Silicone surface activation

Abstract

A method for modifying silicone surfaces to introduce amino groups onto the surface that can be further reacted to introduce desired substituents and activators. The method comprises the step of reacting a molecule containing at least one primary or secondary amino group and at least one additional nucleophilic group selected from OH, SH, COOH, NH2, amide, and NHR, where R is alkyl, or a molecule of the formula Si-(OR 1 )(OR 2 )(OR 3 )R 5 NHR 4 (In the formula, the group R 1 , R 2 , R 3 are independently selected from linear or branched alkyl or alkylene, preferably methyl or ethyl; R 5 is a linear or branched alkyl or alkylene; R 4 is H or an organic group. Advantageously, the surface does not need to be subjected to a pretreatment with an oxidizing agent such as ozone, hydrogen peroxide or plasma treatment prior to the treatment step of the present invention.

Description

[Technical field]

[0001] The present invention is in the field of silicone chemistry, and specifically relates to the surface modification of silicones to introduce amino groups that can further react with desired substituents. [Background technology]

[0002] Silicones, also known as polysiloxanes, are a class of synthetic polymers consisting of an "inorganic" backbone of alternating silicon and oxygen atoms, and two organic side groups attached to the silicon atoms. The most common type is PDMS (polydimethylsiloxane; formula I). ​​Formula I shows a representation of the basic PDMS structure (note that the terminal Si atoms may have one or more -OH groups instead of methyl groups). Silicones are chemically and thermally stable and are widely used in insulation, packaging, building materials, cosmetics, drug delivery systems, and medical devices. Silicones are available in a variety of forms, including elastomers, gels, lubricants, foams, and adhesives. Liquid silicone rubbers and elastomers can be created from linear polymers by crosslinking. Commercially available silicone materials typically also contain various additives that affect properties such as color, rheology, and adhesion. Commercially available silicone elastomers are usually reinforced with inorganic additive fillers such as silica, calcium carbonate, montmorillonite, carbon black, zinc oxide, titanium dioxide, glass and graphene (as well as other materials to improve handling such as boric acid and polyhydroxy alcohols (see, e.g., U.S. Pat. No. 4,252,709)). Medical grade silicone is biocompatible and bioinert, making it a common material in medical implants such as prostheses, artificial heart valves and various catheters.

[0003] [ka]

[0004] Although flexibility and other properties favor silicone for medical applications, its hydrophobic surface makes it prone to bacterial and fungal colonization and biofilm formation. The growth of fungal or bacterial biofilms on silicone surfaces can be problematic for medical applications within the body, as an example of catheter-associated urinary tract infections (CAUTI), the most common hospital-acquired infection causing a significant health burden (Siddiq & Darouiche, NATURE REVIEWS UROLOGY 2012 9 305 D0110.1038 / nrurol.2012.68). Microbial contamination of silicone surfaces can also be a risk factor for applications in the food sector.

[0005] Surface modification: Silicone surfaces can be chemically modified to increase resistance to bacterial colonization and biofilm formation. The most common approach is to introduce certain hydrophilic moieties, e.g., polyethylene glycol (PEG), to reduce hydrophobicity and repel bacteria, retarding colonization and biofilm formation. One alternative approach is to add lipophilic disinfectants or antimicrobials to the polymer matrix that can leach out of the material and kill infectious microorganisms (Kottmann et al. CHEMISTRY-AN ASIAN JOURNAL 2017 12 1168 D0110.1002 / asia.201700244). The drawback of this latter approach is that it becomes less effective as the antimicrobial agent is lost over time, and leaching of bioactive agents can also affect the regulatory status of the device. Coating of silicone surfaces with biocompatible polymers and proteins can also improve biocompatibility and reduce implant-related issues such as scar tissue formation.

[0006] Silicone Coating: Silicones are generally considered chemically inert due to their lack of reactivity, so to coat them, the surface is typically first activated to introduce groups that can be targeted for chemical modification. A commonly used approach is plasma (ozone) treatment to oxidize the surface and introduce -OH groups, but treatment with other oxidizing agents, e.g. H2O2, has also been used for the same purpose (Bracic et al., Bioactive Functionalisation of Silicones with Polysaccharides, Springer Briefs in Molecular Science Biobased Polymers ISBN 978-3-030-02275-4 (eBook) 2018 https: / / doi.org / 10.1007 / 978-3-030-02275-4). This is often followed by a second step in which the surface is treated with a silanizing reagent (R-Si-(OR')3), e.g. APTMS ((3-aminopropyl)trimethoxysilane), to introduce more nucleophilic amino groups to the surface. The silicone surface can then be coated by using the nucleophilic groups on the surface to initiate polymerization of the monomer building blocks, or by reacting with electrophilic moieties of the polymer or other compound used in the coating, or by reacting with a crosslinker and then reacting with electrophilic moieties of the polymer or other compound used in the coating. In this way, a stable coating is obtained in which the polymer coating is covalently bonded to the surface. Alternatively, the oxidized surface can be treated with a silanizing reagent, such as (3-glycidyloxypropyl)trimethoxysilane, to introduce electrophilic epoxy groups to the surface. These can then be further reacted with nucleophilic polymers or other compounds to covalently coat the surface. US Patent Publication No. 2016 / 0130403 (Brook et al.) discloses that hydrophilic or reactive polymers attached to linear or branched hydrophobic silicone polymer chains can be physically adsorbed to the surface silicone elastomer to form a stable hydrophilic coating.U.S. Pat. No. 10,465,055 discloses treating a silicone substrate with a polymer containing at least three reactive sites to facilitate "covalent grafting" of the polymer to the substrate, but assumes that the substrate has accessible Si-H groups, which according to the disclosure can be obtained by immersing the silicone surface in an aqueous hydrogen fluoride solution. Summary of the Invention

[0007] The present invention provides a novel method for the surface modification of silicone materials and for the coating of silicone materials. The present invention provides a simple method for the activation of surfaces so that they can be easily further modified. The method is based on the surprising discovery that the method described herein allows the introduction of amino groups onto silicone surfaces without prior surface oxidation.

