Preparation method of temperature-sensitive antibacterial mimic peptide hydrogel

Abstract

The invention discloses a preparation method of temperature-sensitive antibacterial mimic peptide hydrogel. The preparation method comprises the following steps: S1, preparing oxazoline mimic peptide NPX under the protection of t-butyloxycarboryl; s2, preparation of a t-butyloxycarboryl mimic peptide polymer; s3, removing t-butyloxycarboryl from the polymer; and S4, preparing the mimic peptide hydrogel. A carbonyl translocation strategy is combined, an amino bioactive group is introduced to an oxazoline skeleton to improve biocompatibility, the length of a mimic peptide is regulated and controlled by changing the number and proportion of monomers, so that the antibacterial and cytotoxicity of the hydrogel is regulated and controlled macroscopically, and meanwhile, N-isopropylacrylamide is introduced, so that the hydrogel has thermosensitivity. The biocompatibility is good, and the influence of bacteria on wounds can be inhibited permanently. The defects that biological peptide protein used clinically is low in hydrolytic stability, time-consuming in synthesis, difficult to prepare on a large scale and the like are overcome. In addition, the mimic peptide is used as an antibacterial agent, so that the continuous antibacterial effectiveness is ensured, the drug resistance of bacteria is reduced, and the mimic peptide is suitable for the field of biomedicine.

Description

Technical Field

[0001] The invention belongs to the field of biopolymer materials and relates to a method for preparing a temperature-sensitive antibacterial mimetic peptide hydrogel. Background Art

[0002] Wound infection is one of the major diseases affecting human health and is currently treated mainly with antibiotics. Since antibiotics were introduced more than eighty years ago, they have had a huge impact on our ability to treat bacterial diseases worldwide. However, in the past few decades, only three new classes of antibiotics (lipopeptides, oxazolidinones, and streptococcins) have entered the medical field. The lack of new antibiotics, coupled with the emerging problem of multidrug resistance, requires the exploration of new antimicrobial strategies to treat these infections. Biopeptides play a key role in regulating most biological processes in living organisms and are of considerable biological, medical, and industrial importance. Antimicrobial peptides are an important part of the natural immune defense mechanism of most organisms against pathogens. Antimicrobial peptides are reported to have excellent broad-spectrum antibacterial activity and low biological toxicity. Therefore, the development of antimicrobial peptides provides an effective direction for solving the problem of traditional antibiotic resistance. However, clinical studies have found that as candidate drugs, antimicrobial peptides have various additional disadvantages, including reduced activity in the presence of salts and divalent cations, sensitivity to pH changes, and sensitivity to proteases and other plasma components. Antimicrobial peptides may also have poor pharmacokinetics and high production costs. Therefore, although peptides composed only of D-amino acids are sometimes considered due to their high resistance to proteolysis, this option often significantly increases product cost, and there is currently no published pharmacokinetic evidence that D-peptides have improved bioavailability. Therefore, the use of peptidomimetics to overcome the inherent limitations of peptides is emerging as an important and promising approach to improve the therapeutic potential of antimicrobial peptides.

[0003] Hydrogels are polymer materials with characteristic hydrophilic structures that can store large amounts of water and biofluids in their 3D networks. Hydrogels have a variety of biomimetic properties, such as remarkable flexibility, softness, excellent absorption capacity in a swollen state, non-toxicity, biocompatibility, biodegradability, and tunable mechanical properties. With these properties, hydrogels have irreplaceable advantages in certain specific biomedical applications compared to other nanomaterials (e.g., nanoparticles, nanofibers, films, etc.).

