CN112316203B

CN112316203B - Cationic antibacterial peptide and hyaluronic acid composite hydrogel and preparation method thereof

Info

Publication number: CN112316203B
Application number: CN202011230613.6A
Authority: CN
Inventors: 蒋皓; 饶金安; 张兴华
Original assignee: Huangshi Hanhai New Material Technology Co ltd
Current assignee: Huangshi Hanhai New Material Technology Co ltd
Priority date: 2020-11-06
Filing date: 2020-11-06
Publication date: 2022-08-09
Anticipated expiration: 2040-11-06
Also published as: CN112316203A

Abstract

The invention relates to and provides a cationic antibacterial peptide and hyaluronic acid composite hydrogel and a preparation method thereof, wherein the cationic antibacterial peptide and hyaluronic acid composite hydrogel comprises oxidized hyaluronic acid and cationic antibacterial peptide; the amino acid sequence of the cationic antibacterial peptide is KK (EF) nKK, wherein n is an integer of 3-8; in the amino acid sequence, the carboxyl group of the glutamic acid side chain is modified into hydrazide. The cationic antibacterial peptide and hyaluronic acid composite hydrogel provided by the invention shows broad spectrum and excellent antibacterial performance, can be used for preparing various wound dressings, and has good application prospect; in addition, the cationic antibacterial peptide is prepared by a solid-phase polypeptide synthesis method, so that the synthesis mode is simple, the process of preparing the cationic antibacterial peptide and hyaluronic acid composite hydrogel by combining oxidized hyaluronic acid is simple, and the popularization and application of the cationic antibacterial peptide and hyaluronic acid composite hydrogel in wound dressings are facilitated.

Description

Cationic antibacterial peptide and hyaluronic acid composite hydrogel and preparation method thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a cationic antibacterial peptide and hyaluronic acid composite hydrogel and a preparation method thereof.

Background

One important aspect of the history of human fight against diseases is the history of continuous fight against various pathogens. Cationic antimicrobial peptides or lipopeptides are commonly used for antimicrobial and infection suppression. The antibacterial peptide antigen refers to a basic polypeptide substance with antibacterial activity generated by induction in an insect body, has a molecular weight of about 2000-7000 and consists of 20-60 amino acid residues. Most of the active polypeptides have the characteristics of strong alkalinity, heat stability, broad-spectrum antibiosis and the like.

However, the content of the antibacterial peptide in the animal body is very small. The extraction of the antibacterial peptide from the animal body has low yield, long time consumption, complex process and high cost, and the large-scale production cannot be realized, which becomes the biggest obstacle for restricting the practical application of the antibacterial peptide.

Disclosure of Invention

In view of the above, there is a need to provide a cationic antibacterial peptide and hyaluronic acid composite hydrogel and a method for preparing the same, in view of at least one of the problems mentioned above.

In a first aspect, the present application provides a cationic antimicrobial peptide and hyaluronic acid composite hydrogel, comprising oxidized hyaluronic acid and a cationic antimicrobial peptide; the amino acid sequence of the cationic antibacterial peptide is KK (EF) nKK, wherein n is an integer of 3-8; in the amino acid sequence, the carboxyl of the glutamic acid side chain is modified into hydrazide.

In a second aspect, the present application provides a method for preparing a composite hydrogel of a cationic antimicrobial peptide and hyaluronic acid, comprising the following steps:

preparing oxidized hyaluronic acid;

preparing a cationic antibacterial peptide by using a polypeptide solid-phase synthesis method, wherein the amino acid sequence of the cationic antibacterial peptide is KK (EF) nKK, and n is an integer of 3-8; in the amino acid sequence, the carboxyl of a glutamic acid side chain is modified into hydrazide;

preparing a hyaluronic acid solution according to the oxidized hyaluronic acid, preparing a cationic antibacterial peptide solution according to the cationic antibacterial peptide, uniformly mixing at room temperature, and carrying out self-crosslinking reaction to obtain the cationic antibacterial peptide and hyaluronic acid composite hydrogel.