[0008] Therefore, the present invention presents a novel method for modifying silicone surfaces, thereby making said surfaces suitable for further modification. Specifically, the method presented in this disclosure comprises treating silicone surfaces with substances containing amino groups, selected from a specific group further described below in this disclosure. The inventors have tested different substances under different conditions and settings. These conditions and settings may vary depending on the type of individual surface and / or object to be modified, and what type of subsequent modification, chemically and functionally, is desired and intended. Advantageously, the method can be carried out using readily available and economical reagents, mild conditions, and environmentally friendly solvents.

[0009] Thus, in a first aspect, the present invention relates to a method for modifying a solid silicone surface, in particular a silicone elastomer surface, to introduce amino groups onto said surface, which modification allows said surface to be further modified, said method comprising the steps of: - molecules comprising at least one primary or secondary amino group and at least one further nucleophilic group selected from OH, SH, COOH, and NH2, amide, and NHR, where R is alkyl; - Formula Si-(OR 1 )(OR 2 )(OR 3 )R 5 NHR 4 (In the formula, the group R 1 , R 2 , R 3 are independently selected from linear or branched alkyl or alkylene, preferably methyl or ethyl; R 5 is an optionally further substituted linear or branched alkyl or alkylene; R 4 is H or an organic group; treating the silicone elastomer surface with a material selected from At least in the case where the substance is a silanizing reagent, the surface is not subjected to a pretreatment with an oxidizing agent, such as ozone, hydrogen peroxide or a plasma treatment, prior to the treatment step.

[0010] The term "silicone surface" includes any type of surface of a solid silicone object, including objects derived essentially only from silicone, as well as silicone objects that contain additional components known in the art as common additives and minor components in silicones, such as fillers, reinforcing agents, such as silica, alumina, calcium carbonate, carbon black, zinc oxide, titanium dioxide, glass, graphene crosslinkers, catalysts, boric acid, polyhydroxy alcohols, etc. Thus, silicone objects as used in this disclosure include objects derived from silicone elastomers or silicone rubbers.

[0011] The reagent material may optionally be dissolved in a water-miscible solvent, such as, but not limited to, selected from methanol, ethanol, propanol, isopropanol, 1-butanol, 2-butanol, t-butyl alcohol, glycerol, water or aqueous solutions, and miscible mixtures of one or more of the foregoing, and the silicone surface is then placed in the solvent containing the dissolved reagent.

[0012] A variety of amines have been found to be useful as reagents in the methods of the invention. Non-limiting examples of specific reagents are provided in Table 1.

[0013] [Table 1-1] [Table 1-2]

[0014] Thus, in some embodiments, the reagent is an amine comprising a linear or branched alkane or alkene chain, at least one primary or secondary amine and / or amide group, and at least one additional nucleophilic group selected from OH, SH, COOH, and NH2, amide, and NHR, where R is alkyl. In some embodiments, the reagent is an ester, ether, or alcohol having at least one primary or secondary amine group. The reagent may also be an amine having two or more primary or secondary amino groups, some examples of which are shown in Table 4.

[0015] Useful reagents also include amino acids and amino acid derivatives, such as naturally occurring amino acids and their derivatives, such as their salts and esters. Preferred amino acids are those with nucleophilic groups in the side chain, such as basic amino acids (histidine, lysine) and amino acids with -OH or -SH groups in the side chain (tyrosine, serine, threonine, cysteine). Also useful are small peptides, such as, but not limited to, dipeptides, tripeptides, and other soluble oligopeptides, preferably containing at least one amino acid of the preferred types with a basic side chain -OH or -SH, or polyethyleneimine oligomers or polymers that also contain multiple secondary and primary amino groups.

[0016] Silanization reagent Si-(OR 1 )(OR 2 )(OR 3 )R 5 NHR 4 In embodiments of the invention using R 4 can be selected from H and linear or branched alkyl, which may be further substituted with amide, benzyl, carbamate, and amine. Such reagents may include trimethoxysilane or triethoxysilane in certain embodiments. Specific non-limiting examples include the compounds shown in Table 2.

[0017] [Table 2]

[0018] In some embodiments, the reagent substance has the formula W(X)-NHR 4 (Wherein, W is a molecule containing an aromatic structure; X represents OH, SH, COOH, amide, NH2, or NHR (wherein R is alkyl or alkenyl); R 4 is as defined above. The X group and NHR 4Each of the groups may be independently attached directly to a carbon atom of the aromatic structure or may be attached to an alkyl or alkylene side chain from the aromatic structure. Non-limiting examples include the compounds shown in Table 3.

[0019] [Table 3]

[0020] Table 4 provides a non-limiting list of other useful reagents.