[0004] Traditional thermosensitive antibacterial hydrogels usually use antimicrobial agents such as antibiotic silver nanoparticles, antimicrobial peptides, and metal ions, which are incorporated into hydrogels by physical or chemical methods. The chemical synthesis process is inefficient and has poor stability. In addition, when physically combined with dressings, antimicrobial agents often show an initial burst release, which in turn causes cytotoxicity to normal cells. N-isopropylacrylamide is used to give hydrogels thermosensitivity. The advantages of N-isopropylacrylamide hydrogels are mainly reflected in: 1. Thermosensitivity: N-isopropylacrylamide hydrogels are hydrophilic at room temperature, but they undergo a reversible phase change from hydrophilic to hydrophobic within the body temperature range (approximately 32°C to 37°C). This property allows N-isopropylacrylamide-based porous hydrogels to regulate their water absorption according to the ambient temperature, providing a temperature-responsive control method for the delivery of bioactive molecules. 2. Porous structure: N-isopropylacrylamide-based porous hydrogels have a large specific surface area and pore volume. This porous structure is conducive to cell growth and colonization. This structural characteristic makes the hydrogel have a wide range of application potential in drug delivery, tissue engineering and other fields. 3. Biocompatibility: Poly (N-isopropylacrylamide) is a synthetic polymer. Its biocompatibility can be enhanced and its irritation to biological tissues can be reduced through appropriate surface modification. This biocompatibility makes N-isopropylacrylamide-based hydrogels safer and more reliable in biomedical applications. However, N-isopropylacrylamide hydrogels alone have no antibacterial properties, which limits their application in the healing process of bacterially infected wounds. Summary of the invention

[0005] Purpose of the invention: In view of the problems pointed out in the background technology, the present invention provides a method for preparing a thermosensitive antibacterial mimetic peptide hydrogel, which adopts chemical cross-linking to improve the stability of the hydrogel, and uses mimetic peptides as antibacterial agents, combines the carbonyl translocation strategy to prepare mimetic peptides and N-isopropylacrylamide cross-linking to prepare hydrogels, and uses hydrogels and oxazoline as substrates to make the biomaterial have good biocompatibility.

[0006] Technical solution: The present invention discloses a method for preparing a thermosensitive antibacterial mimetic peptide hydrogel, comprising the following steps: S1. Preparation of oxazoline-mimetic peptide NPX protected by tert-butyloxycarbonyl: ① Tert-butyloxycarbonylglycine and chloroethylamine are reactants, 1H-hydroxybenzotriazole is a polypeptide protective agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is a condensation agent, and triethylamine is a polymerization inhibitor. The mixture is dissolved in an organic solvent A and reacted at room temperature. The product is dried and purified to obtain an intermediate product. ② The intermediate product is reacted with sodium hydroxide under a nitrogen flow, and the product is purified to obtain NPX; S2. Preparation of tert-butyloxycarbonyl peptide analog polymer: tert-butyloxycarbonyl protected oxazoline peptide analog NPX, chloromethylstyrene, and sodium iodide are used as reactants, dissolved in an organic solvent B under a nitrogen stream, heated for reaction, and then a polymerization terminator is added. The obtained product is filtered, vacuum dried, and purified to obtain a copolymer; S3. Preparation of a peptide-mimicking polymer: The polymer in S2 and trifluoroacetic acid are reactants, the reaction mixture is shaken at room temperature, the solvent is removed under a nitrogen stream, and the residue is purified to obtain a peptide-mimicking polymer; S4. Preparation of peptide-mimicking hydrogel: N,N′-methylenebisacrylamide, peptide-mimicking polymer and N-isopropylacrylamide are reactants, ammonium peroxodisulfate is an initiator, and N,N,N′,N′tetramethylethylenediamine is a catalyst. The reaction is carried out under a nitrogen flow, and the resulting hydrogel is extracted, dried in a vacuum and stored.

[0007] Furthermore, in S1, the molar ratio of tert-butyloxycarbonylglycine and ethylchloroamine is 1:1-2; and / or the molar ratio of the intermediate product and sodium hydroxide is 1:1-2.

[0008] Furthermore, in S1, before carrying out the reaction at room temperature, tert-butyloxycarbonylglycine, 1H-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and triethylamine are stirred in a mixture of ice and water at 0°C for more than 20 minutes, and then the temperature is raised to room temperature and chloroethylamine is added and dissolved in organic solvent A and the stirring reaction is continued for more than 6 hours.

