CN110684078B

CN110684078B - Cationic antibacterial peptide modified by dopamine or derivatives thereof, and preparation and application thereof

Info

Publication number: CN110684078B
Application number: CN201910999849.7A
Authority: CN
Inventors: 朱锦涛; 穆巴沙·侯赛因; 蒋皓; 陶娟; 谢盈
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2019-10-21
Filing date: 2019-10-21
Publication date: 2021-07-27
Anticipated expiration: 2039-10-21
Also published as: CN110684078A

Abstract

The invention belongs to the field of antibacterial biomaterials, and particularly relates to a cationic antibacterial peptide modified by dopamine or derivatives thereof, and preparation and application thereof. The amino acid sequence of the antibacterial peptide from the N end to the C end is XX (AB) nXX, wherein N is an integer of 3-8, X is a cationic amino acid, A is a hydrophobic amino acid, and B is an anionic amino acid. The antibacterial peptide containing the dopamine adhesion group prepared by the invention shows broad spectrum and excellent antibacterial performance. The antibacterial peptide containing the dopamine adhesion group provided by the invention has the property of forming hydrogel by ultraviolet irradiation. In addition, the antibacterial peptide containing the dopamine adhesion group can be used for tissue damage repair and antibacterial materials, and has good application prospect and value.

Description

Cationic antibacterial peptide modified by dopamine or derivatives thereof, and preparation and application thereof

Technical Field

The invention belongs to the field of antibacterial biomaterials, and particularly relates to a cationic antibacterial peptide modified by dopamine or derivatives thereof, and preparation and application thereof.

Background

In recent years, overuse or abuse of antibiotics has led to the emergence of multi-drug resistant strains that appear to be resistant to a wide variety of, possibly even all, antibacterial drugs, i.e., the production of "superbacteria". With the emergence of various antibiotic-resistant bacteria, it is becoming more and more difficult to treat bacterial infections with the existing antibiotics, and new solutions are urgently needed to solve the problem. At present, in order to cope with infection caused by drug-resistant bacteria, on one hand, people modify the structure of traditional antibiotics to improve the curative effect and simultaneously reduce the drug resistance of bacteria, and on the other hand, novel antibacterial drugs are continuously developed.

Compared with traditional antibiotics, the antibacterial peptide has the properties of a biological membrane targeting mechanism, broad-spectrum activity, less cytotoxicity and even no drug resistance, is widely concerned and is considered as a potential, effective and broad-spectrum antibacterial agent. At present, some antibacterial peptides, such as natural antibacterial peptides including colistin, polymyxin B, daptomycin, gramicidin and nisin, can be used for treating infection caused by drug-resistant bacteria through preclinical and clinical experiments. However, these antimicrobial peptides have some disadvantages: protease instability and expensive production costs.

Therefore, small-molecule peptide mimics are artificially synthesized to replace natural antibacterial peptides, so that the defects of the natural antibacterial peptides are avoided and the therapeutic effect is improved. The cationic antibacterial peptide is a cationic amphiphilic molecule (containing a plurality of lysines and arginines) containing 12-50 amino acids. Compared with the traditional antibiotics, the cationic antibacterial peptide has the advantages of wide antibacterial spectrum, lower toxic and side effects, good thermal stability, unique antibacterial mechanism and the like. However, most of the artificially synthesized cationic antibacterial peptides have strong antibacterial activity but have certain toxicity to human cells. Therefore, the development of novel cationic antibacterial peptides with high antibacterial activity, low cytotoxicity and low drug resistance is an effective means for solving the problem of bacterial drug resistance, and has important application in the aspects of treating bacterial infectious diseases and promoting wound repair.

Disclosure of Invention

Aiming at the defects or the improvement requirements of the prior art, the invention provides a cationic antibacterial peptide modified by dopamine or derivatives thereof, and preparation and application thereof, wherein the cationic antibacterial peptide is modified by dopamine adhesion groups and comprises hydrophilic cationic amino acids, anionic amino acids with side chains modified by dopamine and hydrophobic amino acids, so that the technical problems that natural antibacterial peptide protease is unstable, and the artificially synthesized cationic antibacterial peptide has strong antibacterial activity but certain toxicity to human cells and the like in the prior art are solved.