[0021] [Table 4-1]

[0022] [Table 4-2]

[0023] [Table 4-3]

[0024] [Table 4-4]

[0025] [Table 4-5]

[0026] As mentioned above, the method of the present invention has the distinct advantage that the silicone surface to be treated according to the present invention does not need to be subjected to a pretreatment with an oxidizing agent, such as ozone, hydrogen peroxide or plasma treatment, before treatment with the reagent according to the method of the present invention. This makes the method of the present invention much simpler than the conventional methods for modifying silicone surfaces described in the Background section above. This applies to any reagent that can be selected, but in particular, when the silanizing reagent defined above is selected, the pre-oxidation step is not applied. When using other reagents, the pre-oxidation step may or may not be applied, but as mentioned above, it is not essential.

[0027] Also, pretreatment with strong alkali such as NaOH, KOH, LiOH, BaOH, NH4OH and CaOH2 can be omitted, and is omitted in a preferred embodiment.

[0028] The method can be advantageously carried out by using any of a variety of common solvents or mixtures of solvents. One advantage of the present invention is the ease of using non-toxic and environmentally friendly solvents, such as common alcohol solvents (solvents that are not highly toxic, are not significant carcinogens, and are not harmful to the environment when disposed of). In some embodiments, an organic solvent is used, which can be selected from, but is not limited to, diethylene glycol, diethyl ether, diethylene glycol, dimethyl ether, methyl t-butyl ether (MTBE), tetrahydrofuran (THF), dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), ethylene glycol, ethanol, propanol, isopropanol, 1-butanol, 2-butanol, t-butyl alcohol, ethyl acetate, and acetone. Alternatively, water or an aqueous solution can be used, or a miscible solution of water and one or more organic solvents can be used. In these embodiments, the silicone surface to be treated is placed in a solvent and the reagents are mixed into the solvent. Tests have shown that this method can be successfully carried out at room temperature, although in some embodiments, higher temperatures may be advantageous for faster reaction. After a period of time, the treated silicone surface is preferably rinsed, preferably with the same solvent used in the reaction or another solvent, preferably with water or an aqueous solution or a non-toxic alcohol or other environmentally friendly solvent.

[0029] In embodiments in which a gas phase reagent is used, the silicone surface is placed in a suitable contained atmosphere, which in some embodiments is air, but in other embodiments is an inert atmosphere such as nitrogen, and the reagent is injected or leaked into the contained atmosphere.

[0030] Applicable reaction conditions vary depending on the particular reagents and solvents selected and can be optimized accordingly. Thus, in some embodiments, the reaction can be carried out at room temperature (about 20-25°C), while in other embodiments, the reaction is carried out at a temperature in the range of about 10°C to 100°C, for example, from about 10°C, or from about 15°C, or from about 20°C, or from about 25°C, or from about 30°C, to about 100°C, or to about 90°C, or to about 70°C, or more preferably to about 50°C, or to about 40°C. In some embodiments, it is useful to apply lower temperatures using applicable solvents, and thus the reaction can be carried out at a temperature in the range of about -30°C to about 10°C.

[0031] The reaction may be accelerated by the application of ultrasound and / or microwave heating.

[0032] The resulting surface-modified silicone with reactive amino groups has the advantage that it can be further modified to obtain silicones with desired modification properties. Thus, in some embodiments, the method of the present invention comprises a further step of further modifying the silicone surface. In one embodiment, the method comprises treating the silicone surface with a diazotransfer reagent (sometimes called an azide transfer reagent) to convert at least a portion of the introduced amino groups into azide groups. These azide groups can then selectively react with alkynes in a 1,3-dipolar cycloaddition (i.e., "click reaction") to give 1,2,3-triazole derivatives (see, for example, US Pat. No. 9,302,997 B2). Such 1,3-dipolar cycloaddition has been used to covalently attach alkyne derivatives of various bioactive molecules, such as proteins, peptides, biotin, polymers, etc., to surfaces (Escorihuela et al. Adv. Mater. Interfaces 2015, 2, 1500135 - https: / / doi.org / 10.1002 / admi.201500135). Thus, in some embodiments of the method, the silicone surface is reacted with a selected azide transfer reagent, followed by a selected desired molecule that binds to the introduced azide group by 1,3-dipolar cycloaddition. The diazo transfer reagent can be selected from, but is not limited to, imidazole-1-sulfonyl azide, triflyl azide, azidotrimethylsilane, and sodium azide. The molecules thus attached can be selected from, but are not limited to, peptides, proteins, biotin, polymers, such as PEG, etc., and other reagents are equally applicable.

[0033] In another embodiment, the method includes treating the surface having the introduced amino groups with a cross-linking agent, preferably selected from, but not limited to, glutaraldehyde, ethylene glycol dimethacrylate, genepin, tannic acid, and polyethylene glycol diglycidyl ether. After the selected cross-linking agent reacts with the modified surface using methods well known in the art, a desired biopolymer can be attached thereto, or alternatively, other polymers, peptides, proteins, or other biologically active molecules having nucleophilic reactive groups present in their structure can be linked thereto. Thus, such surfaces of the invention having introduced cross-linking agents can be reacted with, for example, synthetic polymers (e.g., PEG), proteins, peptides, carbohydrates, small bioactive molecules, such as, but not limited to, bioactive drug molecules, cytokines, or hormones.

[0034] The method of the present invention introduces amino groups to accessible surfaces on treated silicone.The exact structure and nature of the amino group bond to silicone has not yet been elucidated and is under investigation by the inventors.The test included in the following example demonstrates that significant activity remains even after extensive washing, which suggests covalent bonding, although non-covalent bonding may also play a role.

