A peptoid-containing antibacterial hydrogel and preparation method and application thereof

Abstract

The application relates to the field of biological medicine, and discloses an antibacterial hydrogel containing a peptoid as well as a preparation method and application thereof, the preparation method comprising the following steps: step 1, adding a catalyst and dopamine hydrochloride into a hyaluronic acid solution to prepare a hyaluronic acid-dopamine precursor solution through reaction; and step 2, mixing a peptoid solution with the precursor solution, and constant-temperature solidification to obtain the antibacterial hydrogel; the structural formula of the peptoid is as follows. In the application, the hyaluronic acid is modified and grafted by using dopamine, and the benzene rings of dopamine and the benzene rings in the peptoid are used to form a multi-component crosslinking network through the joint action of pi-pi stacking and hydrogen bonding, so that a high-stability hydrogel is prepared. The hyaluronic acid is covered on the hydrogel to have excellent biological functionality, the peptoid is covered on the hydrogel to have excellent antibacterial property, the preparation method is simple, raw materials are easy to obtain, and the hydrogel can be applied to the preparation of medical materials for treating wound infection and chronic wound treatment.

Description

Technical Field

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

[0002] Hydrogels are substances with a three-dimensional network structure formed by the cross-linking of water-soluble polymers through physical or chemical interactions such as intermolecular ionic bonds, covalent bonds, and hydrogen bonds. The hydrophilic and swelling properties of hydrogels give them a more promising application prospect than traditional dressings. By adding bioactive molecules or antibacterial drugs to hydrogels, controlled release, synergistic antibacterial effects, and wound healing promotion can be achieved.

[0003] Traditional antibiotics have drawbacks such as narrow application range, lack of sustained antibacterial activity, and increased bacterial resistance with long-term use. Antimicrobial peptides, on the other hand, typically possess broad-spectrum antibacterial activity and do not induce drug resistance in bacteria.

[0004] CN112316203A discloses a cationic antimicrobial peptide and hyaluronic acid composite hydrogel and its preparation method. The cationic antimicrobial peptide and hyaluronic acid composite hydrogel includes oxidized hyaluronic acid and cationic antimicrobial peptide. The amino acid sequence of the cationic antimicrobial peptide is KK(EF)nKK, where n is an integer from 3 to 8. In the amino acid sequence, the carboxyl group of the glutamic acid side chain is modified into an acylhydrazine. The cationic antimicrobial peptide and hyaluronic acid composite hydrogel provided by this invention exhibits broad-spectrum and excellent antibacterial properties and can be used in the preparation of various wound dressings, showing good application prospects. In addition, since the cationic antimicrobial peptide is prepared by solid-phase peptide synthesis, the synthesis method is relatively simple, and the process of preparing the cationic antimicrobial peptide and hyaluronic acid composite hydrogel by combining it with oxidized hyaluronic acid is also relatively simple, which facilitates the promotion and application of the cationic antimicrobial peptide and hyaluronic acid composite hydrogel in wound dressings.

[0005] CN113214507 A discloses a method for preparing an antibacterial glycopeptide hydrogel, the specific steps of which are as follows: Glucomannan is reacted with sodium periodate, and ethylene glycol is added to terminate the reaction to obtain oxidized glucomannan, which is then dialyzed and lyophilized. This oxidized glucomannan is then mixed with antibacterial peptides to obtain antibacterial peptide glucomannan. Hyaluronic acid is reacted with 3-maleimide propionic acid at room temperature to obtain esterified hyaluronic acid, which is then dialyzed, lyophilized, and then mixed with collagen peptides. An addition reaction is then performed, followed by dialyzed and lyophilized to obtain collagen peptide hyaluronic acid. Finally, the collagen peptide hyaluronic acid is mixed with the antibacterial peptide glucomannan to prepare an antibacterial glycopeptide hydrogel. The hydrogel obtained by this invention exhibits good antibacterial properties against Gram-positive bacteria, Gram-negative bacteria, and fungi. The preparation process of the hydrogel is simple, it is degradable, and has good biocompatibility.

