Elastin-like polypeptide adhesive and its preparation method and application

Abstract

The present invention discloses an elastin-like polypeptide adhesive, its preparation method, and its application. Based on the biocompatible elastin-like polypeptide (SEQ ID NO: 1), the present invention fully explores the enormous potential of mussel adhesion chemistry in the biomedical field. Through chemical modification, an elastin-like polypeptide adhesive is prepared. This adhesive exhibits numerous advantages, including strong adhesion, good biocompatibility, and ease of use.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, in particular to an elastin-like polypeptide adhesive and a preparation method and application thereof. Background Art

[0002] Tissue adhesives are biomedical materials with a certain degree of biocompatibility and adhesion. Compared to traditional fixation and suturing materials used in clinical practice, tissue adhesives offer advantages such as ease of use, minimal trauma, minimal pain during application, and minimal surgical scarring. They can also serve as carriers for the targeted release of hemostatic, antibacterial, and anti-inflammatory drugs, thus holding broad clinical application prospects. However, due to limitations in existing tissue adhesives, such as adhesion strength, biocompatibility, degradability, and safety, several currently used clinically are far from being a viable alternative to traditional fixation materials such as sutures or rivets.

[0003] From the perspective of biomimetics, the present invention takes biocompatible elastin-like polypeptides as its basis, fully exploring the huge potential of mussel adhesion chemistry in the biomedical field. An elastin-like polypeptide adhesive is prepared through a chemical modification method. The adhesive has many advantages such as high adhesion strength, good biocompatibility, and ease of use. It can provide a new option for tissue adhesives, and related products will greatly enrich the range of choices for tissue adhesive users. Summary of the Invention

[0004] The purpose of the present invention is to provide a novel elastin-like polypeptide adhesive and its preparation method and application.

[0005] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides an elastin-like polypeptide, the amino acid sequence of which is [(VPGVG)2VPGKG(VPGVG)2] 25 (as shown in SEQ ID NO: 1). Figure 1 .

[0006] In a second aspect, the present invention provides a method for preparing an elastin-like polypeptide adhesive, comprising the following steps:

[0007] (1) allowing the elastin-like polypeptide to undergo a Schiff base reaction with 2,3,4-trihydroxybenzaldehyde to obtain a pyrogallol-modified elastin-like polypeptide;

[0008] (2) The elastin-like polypeptide modified by pyrogallol is dissolved in water, an oxidant is added, and an oxidative cross-linking reaction is performed to obtain the elastin-like polypeptide hydrogel modified by pyrogallol as an adhesive.

[0009] Furthermore, step (1) is specifically as follows: reacting the elastin-like polypeptide with 2,3,4-trihydroxybenzaldehyde in an acidic medium, adding sodium cyanoborohydride (NaBH3CN) after a period of time to continue the reaction, dialyzing the obtained product, and then freeze-drying to obtain the elastin-like polypeptide modified with pyrogallol.

[0010] Preferably, the elastin-like polypeptide is reacted with 2,3,4-trihydroxybenzaldehyde in a hydrochloric acid solution at a pH of 4-5.

[0011] Preferably, the molar ratio of the elastin-like polypeptide to 2,3,4-trihydroxybenzaldehyde is (0.5-5):(1-200).

[0012] Preferably, the molar ratio of the 2,3,4-trihydroxybenzaldehyde to the sodium cyanoborohydride is 1:(1-20).

[0013] Preferably, the reaction conditions of step (1) are: 4-40° C. for 2-48 hours.

[0014] Step (2) specifically comprises: dissolving the pyrogallol-modified elastin-like polypeptide in an aqueous solution, adding an oxidant, and stirring thoroughly to obtain the pyrogallol-modified elastin-like polypeptide hydrogel.

[0015] Preferably, the oxidant in step (2) is at least one selected from sodium periodate, horseradish peroxidase, hydrogen peroxide and the like, preferably sodium periodate.

[0016] Preferably, the molar ratio of the pyrogallol-modified elastin-like polypeptide to sodium periodate is 1:(1-10).

[0017] Preferably, the reaction conditions of step (2) are: reaction at 4-40° C. for 0.05-3 h.

[0018] The synthesis diagram of the pyrogallol modified elastin-like polypeptide of the present invention is shown in Figure 2 .

[0019] In a third aspect, the present invention provides an elastin-like polypeptide adhesive prepared according to the method.

[0020] In a fourth aspect, the present invention provides any of the following uses of the elastin-like polypeptide or the elastin-like polypeptide adhesive:

[0021] 1) Used in biomaterials engineering;

[0022] 2) Used in tissue engineering;

[0023] 3) Used in medicine and environmental fields.

