilutions of the wash were seeded onto Petri dishes containing the appropriate selective growth medium. Finally, after 24 hours of incubation at 37 °C, the CFU was counted for each sample type.

[162] Considering the number of colonies found in the untreated samples as 100%, the percentage of adhesion inhibition was calculated for each sample treated with the caffeic acid-based solution.

[163] As shown in Figure 10, the caffeic acid-based solution of the invention proved effective in inhibiting surface adhesion of all bacteria considered by at least 80%, regardless of the type of plastic support. The only exception made was polyurethane, which already has excellent anti-adhesive activity against E. coli bacteria. In this specific case, the percentage of bacterial adhesion inhibition was lower (23.2%) when compared to the other materials. Thrombin generation assay (TGA) test

[164] Samples of polyurethane (PU), polyamide (PA), silicone (SI), and polyester (PE), before (NT) and after treatment with a caffeic acid-based solution (CA, n=5 for each treatment type), were subjected to a Thrombin Generation Test (Haemoscan, Groningen, Netherlands). Thrombin is a key enzyme in the coagulation cascade. Its measurement provides direct information about the thrombogenicity of a biomaterial (i.e., its ability to form blood clots). In normal plasma, thrombin is captured in the fibrin mesh and is rapidly inactivated by antithrombin III or other Petition 870260022917, dated 12 / 03 / 2026, pp. 46 / 63 35 / 37 antiproteases. The short half-life of thrombin makes its precise enzymatic determination difficult. The Thrombin Generation Assay is based on a special plasma product that enables the determination of thrombin activity in an incubation medium after it has been exposed to a biomaterial. This method is suitable for evaluating the hemocompatibility of biomaterials and medical devices according to the international standard ISO 10993-4:2002. Specimens were processed following the manufacturer's instructions. Briefly, samples were incubated in modified human plasma (plasma was provided by the manufacturer) with subsequent withdrawals at different time points. The thrombin concentration of the samples was determined from an optical density calibration curve at 405 nm. The thrombin generation curve for each specimen was constructed by plotting the thrombin concentration versus the time points at which the samples were collected.The curve is used to determine the rate of thrombin generation, expressed in cm² of a sample. Reference materials were provided by the manufacturer, in particular: Low-density polyethylene (LDPE, low propensity for thrombin generation) and medical steel (MS, high propensity for thrombin generation). The results shown in Figure 11 denote good overall resistance to thrombus formation by the original material (NT), except for polyester, which tends to behave similarly to medical steel. Surprisingly, treatment with the caffeic acid (CA)-based solution can significantly reduce the thrombotic propensity in all treated materials, inhibiting it by up to about 60% (for polyurethane and polyester).

[165] From the above disclosure, the advantages offered by the present invention will be immediately evident to those skilled in the art. Petition 870260022917, dated 12 / 03 / 2026, page 47 / 63 36 / 37

[166] For example, the invention is able to limit the adhesion of proteins and various bacterial strains to different plastic supports used in the manufacture of medical devices, thus preventing the formation of bacterial colonization and infections.

[167] Also, the invention has been shown to protect the treated plastic support against the formation of blood clots and structured thrombi.

[168] This treatment showed high chemical stability with the plastic substrates and proved effective in modifying the surface interaction properties of plastic polymers, allowing them to modulate the degree of hydrophilicity.

[169] The present invention has proven to be very stable and safe, as confirmed by SEM and NMR analyses.

[170] Last but not least, the invention provided a method that can be performed with conventional devices and machines.