In certain implementations of the second aspect, the step of preparing the oxidized hyaluronic acid comprises:

respectively preparing a hyaluronic acid aqueous solution and a sodium periodate aqueous solution;

dropwise adding the sodium periodate aqueous solution into the hyaluronic acid aqueous solution, reacting at room temperature in a dark place, and adding ethylene glycol to terminate the reaction;

dialyzing and freeze-drying the reaction product after the reaction to obtain the oxidized hyaluronic acid.

With reference to the second aspect and the foregoing implementations, in certain implementations of the second aspect, the mass ratio of the aqueous hyaluronic acid solution to the solute in the aqueous sodium periodate solution is 1: 0.3 to 1.5;

dropwise adding the sodium periodate aqueous solution into the hyaluronic acid aqueous solution, and reacting for 4 hours at room temperature in a dark condition; the oxidation degree of the oxidized hyaluronic acid is 30-60%.

With reference to the second aspect and the foregoing implementation manners, in certain implementation manners of the second aspect, the step of preparing the cationic antimicrobial peptide by using a polypeptide solid phase synthesis method includes:

adopting a solid-phase polypeptide synthesis method, starting from resin, sequentially coupling amino acids from the C end to the N end according to the amino acid sequence of the antibacterial peptide;

coupling hydrazine with carboxyl on a glutamic acid side chain to obtain polypeptide resin;

and cutting the polypeptide resin by TFA to obtain the cationic antibacterial peptide.

With reference to the second aspect and the foregoing implementation manners, in certain implementation manners of the second aspect, the step of preparing an oxidized hyaluronic acid solution according to the oxidized hyaluronic acid, preparing a cationic antibacterial peptide solution according to the cationic antibacterial peptide, uniformly mixing at room temperature, and performing a self-crosslinking reaction to obtain the cationic antibacterial peptide and hyaluronic acid composite hydrogel includes:

dissolving the oxidized hyaluronic acid in a buffer solution to obtain 1-5 wt% of oxidized hyaluronic acid buffer solution with the pH value ranging from 7 to 9;

dissolving the cationic antibacterial peptide in a buffer solution to obtain 1-5 wt% of cationic antibacterial peptide buffer solution with the pH value of 7-9;

and (3) mixing the oxidized hyaluronic acid buffer solution and the cationic antibacterial peptide buffer solution with the same concentration in a volume ratio of 0.6-2: 1, mixing to obtain the cationic antibacterial peptide and hyaluronic acid composite hydrogel.

With reference to the second aspect and the above-described implementations, in certain implementations of the second aspect, the buffer solution is selected from one of:

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.

In a third aspect, the present application provides a method for using a cationic antibacterial peptide and hyaluronic acid composite hydrogel, comprising the cationic antibacterial peptide and hyaluronic acid composite hydrogel as described in the first aspect of the present application, for preparing a wound dressing.

In certain implementations of the third aspect, the wound dressing includes a normal wound dressing, an infectious wound dressing, and a diabetic wound dressing.

The technical scheme provided by the embodiment of the invention has the following beneficial technical effects:

the cationic antibacterial peptide and hyaluronic acid composite hydrogel provided by the invention shows broad spectrum and excellent antibacterial performance, can be used for preparing various wound dressings, and has good application prospect and value; in addition, as the cationic antibacterial peptide is prepared by adopting a solid-phase polypeptide synthesis method, the synthesis mode is simpler, the synthesis cost is lower, the method is suitable for industrial production, the process of preparing the cationic antibacterial peptide and the hyaluronic acid composite hydrogel by combining the oxidized hyaluronic acid is simpler, the process conditions are easy to meet, the large-scale production of the cationic antibacterial peptide is favorably realized, and the popularization and application of the cationic antibacterial peptide and the hyaluronic acid composite hydrogel in the wound dressing are also facilitated.

