CN114668833A

CN114668833A - Preparation method of DNA hydrogel loaded with IL-33, product and application thereof

Info

Publication number: CN114668833A
Application number: CN202210321717.0A
Authority: CN
Inventors: 郑晓峰; 王成世; 李威; 王政昊
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-03-25
Filing date: 2022-03-25
Publication date: 2022-06-28
Anticipated expiration: 2042-03-25
Also published as: CN114668833B

Abstract

The invention discloses a preparation method of DNA hydrogel loaded with IL-33, a product and application thereof, belonging to the field of pharmaceutical preparations. Aims to provide a preparation method of the DNA hydrogel loaded with the IL-33, which has simple preparation process, and the product has the capabilities of slow release, oxidation resistance and self degradation when being applied to wound healing. The technical scheme is that the preparation method of the DNA hydrogel loaded with interleukin IL-33 comprises the following steps: s1) preparing materials; s2) dissolving; s3) constructing a Y monomer and an L monomer; s4) incubating and mixing.

Description

Preparation method of DNA hydrogel loaded with IL-33, product and application thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations, and particularly relates to a preparation method of DNA hydrogel loaded with IL-33, and a product and application thereof.

Background

The wound healing process is generally called wound healing or wound healing, and is realized by the synergistic effect of various processes through the complex combination of regeneration of various tissues at the wound and granulation tissue hyperplasia and scar tissue formation. Wound healing is the process of regenerative repair of wounds, and the basic processes of wound healing include the acute inflammatory phase, the cell proliferation phase, the scarring phase, and the epidermal and other tissue regeneration phases. However, some wounds can be caused to continue to be in an acute inflammatory phase due to their own particularity and aggravate inflammatory processes such as bedsores/pressure sores and burn/scald poor healing; or as complications of diabetes, vascular insufficiency, immunodeficiency and the like, the wound healing is difficult, chronic ulcer and secondary gangrene of the wound occur, and even the life of a patient is threatened in severe cases. At present, an effective treatment method aiming at the wound healing difficulty is lacked, so that the morbidity and mortality of a series of complications caused by the wound healing difficulty are high, the physical and psychological health of a patient is affected, a huge economic burden is brought to the patient, and a huge medical burden is also brought to the country and the society.

The hydrogel is a high water absorption and high water retention material, has wide application range, is applied in various technical fields, has high anti-infection capacity, absorbs wound exudate, keeps the moist balance of a wound, allows gas exchange and loading, protects and transports bioactive molecules, wherein a three-dimensional network formed by single-stranded DNA polymerization is physically crosslinked DNA hydrogel, does not adopt any chemical connector or chemical modification, and has the characteristics of low cytotoxicity, immunogenicity, good biocompatibility and the like, so that the DNA hydrogel has more ideal using effect as one of ideal wound dressings.

The current research indicates that the cytokine immunotherapy has important significance in regulating the immune system, and especially plays an important role in promoting the wound healing difficulty caused by complications caused by diseases such as diabetes and the like. Of these, interleukin-33 (IL-33) is believed to promote wound healing, primarily by virtue of its anti-inflammatory properties through induction of M2 macrophage polarization and proliferation of regulatory T cells. However, due to the short half-life and poor stability of IL-33, its effectiveness in treating skin wounds is not satisfactory, limiting its effectiveness in wound healing therapy.

Disclosure of Invention

In view of the above, the present invention aims to provide a preparation method of a DNA hydrogel loaded with IL-33, and a product and an application thereof, wherein the preparation method adopts a simple process, the product has sustained release, oxidation resistance and self-degradation capability when applied to wound healing, and the product can be particularly used for treating the situation of wound healing difficulty such as diabetes.

The inventor provides a technical scheme for solving the technical problems by continuously reforming and innovating through long-term exploration and attempt, and multiple experiments and endeavors, and provides a preparation method of a DNA hydrogel loaded with interleukin IL-33, which comprises the following steps:

s1) preparing materials: comprises recombinant mouse IL-33 and 5 single-stranded DNAs shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5;

s2) dissolution: dissolving each of the 5 single-stranded DNAs described in S1) in PBS buffer;

s3) construction of monomers: respectively dissolving the single-stranded DNAs shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in S2) in PBS buffer solution to obtain solution, mixing the solution, reacting the solution sequentially through constant temperature treatment and cooling treatment to obtain a Y monomer, and respectively dissolving the single-stranded DNAs shown as SEQ ID No.4 and SEQ ID No.5 in S2) in PBS buffer solution to obtain solution, mixing the solution, reacting sequentially through constant temperature treatment and cooling treatment to obtain an L monomer;

s4) incubation and mixing: mixing and incubating the Y monomer obtained in S3) with the recombinant mouse IL-33 to obtain a product A, mixing and incubating the L monomer obtained in S3) with the recombinant mouse IL-33 to obtain a product B, and mixing the product A and the product B.