[0006] However, the preparation methods of bioactive peptides are complex, the antimicrobial peptide sequences are too long, the preparation cost is high, and they are prone to inactivation. Hydrogels prepared from antimicrobial peptides still suffer from high cost and insufficient antimicrobial properties. At the same time, there are few reports on hydrogels of antimicrobial peptides. Summary of the Invention

[0007] This invention addresses the problem of insufficient antibacterial properties in current antibacterial hydrogels and the tendency for long-term use of antibiotic-containing antibacterial agents to lead to drug-resistant bacteria. This invention provides a method for preparing a peptide-based antibacterial hydrogel that does not require the addition of chemical cross-linking agents. The preparation method is very simple, the raw materials are readily available, and the resulting hydrogel has excellent antibacterial properties and stable mechanical properties that are not easily broken. It can be used in wound infection, chronic wound repair, and other fields.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing a peptide-containing antibacterial hydrogel includes the following steps:

[0010] Step 1: Add a catalyst and dopamine hydrochloride to the hyaluronic acid solution to prepare a hyaluronic acid-dopamine precursor solution.

[0011] Step 2: Mix the peptide-like solution with the precursor solution and solidify at a constant temperature to obtain the antibacterial hydrogel; the structural formula of the peptide-like substance is as follows:

[0012]

[0013] This invention employs dopamine-modified grafted hyaluronic acid, utilizing the π-π stacking and hydrogen bonding between the benzene rings of dopamine and the benzene rings of peptides to form a multi-component cross-linked network. This enhances the stability of the hydrogel and imparts excellent antibacterial properties. The preparation method is very simple, with mild reaction conditions, uncomplicated steps, and readily available raw materials. The positively charged peptides can adsorb and aggregate on the cell membrane surface of negatively charged bacteria, killing them by disrupting the bacterial cell membrane. This provides the hydrogel with broad-spectrum antibacterial properties without inducing drug-resistant bacteria. Hyaluronic acid possesses excellent biocompatibility and human absorbability, playing a crucial role in tissue repair and inflammatory responses. The combination of these two components results in a hydrogel with good biological function, strong antibacterial properties, and low susceptibility to drug-resistant bacteria. It can be widely applied in wound infection and chronic wound treatment.

[0014] The specific method for preparing the peptide is as follows:

[0015] The preparation method of the dipeptide-like peptide-like peptide includes:

[0016] In step 1, 4-(4-methyl-3-pentenyl)-3-cyclohexene-1-carboxaldehyde and 1-(3-aminopropyl)pyrrolidine are mixed and reacted in a solvent, followed by the addition of 4-isopropylphenylacetic acid and benzyl isonitrile to continue the reaction and obtain the crude peptide compound; the reaction formula is as follows. Figure 1 As shown.

[0017] In step 2, the crude product of the peptide compound is removed by rotary evaporation to remove the solvent, purified by column chromatography, and then lyophilized to obtain the peptide.

[0018] 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.

[0019] In step 1, the reaction is carried out at room temperature. 4-(4-methyl-3-pentenyl)-3-cyclohexene-1-carboxaldehyde and 1-(3-aminopropyl)pyrrolidine react for 5-20 min. After adding 4-isopropylphenylacetic acid and benzyl isonitrile, the reaction continues for 12-36 h. The molar ratio of 4-(4-methyl-3-pentenyl)-3-cyclohexene-1-carboxaldehyde, 1-(3-aminopropyl), 4-biphenylacetic acid and isonitrile n-butane is 1:1:1:1. The solvent in step 1 is methanol.

[0020] 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.

[0021] In process 2, the eluent used for column chromatography was a mixed solvent of dichloromethane, methanol, and formic acid;

[0022] The eluent contains dichloromethane, methanol, and formic acid in a volume ratio of 96:3:1.

[0023] Before rinsing, the dipeptide-like peptide compound was dissolved in dichloromethane and subjected to column chromatography.

[0024] More preferably, process 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 and lyophilized to obtain the peptide.

[0025] In step 1, the pH of the reaction solution is 4-5; the reaction time is 1-12 hours. Under acidic conditions, the carboxyl group on hyaluronic acid undergoes an amidation reaction with the amino group of dopamine, thereby grafting dopamine onto the hyaluronic acid.

[0026] After the reaction in step 1 is completed, the reaction solution is purified by dialysis. The molecular weight cutoff of the dialysis bag is 3500-12000 Da. Dialysis is performed for 1-3 days to remove excess reaction catalyst and obtain purified modified hyaluronic acid.

[0027] In step 1, the mass ratio of hyaluronic acid to dopamine hydrochloride is 1:0.1-1. EDC and NHS are used as catalysts to synergistically catalyze the amidation reaction of hyaluronic acid and dopamine. When the dopamine concentration in the reaction system is low, the intermediate product formed by hyaluronic acid and NHS is easily hydrolyzed, releasing NHS back into the solution, thus hindering the reaction. To ensure stable grafting of dopamine onto hyaluronic acid, the two must maintain an appropriate ratio.

