A peptoid-containing antibacterial coating and methods of making the same

Abstract

The application relates to the technical field of medical materials, and discloses an antibacterial coating containing a peptide-like substance and a preparation method thereof, which comprises the following steps: step 1, preparing an antibacterial solution containing a peptide-like substance; step 2, cleaning and drying the surface of a medical material; step 3, immersing the medical material treated in step 2 in the antibacterial solution in step 1 and standing, then taking out and drying the medical material, and preparing the antibacterial coating on the surface of the medical material; the antibacterial solution containing the peptide-like substance comprises the following raw material components according to 100% by mass: 0.1-2% of a peptide-like substance, 5-20% of tannic acid, 0.5-2% of hydrochloric acid, and the rest is an organic solvent; the peptide-like substance has the following structure. In the application, the peptide-like substance compound with excellent antifungal effect is used in cooperation with natural plant polyphenol tannic acid, the hydrogen bond and the pi-pi stacking between the tannic acid and the peptide-like substance are used together, a hydrophobic antibacterial coating with excellent antibacterial effect, stable adhesion and uniformity is formed on the surface of the medical material.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to an antibacterial coating containing peptides and its preparation method. Background Technology

[0002] Today, iatrogenic infections pose a significant threat to human health, potentially leading to serious complications for patients and placing a heavy burden on society. This is because the use of medical devices can damage or penetrate epithelial and mucosal barriers, disrupting the patient's defense mechanisms and providing a platform for microbial growth, making patients susceptible to infection. Once a medical device-related infection occurs, it not only affects the performance of the materials themselves, but if treatment is not timely, it can even lead to systemic infection, and in severe cases, death. To curb drug-resistant bacteria, the development of materials with antimicrobial capabilities has become particularly important.

[0003] Forming an antimicrobial coating on the surface of medical devices can reduce the risk of iatrogenic infections during use. Most antimicrobial coatings are created by directly adding antimicrobial agents, followed by processing to form an antimicrobial functional coating. This process for preparing antimicrobial coatings is complex, time-consuming, and suffers from the problem of the antimicrobial agents not being released stably and for an extended period.

[0004] Ugi reaction can be used to prepare peptide-like compounds (compounds in which the α-carbon substitution of the amino acid in the main chain is transferred to the nitrogen in the main chain, i.e., compounds lacking hydrogen on the nitrogen in the main chain). These compounds have potential biomedical functions and have already shown antifungal, antiviral, and analgesic properties. Due to the amide bond site that can be recognized by proteases, they can exhibit better stability in practical applications compared to traditional antibacterial peptide compounds.

[0005] CN113577237A discloses the application of a peptide-like compound in combating multidrug-resistant, pan-drug-resistant, or pan-drug-resistant bacteria. This peptide-like compound not only kills common pathogens but also exhibits excellent killing activity against multidrug-resistant, pan-drug-resistant, or pan-drug-resistant bacteria. However, such antimicrobial peptides are mainly synthesized through solid-phase synthesis on a supported resin by repeatedly performing "condensation-deprotection-condensation" steps, which is cumbersome, costly, and yields low, making it impossible to prepare large-scale, high-molecular-weight polymeric peptide materials.

[0006] For example, CN110372774A discloses a method for synthesizing a class of isoindolone-substituted α-acyloxyamide dipeptide derivatives. The method involves a three-component Ugi reaction of an amine compound, an aldehyde compound, and an isonitrile compound in a solvent at room temperature under MOF-Cu catalysis to obtain the target compound. This dipeptide derivative can effectively inhibit *Penicillium finger-shaped mold*, *Penicillium italicum*, or *Rhizoctonia solani*. However, research on dipeptide compounds remains insufficient, especially those with excellent antibacterial properties, which still hold significant research value.