[0035] One aspect of the present invention provides silicone objects having a modified surface obtained by the above method. Such objects of the present invention can be manufactured and commercially provided for subsequent use as useful intermediate products, or the silicone objects can be treated using the method of the present invention and further modified by a subsequent step to attach useful moieties thereto selected as above. Thus, the present invention also encompasses silicone objects obtained by the method of the present invention, having one or more molecules attached (preferably covalently attached) to at least a portion of the amino groups introduced into the silicone surface. Such molecules can include, but are not limited to, any of the above, such as, but not limited to, synthetic polymers, proteins, peptides, carbohydrates, and small bioactive molecules, such as bioactive drug molecules, cytokines or hormones, or any combination of one or more of these.

[0036] As used in this disclosure, including the claims, the singular forms of the terms "a," "an," and "the" are to be construed as including the plural forms unless the context indicates otherwise, and vice versa. It should be noted, therefore, that as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0037] Throughout the description and claims, the terms "comprise," "including," "having," "contain," and variations thereof, are to be understood as meaning "including but not limited to," and are not intended to exclude other components.

[0038] The term "at least one" should be understood to mean "one or more," and thus includes both embodiments including one or more components. Furthermore, a dependent claim that refers to an independent claim reciting "at least one" feature has the same meaning both when the feature is recited as "the" and when it is recited as "at least one of."

[0039] It will be understood that modifications to the above-described embodiments of the invention can be made while remaining within the scope of the invention. Features disclosed herein, unless otherwise stated, can be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each disclosed feature represents one example of a generic series of equivalent or similar functionality.

[0040] The use of exemplary language, such as "for instance," "such as," "for example," and the like, is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless specifically recited in the claims. Unless the context clearly indicates otherwise, steps described herein can be performed in any order or simultaneously.

[0041] All features and / or steps disclosed in this specification may be combined in any combination, except where at least some features and / or steps are mutually exclusive. In particular, the preferred features of the invention are applicable to all aspects of the invention and may be used in any combination. [Brief description of the drawings]

[0042] [Figure 1] Figure 1: Calibration curve for the ninhydrin assay for the quantification of free amino groups (-NH2) in samples. [Figure 2A-2C]Figure 2: Effect of reaction time on the three steps of surface modification: A: APTMS, B: glutaraldehyde, and C: chitosan. [Diagram 3] Figure 3: FT-IR spectra of untreated silicone (F), chitosan-coated silicone (G), and chitosan (H). [Figure 4] Figure 4: Samples at various steps. A: Untreated silicone after ninhydrin test, B: After 2-hour long silane treatment with 2% APTMS, C: After 24-hour treatment with 2% APTMS, D: APTMS-treated silicone after ninhydrin test, E: After APTMS and glutaraldehyde treatment, F: Chitosan coating (after APTMS and glutaraldehyde treatment). [Diagram 5] Figure 5: Effect of washing with various solvents on the ninhydrin reaction of silicone samples treated with 2% APTMS. [Figure 6] FIG. 6: FTIR spectrum of a silicone sample that was reacted with APTMS, then with an azide reagent, and finally subjected to a cycloaddition reaction (see Example 6). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0043] Example 1. Silicone samples, ninhydrin assay and FT-IR 1-1. Silicone sample Medical grade silicone elastomer sheets from NuSil Technology (MED82-5010-80 and MED82-5010-40) were used in the experiments. Circular disks of silicone were cut from the sheets using a hollow punch to have a diameter of 1.2 cm, a thickness of 0.2 cm (thin disk) or 0.4 cm (thick disk), and a total surface area of ​​3.0 cm. 2 or 3.8 cm 2The samples were prepared using a 100% ethanol-based solvent. The thin disk sample had a mass of 0.14 g and the thick disk sample had a mass of 0.26 g. The thin disks were used and, unless otherwise stated, were immersed in acetone, sonicated for 30 minutes to remove any surface contaminants, rinsed with deionized water, and dried in an oven at 80° C. prior to use.

[0044] In a second set of experiments, 2 mm thick silicone sheets were made by pouring silicone mixtures prepared from two types of liquid silicone, one containing a catalyst and one containing a crosslinker and a cure inhibitor, into a mould and heating the mould at 100°C to cure the silicone into an elastomer. Three types of silicone membranes with different stiffness (Shore OO) were made. These silicone membranes are called A (CF1350), B (CF15) and C (CF13).

[0045] 1-2. Ninhydrin assay Ninhydrin reagent used to quantify free amino groups (-NH2) in samples: Lithium acetate dihydrate was dissolved in deionized water and the pH was adjusted to 5.2 with glacial acetic acid to give a 4 M lithium acetate buffer solution. Ninhydrin (1.0 g) and hydrindantin (0.15 g) were weighed directly into a round-bottom flask and dissolved in DMSO (37.5 ml). The flask was stoppered with a septum. A syringe fitted with a nitrogen balloon was inserted through the septum into the solution and oxygen was flushed out by inserting another syringe into the septum. Then 12.5 ml of the 4 M lithium acetate buffer was added and the solution was again flushed with nitrogen.