[171] The invention is susceptible to numerous modifications and variations, all within the scope of the appended claims; furthermore, all elements can be replaced with other technically equivalent elements. References 1. EP3972659 - Method for preventing the formation of calcified deposits and for inactivating xenoantigens in biological matrices; 2. EP3383446 - Method for inactivating xenoantigens in biological tissues; 3. Eur J Cardiothorac Surg 2022; ezac583. doi: 10.1093 / ejcts / ezac583. Online before print. 4. Cardiol Cardiovasc Med 2022;6(5):487-492. doi: 10.26502 / fccm.92920287. Petition 870260022917, dated 12 / 03 / 2026, pp. 48 / 63 37 / 37 5. Tissue Eng Part A 2017;23(19-20):1181-1195. doi: 10.1089 / ten.tea.2016.0474. 6. ACS Appl Mater Interfaces 2016;8(40):26570-26577. doi: 10.1021 / acsami.6b08930. 7. Chem Commun (Camb) 2016;52(2):312-315. doi: 10,1039 / c5cc07090b. 8. Polymers (Basel) 2019;11(7):1200. doi: 10.3390 / polym11071200. 9. Biomater Sci 2019;7(12):5035-5043. doi: 10.1039 / c9bm01223k. Petition 870260022917, dated 12 / 03 / 2026, pp. 49 / 63

Claims

1 / 5 CLAIMS 1. Method for imparting antimicrobial properties to a synthetic substrate, characterized in that it comprises the steps of bringing said substrate into contact with a treatment solution based on caffeic acid.

2. Method for imparting antimicrobial properties to a synthetic substrate, according to claim 1, characterized in that the synthetic substrate is represented by a material selected from the group comprising: polyurethane, polyesters, polyamides, polyethylene, silicones, PEEK, polyacrylates, acrylic hydrogels, Teflon, polysiloxane, fluorinated polymers.

3. A method according to claim 1 or 2, characterized in that it comprises a pretreatment step of said surface, wherein said surface is incubated in a pretreatment solution of a C1-C4 alcohol, preferably wherein said pretreatment solution comprises methanol, ethanol, isopropanol or butanol and optionally wherein said pretreatment solution has a concentration of about 100% (v / v) of said C1-C4 alcohol.

4. Method according to claim 3, characterized in that said incubation is continued for a period of time from 2 minutes to 24 hours.

5. Method according to claim 3 or 4, characterized in that prior to said pretreatment step, said pretreatment solution is maintained at a temperature of about -25 °C to -15 °C for a period of time from about 10 minutes to 5 hours.

6. Method, according to any one of claims 1 to 5, characterized in that in said treatment solution caffeic acid Petition 870260022917, dated 12 / 03 / 2026, page 50 / 63 2 / 5 has a concentration of about 1-10 mg / mL.

7. A method according to any one of claims 1 to 6, characterized in that said treatment solution is a C1-C4 alcoholic solution, preferably wherein said treatment solution comprises methanol, ethanol, isopropanol or butanol.

8. A method according to any one of claims 1 to 7, characterized in that said treatment solution further comprises a second component, preferably wherein said second component has a concentration of about 0.1-20 mg / mL.

9. Method according to claim 8, characterized in that said second component is selected from the group comprising: polyphenols and their salts or esters, preferably wherein said polyphenols are selected from the group comprising: resveratrol, aloin, cyanin, epigallocatechin, tannic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, narigenin, gallic acid, hesperidin, quinic acid, eleonolic acid, pinoresinol, luteolin, apigenin, tangeritin, isorhamnetin, kaempferol, myricetin, eriodictiol, theaflavin, thearubigins, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid, salicylic acid, baicalein, 5,7-dihydroxy-4-phenyl coumarin, rutin hydrate, 5,8-dihydroxy-1,4naphthoquinone, 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone, ethyl-3,4-dihydroxycinnamate, butyl gallate, 4-hydroxyl-4-biphenyl-carboxylic acid, oleuropein,garlic acid, magnolol, curcumin, ethyl-3,5-dihydroxybenzoate; phenolic compounds and their salts and derivatives, preferably wherein said phenolic compounds are selected from the group comprising: vanillin, cinnamic acids, phenylalanine, coumarins, xanthones, catechins, flavononides, flavones, chalcones, flavanonols, flavanols, leucoanthocyanidin, anthocyanidin, hydroxycinnamic acids, phenylpropanoids; and their salts or esters; antibiotics or antimicrobial agents, preferably wherein said antibiotics or antimicrobial agents are selected from the group comprising: penicillins, aminoglycosides, carbapenems, glycopeptides and lipoglycopeptides, such as vancomycin, monobactams such as aztreonam, oxazolidinones such as linezolid and tedizolid, rifamycins, streptogramins such as quinupristin and dalfopristin, cephalosporins, tetracyclines, macrolides, fluoroquinolones,sulfonamides; methylated phenols, preferably wherein said methylated phenols are selected from the group comprising: α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol and tocotrienols; fatty acids and their esters; and metal-based solutions, preferably wherein said metal-based solutions are selected from the group comprising: acetates, sulfates, phosphates, chlorides, nitrites, nitrates or carbonates of any from the group comprising: iron, silver, gold, zinc, copper, barium, magnesium and aluminum.