[0028] In step 1, the mass concentration of hyaluronic acid in the reaction solution is 1-10 mg / ml, and the mass concentration of dopamine hydrochloride is 1-10 mg / ml. The mass of dopamine hydrochloride in the reaction system will affect its grafting effect on hyaluronic acid, and thus affect the stable cross-linking sites that can be formed between peptides and dopamine when gel is formed.

[0029] In step 1, the catalyst is EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and / or NHS (N-hydroxysuccinimide); the catalyst mass is 10% to 50% of hyaluronic acid-dopamine.

[0030] In step 1, water is used as the solvent, and in step 2, a mixed solution of ethanol and water is used as the solvent.

[0031] The mass ratio of hyaluronic acid to peptides is 10:1 to 5.

[0032] In step 2, the mass concentration of peptides in the reaction solution is 1-5 mg / ml, and the mass concentration of ethanol is 0.7 g / ml.

[0033] The curing temperature in step 2 is 20-50℃, and the curing time is within 3 hours. The product is then rinsed three times with deionized water to obtain the hydrogel. The reaction conditions in this invention are mild; curing at near room temperature for only 1-3 hours is sufficient to achieve cross-linking of the peptides and the hydrogel, resulting in high reaction efficiency.

[0034] This invention provides an antibacterial hydrogel containing peptides prepared according to the described method. This hydrogel exhibits excellent antibacterial properties, high compressive strength, and good mechanical strength and tensile strength.

[0035] The present invention also provides the application of the aforementioned peptide-containing antibacterial hydrogel in the preparation of medical materials for the treatment of wound infection and chronic wound.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) This invention uses antibacterial molecular peptides containing benzene rings, dopamine and hyaluronic acid as raw materials to prepare antibacterial hydrogels with cross-linked networks. It has broad-spectrum bactericidal properties and good mechanical stability, high compressive strength, and simple preparation method with low cost. It can be applied to the preparation of antibacterial medical materials for complex wounds and slow-healing wounds to reduce the pain of infection for patients. Attached Figure Description

[0038] Figure 1 This is the synthesis reaction formula for peptides in this invention.

[0039] Figure 2 The image shows the 1H NMR spectrum of the peptide prepared in the examples.

[0040] Figure 3 The mass spectrometry results of the peptides prepared in the examples are shown below.

[0041] Figure 4 The compressive strength diagrams are for the hydrogels prepared in Example 1 and Comparative Examples 1-3. Detailed Implementation

[0042] 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 merely illustrative and 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 invention, should be covered within the protection scope of this invention.

[0043] The raw materials used in the following specific embodiments are all purchased from the market, and the preparation method of the dipeptide-like peptide-like peptide includes:

[0044] In step 1, 4-(4-methyl-3-pentenyl)-3-cyclohexene-1-carboxaldehyde and 1-(3-aminopropyl)pyrrolidine are mixed and reacted in a solvent, followed by the addition of 4-isopropylphenylacetic acid and benzyl isonitrile to continue the reaction and obtain the crude peptide compound; the reaction formula is as follows. Figure 1 As shown.

[0045] In step 2, the crude product of the peptide compound is removed by rotary evaporation to remove the solvent, purified by column chromatography, and then lyophilized to obtain the peptide.

[0046] 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.

[0047] In step 1, the reaction is carried out at room temperature. 4-(4-methyl-3-pentenyl)-3-cyclohexene-1-carboxaldehyde and 1-(3-aminopropyl)pyrrolidine react for 5-20 min. After adding 4-isopropylphenylacetic acid and benzyl isonitrile, the reaction continues for 12-36 h. The molar ratio of 4-(4-methyl-3-pentenyl)-3-cyclohexene-1-carboxaldehyde, 1-(3-aminopropyl), 4-biphenylacetic acid and isonitrile n-butane is 1:1:1:1. The solvent in step 1 is methanol.

[0048] 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.

[0049] In process 2, the eluent used for column chromatography was a mixed solvent of dichloromethane, methanol, and formic acid;

[0050] The eluent contains dichloromethane, methanol, and formic acid in a volume ratio of 96:3:1.

[0051] Before rinsing, the dipeptide-like peptide compound was dissolved in dichloromethane and subjected to column chromatography.