[0009] Furthermore, in S2, the molar ratio of the oxazoline mimetic peptide NPX protected by the tert-butyloxycarbonyl group, chloromethylstyrene, and sodium iodide is 20 to 80:1:2; And / or adding a polymerization terminator after the heating reaction in S2, the heating temperature is 70-90° C., and the time is more than 8 hours.

[0010] Furthermore, in S2, the polymerization terminator is a methanol solution of potassium hydroxide.

[0011] Furthermore, in S3, the reaction mixture is oscillated at room temperature for more than 2 hours.

[0012] Furthermore, the molar ratio of the N,N′-methylenebisacrylamide, the peptidomimetic polymer, N-isopropylacrylamide, ammonium peroxodisulfate and N,N,N′,N′tetramethylethylenediamine is 0.3:0.1 to 10:0.1 to 10:0.1:0.2.

[0013] Furthermore, in S4, the vacuum drying temperature is 20-50°C.

[0014] Furthermore, the organic solvent A and the organic solvent B are any one of methanol, dichloromethane and acetonitrile.

[0015] Furthermore, the nitrogen circulation condition is to first evacuate the air and then fill it with nitrogen, and repeat this process for 5 to 10 times, each time for 3 to 5 minutes, and then maintain the nitrogen circulation environment.

[0016] Beneficial Effects

[0017] The invention overcomes the shortcomings of commonly used thermosensitive antibacterial hydrogels carrying antibacterial agents and antibacterial peptides, uses glycine-dependent polyoxazoline, a glycine pseudopeptide, as a synthetic mimetic of biological peptides to improve the biocompatibility and stability of the mimetic peptides, and simultaneously chemically cross-links with N-isopropylacrylamide to improve the mechanical properties of the hydrogel, adjusts the phase transition temperature of the hydrogel to a temperature suitable for the human body, improves the biocompatibility of the hydrogel and promotes wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the antibacterial effect of the comparative examples and embodiments of the present invention after the antibacterial test on Staphylococcus aureus; Figure 2 The antibacterial effect diagram of the comparative example and the embodiment of the present invention after being immersed in PBS solution for different time periods; Figure 3 The hydrogel size of Comparative Example 1 of the present invention at 22°C and 37°C; Figure 4 The hydrogel size of Comparative Example 2 of the present invention at 22°C and 37°C; Figure 5 Shrinkage rate diagram of comparative example and experimental example of the present invention. DETAILED DESCRIPTION

[0019] To further illustrate the present invention, the following is a detailed description of a method for preparing a thermosensitive antibacterial mimetic peptide hydrogel provided by the present invention in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0020] In addition, it should be understood that after reading the contents of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0021] The present invention discloses a method for preparing a thermosensitive antibacterial mimetic peptide hydrogel, comprising the following steps: (1) Preparation of tert-butyloxycarbonyl protected oxazoline peptide mimetic (NPX): ① Tert-butyloxycarbonylglycine and chloroethylamine are reactants, 1H-hydroxybenzotriazole is a polypeptide protective agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is a condensation agent, and triethylamine is a polymerization inhibitor. They are dissolved in organic solvent A and reacted at room temperature. The product is washed and purified to obtain an intermediate product. Before reacting at room temperature, tert-butyloxycarbonylglycine, 1H-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and triethylamine are stirred in a 0°C ice-water mixture for more than 20 minutes, and then the temperature is raised to room temperature, and chloroethylamine is added and dissolved in organic solvent A and the reaction is continued by stirring for more than 6 hours. The molar ratio of tert-butyloxycarbonylglycine and chloroethylamine is 1:1-2, and the molar ratio of the intermediate product and sodium hydroxide is 1:1-2.

[0022] ② The intermediate product and sodium hydroxide are reacted under nitrogen flow, and the product is purified to obtain NPX. In this embodiment, the nitrogen flow condition is to first evacuate and then fill with nitrogen, and repeat this process 5 to 10 times, each time for 3 to 5 minutes, and then maintain the nitrogen flow environment.