10. A method according to any one of claims 1 to 9, characterized in that said treatment solution is adjusted to a pH of about 2.5-9.0, preferably about 5.5-8.

0.

11. Method, according to any one of claims 1 to 10, characterized in that said treatment comprises at least one treatment cycle in which i) said synthetic substrate is incubated in said treatment solution and then ii) said synthetic substrate is washed. Petition 870260022917, dated 12 / 03 / 2026, p. 52 / 63 4 / 5 12. Method according to claim 11, characterized in that said treatment comprises at least one cycle carried out at pH 5.5, optionally wherein said treatment further comprises at least one cycle carried out at pH 8.

0.

13. Method, according to claim 11 or 12, characterized in that said treatment comprises from 1 to 5 treatment cycles carried out at pH 5.5, and / or in that said treatment comprises from 1 to 5 treatment cycles carried out at pH 8.0, and / or in that said treatment is carried out in the dark.

14. Method, according to any one of claims 11 to 13, characterized in that said step i) is performed for a period of time from about 5 to 25 minutes, and / or in that said step ii) is performed for a period of time from about 2 to 120 minutes.

15. A method according to any one of claims 11 to 14, characterized in that said washing is carried out with a buffer solution, preferably wherein the washing solution is selected from the group comprising: PBS (phosphate buffer), bicarbonate buffer, Dulbecco's Phosphate Buffered Saline, TBE (tris / borate / EDTA buffer), TE (Tris / EDTA) buffer, Tris-buffered saline (TBS), SSC (sodium chloride / sodium citrate) and SSPE (sodium chloride / sodium phosphate / EDTA).

16. Method, according to any one of claims 11 to 15, characterized in that said treatment method further comprises a drying step, preferably in which said drying step is carried out at a temperature of about 30-45 °C, optionally in which said drying step is carried out for a period of time of about 1 minute to 5 hours.

17. Method, according to any one of claims 1 to 16, Petition 870260022917, dated 12 / 03 / 2026, p. 53 / 63 5 / 5 characterized in that it can also provide said synthetic substrate with one or more properties selected from the group comprising: inhibition of surface adhesion to serum proteins, resistance to bacterial adhesion to tissue, inhibition of thrombin generation.

18. Method, according to any one of claims 1 to 17, characterized in that the said antimicrobial properties are effective against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterococcus faecalis, Listeria monocytogenes, Salmonella enterica typhimurium, Streptococcus viridans, Mycobacterium chelonae, Candida, Aspergillus brasiliensis.

19. Synthetic substrate, characterized in that it is obtained using the method defined in any one of claims 1 to 18.

20. Medical device, characterized in that it comprises the synthetic substrate defined in claim 19, preferably wherein said medical device is selected from the group comprising: catheters, such as vascular catheters, urinary catheters, embolic protection filters, abdominal wall repair mesh, syringes, kits intended for various types of use, laboratory tubes, blood bags, instruments, gloves, trays, thermometers and sutures. Petition 870260022917, dated 12 / 03 / 2026, pp. 54 / 63

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