[0052] Further preferably, step 2 specifically includes the following steps: After removing the solvent from the crude peptide product by rotary evaporation, dissolve it in dichloromethane, and perform gradient purification elution with an eluent to obtain a purified product. The purified product is then removed by vacuum extraction and lyophilized to obtain the peptide. The NMR spectrum of the peptide is shown below. Figure 2 As shown, the mass spectrometry is as follows Figure 3 As shown.

[0053] Example 1

[0054] Step 1: Dissolve 250 mg of hyaluronic acid in 50 ml of deionized water, add 200 mg of EDC and 110 mg of NHS sequentially, stir evenly for 20 min, add 100 mg of dopamine hydrochloride to the solution, keep the pH of the solution at 4-5, stir the reaction solution at room temperature for 10 h under nitrogen protection, and purify the solution by dialyzing (molecular weight cutoff 12000 Da) for 2 days to obtain the hyaluronic acid-dopamine precursor solution for gelation.

[0055] Step 2: Add 25 mg of the dipeptide to 10 ml of 0.7 g / ml ethanol aqueous solution and stir at room temperature for 30 min until dissolved.

[0056] Step 3: Mix the dipeptide-like solution and the hyaluronic acid-dopamine solution thoroughly, pour the mixture into a 24-well plate, and cure at 40°C for 1 hour. Then rinse twice with deionized water to obtain an antibacterial hydrogel containing the dipeptide-like substance. Comparative Example 1:

[0057] Step 1: Dissolve 250 mg of hyaluronic acid in 50 ml of deionized water, add 200 mg of EDC and 110 mg of NHS to the solution, stir evenly for 20 min, then add 100 mg of dopamine hydrochloride to the solution, keep the pH of the solution at 4-5, stir the reaction solution at room temperature for 10 h under nitrogen protection, and purify the solution by dialyzing (molecular weight cutoff 12000 Da) for 2 days to obtain the hyaluronic acid-dopamine precursor solution for gelation.

[0058] Step 2: Add 25 mg of the dipeptide to 10 ml of 0.7 g / ml ethanol aqueous solution and stir at room temperature for 30 min until dissolved.

[0059] Step 3: Mix the dipeptide-like solution with the hyaluronic acid-dopamine solution evenly, pour the mixture into a 24-well plate, and cure it at 40°C for 10 minutes. Then rinse it twice with deionized water to obtain an antibacterial hydrogel containing dipeptides.

[0060] Comparative Example 2:

[0061] Step 1: Dissolve 250 mg of hyaluronic acid in 50 ml of deionized water, add 200 mg of EDC and 110 mg of NHS to the solution, stir evenly for 20 min, then add 50 mg of dopamine hydrochloride to the solution, keep the pH of the solution at 4-5, stir the reaction solution at room temperature for 10 h under nitrogen protection, and purify the solution by dialyzing (molecular weight cutoff 12000 Da) for 2 days to obtain the hyaluronic acid-dopamine precursor solution for gelation.

[0062] Step 2: Add 25 mg of the dipeptide to 10 ml of 0.7 g / ml ethanol aqueous solution and stir at room temperature for 30 min until dissolved.

[0063] Step 3: Mix the dipeptide-like solution with the hyaluronic acid-dopamine solution evenly, pour the mixture into a 24-well plate, and cure it at 40°C for 1 hour. Then rinse it twice with deionized water to obtain an antibacterial hydrogel containing dipeptides.

[0064] Example 2:

[0065] (1) Dissolve 250 mg of hyaluronic acid in 50 ml of deionized water, add 200 mg of EDC and 110 mg of NHS to it in sequence, stir evenly for 20 min, add 100 mg of dopamine hydrochloride to the solution, keep the solution pH at 4-5, stir the reaction solution at room temperature for 10 h under nitrogen protection, and dialyze the solution to purify it (molecular weight cutoff 12000 Da) for 2 days to obtain the hyaluronic acid-dopamine precursor solution for gelation.

[0066] (2) Add 100mg of dipeptide to 10ml of 0.7g / ml ethanol aqueous solution and stir at room temperature for 30min until dissolved.

[0067] (3) Mix the dipeptide-like solution with the hyaluronic acid-dopamine solution until homogeneous, pour into a 24-well plate, and cure at 40℃ for 1 hour. Rinse twice with deionized water to obtain an antibacterial hydrogel containing dipeptides. Compressive strength test:

[0068] The hydrogels formed in the examples and comparative examples were prepared into test samples with a diameter of 8 mm and a height of 15 mm, respectively. These samples were placed on a universal testing machine (UTM, M350, Testometric Ltd.) and tested at a compression speed of 1 mm / min. The compressive strength of the gel was characterized by the average compressive load measured at the time of gel rupture. The results are as follows: Figure 4 As shown.