[0023] (2). Preparation of tert-butyloxycarbonyl peptide analog polymer: Oxazoline peptide analog protected by tert-butyloxycarbonyl, chloromethylstyrene, and sodium iodide are used as reactants and dissolved in organic solvent B under nitrogen flow. After heating reaction, a polymerization terminator is added. The heating temperature is 70-90°C and the time is more than 8 hours. The polymerization terminator is a methanol solution of potassium hydroxide. The obtained product is filtered and vacuum dried and purified to obtain a copolymer. The molar ratio of tert-butyloxycarbonyl protected oxazoline peptide analog NPX, chloromethylstyrene, and sodium iodide is 20-80:1:2.

[0024] (3) Preparation of peptide-mimicking polymers: The polymer and trifluoroacetic acid are reactants, and the reaction mixture is shaken at room temperature for more than 2 hours. After removing the solvent under a nitrogen flow, the residue is purified to obtain a peptide-mimicking polymer. The nitrogen flow condition is to first evacuate the air and then fill it with nitrogen, and repeat this process 5 to 10 times, each time for 3 to 5 minutes, and then maintain the nitrogen flow environment.

[0025] (4). Preparation of peptidomimetic hydrogel: N, N′-methylenebisacrylamide, peptidomimetic polymer and N-isopropylacrylamide are reactants, ammonium peroxodisulfate is initiator, N, N, N′, N′tetramethylethylenediamine is catalyst, and the reaction is carried out under nitrogen flow. The obtained hydrogel is extracted and stored after vacuum drying. The molar ratio of N, N′-methylenebisacrylamide, peptidomimetic polymer, N-isopropylacrylamide, ammonium peroxodisulfate and N, N, N′, N′tetramethylethylenediamine is 0.3:0-10:0-10:0.1:0.2. The vacuum drying temperature is 20-50°C.

[0026] The nitrogen circulation condition is to first evacuate the air and then fill it with nitrogen, and repeat this process for 5 to 10 times, each time for 3 to 5 minutes, and then maintain the nitrogen circulation environment.

[0027] In the above steps, the organic solvent A and the organic solvent B are any one of methanol, dichloromethane and acetonitrile. Comparative Example 1

[0028] Using tert-butyloxycarbonylglycine and chloroethylamine as reactants, an intermediate is prepared, and then NPX is prepared under alkaline conditions. Using NPX, chloromethylstyrene, and sodium iodide as reactants, a peptidomimetic polymer is prepared. After removing the protecting group of the polymer, N,N′-methylenebisacrylamide, a peptidomimetic polymer, N-isopropylacrylamide, ammonium peroxodisulfate, and N,N,N′,N′tetramethylethylenediamine (molar ratio of 0.3:0:10:0.1:0.2) are used as reactants for cross-linking to form Comparative Example 1.

[0029] In Comparative Example 1, the value of the peptidomimetic polymer is 0, which means that the peptidomimetic polymer is not used in the synthesis of the hydrogel. Comparative Example 2

[0030] An intermediate was prepared using tert-butyloxycarbonylglycine and chloroethylamine as reactants, and then NPX was prepared under alkaline conditions. NPX, chloromethylstyrene, and sodium iodide were used as reactants to prepare a peptidomimetic polymer. After removing the protecting groups of the polymer, N,N′-methylenebisacrylamide, a peptidomimetic polymer, N-isopropylacrylamide, ammonium peroxodisulfate, and N,N,N′,N′tetramethylethylenediamine (molar ratio of 0.3:10:0:0.1:0.2) were used as reactants for cross-linking to form comparative example 2.