[0024] In a fifth aspect, the present invention provides a method for using the elastin-like polypeptide adhesive, which comprises applying the elastin-like polypeptide adhesive to a surface of a material and curing it at 10-40° C. for 0.1-3 hours. After a period of curing, a good adhesion effect is achieved.

[0025] Compared with existing adhesives, the elastin-like polypeptide adhesive provided by the present invention has at least the following advantages:

[0026] (1) The present invention is based on biocompatible elastin-like polypeptides, fully exploring the huge potential of mussel adhesion chemistry in the biomedical field. An elastin-like polypeptide adhesive is prepared by chemical modification. It has many advantages such as good adhesion strength, good biocompatibility, and ease of use.

[0027] (2) The elastin-like polypeptide used as the raw material of the present invention is an artificial polypeptide with elastin characteristics, designed and synthesized based on the amino acid sequence of natural elastin. Natural elastin is an important extracellular matrix protein whose primary function is to impart elasticity and reversible deformation to the tissues or organs in which it resides. Consequently, large quantities of elastin are present in organs that are frequently deformed by stress, such as the skin, arteries, and lungs. In addition to excellent mechanical properties, elastin-like polypeptides also possess excellent biocompatibility and non-immunogenicity. Through genetic engineering and fermentation engineering techniques, they can be produced on a large scale, offering advantages such as low cost, high yield, and ease of purification. Therefore, elastin-like polypeptides are gradually becoming a new tissue engineering material, replacing existing polymers, and are becoming one of the most promising biomaterials.

[0028] (3) Natural elastin is one of the most stable proteins in the body and has a very long half-life. Elastic fibers composed of elastin are highly cross-linked in natural tissues and thus stably exist in tissues and organs. Elastin currently on the market is usually obtained from cow and pig tissues rich in elastin. However, the elastin extracted and purified by this process is insoluble (the structure is highly cross-linked), has high instability between batches, and has a potential risk of disease transmission. Therefore, it is difficult to develop it as a standard biomaterial. Therefore, elastin-like polypeptide biomaterials constructed and expressed by genetic engineering methods have great potential. In the future, it can be widely used as regenerative medicine and tissue engineering materials in medical industries such as bioadhesives, cartilage repair, nerve regeneration, soft tissue reconstruction, and cardiovascular tissue engineering.

[0029] (4) The modified chemical group used, 2,3,4-trihydroxybenzaldehyde, is an inexpensive and readily available chemical modifier. Thanks to the high reactivity of the aldehyde group and the amino group, the reaction proceeds easily and rapidly, without the need for a catalyst, and the reaction product is non-toxic. Furthermore, the modified pyrogallol exhibits excellent adhesion to a variety of surfaces through various interactions, including covalent crosslinking, hydrogen bonding, metal coordination, and hydrophobic interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure shows the characterization results of the elastin-like polypeptide of the present invention and its molecular weight.

[0031] Figure 2 Schematic diagram of the synthesis of the pyrogallol-modified elastin-like polypeptide of the present invention.

[0032] Figure 3 These are photos of the adhesion effects of the adhesive on glass, polytetrafluoroethylene, and pigskin in a preferred embodiment of the present invention.

[0033] Figure 4 This is a photograph of the adhesion effect of the adhesive on mouse tissues and organs in a preferred embodiment of the present invention.

[0034] Figure 5 This is a microscope photograph of fibroblasts cultured with adhesive in a preferred embodiment of the present invention.

[0035] Figure 6 It is the survival rate of fibroblasts cultured with adhesive in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0036] The present invention provides a method for preparing an elastin-like polypeptide adhesive having good biocompatibility and bonding strength.

[0037] The present invention adopts the following technical solutions:

[0038] (1) Using elastin-like polypeptide as raw material, a pyrogallol-modified elastin-like polypeptide derivative is obtained by reacting with 2,3,4-trihydroxybenzaldehyde;

[0039] (2) dissolving the pyrogallol-modified elastin-like polypeptide derivative in an aqueous solution, adding an oxidant, mixing uniformly, and performing an oxidative cross-linking reaction to obtain a pyrogallol-modified elastin-like polypeptide hydrogel;

[0040] (3) Apply the hydrogel evenly on the surface of the material and solidify it for a period of time to obtain a good adhesion effect.

[0041] In the above step (1), the elastin-like polypeptide is fully dissolved in a hydrochloric acid solution, and then 2,3,4-trihydroxybenzaldehyde is added to the above solution to react, and the pH is maintained between 4 and 5. After a period of reaction, sodium cyanoborohydride is added to continue the reaction. After the reaction is completed, the product is dialyzed in deionized water and freeze-dried to obtain a pyrogallol-modified elastin-like polypeptide derivative.