According to a specific embodiment of the preparation method of the interleukin IL-33 loaded DNA hydrogel, the PBS buffer solution in S2) is 1xPBS buffer solution.

According to a preferred embodiment of the preparation method of the DNA hydrogel loaded with interleukin IL-33, 1-2 mmol/L MgCl is added into the PBS buffer solution₂The pH value of the PBS buffer solution is 7.2-8.0, and the final concentration of the 5 kinds of single-stranded DNA respectively dissolved in the PBS buffer solution in S2) is 1-2 mmol/L.

According to a further embodiment of the method for preparing the interleukin IL-33 loaded DNA hydrogel, the constant temperature treatment in S3) is: standing the mixed solution for 1-3 min at 95 ℃; the cooling treatment in S3) is: and reducing the temperature of the mixed solution after constant temperature treatment by 0.1 ℃ every 0.06s at 95 ℃ until the temperature is reduced to 25 ℃, and then storing the obtained Y monomer and L monomer at the storage temperature of 4-8 ℃.

According to a further embodiment of the method for preparing the DNA hydrogel loaded with interleukin IL-33, the concentration of the mixed solution obtained by mixing the dissolving solutions obtained by respectively dissolving the single-stranded DNAs shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in the PBS buffer solution is 250 to 300. mu. mol/L, and the concentration of the mixed solution obtained by mixing the dissolving solutions obtained by respectively dissolving the single-stranded DNAs shown as SEQ ID No.4 and SEQ ID No.5 in the PBS buffer solution is 375 to 500. mu. mol/L.

According to a further embodiment of the method for preparing the DNA hydrogel loaded with the interleukin IL-33, the Y monomer or/and the L monomer constructed in S3) is processed by a non-denaturing polyacrylamide gel electrophoresis method or a liquid chromatography-mass spectrometry combined measuring method, and is used for judging whether the Y monomer or/and the L monomer are successfully constructed.

According to a further embodiment of the method for preparing a DNA hydrogel loaded with interleukin IL-33, the ratio of the concentration of the Y monomer to the concentration of the L monomer in S3) is 2:3, the volume ratio of the Y monomer to the mouse IL-33 in S4) is 24:1, and the volume ratio of the L monomer to the mouse IL-33 is 24: 1.

According to a further embodiment of the method for preparing the DNA hydrogel loaded with the interleukin IL-33, the concentration of the recombinant mouse IL-33 in S1) is 0.5-1 ng/muL.

According to a further embodiment of the preparation method of the DNA hydrogel loaded with interleukin IL-33, in the step S4), the product A and the product B are mixed by shaking for 1min to 2min at 20 to 30 ℃.

The invention also provides an IL-33-loaded DNA hydrogel, and the IL-33-loaded DNA hydrogel is obtained by the preparation method.

The invention also provides an application of the DNA hydrogel loaded with the IL-33 in wound treatment, and the DNA hydrogel loaded with the IL-33 is obtained by the preparation method.

Compared with the prior art, one of the technical solutions has the following advantages:

a) the process is simple, the obtained DNA hydrogel loaded with the IL-33 has a slow release effect on the IL-33, the characteristics of short half-life period and poor stability of the IL-33 can be improved, the concentration of the IL-33 at the wound can be kept for a long time by one-time administration, the conditions of continuous anti-inflammation and wound healing promotion are met, the administration period is shortened, and the treatment effect which can be achieved by one-time administration is improved.

b) The product obtained by the preparation method of the invention also has antioxidant capacity, and can remove high active oxygen in the wound environment when the product is used for wound healing treatment, thereby promoting wound healing recovery, in addition, the product can be self-degraded in the treatment process, and the DNA hydrogel has better biocompatibility, so the self-degraded DNA hydrogel can be replaced by newly formed tissues, bioactive molecules are pertinently delivered to the wound without changing dressing, and hydrolyzed DNA micromolecules can be reused by surrounding tissues, and the physically crosslinked DNA hydrogel is soft, can adapt to various irregular wounds with irregular shapes and depths, can not generate further mechanical injury, and provides a scaffold for cells of new tissues.