[0031] In Comparative Example 2, the value of N-isopropylacrylamide is 0, that is, N-isopropylacrylamide is not used when synthesizing the hydrogel. Experimental Example 1

[0032] The above-mentioned thermosensitive antibacterial peptide mimetic hydrogel was prepared using the preparation method, specifically using N,N′-methylenebisacrylamide, peptide mimetic polymer, N-isopropylacrylamide, ammonium peroxydisulfate, and N,N,N′,N′tetramethylethylenediamine (molar ratio of 0.3:1:9:0.1:0.2) as reactants for cross-linking to form experimental example 1.

[0033] During the experiment: The molar ratio of tert-butyloxycarbonylglycine and ethyl chloride is 1:1.3.

[0034] The molar ratio of the intermediate product and the sodium hydroxide input is 1:1.

[0035] The molar ratio of the tert-butyloxycarbonyl-protected oxazoline peptide analog NPX, chloromethylstyrene, and sodium iodide is 50:1:2.

[0036] The organic solvent A and the organic solvent B are selected as: dichloromethane and acetonitrile respectively.

[0037] After the heating reaction, a polymerization terminator is added, wherein the heating temperature is: 80°C. Experimental Example 2

[0038] The above-mentioned thermosensitive antibacterial peptide mimetic hydrogel was prepared by the preparation method, specifically using N,N′-methylenebisacrylamide, peptide mimetic polymer, N-isopropylacrylamide, ammonium peroxydisulfate, and N,N,N′,N′tetramethylethylenediamine (molar ratio of 0.3:5:5:0.1:0.2) as reactants for cross-linking to form Example 2.

[0039] The other experimental parameters are the same as those in Experimental Example 1 and will not be described in detail here. Experimental Example 3

[0040] The above-mentioned thermosensitive antibacterial peptide mimetic hydrogel was prepared using the preparation method, specifically using N,N′-methylenebisacrylamide, peptide mimetic polymer, N-isopropylacrylamide, ammonium peroxydisulfate, and N,N,N′,N′tetramethylethylenediamine as reactants (molar ratio of 0.3:9:1:0.1:0.2) to cross-link to form Experimental Example 3.

[0041] The other experimental parameters are the same as those in Experimental Example 1 and will not be described in detail here.

[0042] The present application conducts the following experiments on the above two comparative examples and three experimental examples. Figure 1 The antibacterial effect diagram of the comparative example and the embodiment after the antibacterial test of Staphylococcus aureus is shown. It can be seen from the figure that the thermosensitive antibacterial mimetic peptide hydrogel has a good antibacterial rate. Since no mimetic peptide polymer is added in comparative example 1, it does not have an antibacterial rate.

[0043] See also Figure 2 , the antibacterial effect diagram of the comparative example and the experimental example after soaking in PBS solution for different time periods, from which it can be seen that the present invention has good sustained antibacterial property.

[0044] See also Figure 3 , Figure 3 The hydrogel sizes of Comparative Example 1 at room temperature 22°C and 37°C are different at different temperatures, indicating that the hydrogel has a certain temperature sensitivity in the presence of N-isopropylacrylamide.

[0045] See also Figure 4The hydrogel sizes of Comparative Example 2 at room temperature 22°C and 37°C are the same at different temperatures, indicating that the synthesized hydrogel is not temperature-sensitive when N-isopropylacrylamide is not added.

[0046] See also Figure 5 , the shrinkage rate diagram of the comparative example and the experimental example, the size of the hydrogel is detected at room temperature 22°C and 37°C for both the comparative example and the experimental example, and the shrinkage rate diagram is obtained by calculating the volume change of the hydrogel. It can be seen from the diagram that the shrinkage volume of the present invention increases with the increase of N-isopropylacrylamide.