Additional aspects and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a schematic flow chart of a method for preparing a composite hydrogel of a cationic antimicrobial peptide and hyaluronic acid according to an embodiment of the present application;

FIG. 2 is a schematic flow chart of a process for preparing oxidized hyaluronic acid according to an embodiment of the present application;

FIG. 3 is a schematic flow chart of a method for preparing cationic antimicrobial peptides by using a polypeptide solid phase synthesis method according to an embodiment of the present invention;

FIG. 4 is a schematic flow chart of a method for obtaining a composite hydrogel of cationic antimicrobial peptide and hyaluronic acid through self-crosslinking reaction according to an embodiment of the present application;

FIG. 5 is a molecular structural formula of a cationic antimicrobial peptide according to an embodiment of the present invention;

FIG. 6 is a molecular structural formula of an oxidized hyaluronic acid according to an embodiment of the present application;

FIG. 7 is a schematic diagram of a cross-linking reaction of an antibacterial peptide with oxidized hyaluronic acid according to an embodiment of the present application;

FIG. 8 is an SEM image of a composite hydrogel of a cationic antibacterial peptide and hyaluronic acid in an embodiment of the present application;

FIG. 9 is a comparison graph of bacteriostatic experimental results of the cationic antibacterial peptide and hyaluronic acid composite hydrogel in the embodiment of the present application.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Possible embodiments of the invention are given in the figures. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein by the accompanying drawings. The embodiments described by way of example with reference to the figures are intended to provide a more complete understanding of the disclosure of the present invention and are not to be construed as limiting the invention. Furthermore, if a detailed description of known technologies is not necessary for illustrating the features of the present invention, such technical details may be omitted.

It will be understood by those skilled in the relevant art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

As used herein, the singular forms "a", "an", "the" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. It is to be understood that the term "and/or" as used herein is intended to include all or any and all combinations of one or more of the associated listed items.

The technical solution of the present invention and how to solve the above technical problems will be described in detail with specific examples.

Embodiments of a first aspect of the present application provide a cationic antimicrobial peptide and hyaluronic acid composite hydrogel, comprising oxidized hyaluronic acid and a cationic antimicrobial peptide; the amino acid sequence of the cationic antibacterial peptide is KK (EF) nKK, wherein n is an integer of 3-8; in the amino acid sequence, the carboxyl group of the glutamic acid side chain is modified into hydrazide. FIG. 1 shows the molecular structure of a cationic antimicrobial peptide provided in the examples of the present application.

The embodiment of the second aspect of the present application provides a method for preparing a composite hydrogel of a cationic antibacterial peptide and hyaluronic acid, as shown in fig. 1, comprising the following steps:

s100: preparing oxidized hyaluronic acid.

S200: preparing a cationic antibacterial peptide by using a polypeptide solid-phase synthesis method, wherein the amino acid sequence of the cationic antibacterial peptide is KK (EF) nKK, and n is an integer of 3-8; in the amino acid sequence, the carboxyl group of the glutamic acid side chain is modified into hydrazide.

S300: preparing an oxidized hyaluronic acid solution according to oxidized hyaluronic acid, preparing a cationic antibacterial peptide solution according to cationic antibacterial peptide, uniformly mixing at room temperature, and performing self-crosslinking reaction to obtain the cationic antibacterial peptide and hyaluronic acid composite hydrogel.

Alternatively, in certain implementations of embodiments of the first aspect of the present application, as shown in fig. 2, the step of preparing oxidized hyaluronic acid comprises:

s110: preparing hyaluronic acid aqueous solution and sodium periodate aqueous solution respectively.

S120: and (3) dropwise adding a sodium periodate aqueous solution into a hyaluronic acid aqueous solution, reacting at room temperature in a dark condition, and adding ethylene glycol to terminate the reaction.

S130: dialyzing and freeze-drying the reaction product after the reaction to obtain the oxidized hyaluronic acid.