[0007] On the one hand, research is needed on the antibacterial properties of dipeptide compounds themselves, and on the other hand, research is needed on how antibacterial compounds can exist stably on the surface of materials for a long time and release effective antibacterial effects. Both aspects of research are crucial. Summary of the Invention

[0008] This invention addresses the problems of poor antibacterial performance or insufficient stability of existing antibacterial coatings by providing a method for preparing a peptide-containing antibacterial coating. The method combines peptides with a biphenyl ring structure and tannic acid to coat the coating, resulting in excellent antibacterial effect and strong adhesion. The coating can be stably applied to the surface of medical materials for a long time, achieving a strong ability to resist / kill methicillin-resistant Staphylococcus aureus.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A method for preparing a peptide-containing antibacterial coating includes the following steps:

[0011] Step 1: Prepare an antibacterial solution containing peptides;

[0012] Step 2: Clean and dry the surface of the medical materials;

[0013] Step 3: Immerse the medical material treated in Step 2 into the antibacterial solution in Step 1 and let it stand. Then, take out the medical material, dry it, and obtain the antibacterial coating on the surface of the medical material.

[0014] The antibacterial solution containing peptides comprises, by mass percentage (100%), the following raw material components: 0.1-2% peptides, 5-20% tannic acid, 0.5-2% hydrochloric acid, and the remainder being organic solvents.

[0015] The peptide-like substance has the following structure:

[0016] .

[0017] This invention utilizes peptide compounds with excellent antifungal properties. Due to their protease-recognizable sites, they exhibit better stability than traditional antimicrobial peptides in practical applications. Combined with natural plant polyphenols, tannins, the phenolic hydroxyl groups in tannins can form hydrogen bonds with the carbonyl groups in the peptide structure, and can also form π-π stacking with the benzene rings in the peptides. Through the combined effects of hydrogen bonding and π-π stacking, a stable complex is formed. The peptides possess strong hydrophobic properties, which improve the hydrophilicity of tannins, forming a stable coating. Simultaneously, the trihydroxyphenyl and dihydroxyphenyl groups in tannins have strong surface adhesion, enhancing the adhesion between the coating and the substrate. Finally, the tannin-peptide co-deposit is applied to the surface of the biomedical material via dip-coating, forming a highly hydrophobic, excellent antibacterial, and uniformly adherent antibacterial coating.

[0018] The preparation method of the peptide-like substance includes the following steps:

[0019] Step 1: 4-(4-methyl-3-pentenyl)-3-cyclohexen-1-carboxaldehyde and 1-(3-aminopropyl)pyrrolidine are mixed and reacted in a solvent, followed by the addition of 4-biphenylacetic acid and isonitrile-n-butane to continue the reaction and obtain the crude peptide compound; the reaction formula is as follows. Figure 1 As shown.

[0020] Step 2: The crude peptide compound is removed by rotary evaporation to remove the solvent, purified by column chromatography, and then lyophilized to obtain the peptide.

[0021] The peptide-like compound is prepared using the Ugi method, in which an aldehyde and a ketone first undergo dehydration condensation to form an imine. Subsequently, the imine ion undergoes nucleophilic addition with an isonitrile to generate a nitrileonium ion. The carboxylate anion then reacts with the carbon atom of the isonitrile to form another imine intermediate. Finally, a Mumm rearrangement reaction occurs to generate the stable peptide-like compound. The entire Ugi reaction produces only water as a byproduct, and the reaction conditions are mild with high yields.

[0022] In step 1, the reaction is carried out at room temperature, with 4-(4-methyl-3-pentenyl)-3-cyclohexene-1-carboxaldehyde and 1-(3-aminopropyl)pyrrolidine reacting for 5-20 min; after adding 4-biphenylacetic acid and isonitrile n-butane, the reaction continues for 12-36 h.

[0023] The molar ratio of 4-(4-methyl-3-pentenyl)-3-cyclohexene-1-carboxaldehyde, 1-(3-aminopropyl), 4-biphenylacetic acid and isonitrile n-butane is a stoichiometric ratio, such as 1:1:1:1.

[0024] The solvent in step 1 is methanol.

[0025] Preferably, to facilitate uniform mixing of raw materials, all raw materials are dissolved separately in a solvent before the reaction, and then the mixture is reacted.

