Polyphenol-based tissue adhesive for promoting wound healing and preparation method thereof

Abstract

The invention discloses a polyphenol-based tissue adhesive for promoting wound healing and a preparation method thereof, and relates to the technical field of medical tissue adhesives. The preparation method of the tissue adhesive comprises the following steps: step 1, preparing a polyphenol compound and recombinant mussel mucin into a solution, and uniformly stirring for later use, namely a solution A; 2, preparing a temperature response type high polymer material into a solution for later use, namely a solution B; and 3, mixing the solution A prepared in the step 1 and the solution B prepared in the step 2, fully and uniformly mixing, shaking and centrifuging, and discarding the supernatant. By selecting the temperature response type high polymer material, the tissue adhesive is endowed with the characteristic of temperature sensitivity, so that the modulus of the adhesive at the human body temperature is improved, and the colloid contraction can further promote wound closure. The mussel mucin is introduced to stimulate collagen regeneration of the wound part, activate the immune mechanism and remodel the wound microenvironment so as to achieve the purpose of promoting healing.

Description

Technical Field

[0001] The present invention relates to the technical field of medical tissue adhesives, and particularly relates to a polyphenol-based tissue adhesive for promoting wound healing and a preparation method thereof. Background Art

[0002] Existing medical adhesives, such as fibrin glue, although having certain adhesiveness and biocompatibility, have unstable performance in a humid environment, limited adhesion strength, and cannot achieve rapid curing and reuse. Polyphenolic compounds can form various covalent and non-covalent interactions with human tissues relying on high-density phenolic hydroxyl groups, enhancing the adhesion effect with tissues. However, the repair effect of a single polyphenol-based material on wounds is still limited, and new functional components need to be introduced to enhance the repair effect of the adhesive on wounds.

[0003] As a key component in natural mussels, mussel adhesive protein not only maintains underwater adhesion but also has the functions of repair and anti-inflammation. The content of 3,4-dihydroxy-L-phenylalanine (DOPA) in mussel adhesive protein is relatively high, and DOPA can form a microscopic network biological scaffold through oxidative cross-linking under physiological conditions. This scaffold structure can attract negatively charged cells (such as epidermal cells and fibroblasts), promote cell adhesion and growth, and ultimately achieve the purpose of promoting wound healing. Related research also shows that mussel adhesive protein has a promoting effect on wound healing and repair.

[0004] In addition, some polymer materials with a lower critical phase transition temperature can remain hydrophilic at a lower temperature or room temperature, and become hydrophobic when close to or higher than the human body temperature. This property can be used to prepare adhesive materials, so that the adhesive becomes hydrophobic and shrinks at the human body temperature. Through its unique mechanical contraction property, simulating the wound contraction mechanism in the natural healing process of the skin, it can effectively reduce the wound area and promote wound healing.

[0005] Therefore, it is of great significance to prepare such a tissue adhesive with temperature-responsive characteristics, stable adhesion and effective promotion of wound healing. Summary of the Invention

[0006] To overcome the technical defects of existing tissue adhesives, the present invention provides a polyphenol-based tissue adhesive for promoting wound healing and its preparation method. The adhesive is an adhesive based on polyphenol-temperature-responsive polymer-mussel adhesive protein. Among them, polyphenol materials can rapidly form aggregates with certain adhesion properties through hydrogen bonds and hydrophobic interactions with some temperature-responsive polymer materials. However, although the adhesive formed by polyphenol-polymer materials can be used to adhere to and close the wound site, its effect on promoting wound repair is still limited. The primary object of the present invention is to provide a polyphenol-based tissue adhesive for promoting wound healing and its preparation method. By adding recombinant mussel adhesive protein to the tissue adhesive composed of polyphenol-polymer materials, a medical tissue adhesive material with enhanced repair function is obtained. Recombinant mussel adhesive protein provides a good attachment matrix for newborn cells, promotes the adhesion and proliferation of fibroblasts, keratinocytes, etc., while reducing the inflammatory response and stimulating the synthesis of new collagen, thereby achieving the purpose of promoting wound healing.