c) When the invention is preparedMg in PBS buffer used²⁺Can enhance the stability of DNA strands, and Cl^-The content of the DNA chain in a human body is more, so that the product meets the characteristic of a biological material, the product is safer and more reliable, and the stability of the DNA chain can be further improved when the pH is 7.2-8.0.

d) According to the invention, whether the Y monomer or/and the L monomer is successfully constructed is judged by a non-denaturing polyacrylamide gel electrophoresis method or a liquid chromatography-mass spectrometry combined measuring method, the judging method is mature, the operation steps are simple, and the reliability of the judging result is high.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and that those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.

FIG. 1 is a schematic flow chart of a method for preparing an IL-33-loaded DNA hydrogel according to the present invention.

FIG. 2 is an electrophoresis chart of the Y monomer and the L monomer verified by the non-denaturing polyacrylamide gel electrophoresis method in the present invention.

FIG. 3 is a comparative reference chart of the viscosity, i.e., the adhesion ability, of the DNA hydrogel used in the present invention with distilled water.

FIG. 4 is a schematic view of the effect of the DNA hydrogel used in the present invention under a transmission electron microscope.

FIG. 5 is a schematic view of the effect of the DNA hydrogel used in the present invention under a scanning electron microscope.

FIG. 6 is a graph showing the results of an experiment conducted in example 6 of the present invention.

Detailed Description

The following description will be given with reference to specific examples.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of embodiments of the invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention.

Example 1

See fig. 1. The preparation method of the DNA hydrogel loaded with IL-33 described in the embodiment comprises the following steps:

s1) preparing materials: comprises recombinant mouse IL-33 and 5 single-stranded DNAs shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, wherein the concentration of the recombinant mouse IL-33 is 0.5 ng/mu L;

s2) dissolution: dissolving the 5 single-stranded DNAs described in S1) in 1xPBS buffer solution to obtain solutions each having a final concentration of 1mmol/L, wherein MgCl is added to the 1xPBS buffer solution in an amount of 1mmol/L₂The pH value of the 1xPBS buffer solution is 7.2;

s3) construction of monomers: respectively dissolving single-stranded DNA shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in S2) in S2) to obtain a solution, mixing the solution to obtain a mixed solution with a concentration of 250 mu mol/L, performing constant temperature treatment, namely standing the mixed solution at 95 ℃ for 2min, and then performing cooling treatment, namely reducing the temperature of the mixed solution after constant temperature treatment at 95 ℃ by 0.1 ℃ every 0.06S for 700 times in total, and then reducing the temperature to 25 ℃ to obtain a Y monomer with a concentration of 160 mu mol/L; respectively dissolving single-stranded DNA shown as SEQ ID No.4 and SEQ ID No.5 in S2) in the 1xPBS buffer solution described in S2) to obtain a mixed solution with a concentration of 375 mu mol/L, mixing, performing constant temperature treatment, namely standing the mixed solution at 95 ℃ for 1min, then performing temperature reduction treatment, namely reducing the temperature of the mixed solution after constant temperature treatment at 95 ℃ by 0.1 ℃ every 0.06S for 700 times in total to obtain L monomers with a concentration of 240 mu mol/L, and then storing the obtained Y monomers and the L monomers at 4 ℃;

s4) judging whether the Y monomer or/and the L monomer is successfully constructed by a non-denaturing polyacrylamide gel electrophoresis method or a liquid chromatography-mass spectrometry combined determination method;

s5) incubation and mixing: mixing and incubating the Y monomer obtained in S3) with recombinant mouse IL-33 at 20 ℃ to obtain a product A, wherein 24 mu L of Y monomer and 1 mu L of IL-33 are required; then mixing and incubating the L monomer obtained in S3) with the recombinant mouse IL-33 at 20 ℃ to obtain a product B, wherein 24 mu L of the L monomer and 1 mu L of the IL-33 are needed, and then mixing the product A and the product B by shaking at 20 ℃ for 1min through a shaking table.