Optionally, the mass ratio of the solutes in the hyaluronic acid aqueous solution and the sodium periodate aqueous solution is 1: 0.3 to 1.5. That is, after the aqueous solution of sodium periodate was added dropwise to the aqueous solution of hyaluronic acid in S120, the mass ratio of hyaluronic acid to sodium periodate of the obtained mixture was 1: 0.3 to 1.5. And dropwise adding the sodium periodate aqueous solution into the hyaluronic acid aqueous solution, and reacting for 4 hours at room temperature in a dark condition. The oxidation degree of the oxidized hyaluronic acid is 30-60%.

Alternatively, in certain implementations of embodiments of the first aspect of the present application, as shown in fig. 3, the step of preparing the cationic antimicrobial peptide by using a polypeptide solid phase synthesis method specifically includes:

s210: by adopting a solid-phase polypeptide synthesis method, starting from resin, amino acids are coupled from the C end to the N end in sequence according to the amino acid sequence of the antibacterial peptide.

S220: and (3) coupling hydrazine with carboxyl on a glutamic acid side chain to obtain the polypeptide resin.

S230: and (3) cutting the polypeptide resin by TFA to obtain the cationic antibacterial peptide.

In one embodiment of the present application, a specific example of the preparation of cationic antimicrobial peptide is provided as follows:

(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), the amino acid sequence obtained by sequential condensation (Fmoc-Lys (BOC) -OH, Fmoc-Phe-OH, Fmoc-Glu (OAll) -OH, Fmoc-Lys (BOC) -OH) was KKEFEFEFEFKK.

(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. 0.2 g hydrazine hydrochloride, 1.4 g PyBOP are weighed out in 10mL DMF and the solution is transferred to the above apparatus for peptide synthesis containing the treated resin, 1mL Diisopropylethylamine (DIPEA) is added and shaken at room temperature for 24 hours, and the resin is then 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 by using preparative MPLC, finally adding deionized water for dissolution, performing freeze drying, and collecting white powder to obtain the cationic antimicrobial peptide.

Optionally, in another implementation manner of the embodiment of the first aspect of the present application, the S300 is a step of preparing a oxidized hyaluronic acid solution according to oxidized hyaluronic acid, a cationic antimicrobial peptide solution according to cationic antimicrobial peptide, uniformly mixing at room temperature, and obtaining a composite hydrogel of cationic antimicrobial peptide and hyaluronic acid through a self-crosslinking reaction, as shown in fig. 4, and specifically includes the following contents:

s310: dissolving oxidized hyaluronic acid in a buffer solution to obtain 1-5 wt% of oxidized hyaluronic acid buffer solution with the pH value ranging from 7 to 9.

S320: dissolving the cationic antibacterial peptide into a buffer solution to obtain the cationic antibacterial peptide buffer solution with the pH value of 7-9 and the weight percentage of 1-5%.

S330: the method comprises the following steps of (1) mixing oxidized hyaluronic acid buffer solution and cationic antibacterial peptide buffer solution with the same concentration in a volume ratio of 0.6-2: 1, mixing to obtain the cationic antibacterial peptide and hyaluronic acid composite hydrogel. Fig. 5 and fig. 6 show chemical structural formulas of a cationic antimicrobial peptide and oxidized hyaluronic acid provided herein, and fig. 7 shows that a portion a and a portion B can react to form a hydrazone bond under weak base conditions, thereby forming a crosslinked hydrogel, and the microstructure of the crosslinked hydrogel can refer to fig. 8.

It should be noted that, in the above three steps, S310 and S320 are not strictly distinguished in sequence, and may be performed simultaneously.

Optionally, the selection range of the buffer solution is wide, and the buffer solution may be specifically selected from one of the following buffer solutions: 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.