[0042] In the above method, the amino acid sequence of the elastin-like polypeptide is [(VPGVG)2VPGKG(VPGVG)2] 25 , its concentration is 0.5% to 10%.

[0043] In the above method, the molar ratio of the elastin-like polypeptide to 2,3,4-trihydroxybenzaldehyde is (0.5-5):(1-200), and the molar ratio of 2,3,4-trihydroxybenzaldehyde to sodium cyanoborohydride (NaBH3CN) is 1:(1-20).

[0044] In the above method, the reaction temperature is 4 to 40° C., and the reaction time is 2 to 48 hours.

[0045] In the above step (2), the elastin-like polypeptide modified by pyrogallol is first dissolved in an aqueous solution, and then an oxidizing agent, sodium periodate, is added and stirred thoroughly. The elastin-like polypeptide hydrogel modified by pyrogallol is obtained through an oxidative cross-linking reaction.

[0046] In the above method, the molar ratio of the pyrogallol-modified elastin-like polypeptide to sodium periodate is 1:(1-10).

[0047] In the above method, the temperature of the oxidative cross-linking reaction is 4 to 40° C., and the cross-linking time is 0.05 to 3 hours.

[0048] In the above step (3), the elastin-like polypeptide hydrogel modified with pyrogallol is applied to the surface of the material and solidified after a period of time to obtain a good adhesion effect.

[0049] In the above method, the curing temperature is 10-40° C., and the curing time is 0.1-3 hours.

[0050] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0051] Example 1 Preparation of Elastin Polypeptide Adhesive

[0052] (1) Using elastin-like polypeptide as raw material, pyrogallol-modified elastin-like polypeptide derivatives were obtained by reacting with 2,3,4-trihydroxybenzaldehyde.

[0053] 1g of elastin-like polypeptide was added to 100mL of aqueous solution and stirred thoroughly until completely dissolved, yielding a colorless, clear solution. The pH was adjusted to 4 with 1M hydrochloric acid. Then, 10mL of a DMF solution containing 2g of 2,3,4-trihydroxybenzaldehyde was added dropwise to the solution and stirred at room temperature for 6 hours. 2g of NaBH3CN powder was then added and stirred at room temperature for 6 hours. After the reaction, the mixed solution was dialyzed at room temperature for 3 days. Finally, the product was freeze-dried to yield a pyrogallol-modified elastin-like polypeptide derivative.

[0054] (2) dissolving the pyrogallol-modified elastin-like polypeptide derivative in an aqueous solution, adding an oxidant, and mixing uniformly. The pyrogallol-modified elastin-like polypeptide hydrogel can be obtained by an oxidative cross-linking reaction.

[0055] Dissolve 1g of the pyrogallol-modified elastin-like polypeptide derivative in 10mL of aqueous solution and stir thoroughly until completely dissolved. Then, add 0.5mL of a 2% sodium periodate solution. Stir thoroughly at room temperature to initiate an oxidative cross-linking reaction, resulting in a hydrogel with a certain degree of biomaterial adhesion.

[0056] (3) Apply the hydrogel evenly on the surface of the material and solidify it for a period of time to obtain a good adhesion effect.

[0057] Take 1 mL of the hydrogel and apply it on the surface of the material. After 10 minutes of curing time, a good adhesion effect can be achieved.

[0058] Example 2 Preparation of Elastin-like Polypeptide Adhesive

[0059] (1) Using elastin-like polypeptide as raw material, pyrogallol-modified elastin-like polypeptide derivatives were obtained by reacting with 2,3,4-trihydroxybenzaldehyde.

[0060] 0.3 g of elastin-like polypeptide was added to 50 mL of aqueous solution and stirred thoroughly until completely dissolved, yielding a colorless, clear solution. The pH was adjusted to 4 with 1 M hydrochloric acid. Then, 5 mL of a DMF solution containing 1 g of 2,3,4-trihydroxybenzaldehyde was added dropwise to the solution and stirred at room temperature for 6 hours. 1 g of NaBH3CN powder was then added and stirred at room temperature for 6 hours. After the reaction, the mixed solution was dialyzed at room temperature for 3 days. Finally, the product was freeze-dried to yield a pyrogallol-modified elastin-like polypeptide derivative.

[0061] (2) dissolving the pyrogallol-modified elastin-like polypeptide derivative in an aqueous solution, adding an oxidant, mixing uniformly, and performing an oxidative cross-linking reaction to obtain a pyrogallol-modified elastin-like polypeptide hydrogel;

[0062] 0.5g of a pyrogallol-modified elastin-like polypeptide derivative was dissolved in 5mL of aqueous solution and stirred thoroughly until completely dissolved. Then, 0.5mL of a 2% sodium periodate solution was added. At room temperature, stirring was continued to induce an oxidative cross-linking reaction, resulting in a hydrogel with a certain degree of biomaterial adhesion.