Example 2

s1) preparing materials: comprises recombinant mouse IL-33 and 5 single-stranded DNAs shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, wherein the concentration of the recombinant mouse IL-33 is 1 ng/mu L;

s2) dissolution: respectively dissolving the 5 single-stranded DNAs in S1) in a 1xPBS buffer solution to obtain solution with the final concentration of 2mmol/L, wherein the 1xPBS buffer solution is added with 2mmol/L MgCl2, and the pH value of the 1xPBS buffer solution is 8.0;

s3) construction of monomers: respectively dissolving single-stranded DNA shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in S2) in the 1xPBS buffer solution described in S2) to obtain a dissolved solution, mixing the dissolved solution to obtain a mixed solution with the concentration of 300 mu mol/L, performing constant temperature treatment, namely standing the mixed solution for 3min at 95 ℃, and then performing cooling treatment, namely reducing the temperature of the mixed solution after constant temperature treatment by 0.1 ℃ every 0.06S at 95 ℃, performing 700 times in total, and then reducing the temperature to 25 ℃ to obtain a Y monomer with the concentration of 180 mu mol/L; respectively dissolving single-stranded DNA shown as SEQ ID No.4 and SEQ ID No.5 in S2) in the 1xPBS buffer solution described in S2) to obtain a mixed solution with the concentration of 500 mu mol/L, mixing, performing constant temperature treatment, namely standing the mixed solution at 95 ℃ for 3min, then performing temperature reduction treatment, namely reducing the temperature of the mixed solution after constant temperature treatment at 95 ℃ by 0.1 ℃ every 0.06S for 700 times in total to obtain L monomers with the concentration of 270 mu mol/L, and then storing the obtained Y monomers and the L monomers at 8 ℃;

s5) incubation and mixing: mixing and incubating the Y monomer obtained in S3) with recombinant mouse IL-33 at 30 ℃ to obtain a product A, wherein 24 mu L of Y monomer and 1 mu L of IL-33 are required; then mixing and incubating the L monomer obtained in S3) with the recombinant mouse IL-33 at 30 ℃ to obtain a product B, wherein 24 mu L of the L monomer and 1 mu L of the IL-33 are needed, and then mixing the product A and the product B by shaking at 30 ℃ for 2min through a shaking table.

Example 3

The non-denaturing polyacrylamide gel electrophoresis method described in this example is used to determine whether the Y monomer and the L monomer described in S3) in example 1 were successfully constructed, and includes the steps of:

a) preparing PAGE gel electrophoresis buffer;

b) preparing separation gel and concentrated gel required by the non-denatured polyacrylamide gel electrophoresis;

c) performing non-denaturing polyacrylamide gel electrophoresis;

d) analyzing the electrophoresis result, and judging whether the Y monomer and the L monomer are successfully constructed according to a general standard analysis method.

Example 4

The measurement method using the combination of liquid chromatography and mass spectrometry described in this embodiment is used to determine whether the Y monomer and the L monomer described in S3) in embodiment 1 were successfully constructed, the Y monomer and the L monomer are processed by the combination of liquid chromatography and mass spectrometry, and the processing result is analyzed according to a general standard analysis method, so as to determine whether the Y monomer and the L monomer were successfully constructed.

Example 5

An IL-33-loaded DNA hydrogel as described in this example was prepared by the preparation method described in example 1.

Example 6

See fig. 6. The application of the DNA hydrogel loaded with IL-33 in wound treatment, which is described in the embodiment and can be particularly used for treating wounds of diabetic patients, the DNA hydrogel loaded with IL-33 in the embodiment is prepared by the preparation method described in the embodiment 1.

Example 7

In the experiment for healing the whole wound of the diabetic mouse by using the IL-33-transfected DNA hydrogel, the data obtained in the experiment is the mean value + -SEM of the results obtained from 6 independent experiments, and the experiment comprises the following steps:

(A) representative wound photographs on

days

0, 3, 7, and 14 for the blank, hydrogel, IL-33, and IL-33 loaded DNA hydrogel groups;

(B) graphs of wound healing traces on

days

(C) analyzing and comparing the wound healing rates of the blank group, the hydrogel group, the IL-33 group and the DNA hydrogel group loaded with the IL-33 on 0, 3, 7 and 14 days;

(D) sections of each of the groups of fresh granulation tissue observed under HE staining for the blank group, the hydrogel group, the IL-33 group and the IL-33-loaded DNA hydrogel group, wherein the length of the arrow indicates the thickness of the granulation tissue, and the scale bar in the figure is 200 μm;

(E) the blank, hydrogel, IL-33, and IL-33-loaded DNA hydrogel groups were analyzed for thickness of granulation tissue on day 14.