To achieve the above objects, according to one aspect of the present invention, there is provided a dopamine or derivative modified cationic antibacterial peptide, the amino acid sequence of the antibacterial peptide from N-terminus to C-terminus is xx (ab) nXX, wherein N is an integer of 3 to 8, X is a cationic amino acid, a is a hydrophobic amino acid, and B is an anionic amino acid;

the carboxyl on the side chain of the anionic amino acid in the amino acid sequence is coupled with dopamine or dopamine derivative.

Preferably, the carboxyl group on the side chain of the anionic amino acid in the amino acid sequence is coupled with the amino group in the structural formula of dopamine or dopamine derivative.

Preferably, the cationic amino acid is lysine, arginine or histidine; the anionic amino acid is aspartic acid or glutamic acid; the hydrophobic amino acid is alanine, valine, leucine, isoleucine, phenylalanine or tryptophan.

Preferably, the dopamine derivative is one or more of alpha-methyldopamine, norepinephrine, levodopa, alpha-methyldopa, and droxidopa.

According to another aspect of the invention, a solid phase polypeptide synthesis method is adopted, starting from resin, amino acids are sequentially coupled from the C end to the N end according to the amino acid sequence (XX (AB) nXX), then dopamine or derivatives thereof are coupled with carboxyl on the side chain of anionic amino acid, and the obtained polypeptide resin is cut to obtain the cationic antibacterial peptide modified by dopamine or derivatives thereof.

According to another aspect of the present invention, there is provided a use of the cationic antimicrobial peptide modified with dopamine or its derivative for preparing a medicament for inhibiting bacterial growth.

Preferably, the cationic antibacterial peptide is prepared into an aqueous solution and then is irradiated under ultraviolet light or dissolved in a buffer solution to prepare hydrogel for preparing the medicine for inhibiting the growth of bacteria.

According to another aspect of the present invention, there is provided a use of the cationic antimicrobial peptide modified with dopamine or its derivative for preparing an antimicrobial coating.

Preferably, the cationic antibacterial peptide is prepared into an aqueous solution and then is irradiated under ultraviolet light or dissolved in a buffer solution to prepare hydrogel for preparing the antibacterial coating.

According to another aspect of the invention, the application of the cationic antibacterial peptide modified by dopamine or derivatives thereof in preparing a tissue injury repair medicine is provided.

Preferably, the cationic antibacterial peptide is prepared into an aqueous solution and then irradiated under ultraviolet light or dissolved in a buffer solution to prepare hydrogel for preparing the medicine for repairing tissue damage.

In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:

(1) the invention firstly provides a cationic antibacterial peptide with a simple amino acid sequence, which is modified by dopamine or derivatives thereof, the cationic antibacterial peptide has high-efficiency broad-spectrum killing effect on gram-negative bacteria and gram-positive bacteria, and the dopamine or derivatives thereof on the side chain of the polypeptide provides good adhesion performance.

(2) The invention also provides application of the cationic peptide modified by dopamine or derivatives thereof in preparation of medicines for inhibiting bacterial growth, antibacterial coatings or medicines for repairing tissue injuries, and the cationic peptide is prepared into hydrogel during application. Modifying dopamine or derivatives thereof on the side chain of the antibacterial polypeptide chain, and polymerizing the dopamine or derivatives thereof under the action of ultraviolet irradiation to form hydrogel; or dissolving the cationic antibacterial peptide in a buffer solution, and performing ultrasonic treatment to obtain the hydrogel. The cationic peptide is modified with dopamine or derivatives thereof, and also has good adhesion performance. Therefore, the invention also provides a cationic peptide hydrogel with good adhesion performance and broad-spectrum antibacterial function.

(3) The cationic antibacterial peptide and the hydrogel formed by the cationic antibacterial peptide have good adhesion performance and broad-spectrum antibacterial function, so the cationic antibacterial peptide has wide application prospect in the fields of long-acting bacteriostasis, bacteriostatic coatings and wound repair.

Drawings

FIG. 1 is a transmission electron microscope image of the dopamine modified cationic antimicrobial peptide obtained in example 1 of the present invention.