[0046] 1-3. Calibration of the ninhydrin assay with glucosamine hydrochloride A 25 mL solution of CH3COOH / H2O (1:250 v / v) was prepared by adding 0.1 mL of CH3COOH to 25 mL of H2O. A stock solution with a concentration of 1 mg / ml was then prepared by adding 0.25 g of chitosan to the solution and stirring the solution with a magnetic stirrer until the chitosan was completely dissolved. This stock solution was then used to prepare standard solutions with six concentrations: 15.62 μg / ml, 31.25 μg / ml, 62.5 μg / ml, 125 μg / ml, 250 μg / ml, and 500 μg / ml.

[0047] A similar process was carried out to prepare glucosamine standard solutions.

[0048] A blank was prepared by mixing 1 ml of deionized water with 1 ml of ninhydrin reagent. The test tube was then heated in a water bath (90 °C) for 30 min to complete the reaction. After cooling in another water bath, the solution was diluted with 5 ml of a 50% (v / v) EtOH / H2O mixture to a final volume of 7 ml. These solutions were vortexed again for 15 s to oxidize the excess hydrindatin. The absorbance at 570 nm was measured with a UV-vis spectrophotometer (GENESYS 150). The results were used to construct a calibration curve (Figure 1).

[0049] [Table 5]

[0050] 1-4. Ninhydrin assay of silicone disk samples to quantify amino groups Treated or untreated disks were placed in test tubes containing 1 ml of ninhydrin reagent and 1 ml of water. The test tubes were heated in a water bath (90°C) for 30 min to complete the ninhydrin reaction. The disks were then removed, the solutions were cooled, and then diluted with 5 ml of a 50% (v / v) ethanol / water mixture to a final volume of 7 ml. The absorbance of the diluted solutions was measured in a 1 cm path length cuvette by UV spectrophotometer. In case of strong responses (absorbance >1-2), the solutions were further diluted with 50% (v / v) ethanol / water to allow measurements in the optimal 0-2 absorbance range.

[0051] 1-5.FT-IR The surface functionalization and coating were investigated by a Nicolet iZ10 FT-IR spectrophotometer (Thermo scientific) in reflectance mode and the spectra were analyzed by OMNIC software.

[0052] Example 2. Siliconization with APTMS in toluene, cross-linking with glutaraldehyde, and coating with chitosan 2-1. Treatment with APTMS dissolved in toluene In this experiment, 1%, 2% and 4% solutions of (3-aminopropyl)trimethoxysilane (APTMS) were prepared by adding 0.5, 1 and 2 mL of APTMS to 49.5, 49 and 48 mL of toluene, respectively.

[0053] 1 ml of (3-aminopropyl)trimethoxysilane (APTMS) was diluted with 50 ml of toluene to obtain a 2% APTMS solution. A thin silicone disk was passed through a 19G BD Microlance syringe needle and pressed down to be completely immersed in the APTMS solution. The disk was then immersed in the APTMS solution in an Erlenmeyer flask, and the solution was stirred with a magnetic stirrer. After a set treatment time (20 min, 40 min, 1 h, 1.5 h, 2 h or 24 h), the disk was removed and thoroughly rinsed with water, followed by washing six times on each side using a transfer pipette to remove excess reagent. It was then dried in an oven (80 °C) for 2 h. The response to ninhydrin increased up to 24 h (Figure 2A). However, such a long reaction time also had a negative effect on the properties of the silicone material. The treated disks became white and brittle after 24 h of reaction, indicating that significant covalent crosslinking had occurred. Therefore, a 2-hour reaction appears to be optimal since the response in the ninhydrin assay was approximately 65% ​​of the 24-hour response. No effect on the properties of the silicone material was observed with a 2-hour treatment.

[0054] 2-2. APTMS-treated silicone with glutaraldehyde crosslinker Silicone disks treated with a 2% solution of APTMS in toluene for 2 hours and rinsed were immersed in a 25% solution of glutaraldehyde in H2O with occasional agitation. After 20 minutes, 40 minutes, 1 hour, 1.5 hours, 2 hours or 3 hours, the disks were removed from the solution, washed four times on each side with methanol using a transfer pipette and air-dried overnight. The sheets were analyzed by FT-IR and the ninhydrin test. All tests were performed in triplicate. Prior to coating of the amino-functionalized silicone with chitosan, the samples must be treated with a suitable cross-linking agent capable of reacting with the amino groups of the silicone surface and with polysaccharides. Glutaraldehyde is a cross-linking agent that reacts readily with primary amino groups to form imines. The imines can then be reduced to form stable dialkylamines. The response to the ninhydrin test should decrease as the reaction proceeds, since the reagent blocks the amino groups of the silicone surface. The ninhydrin response of the samples decreased with increasing reaction time in glutaraldehyde solution because the amino groups were converted to imines. After 3 hours of reaction, there was almost no response to the ninhydrin test. Thus, the cross-linking reaction was nearly complete after 3 hours of cross-linking (Figure 2B).

[0055] 2-3. Chitosan treatment of silicone treated with APTMS and glutaraldehyde Silicone disks treated with 2% APTMS solution in toluene for 2 h and rinsed were immersed in 25% glutaraldehyde solution as above and immersed in 2% (v / v) acetic acid / H2O solution containing 0.04 g / ml chitosan and 0.001 g / ml NaBH4 for 2 min, 40 min, 1 h, 1.5 h or 2 h. This was done at room temperature with occasional stirring with a needle. They were then removed from the solution, immersed in deionized water, rinsed thoroughly, washed 10 times on each side using a plastic pipette with the same solvent, and dried in an oven (80 °C) for 1 h. The sheets were analyzed by FT-IR and ninhydrin test. All tests were performed in triplicate.