According to the experimental results, in (A), the IL-33 loaded DNA hydrogel group has a significantly better wound treatment effect on diabetic mice on days 7 and 14 than the blank group, the hydrogel group and the IL-33 group; in the step (B), the DNA hydrogel group loaded with IL-33 has better healing effect on wound healing tracks of diabetic mice in wound treatment at 0, 3, 7 and 14 days; in (C), the levels of significance were all <0.001 for the wound healing rate analyses comparing the blank group at day 7 with the DNA hydrogel group loaded with IL-33 and the blank group at day 14 with the DNA hydrogel group loaded with IL-33, and the levels of significance for the wound healing rate analyses comparing the hydrogel group at day 7 with the DNA hydrogel group loaded with IL-33, the DNA hydrogel group at day 7 with IL-33, the hydrogel group at day 14 with the DNA hydrogel group loaded with IL-33, and the DNA hydrogel group at day 14 with the DNA hydrogel group loaded with IL-33 were all < 0.01; in (D), the thickness of granulation tissue is the largest in the IL-33-loaded DNA hydrogel group; in (E), the significance levels of the thickness quantification results of granulation tissue of blank group and DNA hydrogel group of IL-33, hydrogel group and DNA hydrogel group of IL-33, and IL-33 group and DNA hydrogel group of IL-33 on day 14 were all p < 0.001.

From the experiment, the IL-33 loaded DNA hydrogel group has better healing capacity and healing effect on all aspects of wounds of the diabetic mice than the blank group, the hydrogel group and the IL-33 group.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

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Claims

1. A method for preparing an IL-33 loaded DNA hydrogel, which is characterized by comprising the following steps:

2. The method for preparing an IL-33-loaded DNA hydrogel according to claim 1, wherein the PBS buffer in S2) is 1xPBS buffer.

3. The method for preparing the IL-33-loaded DNA hydrogel according to claim 2, wherein 1-2 mmol/L MgCl is added to the PBS buffer solution₂The pH value of the PBS buffer solution is 7.2-8.0, and the final concentration of the 5 kinds of single-stranded DNA respectively dissolved in the PBS buffer solution in S2) is 1-2 mmol/L.

4. The method for preparing an IL-33-loaded DNA hydrogel according to claim 1, wherein the isothermal treatment in S3) is: standing the mixed solution for 1-3 min at 95 ℃; the cooling treatment in S3) is: and reducing the temperature of the mixed solution after constant temperature treatment by 0.1 ℃ every 0.06s at 95 ℃ until the temperature is reduced to 25 ℃, and then storing the obtained Y monomer and L monomer at the storage temperature of 4-8 ℃.

5. The method for preparing the DNA hydrogel loaded with IL-33 according to claim 1, wherein the concentration of the mixed solution obtained by mixing the single-stranded DNAs shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 dissolved in PBS buffer solution is 250 to 300. mu. mol/L, and the concentration of the mixed solution obtained by mixing the single-stranded DNAs shown as SEQ ID No.4 and SEQ ID No.5 dissolved in PBS buffer solution is 375 to 500. mu. mol/L.

6. The method of claim 1, wherein the Y monomer and/or the L monomer constructed in S3) is processed by native polyacrylamide gel electrophoresis or liquid chromatography-mass spectrometry to determine whether the Y monomer and/or the L monomer is successfully constructed.

7. The method of claim 1, wherein the ratio of the concentration of the Y monomer to the concentration of the L monomer in S3) is 2:3, the volume ratio of the Y monomer to the mouse IL-33 in S4) is 24:1, and the volume ratio of the L monomer to the mouse IL-33 is 24: 1.

8. The method for preparing the IL-33-transfected DNA hydrogel of claim 7, wherein the concentration of the recombinant mouse IL-33 in S1) is 0.5-1 ng/μ L.

9. The method for preparing DNA hydrogel loaded with IL-33 according to claim 1, wherein the product A and the product B in S4) are mixed by shaking the shaking table at 20-30 ℃ for 1-2 min.

10. An IL-33-loaded DNA hydrogel produced by the production method according to any one of claims 1 to 9.

11. Use of an IL-33 loaded DNA hydrogel in wound treatment, wherein the IL-33 loaded DNA hydrogel is obtained by the preparation method of any one of claims 1 to 9.