FIG. 2 is a circular dichroism diagram of the dopamine modified cationic antimicrobial peptide obtained in example 1 of the present invention.

FIG. 3 is a scanning electron microscope image and a photograph of the dopamine modified cationic antimicrobial peptide hydrogel obtained in example 21 of the present invention.

FIG. 4 is a graph showing the antibacterial activity of the cationic antibacterial peptide obtained in example 24 of the present invention.

FIG. 5 shows the cell viability of the dopamine modified cationic antimicrobial peptide obtained in example 1 of the present invention after 24 hours of incubation with NIH-3T3 cells.

FIG. 6 shows the skin wound healing effect of the cationic antimicrobial peptide hydrogel obtained in example 33 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The dopamine-modified cationic antibacterial peptide has an amino acid sequence XX (AB) nXX from the N end to the C end, wherein N is an integer of 3-8, X is a cationic amino acid, A is a hydrophobic amino acid, and B is an anionic amino acid. The carboxyl on the side chain of the anionic amino acid in the amino acid sequence is coupled with dopamine or dopamine derivative. The dopamine and the derivatives thereof have good surface adhesion performance, and the dopamine and the derivatives thereof can be subjected to polymerization crosslinking under the action of ultraviolet irradiation.

In some embodiments, the carboxyl group on the side chain of the anionic amino acid in the amino acid sequence is coupled to an amino group in the dopamine formula or an amino group in the dopamine derivative formula.

In some embodiments, the cationic amino acid is lysine, arginine, or histidine; the anionic amino acid is aspartic acid or glutamic acid; the hydrophobic amino acid is alanine, valine, leucine, isoleucine, phenylalanine or tryptophan.

In some embodiments, the dopamine derivative is alpha-methyldopamine, norepinephrine, levodopa, alpha-methyldopa, or droxidopa.

In a preferred embodiment, the antibacterial peptide is substituted with NH at the C-terminus₂End-capped to provide a C-terminal amide.

The invention also provides a preparation method of the cationic antibacterial peptide, which comprises the steps of adopting a solid phase polypeptide synthesis method, starting from resin, sequentially coupling amino acids from a C end to an N end according to the amino acid sequence (XX (AB) nXX), then coupling dopamine or derivatives thereof with carboxyl on an anionic amino acid side chain, and cutting the obtained polypeptide resin through TFA to obtain the cationic antibacterial peptide modified by the dopamine or derivatives thereof. Wherein the resin can be Rink Amide resin, MBHA resin or Sieber Amide resin.

The cationic antibacterial peptide modified by dopamine or derivatives thereof provided by the invention can be used for preparing medicines for inhibiting bacterial growth, antibacterial coatings or medicines for repairing tissue injuries. In some embodiments, the cationic antimicrobial peptides are formed into a hydrogel prior to the application.

Firstly, the cationic antibacterial peptide modified by dopamine or derivatives thereof provided by the invention is prepared into antibacterial peptide aqueous solutions with different pH values, and the aqueous solutions are subjected to sol-gel conversion under the irradiation of ultraviolet light to form hydrogel. In some embodiments, the hydrogel is obtained by dissolving the cationic antimicrobial peptide in aqueous solutions with different pH values and then irradiating the aqueous solutions under 254nm ultraviolet light.

In some embodiments, the antimicrobial peptide is formulated as a 1 wt% to 5 wt% aqueous solution, and the pH of the aqueous solution is adjusted to between 3-11 using an acid or a base; irradiating for 2-10 hours under 254nm ultraviolet light to obtain the antibacterial peptide hydrogel.

In some embodiments, the hydrogel can be prepared by dissolving the cationic antimicrobial peptide in a buffer solution, preparing the buffer solution with the mass fraction of 1 wt% to 5 wt% and the pH value of 7-9, and performing ultrasonic treatment or heating and cooling. The purpose of ultrasonic treatment or heating is to promote the dissolution of the cationic peptide in the buffer solution, and the cationic peptide can be in a gel state when the dissolution reaches a certain concentration. Preparing 1-5 wt% of buffer solution with pH value of 7-9, and performing ultrasonic treatment for 20-40s to obtain hydrogel; or heating the buffer solution to 50-60 ℃ and then cooling to room temperature (20-30 ℃) to obtain the hydrogel. The buffer solution can be selected from disodium hydrogen phosphate-citric acid buffer solution, phosphate-sodium hydroxide buffer solution, barbituric acid-hydrochloric acid buffer solution, Tris-hydrochloric acid buffer solution, boric acid-borax buffer solution, glycine-sodium hydroxide buffer solution, PBS buffer solution or HBSS buffer solution.