[0026] In step 2, the eluent used for column chromatography is a mixed solvent of dichloromethane, methanol, and formic acid;

[0027] The volume ratio of dichloromethane, methanol, and formic acid in the rinsing agent is 96:3:1.

[0028] Before elution, the peptide-like compound was dissolved in dichloromethane and subjected to column chromatography.

[0029] More preferably, step 2 specifically includes the following steps: after removing the solvent by rotary evaporation, the crude peptide product is dissolved in dichloromethane, purified by gradient elution with an eluent to obtain a purified product, and the solvent is removed by vacuum evaporation and lyophilized to obtain the peptide.

[0030] Preferably, the antibacterial solution containing peptides comprises, by mass percentage (100%), the following raw material components: 0.8-1.2% peptides, 8-10% tannic acid, 0.5-1% hydrochloric acid, and the remainder being organic solvents.

[0031] Preferably, the pH of the antibacterial solution is 3-5. Under acidic conditions, the ionization of tannic acid is inhibited, which is beneficial to the stability of the hydrogen bond between peptides and tannic acid.

[0032] Preferably, the mass ratio of peptides to tannic acid in the antibacterial solution is 10:0.5-2. Experiments show that at this mass ratio, the peptides and tannic acid form a high bonding strength through π-π stacking and hydrogen bonding, resulting in a uniform and stable coating with excellent antibacterial properties. More preferably, the mass ratio of peptides to tannic acid is 10:1.

[0033] Preferably, the medical material is a medical catheter; preferably, to facilitate immersion of the catheter in the antibacterial lubricating solution, the medical catheter is pre-cut to fit the length of the container containing the solution for easier operation;

[0034] Preferably, the medical material is one or more of TPU, Pebax, and PTFE. These materials have low surface energy, better adhesion, and are less prone to detachment. TPU and Pebax are preferred materials.

[0035] Preferably, the organic solvent includes one or more of methanol, ethanol, acetone, and dimethyl sulfoxide. Ethanol is further preferred because it is volatile and has low biotoxicity.

[0036] Preferably, the cleaning in step 2 includes one or any two or more of the following methods: ethanol wiping, ultrasonic vibration, and low-temperature plasma cleaning.

[0037] Preferably, in step 3, the immersion speed of the medicinal material is 0.5-2 cm / s, and it is soaked in the solution for more than 5 seconds. When taking it out, the lifting speed is 1-3 cm / s. The immersion and removal speeds should not be too fast, so that the coating obtained is more uniform.

[0038] The drying process involves drying at 60-80°C for at least 1 hour. Preferably, after preparing the antibacterial solution, all raw materials are mixed and stirred at room temperature for 2-6 hours.

[0039] The present invention also provides a peptide-containing antibacterial coating or a medical material containing a peptide-containing antibacterial coating prepared according to the preparation method described above.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) The antibacterial solution prepared by the present invention using peptides and tannic acid not only has excellent antibacterial properties, but also has strong hydrophobicity and strong adhesion. It can be stably coated on the surface of medical materials for a long time to achieve effective sterilization. It can be applied to the field of medical technology to achieve antibacterial effect and reduce the probability of iatrogenic infection.

[0042] (2) The peptide preparation method used in this invention is simple, and the medical materials or catheters prepared with antibacterial solutions containing peptides and tannic acid have a coating that adheres firmly to the surface of the catheter and can still adhere effectively under external force, showing a long-lasting antibacterial effect, and can be applied to various medical devices. Attached Figure Description

[0043] Figure 1 The reaction formula for the peptide-like compound prepared as an example.

[0044] Figure 2 H of the peptide-like compound prepared for the example 1 NMR spectrum.

[0045] Figure 3 The mass spectrum of the peptide-like compound prepared for the preparation example.

[0046] Figure 4 The image shows the appearance of the antibacterial lubricated medical catheters prepared in the examples and comparative examples. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the technical solutions of this invention, should all be covered within the protection scope of this invention. The raw materials used in the following specific embodiments are all commercially available.