[0063] (3) Apply the hydrogel evenly on the surface of the material and solidify it for a period of time to obtain a good adhesion effect.

[0064] 0.5 mL of the hydrogel was applied to the surface of the material and a good adhesion effect was achieved after 5 minutes of curing time. The results were similar to those in Example 1.

[0065] The photos of the adhesive prepared in Example 1 of the present invention on glass, polytetrafluoroethylene and pigskin are shown in Figure 3 .

[0066] Experimental method: Take 0.5mL of elastin-like polypeptide derivative solution and 50μL of periodic acid solution (concentration 2% w / v) and evenly mix them. Then apply them to the surface of a test plate (area area 2.5cm×2.5cm). The two plates are pressed together for 5 minutes. The resulting adhesive force can lift a weight of at least 200g.

[0067] The photo of the adhesive prepared in Example 1 of the present invention on the adhesion effect of mouse tissues and organs is shown in Figure 4 .

[0068] Experimental method: Take 0.2mL of elastin-like polypeptide derivative solution and mix it evenly with 20μL of periodic acid solution (concentration 2% w / v), and apply it on the surface of freshly extracted mouse tissues and organs, including muscles, bones, skin, heart, liver, lungs, etc. The resulting adhesive force can firmly adsorb on the moist surface of fresh tissues and organs.

[0069] Microscopic photographs of fibroblasts cultured with the adhesive prepared in Example 1 of the present invention are shown in FIG. Figure 5 .

[0070] Experimental method: Before planting fibroblasts, the adhesive was soaked in 75% ethanol overnight for disinfection, then washed with PBS to remove the ethanol and soaked in RPMI 1640 medium for 2 hours. Then mouse fibroblast L929 cells were cultured at 2×10 6 Cells were seeded on the adhesive surface at a density of 10 cells / mL and then cultured in an incubator at 37°C and 5% CO2. After 3 days, the cells were stained with a Calflavin-AM live cell detection kit (Beyotime Biotechnology / Beyotime Biotechnology Co., Ltd.) to observe the cell morphology and take pictures.

[0071] The survival rate of the fibroblasts (mouse fibroblast L929) cultured with the adhesive prepared in Example 1 of the present invention is shown in FIG. Figure 6 .

[0072] Experimental method: A standard Calflavin-AM live-dead cell detection kit (Beyotime Biotechnology / Beyotime Biotechnology Co., Ltd.) was used for detection and the cell survival rate was calculated.

[0073] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. An elastin-like polypeptide, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. A method for preparing an elastin-like polypeptide adhesive, characterized in that: The following steps are involved: (1) reacting the elastin-like polypeptide according to claim 1 with 2,3,4-trihydroxybenzaldehyde in an acidic medium, adding sodium cyanoborohydride after a period of time to continue the reaction, dialyzing the resulting product, and then freeze-drying to obtain a pyrogallol-modified elastin-like polypeptide; The molar ratio of the elastin-like polypeptide to 2,3,4-trihydroxybenzaldehyde is (0.5-5): (1-200); The molar ratio of the 2,3,4-trihydroxybenzaldehyde to the sodium cyanoborohydride is 1:(1-20); (2) dissolving the elastin-like polypeptide modified by pyrogallol in water, adding an oxidant, and performing an oxidative cross-linking reaction to obtain the elastin-like polypeptide hydrogel modified by pyrogallol as an adhesive; The oxidant is selected from at least one of sodium periodate, horseradish peroxidase, and hydrogen peroxide; The molar ratio of the pyrogallol-modified elastin-like polypeptide to sodium periodate is 1:(1-10).

3. The method according to claim 2, characterized in that Step (1) reacting the elastin-like polypeptide with 2,3,4-trihydroxybenzaldehyde in a hydrochloric acid solution at pH 4-5.

4. The method according to claim 2, characterized in that The reaction conditions of step (1) are: 4-40°C for 2-48 hours.

5. The method according to claim 2, characterized in that The reaction conditions of step (2) are: 4-40°C for 0.05-3h.

6. An elastin-like polypeptide adhesive prepared according to the method of any one of claims 2 to 5.

7. Any of the following uses of the elastin-like polypeptide according to claim 1 or the elastin-like polypeptide adhesive according to claim 6: 1) Used in biomaterials engineering; 2) Used in tissue engineering; 3) Used in medicine and environmental fields; The applications are for non-disease diagnosis and treatment purposes.

8. The method for using the elastin-like polypeptide adhesive according to claim 6, characterized in that: The elastin-like polypeptide adhesive is applied to the surface of the material and cured at 10-40° C. for 0.1-3 hours.

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