The cationic antibacterial peptide modified by the dopamine adhesion group comprises a hydrophilic cationic amino acid, a side chain modified anionic amino acid and a hydrophobic amino acid. The antibacterial peptide containing the dopamine adhesion group prepared by the invention has the key factors that the natural antibacterial peptide plays an antibacterial role: positive charge, hydrophobic domain, amphiphilicity, beta-sheet secondary conformation, etc., thus exhibiting a broad spectrum and excellent antibacterial properties. The antibacterial peptide containing the dopamine adhesion group provided by the invention has the property of forming hydrogel by ultraviolet irradiation. In addition, the antibacterial peptide containing the dopamine adhesion group can be used for tissue damage repair and antibacterial materials, and has good application prospect and value.

The following are examples:

example 1

The amino acid sequence of the cationic antibacterial peptide is KKFEFEFEFEKK, the structural formula of the antibacterial peptide is KKFE (DA) FE (DA) KK-NH2, and DA is dopamine.

Note: in the formula of the antimicrobial peptide, the C-terminal contains-NH 2, and it is understood by those skilled in the art that the carboxyamidation of the antimicrobial peptide belongs to the conventional treatment method of the antimicrobial peptide. And E (DA) shows that dopamine is modified on the side chain of glutamic acid through an amido bond.

The preparation method of the antibacterial peptide comprises the following steps:

(1) 0.3 g Rink Amide-AM resin was weighed into a polypeptide synthesis apparatus, and dried N, N-Dimethylformamide (DMF) was added and shaken for 2 hours to swell the resin. The resin was then deprotected with 10mL of 20% piperidine (volume fraction) in DMF and the procedure was repeated twice for 20 minutes each. The resin was washed 3 times repeatedly with 10mL DMF each for 5 min. Adding a little resin into an ethanol solution of ninhydrin and phenol, heating to boil, observing the color change of the resin, if the resin turns blue or even blackens, indicating that the protecting group of the resin is successfully removed, performing the coupling of the first amino acid, and if the color of the resin does not obviously change, continuing the operation of removing the protecting group of the resin.

(2) 0.3 g of Fmoc-Lys (BOC) -OH, 0.35 g of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) were weighed out and dissolved in 10mL of DMF, and the solution was transferred to the above apparatus for polypeptide synthesis containing the treated resin, followed by addition of 216. mu.L of Diisopropylethylamine (DIPEA), shaking at room temperature for 4 hours, and then washing the resin 3 times with 10mL of DMF, each for 5 minutes. The above operation is repeated. And adding a small amount of resin into the ethanol solution of ninhydrin and phenol, heating to boil, observing the color change of the resin, wherein if the color of the resin is not obviously changed, the first amino acid is completely coupled with the resin, and if the resin is blue or even black, the first amino acid is not completely reacted with the resin and needs to be repeatedly connected.

(3) The protecting group Fmoc was removed with 10mL of 20% piperidine (volume fraction) in DMF and reacted twice for 20 min each. And then repeatedly washing the resin by using 10mL of N, N-dimethylformamide for 3 times, wherein each time lasts for 5 minutes, taking a little of resin, adding the resin into an ethanol solution of ninhydrin and phenol, heating to boiling, observing the color change of the resin, if the resin turns blue or even blackens, indicating that the protecting group of the first amino acid is successfully removed, performing coupling of the second amino acid, and if the color of the resin does not obviously change, continuing the operation of removing the protecting group of the first amino acid.

(4) Referring to step (2) and step (3), Fmoc-Lys (BOC) -OH, Fmoc-Glu (OAll) -OH, Fmoc-Phe-OH, Fmoc-Lys (BOC) -OH, and Fmoc-Lys (BOC) -OH are condensed in this order. The resulting amino acid sequence was KKFEFEFEFEKK.