[0047] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a thermosensitive antibacterial mimetic peptide hydrogel, characterized in that: The following steps are involved: S1. Preparation of oxazoline-mimetic peptide NPX protected by tert-butyloxycarbonyl: ① Tert-butyloxycarbonylglycine and chloroethylamine are reactants, 1H-hydroxybenzotriazole is a polypeptide protective agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is a condensation agent, and triethylamine is a polymerization inhibitor. The mixture is dissolved in an organic solvent A and reacted at room temperature. The product is dried and purified to obtain an intermediate product. ② The intermediate product is reacted with sodium hydroxide under a nitrogen flow, and the product is purified to obtain NPX; S2. Preparation of tert-butyloxycarbonyl peptide analog polymer: tert-butyloxycarbonyl protected oxazoline peptide analog NPX, chloromethylstyrene, and sodium iodide are used as reactants, dissolved in an organic solvent B under a nitrogen stream, heated for reaction, and then a polymerization terminator is added. The obtained product is filtered, vacuum dried, and purified to obtain a copolymer; S3. Preparation of a peptide-mimicking polymer: The polymer in S2 and trifluoroacetic acid are reactants, the reaction mixture is shaken at room temperature, the solvent is removed under a nitrogen stream, and the residue is purified to obtain a peptide-mimicking polymer; S4. Preparation of peptide-mimicking hydrogel: N,N′-methylenebisacrylamide, peptide-mimicking polymer and N-isopropylacrylamide are reactants, ammonium peroxodisulfate is an initiator, and N,N,N′,N′tetramethylethylenediamine is a catalyst. The reaction is carried out under a nitrogen flow, and the resulting hydrogel is extracted, dried in a vacuum and stored.

2. The method for preparing a thermosensitive antibacterial mimetic peptide hydrogel according to claim 1, characterized in that: In the above S1, the molar ratio of tert-butyloxycarbonylglycine and ethylchloroamine added is 1:1-2; and / or the molar ratio of the intermediate product and sodium hydroxide added is 1:1-2.

3. A method for preparing a thermosensitive antibacterial mimetic peptide hydrogel according to claim 1 or 2, characterized in that: In S1, before the reaction at room temperature, tert-butyloxycarbonylglycine, 1H-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and triethylamine are stirred in a mixture of ice and water at 0°C for more than 20 minutes, and then the temperature is raised to room temperature and ethyl chloride is added and dissolved in organic solvent A, and the reaction is continued by stirring for more than 6 hours.

4. The method for preparing a thermosensitive antibacterial mimetic peptide hydrogel according to claim 1, characterized in that: In S2, the molar ratio of the oxazoline mimetic peptide NPX, chloromethylstyrene, and sodium iodide under the protection of tert-butyloxycarbonyl is 20-80:1:2; And / or adding a polymerization terminator after the heating reaction in S2, the heating temperature is 70-90° C., and the time is more than 8 hours.

5. A method for preparing a thermosensitive antibacterial mimetic peptide hydrogel according to claim 1 or 4, characterized in that: In S2, the polymerization terminator is a methanol solution of potassium hydroxide.

6. A method for preparing a thermosensitive antibacterial mimetic peptide hydrogel according to claim 1 or 4, characterized in that: In S3, the reaction mixture is shaken at room temperature for more than 2 hours.

7. The method for preparing a thermosensitive antibacterial mimetic peptide hydrogel according to claim 1, characterized in that: The molar ratio of the N,N'-methylenebisacrylamide, the peptide-mimicking polymer, N-isopropylacrylamide, ammonium peroxodisulfate and N,N,N',N'tetramethylethylenediamine is 0.3:0.1-10:0.1-10:0.1:0.

2.

8. The method for preparing a thermosensitive antibacterial mimetic peptide hydrogel according to claim 1, characterized in that: In the step S4, the vacuum drying temperature is 20-50°C.

9. The method for preparing a thermosensitive antibacterial mimetic peptide hydrogel according to claim 1, characterized in that: The organic solvent A and the organic solvent B are any one of methanol, dichloromethane and acetonitrile.

10. The method for preparing a thermosensitive antibacterial mimetic peptide hydrogel according to any one of claims 1 to 9, characterized in that: The nitrogen circulation condition is to first evacuate the air and then fill it with nitrogen, and repeat this process for 5 to 10 times, each time for 3 to 5 minutes, and then maintain the nitrogen circulation environment.

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