[0056] Chitosan polymer chains contain many amino groups (-NH2), and when the silicone sample treated with glutaraldehyde was immersed in a chitosan solution, the response in the ninhydrin test should increase again. An increase in the ninhydrin response was observed (Figure 2C). The response increased rapidly in the first hour, but then slowed down and approached a plateau by 2 hours. To obtain significant measurements and fit the calibration curve, the samples were diluted 10-fold after the ninhydrin reaction.

[0057] Figure 2 shows the effect of reaction time on the response in the ninhydrin assay for all three steps of coating. A: APTMS, B: glutaraldehyde, and C: chitosan. All measurements were performed in triplicate. Standard error bars are shown. After the ninhydrin assay of APTMS-treated disks (A) and glutaraldehyde-treated disks (B), the ninhydrin reagent was diluted 7-fold (to 7 ml). After the ninhydrin reaction of the chitosan-treated disks, the reagent was diluted 70-fold (first to 7 ml, then 10-fold) so that the measured absorbance fell within the calibration curve.

[0058] 2-4.FT-IR Results The untreated silicone disks were measured to identify characteristic peaks that could be compared to the spectra of treated silicones. The untreated silicone sample had a peak at 2961 cm -1 A characteristic silicone peak of (CH stretch) was observed (Figure 3F). Additional peaks were observed in the spectrum of chitosan-coated silicone, which were similar to those observed in the spectrum of pure chitosan. The characteristic peak of chitosan is: 3292 cm -1 (NH and CH stretching), 2871 cm -1 (CH3 symmetric stretching), 1647cm -1 (C=O stretching), and 1375 cm -1 (CN stretching) (Figure 3H). The characteristic peaks of chitosan-coated silicone similar to chitosan (Figure 3G) are: 3262 cm -1 (NH and CH stretching), 2961 cm -1 (CH3 symmetric stretching), 1558cm-1 (C=O stretching), and 1402.52 cm -1 (CN expansion and contraction).

[0059] FIG. 3 shows the FT-IR spectra of F: untreated silicone, G: chitosan-coated silicone, and H: chitosan.

[0060] Visual Analysis Digital images showed the appearance of the silicone disc samples after various treatments. Untreated silicone had an opaque grayish color that darkened after treatment with ninhydrin reagent (Figure 4A). There was no change in appearance after 2 h treatment with 2% APTMS solution (Figure 4B), but after 24 h treatment the samples became white and brittle (Figure 4C). Discs after 2 h treatment with 2% APTMS turned blue after treatment with ninhydrin reagent (Figure 4D). APTMS-treated discs turned brownish orange after treatment with glutaraldehyde (Figure 4E) and further changed to a reddish color after treatment with chitosan.

[0061] The results are shown in Figure 4, which shows A: untreated silicone after ninhydrin test, B: after 2 hour long silane treatment with 2% APTMS, C: after 24 hour treatment with 2% APTMS, D: APTMS treated silicone after ninhydrin test, E: glutaraldehyde treatment, F: chitosan coating.

[0062] Example 3. Stability of amino group modification of silicone Silicone disk samples treated with 2% APTMS for 2 hours were washed extensively with various solvents in an attempt to extract reagents that were not tightly (covalently) attached to the silicone (Figure 5). The ninhydrin response was similar (~1.2) when the first wash was done with water, methanol, and toluene, but was initially reduced by 50% (~0.6) when the first wash was done with water, methanol, and toluene. Washing with ethanol followed. Extensive washing for up to one week further reduced the ninhydrin response, but this occurred mainly in the first 24 hours, with only limited reduction after another week of washing. Samples were heated in water (90°C) for 20 minutes after washing with various solvents. Heating reduced the initial response of all samples by about 50%.

[0063] Figure 5 shows the effect of washing with various solvents on the ninhydrin reaction of silicone samples treated with 2% APTMS. The Y scale shows absorbance at 570 nm and the X scale shows time in days.

[0064] Example 4. Siliconization with pure APTMS and with various APTMS solutions Procedure for toluene solution: Silicone disks were immersed in a 2% (v / v) APTMS toluene standard solution for 20 min at room temperature. The disks were then thoroughly rinsed with anhydrous toluene to remove excess reagent residues. Silicone samples were then dried in an oven (80° C.) and the ninhydrin assay was performed as described in Example 1.

[0065] Procedure for deionized water solution: The samples were immersed in a 5% APTMS (heated to 85°C) solution for 5 or 30 minutes, dried with nitrogen gas, and then held at 65°C for 10 minutes.

[0066] Procedure for pure APTMS liquid: The silicone samples were immersed in 97% APTMS for 5 min, then washed with deionized water and dried in an oven at 80 °C.

[0067] Procedure for ethanol and ethyl acetate solutions: Anhydrous solutions were prepared by mixing APTMS with absolute ethanol or ethyl acetate to give a 1% solution. The solution was stirred at room temperature under nitrogen for 10 minutes. A silicone disk was then placed in the solution at room temperature under nitrogen. After 20 minutes, the substrate was washed with ethanol and blown dry with nitrogen. The results are shown in Table 6.