[0048] Preparation Example: Preparation of Peptide-like Compounds

[0049] Step 1: Add 500 μL of 4-(4-methyl-3-pentenyl)-3-cyclohexene-1-carboxaldehyde (dissolved in methanol, 0.4 M) to a methanol solution containing 500 μL of an equimolar concentration of 1-(3-aminopropyl)pyrrolidine. Stir and react for 15 minutes. Then, add 500 μL of 4-biphenylacetic acid (0.4 M methanol solution) and isonitrile-n-butane (0.4 M methanol solution) sequentially. Stir and react at room temperature for 24 hours. The reaction formula is as follows. Figure 1 As shown.

[0050] Step 2: The crude peptide product solution prepared above was removed from the methanol solvent by rotary evaporation under vacuum. It was then reconstituted in dichloromethane. The resulting crude dichloromethane product solution was then purified using an automated column chromatography system. The solvent ratio for gradient purification was dichloromethane:methanol:formic acid = 96:3:1. The target solution obtained after gradient elution and purification was then vacuum-dried in a vacuum oven to remove the solvent. The purified product was then further lyophilized to remove moisture and stored at -4°C. Product H 1 NMR spectrum as follows Figure 2 As shown, the mass spectrometry is as follows Figure 3 As shown.

[0051] Example 1

[0052] Step 1: Weigh 1g of tannic acid and 8.8g of anhydrous ethanol and add them to a 50ml centrifuge tube. Add a small magnetic stir bar and stir at room temperature for 10min to completely dissolve the tannic acid. Add 0.1g of hydrochloric acid to adjust the pH to 4.5 to form a precursor solution. Then weigh 0.1g of peptide and add it to the above precursor solution. Stir at room temperature for 3h to obtain an antibacterial solution.

[0053] Step 2: Cut the medical TPU drainage tube into 20 cm lengths, fix its upper end to the hydrophilic coating machine fixture, moisten the lint-free paper with anhydrous ethanol, and wipe the tube three times from the upper end to the lower end with the moistened lint-free paper.

[0054] Step 3: Pour the solution prepared in Step 1 into the corresponding solution tube of the hydrophilic coating machine. Immerse the catheter in the coating solution at a speed of 1 cm / s, let it stand for 5 seconds, and then pull it out at a speed of 2 cm / s. Then place it in a vacuum drying oven and dry it at 70°C for 1 hour to obtain a TPU medical catheter with a peptide-like antibacterial coating.

[0055] The antibacterial properties and stability of the medical catheters were tested. The antibacterial property test method is as follows:

[0056] Under aseptic conditions, the experimental samples and uncoated control samples were cut into 2mm*3mm pieces. Methicillin-resistant Staphylococcus aureus was diluted with PBS to a concentration of 10. 6Prepare a CFU / ml bacterial suspension. Take a sterile culture dish and use sterile forceps to pick up two experimental samples. Incubate the samples in a water bath at 20±1℃ for 5 minutes. Add 0.1ml of bacterial suspension to each sample. After the samples have been in contact with the colonies for 10 minutes, transfer the contaminated samples to a test tube containing 5ml of PBS. Mix well, shake, and elute. Take 1ml of the sample solution and inoculate it onto agar medium. Incubate at 37℃ for 1 day, 3 days, and 7 days, respectively. Remove the culture dishes, photograph, and count the bacteria. Use a catheter without antibacterial coating as a blank control group. Repeat the experiment three times and calculate the inhibition rate.

[0057] The stability performance test method is as follows:

[0058] The coated area of ​​the catheter was immersed in a 1% m / v Congo red solution. After 30 seconds, it was removed and the surface was cleaned with pure water to remove any residual solution. The Congo red staining was observed. Then, the catheter was placed under a silicone sheet with a constant clamping force of 300g, and the sample was pulled out at a speed of 5mm / s. This test was repeated 3 times to observe the peeling of the coating after Congo red staining and to characterize the stability of the coating.