[0007] Specifically, it includes the following technical features: First, a preparation method of a polyphenol-based tissue adhesive for promoting wound healing is provided, including the following steps: Step 1: Prepare a solution by mixing a polyphenol compound and recombinant mussel adhesive protein, stir evenly, and set aside, which is called solution A; Step 2: Prepare a solution of the temperature-responsive polymer material and set aside, which is called solution B; Step 3: Mix the solution A prepared in Step 1 and the solution B prepared in Step 2, mix well, centrifuge with shaking, and discard the supernatant to obtain a polyphenol-based tissue adhesive for promoting wound healing.

[0008] Preferably, in Step 1, the polyphenol compound and recombinant mussel adhesive protein are prepared into a solution by adding water.

[0009] Preferably, in Step 2, the temperature-responsive polymer material is prepared into a solution by adding water.

[0010] Furthermore, in Step 1, the polyphenol compound includes at least one of catechol, dopamine, 1-(3,4-dihydroxyphenyl)-2-aminoethanol, flavonol, ellagic acid, chrysin, hesperetin, kaempferol, morin, catechin, luteolin, quercetin, pyrogallol, gallic acid, baicalin, 1,2,4-benzenetriol, tannic acid, epigallocatechin gallate, epicatechin gallate, epigallocatechin.

[0011] Further, in step two, the temperature-responsive polymer material includes at least one of poly(N-isopropylacrylamide), poly(N,N-diethylacrylamide), poly(dimethylaminoethyl methacrylate), Pluronic F127 (PF127), poly(N-vinylcaprolactam), and poly(oligoethylene glycol monomethyl ether (meth)acrylate).

[0012] Further, in step one, the recombinant mussel adhesive protein is type V.

[0013] Preferably, in step one, the recombinant mussel adhesive protein is type V (Mfp5).

[0014] Further, the concentration of the polyphenol compound in the solution of step one is 10 - 500 mg / mL.

[0015] Further, the concentration of the recombinant mussel adhesive protein in the solution of step one is 1 - 50 mg / mL.

[0016] Further, in step one, the molecular weight of the recombinant mussel adhesive protein is 8 - 10 kDa.

[0017] Further, the concentration of the temperature-responsive polymer material in the solution of step two is 5 - 200 mg / mL.

[0018] Further, the concentration of the recombinant mussel adhesive protein in the solution of step one is 1 - 10 mg / mL.

[0019] In a second aspect, a polyphenol-based tissue adhesive for promoting wound healing is provided, which is prepared by the method for preparing a polyphenol-based tissue adhesive for promoting wound healing described in the first aspect.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a polyphenol-based tissue adhesive for promoting wound healing and a preparation method thereof. By selecting a polyphenol compound and a temperature-responsive polymer material as the condensate matrix and introducing a recombinant mussel adhesive protein, the temperature-responsive polymer material endows the tissue adhesive with temperature-sensitive properties, making the modulus of the tissue adhesive increase at human body temperature, and the colloid shrinkage can further promote wound closure. The introduction of the recombinant mussel adhesive protein can stimulate collagen regeneration at the wound site, activate the immune mechanism, and reshape the wound microenvironment to achieve the purpose of promoting healing. At the animal experiment level, it can be clearly observed that the introduction of the recombinant mussel adhesive protein effectively accelerates the wound healing process. Description of the Drawings

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other accompanying drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 It is a graph of the modulus change of the polyphenol-based tissue adhesive for promoting wound healing prepared in Example 1 of the present invention; Figure 2 It is a graph of the modulus change of the polyphenol-based tissue adhesive for promoting wound healing prepared in Comparative Example 6 of the present invention; Figure 3 It is an effect diagram of wound repair of the polyphenol-based tissue adhesive for promoting wound healing prepared in Examples 1-3 and Comparative Example 1 of the present invention; Figure 4 It is an effect diagram of wound repair of the polyphenol-based tissue adhesive for promoting wound healing prepared in Comparative Examples 1-2 of the present invention. Detailed implementation manners

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0024] It should be understood that when used in this specification and the appended claims, the terms "comprises" and "comprising" indicate the presence of the described features, wholes, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations.