[0068] [Table 6]

[0069] Example 5. Azide transfer reaction and azide-alkyne cycloaddition via modified silicone Azide transfer reaction protocol: A silicone disk sample treated with 2% APTMS in toluene for 2 hours was passed through a syringe needle and immersed in 50 mL of MeOH in a 100 ml round bottom flask. Imidazole sulfonyl azide (50 mg) was then added to the reaction medium with stirring. This was followed by the addition of 50 mg of K2CO3 dissolved in 1 mL of deionized water and 5.75 mg of CuSO4·5H2O dissolved in 1 mL of deionized water. The flask was stoppered and the reaction medium was stirred for 16-20 hours. After this reaction time, the sample was washed with acetone, then with water, again with acetone, and finally, dried at room temperature for 1 hour. FTIR was used to analyze the treated disks. 2097 cm -1 The spectrum of the treated disk was consistent with the introduction of azide groups on the surface, since new peaks were observed at .

[0070] Cycloaddition reaction protocol: 15 mL of DMSO was added to a 25 mL round-bottom flask equipped with a reflux condenser. The solvent was heated until it reached 50 °C. Then, the azide-functionalized disk (passed through a needle) was added to the reaction medium and stirred with a magnetic stirrer. Then, 1.5 mg of CuSO4 and 4.7 mg of sodium ascorbate were dissolved separately in 1 mL of deionized water and added to the reaction medium. Finally, 23.3 mg of N,N,N-trimethylprop-2-yn-l-aminium were added under a nitrogen atmosphere. The solution was then stirred at 50 °C for 48 h. After this time, the sample was removed from the medium and washed at least three times with deionized water. Afterwards, the sample was dried in the open air. The treated disk was analyzed by FTIR. After the cycloaddition reaction, the azide peak disappeared, but the 2922 cm peak in the spectrum was still visible, consistent with triazole functionalization. -1 A new peak in the CH stretching region was observed.

[0071] FIG. 6 shows the FTIR spectra for the study of the azide-alkyne cycloaddition reaction.

[0072] Example 6. Silicone modification with ethanolamine in various solvents Cleaning the Silicone Disc A silicone disk was cut from a 0.4 cm thick silicone sheet. The silicone disk was immersed in deionized water in an Erlenmeyer flask and shaken. The water wash was replaced and the disk was soaked at room temperature for 1 h. This was repeated twice. Finally, the disk was dried in an oven at 80 °C for 2 h.

[0073] Ethanolamine (EA) was mixed with water, isopropanol, ethanol, or ethyl acetate to prepare a treatment solution of the desired concentration. The disks were immersed in the treatment solution and gently agitated every hour (thick disks did not float in the solution, so there was no need to pass a needle through them).

[0074] After the reaction, the disks were removed and washed thoroughly in water four times (total washing time 15 min), and then subjected to the ninhydrin assay as described above (Example 1). The results are shown in Table 7.

[0075] [Table 7]

[0076] Example 7. Silicone modification with APTMS In this example, silicone disks from the prepared sheets A, B, and C were modified with APTMS. A 12 mm hollow steel punch was used to prepare silicone disks, which were washed with deionized water and finally dried in an oven at 80 °C. The samples (silicone disks) were added to 50 ml of APTMS solution or solvent only (control) and stirred at room temperature for 1 h. The supernatant was then discarded and the samples were washed six times with toluene (in this case, an appropriate amount of toluene (20 mL) was added to each disk to completely cover the disk, shaken for about 30 seconds, and then the solution was replaced with 20 ml of fresh toluene, which was repeated six times). Next, to remove any residual traces, the samples were washed six times with methanol and deionized water, respectively, as described above. Finally, the samples were dried in an oven at 80 °C and used for the ninhydrin assay.

[0077] The ninhydrin assay using silicone disks was performed in the same manner as the calibration standards. The disks were immersed in 1 ml of water and 1 ml of ninhydrin reagent in a test tube. The test tubes were then heated in a water bath (90°C) for 30 min to complete the reaction. The solution was then diluted with 26 ml of a 50% (v / v) EtOH / H2O mixture before UV measurement. All tests were performed in triplicate. The results are shown in Table 8.

[0078] [Table 8]

[0079] Example 8. Silicone modification with solutions of DAP (1,3-diaminopropane), EA (ethanolamine), A12P (3-amino-1,2-propanediol), 3AP (3-amino-1-propanol), and EDA (ethylenediamine) in isopropanol In this experiment, five different reagents were prepared by adding 2.2 mL each of DAP, EA, A12P, 3AP, and EDA to 20 mL of isopropanol.

[0080] Disks were made from Type A silicone sheets using a 12 mm hollow steel punch as above and washed twice with 20 mL each of isopropanol and water (30 min) as before.

[0081] Samples (silicone discs) were added separately to DAP, EA, A12P, 3AP and EDA solutions (three discs for each treatment) and stirred for 24 h. The supernatant was then discarded and the samples were washed twice with isopropanol and water as described above. Finally, the samples were dried in an oven at 80 °C and used for ninhydrin analysis.

[0082] Ninhydrin assays were performed as in Example 8, except that the solutions were diluted with 40 mL of 50% (v / v) EtOH / H2O mixture after heating. All tests were performed in triplicate. The results are shown in Table 9.