[0059] Example 2

[0060] Step 1: Weigh 1g of tannic acid and 8.8g of anhydrous ethanol and add them to a 50ml centrifuge tube. Add a small magnetic stir bar and stir at room temperature for 10min to completely dissolve the tannic acid. Add 0.1g of hydrochloric acid to adjust the pH to 4.5 to form a precursor solution. Then weigh 0.1g of peptide and add it to the above precursor solution. Stir at room temperature for 3h to obtain an antibacterial solution.

[0061] Step 2: Cut the medical Pebax catheter into 20 cm lengths, fix its upper end to the hydrophilic coating machine fixture, moisten the lint-free paper with anhydrous ethanol, and wipe the catheter three times from the upper end to the lower end with the moistened lint-free paper.

[0062] Step 2: Pour the prepared antibacterial solution into the corresponding solution tube of the hydrophilic coating machine. Immerse the catheter in the coating solution at a speed of 1 cm / s, let it stand for 5 seconds, and then pull it out at a speed of 2 cm / s. Then place it in a vacuum drying oven and dry it at 70°C for 1 hour to obtain a medical Pebax catheter with a peptide-like antibacterial coating.

[0063] The antibacterial properties and stability of the medical catheters were tested. The testing process is as shown in Example 1, and the test results are shown in Table 1.

[0064] Comparative Example 1

[0065] Step 1: Weigh 1g of tannic acid and 8.8g of anhydrous ethanol and add them to a 50ml centrifuge tube. Add a small magnetic stir bar and stir at room temperature for 10min to completely dissolve the tannic acid. Add 0.1g of hydrochloric acid to adjust the pH to 4.5 to form a precursor solution. Then weigh 0.1g of peptide and add it to the above precursor solution. Stir at room temperature for 3h to obtain an antibacterial solution.

[0066] Step 2: Cut the medical PTFE catheter into 20 cm lengths, fix its upper end to the hydrophilic coating machine fixture, moisten the lint-free paper with anhydrous ethanol, and wipe the catheter three times from the upper end to the lower end with the moistened lint-free paper.

[0067] Step 3: Pour the solution prepared in Step 1 into the corresponding solution tube of the hydrophilic coating machine. Immerse the catheter in the coating solution at a speed of 1 cm / s, let it stand for 5 seconds, and then pull it out at a speed of 2 cm / s. Then place it in a vacuum drying oven and dry at 70℃ for 1 hour to obtain a medical PTFE catheter with a peptide-like antibacterial coating. The antibacterial properties and stability of the medical catheter were tested, as described in Example 1, and the test results are shown in Table 1.

[0068] Comparative Example 2

[0069] Step 1: Weigh 1g of tannic acid and 8.85g of anhydrous ethanol and add them to a 50ml centrifuge tube. Add a small magnetic stir bar and stir at room temperature for 10min to completely dissolve the tannic acid. Add 0.1g of hydrochloric acid to adjust the pH to 4.5 to form a precursor solution. Then weigh 0.05g of peptide and add it to the above precursor solution. Stir at room temperature for 3h to obtain an antibacterial solution.

[0070] Step 2: Cut the medical TPU catheter into 20 cm lengths, fix the upper end of the catheter to the hydrophilic coating machine fixture, moisten the lint-free paper with anhydrous ethanol, and wipe the catheter three times from the upper end to the lower end with the moistened lint-free paper.

[0071] Step 3: Pour the prepared solution into the corresponding solution tube of the hydrophilic coating machine. Immerse the catheter in the coating solution at a speed of 1 cm / s, let it stand for 5 seconds, and then pull it out at a speed of 2 cm / s. Then place it in a vacuum drying oven and dry at 70℃ for 1 hour to obtain a medical TPU catheter with a peptide-like antibacterial coating. The antibacterial properties and stability of the medical catheter were tested, as described in Example 1, and the test results are shown in Table 1.