(5) 0.05 g of tetrakis (triphenylphosphine) palladium is weighed, dissolved by 10mL of dichloromethane, and then the solution is transferred to the polypeptide synthesis device containing the treated resin, 646 mu L of phenylsilane is added, the lysine protecting group on the resin is removed, the reaction is carried out twice, each time lasts for 30 minutes, and then the resin is repeatedly washed by 10mL of DMF for 3 times, each time lasts for 5 minutes. 0.5 g dopamine hydrochloride, 1.4 g PyBOP are weighed out in 10mL DMF and the solution is transferred to the peptide synthesizer containing the treated resin as above, 1mL Diisopropylethylamine (DIPEA) is added and shaken at room temperature for 24 hours, and the resin is washed 3 times with 10mL DMF for 5 minutes each.

(8) The resin was washed 3 times with 10mL of dichloromethane for 5 minutes, then 3 times with 10mL of methanol for 5 minutes, and then 3 times with 10mL of dichloromethane for 5 minutes.

(9) The polypeptide is cleaved from the resin, and the specific process is as follows: firstly, preparing a lysate: 9.5mL trifluoroacetic acid +0.25mL triisopropylsilane +0.25mL deionized water. Adding the lysate into the polypeptide synthesizer containing the treated resin, reacting for 3 hours, filtering the resin, removing the solvent in the resin by rotary evaporation, adding ether, and immediately generating a purple red precipitate. And then centrifuging the suspension twice at the rotation speed of 5000rpm for 10 minutes, removing the supernatant, adding methanol for dissolution, purifying the product through preparative MPLC, finally adding deionized water for dissolution, performing freeze drying, and collecting light brown powder to obtain the antibacterial peptide. FIG. 1 is a transmission electron microscope image of the dopamine modified cationic antimicrobial peptide obtained in example 1 of the present invention. FIG. 2 is a circular dichroism diagram of the dopamine modified cationic antimicrobial peptide obtained in example 1 of the present invention.

Parameters, conditions, and the like used in examples 2 to 12 are shown in table 1, and parameters, conditions, processing means, and the like not described are the same as those in example 1 except for specific parameters, condition settings, and the like shown in the table.

Example 13 to example 17

Examples 13 to 17, the parameters, conditions and the like used were the same as in example 1 except that dopamine was replaced with one of α -methyldopamine, norepinephrine, levodopa, α -methyldopa and droxidopa, respectively.

Example 18

The antimicrobial peptide prepared in example 1 was prepared into an aqueous solution with a mass fraction of 2%, and the pH was adjusted to 2.5 with dilute hydrochloric acid. And irradiating for 6 hours under an ultraviolet lamp of 254nm, and then carrying out sol-gel conversion on the solution to obtain the antibacterial peptide hydrogel.

Example 19

The antimicrobial peptide prepared in example 1 was prepared into an aqueous solution with a mass fraction of 2%, and the pH value was adjusted to 7.5 using a dilute sodium hydroxide solution. And irradiating for 6 hours under an ultraviolet lamp of 254nm, and then carrying out sol-gel conversion on the solution to obtain the antibacterial peptide hydrogel.

Example 20

The antimicrobial peptide prepared in example 1 was prepared into an aqueous solution with a mass fraction of 2%, and the pH value was adjusted to 10.5 using a dilute sodium hydroxide solution. And irradiating for 6 hours under an ultraviolet lamp of 254nm, and then carrying out sol-gel conversion on the solution to obtain the antibacterial peptide hydrogel.

Example 21

The antimicrobial peptide prepared in example 1 was prepared into a HBSS buffer solution with a pH of 7.4 at a mass fraction of 2%, and the antimicrobial peptide hydrogel was obtained after 30 seconds of ultrasonic treatment. FIG. 3 is a scanning electron microscope image and a photograph of the dopamine modified cationic antimicrobial peptide hydrogel obtained in example 21 of the present invention.