Based on the same inventive concept, embodiments of the second aspect of the present application provide a method for applying the cationic antibacterial peptide and hyaluronic acid composite hydrogel, comprising the cationic antibacterial peptide and hyaluronic acid composite hydrogel according to claim 1, wherein the cationic antibacterial peptide and hyaluronic acid composite hydrogel are used for preparing a wound dressing. As shown in fig. 9, the experimental group using the cationic antimicrobial peptide and hyaluronic acid composite hydrogel provided herein was able to significantly inhibit escherichia coli and staphylococcus aureus, compared to the unused control group. Alternatively, the wound dressing includes a general wound dressing, an infectious wound dressing, and a diabetic wound dressing. The wounds are wide in category, and can be applied to common wounds such as knife cut scratches or infectious wounds such as traumatic wounds with necrotic tissues, and various wounds of patients with diabetes constitution.

Those of skill in the art will understand that various operations, methods, steps in the flow, measures, schemes discussed in this application can be alternated, modified, combined, or deleted. Further, other steps, measures, or schemes in various operations, methods, or flows that have been discussed in this application can be alternated, altered, rearranged, broken down, combined, or deleted. Further, steps, measures, schemes in the prior art having various operations, methods, procedures disclosed in the present application may also be alternated, modified, rearranged, decomposed, combined, or deleted.

The terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present application, "a plurality" means two or more unless otherwise specified.

In the description of the present application, it is to be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present application can be understood in a specific case by those of ordinary skill in the art.

In the description herein, particular features, structures, materials, or characteristics may be combined in any suitable manner in any one or more embodiments or examples.

It should be understood that, although the steps in the flowcharts of the figures are shown in order as indicated by the arrows, the steps are not necessarily performed in order as indicated by the arrows. The steps are not performed in the exact order shown and may be performed in other orders unless explicitly stated herein. Moreover, at least a portion of the steps in the flow chart of the figure may include multiple sub-steps or multiple stages, which are not necessarily performed at the same time, but may be performed at different times, which are not necessarily performed in sequence, but may be performed alternately or alternately with other steps or at least a portion of the sub-steps or stages of other steps.

The foregoing is only a partial embodiment of the present application, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the principle of the present application, and these modifications and decorations should also be regarded as the protection scope of the present application.

Claims

1. A preparation method of a cationic antibacterial peptide and hyaluronic acid composite hydrogel is characterized by comprising the following steps:

preparing oxidized hyaluronic acid;

dissolving the oxidized hyaluronic acid in a buffer solution to obtain 1-5 wt% of oxidized hyaluronic acid buffer solution with the pH value ranging from 7 to 9; dissolving the cationic antibacterial peptide in a buffer solution to obtain 1-5 wt% of cationic antibacterial peptide buffer solution with the pH value of 7-9; and (3) mixing the oxidized hyaluronic acid buffer solution and the cationic antibacterial peptide buffer solution with the same concentration in a volume ratio of 0.6-2: 1, uniformly mixing at room temperature, and carrying out self-crosslinking reaction to obtain the cationic antibacterial peptide and hyaluronic acid composite hydrogel.

2. The method according to claim 1, wherein the step of preparing oxidized hyaluronic acid comprises:

dialyzing and freeze-drying the reaction product to obtain the oxidized hyaluronic acid.

3. The method according to claim 2, wherein the mass ratio of the aqueous hyaluronic acid solution to the solute in the aqueous sodium periodate solution is 1: 0.3 to 1.5;

4. The method according to claim 1, wherein the step of preparing the cationic antibacterial peptide by using a polypeptide solid phase synthesis method comprises:

and (3) cutting the polypeptide resin by TFA to obtain the cationic antibacterial peptide.

5. The method according to claim 1, wherein the buffer solution is selected from one of:

6. A cationic antibacterial peptide and hyaluronic acid composite hydrogel, which is prepared by the method for preparing the cationic antibacterial peptide and hyaluronic acid composite hydrogel according to any one of claims 1 to 5.

7. The method for using the cationic antibacterial peptide and hyaluronic acid composite hydrogel according to claim 6, wherein the cationic antibacterial peptide and hyaluronic acid composite hydrogel is used for preparing a wound dressing.

8. The method of use of claim 7, wherein the wound dressing is an infectious wound dressing.

9. The method of use of claim 7, wherein the wound dressing is a diabetic wound dressing.