[0072] Comparative Example 3

[0073] Step 1: Weigh 2g of tannic acid and 7.8g of anhydrous ethanol and add them to a 50ml centrifuge tube. Add a small magnetic stir bar and stir at room temperature for 10min to completely dissolve the tannic acid. Add 0.1g of hydrochloric acid to adjust the pH to 4.5 to form a precursor solution. Then weigh 0.1g of peptide and add it to the above precursor solution. Stir at room temperature for 3h to obtain an antibacterial solution.

[0074] Step 2: Cut the medical TPU drainage tube into 20 cm lengths, fix its upper end to the hydrophilic coating machine fixture, moisten the lint-free paper with anhydrous ethanol, and wipe the tube three times from the upper end to the lower end with the moistened lint-free paper.

[0075] Step 3: Pour the solution prepared in Step 1 into the corresponding solution tube of the hydrophilic coating machine. Immerse the catheter in the coating solution at a speed of 1 cm / s, let it stand for 5 seconds, and then pull it out at a speed of 2 cm / s. Then place it in a vacuum drying oven and dry it at 70°C for 1 hour to obtain a TPU medical catheter with a peptide-like antibacterial coating.

[0076] Table 1. Antibacterial rate and coating stability of different catheters in the examples and comparative examples.

[0077]

[0078] The results from Example 1 and Comparative Examples 2 and 3 show that catheters containing a tannic acid-peptide coating exhibit good antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). For the coating system, the concentrations of peptides and tannic acid must be maintained at a certain level; excessive tannic acid content or insufficient peptide content will reduce the antibacterial performance of the coating. Medical catheters with a tannic acid-peptide coating exhibit highly efficient antibacterial activity at the optimal peptide ratio.

[0079] The coating peeling stability results of Examples 1-2 and Comparative Examples 1-3 are as follows: Figure 4 As shown, from Figure 4 As can be seen, in Comparative Example 1, when the coating was applied to PTFE medical catheters, the coating had poor adhesion and stability due to the large surface energy of PTFE catheters, and the coating would peel off. However, for TPU and Pebax medical catheters, which have wide applications, the coating can form a stable adhesion and give the catheter good antibacterial properties.

Claims

1. A method for preparing a peptide-containing antibacterial coating, characterized in that, Includes the following steps: Step 1: Prepare an antibacterial solution containing peptides; Step 2: Clean and dry the surface of the medical material; the medical material is one or both of TPU and Pebax. Step 3: Immerse the medical material treated in Step 2 into the antibacterial solution in Step 1 and let it stand. Then, take out the medical material, dry it, and obtain the antibacterial coating on the surface of the medical material. The antibacterial solution containing peptides comprises, by mass percentage (100%), the following raw material components: 0.1-2% peptides, 5-20% tannic acid, 0.5-2% hydrochloric acid, and the remainder being organic solvents. The peptide-like substance has the following structure: 。 2. The method for preparing the peptide-containing antibacterial coating according to claim 1, characterized in that, The medical material is a medical catheter.

3. The method for preparing the peptide-containing antibacterial coating according to claim 1, characterized in that, The organic solvent includes one or more of methanol, ethanol, acetone, and dimethyl sulfoxide.

4. The method for preparing the peptide-containing antibacterial coating according to claim 1, characterized in that, Step 2 cleaning includes one or more of the following methods: ethanol wiping, ultrasonic vibration, and low-temperature plasma cleaning.

5. The method for preparing the peptide-containing antibacterial coating according to claim 1, characterized in that, Step 3: The immersion speed of the TCM materials is 0.5-2 cm / s, and they are soaked in the solution for more than 5 seconds. When taking them out, the lifting speed is 1-3 cm / s. The drying is carried out at 60-80℃ for more than 1 hour.

6. The method for preparing the peptide-containing antibacterial coating according to claim 1, characterized in that, After mixing all the raw materials, the antibacterial solution is prepared and stirred at room temperature for 2-6 hours.

7. The method for preparing the peptide-containing antibacterial coating according to claim 1, characterized in that, The pH of the antibacterial solution is 3-5.

8. The peptide-containing antibacterial coating prepared by the preparation method according to any one of claims 1-7.

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