[0083] [Table 9]

Claims

1. A method for modifying a silicone solid surface to introduce amino groups into the surface to make it suitable for further modification, the method comprising treating the silicone elastomer surface with a reagent substance, The aforementioned reagent substance is - Molecules represented by the formula W(X)-NHR1, such as polyethyleneimine, O-(2-aminoethyl)polyethylene glycol, O,O'-bis(3-aminopropyl)polyethylene glycol, and poly-L-ornithine, where W is a linear or branched molecule having the structure -((CY1)n-Z-(CY2)m)l-, where Y1 and Y2 are independently selected from H, OH, SH, NH2, alkyl group (CH2)kCH3, n, m, and l are independently integers from 1 to 10, k is an integer from 1 to 5, Z is selected from -O- and -NH-, and X is optionally present but required if Y1 and Y2 are non-nucleophilic groups, and represents OH, COOH, amide, NH2, or NHR (where R is alkyl); - Molecules represented by the formula W(X)-NHR4, such as 4-aminophenol, 3-aminophenol, 4-aminobenzyl alcohol, 4-aminophenethyl alcohol, 4-hydroxybenzylamine, tyramine, tyrosine, and L-tyrosine methyl ester, where W is a molecule containing an aromatic structure, X represents OH, NH2, or NHR (where R is alkyl), and R4 is H, or a linear or branched alkyl which may be further substituted with amide, benzyl, carbamate, and amine; - Ethanolamine, 2-(ethylamino)ethanol, diethanolamine, 2-amino-1-propanol, 3-methylamino-1-propanol, 3-amino-1,2-propanediol, 2-(methylamino)ethanol, 2-hydroxyethylhydrazine, 3-amino-1-propanol, 2-amino-1,3-propanediol, 3-amino-2-methylpropan-1-ol, 2-(2-aminoethoxy)ethylamine, 3,3'-azandiyldipropan-1-ol, 3-[(2-aminoethyl)amino]-1-propanol, N, N'-bis(2-hydroxyethyl)ethylenediamine, 3-(2-hydroxyethylamino)-1-propanol, ethylenediamine, N-methylethylenediamine, N,N'-diethylethylenediamine, glycinamide, triethylenetetramine, tetraethylenepentamine, N-(2-hydroxyethyl)ethylenediamine, 3-[(2-aminoethyl)amino]-1-propanol, tris(2-aminoethyl)amine, 1,1-diamino-3,6,9-trioxaundecane, 1,3-diaminopropane, 1,3-diamino-2-propano 1,2-diaminopropane, 2,2-dimethyl-1,3-propanediamine, 3-aminopropanamide, bis(3-aminopropyl)amine, N,N'-bis(3-aminopropyl)-1,3-propanediamine, N,N'-bis(2-aminoethyl)-1,3-propanediamine, 1,2-bis(3-aminopropylamino)ethane, 1,3-diamino-N-(2-hydroxyethyl)-propane, 3-(3-aminopropoxy)propylamine, tris(3-aminopropyl)amine, 4,9-dioxa-1,12-dodecanediamine, 4-amino-1-butanol, piperazine, 1-(2-hydroxyethyl)piperazine, 4-hydroxypiperidine, 1,4-diaminobutane, spermidine, 2,2'-(ethylenedioxy)bis(ethylamine), glycine, serine, serine methyl ester, serine ethyl ester, serine isopropyl ester, threonine, threonine methyl ester, lysine, lysine ethyl ester, lysine methyl ester, ornithine, tyrosine, l-tyrosine methyl ester, and two or more oligopeptides of the above amino acids and / or amino acid derivatives. Being selected from; and The reagent substance is optionally dissolved in a solvent, the silicone elastomer surface is placed in the solvent containing the dissolved reagent, and the solvent is preferably a water-miscible solvent selected from methanol, ethanol, propanol, isopropanol, 1-butanol, 2-butanol, t-butyl alcohol, glycerol, water or aqueous solution, and one or more miscible mixtures thereof; A method characterized by the following.

2. The method according to claim 1, wherein the silicone surface is not subjected to pretreatment with an oxidizing agent such as ozone, hydrogen peroxide, or plasma treatment before the processing step.

3. The aforementioned surface is, for example, NaOH, KOH, LiOH, BaOH, NH ４ OH and CaOH ２ The method according to either claim 1 or 2, wherein the method is not pretreated with a strong alkali such as the above.

4. The method according to claim 1 or 2, wherein the reagent is dissolved in a solvent and the silicone surface is placed in the solvent containing the dissolved reagent, or the reagent is in gaseous form and is brought into contact with the silicone surface in a gaseous atmosphere.

5. The method according to claim 1 or 2, further comprising the step of treating the silicone surface with an azide transfer reagent to convert at least some of the introduced amino groups into azide groups.

6. The method according to claim 5, wherein the azide transfer reagent is selected from imidazole-1-sulfonyl azide, trifuryl azide, azidotrimethylsilane, and sodium azide.

7. The method according to claim 1 or 2, further comprising the step of treating the silicone surface with a crosslinking agent preferably selected from glutaraldehyde, ethylene glycol dimethacrylate, genepine, tannic acid, and polyethylene glycol diglycidyl ether.

8. The method according to claim 1 or 2, further comprising the step of treating the silicone surface with a bioactive molecule selected from chitosan, chitosan derivatives, oligopeptides, and proteins to form a bioactive coating.

9. A silicone object having a modified surface containing reactive amino groups, obtained by the method described in claim 1.

10. A silicone object having a modified surface obtained by the method of claim 9, wherein one or more molecules selected from synthetic polymers, proteins, peptides, carbohydrates, and small bioactive molecules, such as antibiotics, cytokines, or hormones, are bonded to at least some of the amino groups introduced on the silicone surface.

11. The silicone object according to claim 9 or 10, wherein the silicone surface is further modified to alter its physicochemical or biological properties, for example, to make the surface more hydrophilic, more hydrophobic, more antibacterial to resist bacterial colonization, and / or more biocompatible.

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