[0069] Depend on Figure 4 It can be seen that in Comparative Example 1, the cross-linking time was relatively short, and the gel was not fully cross-linked to form a tight network, resulting in a decrease in its strength. In Comparative Example 2, the dopamine content was reduced, and the number of cross-linking sites that could be formed in the cross-linking system decreased, leading to a decrease in gel strength. In Example 2, since the hyaluronic acid-dopamine content and cross-linking time were the same, the gel network was already fully cross-linked, and the increase in dipeptide content had little effect on the strength.

[0070] Antibacterial performance test:

[0071] The in vitro antibacterial assay for hydrogels against Staphylococcus aureus and Escherichia coli involved adhering the hydrogels formed in Examples 1-2 and Comparative Examples 1-2 onto sterilized LB agar medium, adding diluted bacterial suspension to the medium, and incubating overnight at 37°C. The antibacterial test results are shown in Table 1.

[0072] Table 1. Results of antibacterial performance test

[0073] Staphylococcus aureus inhibition rate 97.4％ 72％ 63％ 98.3％ Escherichia coli inhibition rate 98.1％ 73.2％ 60.4％ 99.3％

[0074] As can be seen from Table 1, the dipeptide-like hydrogels prepared in Examples 1 and 2 have good antibacterial effects. In Comparative Example 1, due to the short cross-linking time, some dipeptides failed to completely cross-link with the modified hyaluronic acid, resulting in poor antibacterial properties and strength of the hydrogel.

[0075] In Comparative Example 2, due to the low amount of dopamine, the hyaluronic acid catalyzed by EDC / NHS will undergo a certain degree of hydrolysis, resulting in a lower content of grafted dopamine. Ultimately, this reduces the number of sites for cross-linking with peptides, leading to a lower content of peptides in the gel, poorer cross-linking degree of the formed hydrogel, and poorer antibacterial properties.

Claims

1. A method for preparing a peptide-containing antibacterial hydrogel, characterized in that, Including the following steps: Step 1: Add a catalyst and dopamine hydrochloride to a hyaluronic acid solution to prepare a hyaluronic acid-dopamine precursor solution; the mass ratio of hyaluronic acid to dopamine hydrochloride in Step 1 is 1:0.1~1; the mass concentration of hyaluronic acid in the reaction solution of Step 1 is 1-10 mg / ml, and the mass concentration of dopamine hydrochloride is 1-10 mg / ml. Step 2: Mix the peptide solution with the precursor solution, cure at a constant temperature for 1-3 hours, and then wash to obtain the antibacterial hydrogel; The structural formula of the peptide is as follows: 。 2. The method for preparing the peptide-containing antibacterial hydrogel according to claim 1, characterized in that, In step 1, the pH of the reaction solution is 4-5; the reaction time is 1-12 hours.

3. The method for preparing the peptide-containing antibacterial hydrogel according to claim 1, characterized in that, After the reaction in step 1 is completed, the reaction solution is purified by dialysis. The molecular weight cutoff of the dialysis bag is 3500-12000 Da, and the dialysis is performed for 1-3 days.

4. The method for preparing the peptide-containing antibacterial hydrogel according to claim 1, characterized in that, In step 1, the catalyst is EDC and / or NHS; the catalyst mass is 10% to 50% of hyaluronic acid-dopamine.

5. The method for preparing the peptide-containing antibacterial hydrogel according to claim 1, characterized in that, In step 1, water is used as the solvent, and in step 2, a mixed solution of ethanol and water is used as the solvent.

6. The method for preparing the peptide-containing antibacterial hydrogel according to claim 1, characterized in that, The mass ratio of hyaluronic acid to peptides is 10:1~5; The mass concentration of peptides in the reaction solution in step 2 is 1-5 mg / ml.

7. The method for preparing the peptide-containing antibacterial hydrogel according to claim 1, characterized in that, The curing temperature in step 2 is 20-50℃, and the cleaning method is to rinse with deionized water 1-5 times.

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

9. The application of the peptide-containing antibacterial hydrogel according to claim 8 in the preparation of medical materials for the treatment of wound infection or chronic wound.

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