Example 22

The antimicrobial peptide prepared in example 1 was prepared into a 2% mass-fraction PBS buffer solution with pH of 7.4, and the antimicrobial peptide hydrogel was obtained after sonication for 30 seconds.

Example 23

The antimicrobial peptide prepared in example 1 was prepared into 2% by mass of Tris-hydrochloric acid buffer solution with pH of 7.4, and the antimicrobial peptide hydrogel was obtained after sonication for 30 seconds.

Example 24

Determination of cationic antimicrobial peptide activity: the antimicrobial peptide prepared in example 1 was formulated into a stock solution at a concentration of 8192. mu.g/mL for use. The Minimum Inhibitory Concentration (MIC) is an important parameter for evaluating the antibacterial performance of an antibacterial agent. In the experiment, the antibacterial performance of the antibacterial peptide on gram-negative bacteria escherichia coli is evaluated by a trace broth dilution method.

The experimental procedure was as follows:

(1) adding 10mL of LB bone broth culture medium into a culture dish;

(2) taking a sterile 96-well plate, adding 200 mu L of antibacterial peptide with the concentration of 4096 mu g/mL into the first well, adding 100 mu L of LB bone broth culture medium into the second to fourteen wells respectively, sucking 100 mu L from the first well, adding the mixture into the second well, uniformly mixing, sucking 100 mu L to the third well, and repeating the steps, sucking 100 mu L from the fourteenth well and discarding. The concentration of the antibacterial peptide in each hole is as follows in sequence: 8192,4096,2048,1024,512,256,128,64,32,16,8,4,2, 1. mu.g/mL, 200. mu.L of bacterial liquid was added to the fifteenth well, and 200. mu.L of LB bone broth was added to the sixteenth well.

(3) In the first toAdding 100 μ L of activated bacteria solution into each of fourteen wells to make final bacteria solution concentration about 5 × 10⁵CFU/mL, the concentration of antibacterial peptide in the first to fourteen holes is 4096,2048,1024,512,256,128,64,32,16,8,4,2,1 and 0.5 mu g/mL respectively. The inoculated 96-well plate is placed in an incubator at 37 ℃ for culture, and the minimum concentration of the antibacterial peptide inhibiting the growth of the strain is observed to be 16 mu g/mL after 18 hours.

FIG. 4 is a graph showing the antibacterial activity of the cationic antibacterial peptide obtained in example 24 of the present invention. It can be seen that the antibacterial peptide showed a good inhibitory effect against gram-negative bacteria (E.coli).

Examples 25 to 31 the minimum inhibitory concentration of the antimicrobial peptide was determined by the same experimental method as in example 24, and the results are shown in table 2:

example number	Test sample	Bacterial types	Minimum inhibitory concentration
				25	Example 1 antimicrobial peptides	Staphylococcus aureus	64μg/mL
26	Example 2 antimicrobial peptides	Escherichia coli	16μg/mL
				27	Example 2 antimicrobial peptides	Staphylococcus aureus	64μg/mL
28	Antibacterial peptide prepared in example 8	Escherichia coli	16μg/mL
				29	Antibacterial peptide prepared in example 8	Staphylococcus aureus	32μg/mL
30	Antibacterial peptide prepared in example 11	Escherichia coli	8μg/mL
				31	Antibacterial peptide prepared in example 11	Staphylococcus aureus	32μg/mL

Example 32

And detecting by using a CCK-8 kit. Mouse fibroblasts (NIH-3T3) were seeded into 96-well plates and the cell density was adjusted to 5X 10⁴At 24h after plating, the antimicrobial peptide of example 1 (at concentrations of 10, 100 and 1000. mu.g/mL in this order) was added to a 96-well plate. Incubate at 37 ℃ for 24h, 48h, 72h, respectively. After completion of incubation, cells were washed 2 times with PBS to remove free antimicrobial peptides, 90. mu.L of PBS and 10. mu.L of CCK-8 reagent were added to each well of a 96-well plate, and incubated in an incubator at 37 ℃1.5-2 h. The solution in the plate was observed to turn orange yellow, and the absorbance (wavelength of 450 nm) of the solution in each well was measured with a microplate reader.

FIG. 5 shows the cell viability of the dopamine modified cationic antimicrobial peptide obtained in example 1 of the present invention after 24 hours of incubation with NIH-3T3 cells. As can be seen from FIG. 5, the cationic antimicrobial peptide prepared by the invention has no toxic or side effect on normal cells and has good biological safety.

Example 33

The antibacterial peptide hydrogel prepared by the invention is used for repairing skin injury, and the beneficial effects are proved by the way of the embodiment:

(1) modeling of the skin injury animal model: selecting 18-20g Balb/c female mice, breeding for one week after quarantine is qualified, and adapting to the environment. Then, the female mouse is anesthetized by chloral hydrate, the hair on the back of the female mouse is removed on a cleaning operation table, the operation position is exposed, the operation position is cleaned and disinfected by alcohol, and a biopsy device with the diameter of 7mm is used for punching a round wound with the full thickness on the back of the female mouse, so that the modeling full-thickness skin injury is successful.

(2) Repairing skin damage: to verify the effect of the antimicrobial peptide hydrogel in repairing skin lesions, the full-thickness skin lesion model female mice were divided into three groups, namely: group A, blank control group, wound surface was not treated; group B, 3M wound repair liquid; group C, antimicrobial peptide hydrogels prepared according to the method of example 22. Wound healing was measured by observation and photographing at 3 rd, 5 th, 8 th, 10 th, 12 th, and 14 th days after operation. On day 16, the female mice were sacrificed, wound skin was harvested and fixed, and H & E stained after paraffin embedding.

FIG. 6 shows the skin wound healing effect of the cationic antimicrobial peptide hydrogel obtained in this example. The experimental result shows that the antibacterial peptide hydrogel prepared by the invention can inhibit bacterial breeding and shorten the inflammatory phase, can quickly repair skin injury, has obvious new capillaries and hyperplastic fibroblasts at the wound surface part, is beneficial to hyperplastic repair of the wound surface, and shows excellent repair effect.

The embodiments described above are intended to facilitate one of ordinary skill in the art in understanding and using the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should understand that improvements and modifications can be made without departing from the scope of the present invention.

Claims

1. The cationic antibacterial peptide modified by dopamine or derivatives thereof is characterized in that the cationic antibacterial peptide is KKFE (DA) FE (DA) KK-NH₂、KKAE(DA)AE(DA)AE(DA)AE(DA)KK-NH₂、KKFE(DA)FE(DA)FE(DA)FE(DA)FE(DA)FE(DA)KK-NH₂Or RRFE (DA) FE (DA) RR-NH₂And DA is dopamine, and the dopamine is coupled with carboxyl of a glutamic acid side chain in the antibacterial peptide.

2. The method for preparing the cationic antibacterial peptide according to claim 1, wherein the cationic antibacterial peptide modified by dopamine or derivatives thereof is obtained by a solid-phase polypeptide synthesis method, wherein amino acids are sequentially coupled from the C end to the N end from resin, then the dopamine or derivatives thereof are coupled with carboxyl on the side chain of anionic amino acid, and the obtained polypeptide resin is cut.

3. Use of the dopamine or derivative thereof-modified cationic antimicrobial peptide of claim 1 in the preparation of a medicament for inhibiting bacterial growth.

4. The use according to claim 3, wherein the cationic antimicrobial peptide is prepared into an aqueous solution, and then irradiated under ultraviolet light or dissolved in a buffer solution to prepare a hydrogel for preparing a medicament for inhibiting bacterial growth.

5. Use of the dopamine or derivative thereof modified cationic antimicrobial peptide of claim 1 for the preparation of an antimicrobial coating.

6. The use according to claim 5, wherein the cationic antimicrobial peptide is prepared into an aqueous solution, and then irradiated under ultraviolet light or dissolved in a buffer solution to prepare a hydrogel for preparing an antimicrobial coating.

7. The use of the dopamine or derivative thereof-modified cationic antimicrobial peptide of claim 1 in the preparation of a medicament for repairing tissue damage.

8. The use of claim 7, wherein the cationic antimicrobial peptide is prepared into an aqueous solution, and then is irradiated under ultraviolet light or dissolved in a buffer solution to prepare hydrogel for preparing a medicine for repairing tissue damage.