[0025] It should also be understood that the terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in this specification of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" are intended to include the plural forms.

[0026] It should be further understood that the term " / and" as used in this specification of the present invention and the appended claims refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0027] To more fully understand the technical content of the present invention, the technical solutions of the present invention will be further introduced and illustrated below with specific embodiments.

[0028] In Step 1 of the examples and comparative examples of the present invention, the molecular weight of the recombinant mussel adhesive protein is 9 kDa.

[0029] Example 1 A polyphenol-based tissue adhesive for promoting wound healing, comprising a polyphenol compound, a recombinant mussel adhesive protein, and a temperature-responsive polymer material; the polyphenol compound is tannic acid, the recombinant mussel adhesive protein is Mfp5, and the temperature-responsive polymer material is PF127.

[0030] The preparation method of the polyphenol-based tissue adhesive for promoting wound healing includes the following steps: Step 1: Mix the polyphenol compound and the recombinant mussel adhesive protein with water to form a solution such that the concentration of the polyphenol compound in the solution is 250 mg / mL and the concentration of the recombinant mussel adhesive protein in the solution is 10 mg / mL. After stirring evenly, set aside and call it Solution A; Step 2: Dissolve the temperature-responsive polymer material in water to form a solution such that the concentration of the temperature-responsive polymer material in the solution is 100 mg / mL. Set aside and call it Solution B; Step 3: Mix Solution A prepared in Step 1 and Solution B prepared in Step 2, mix well, centrifuge by shaking, and discard the supernatant to obtain the polyphenol-based tissue adhesive for promoting wound healing.

[0031] Example 2 A polyphenol-based tissue adhesive for promoting wound healing, comprising a polyphenol compound, a recombinant mussel adhesive protein, and a temperature-responsive polymer material; the polyphenol compound is tannic acid, the recombinant mussel adhesive protein is Mfp5, and the temperature-responsive polymer material is PF127.

[0032] The preparation method of the polyphenol-based tissue adhesive for promoting wound healing includes the following steps: Step 1: Mix the polyphenol compound and the recombinant mussel adhesive protein with water to form a solution such that the concentration of the polyphenol compound in the solution is 250 mg / mL and the concentration of the recombinant mussel adhesive protein in the solution is 5 mg / mL. After stirring evenly, set aside and call it Solution A; Step 2: Dissolve the temperature-responsive polymer material in water to form a solution such that the concentration of the temperature-responsive polymer material in the solution is 100 mg / mL. Set aside and call it Solution B; Step 3: Mix Solution A prepared in Step 1 and Solution B prepared in Step 2, mix well, centrifuge by shaking, and discard the supernatant to obtain the polyphenol-based tissue adhesive for promoting wound healing.

[0033] Example 3 A polyphenol-based tissue adhesive for promoting wound healing, comprising a polyphenol compound, recombinant mussel adhesive protein, and a temperature-responsive polymer material; the polyphenol compound is tannic acid, the recombinant mussel adhesive protein is Mfp5, and the temperature-responsive polymer material is PF127.

[0034] The preparation method of the polyphenol-based tissue adhesive for promoting wound healing comprises the following steps: Step 1: Mix the polyphenol compound and the recombinant mussel adhesive protein with water to form a solution such that the concentration of the polyphenol compound in the solution is 250 mg / mL and the concentration of the recombinant mussel adhesive protein in the solution is 1 mg / mL. After stirring evenly, set aside, and call it Solution A; Step 2: Dissolve the temperature-responsive polymer material in water to form a solution such that the concentration of the temperature-responsive polymer material in the solution is 100 mg / mL. Set aside, and call it Solution B; Step 3: Mix the Solution A prepared in Step 1 and the Solution B prepared in Step 2, mix well, centrifuge with vibration, and discard the supernatant to obtain the polyphenol-based tissue adhesive for promoting wound healing.

[0035] Comparative Example 1 Comparative Example 1 provides a preparation method of a polyphenol-based tissue adhesive for promoting wound healing. Compared with Example 1, the difference is that the recombinant mussel adhesive protein is not added in Step 1, and the other conditions are the same.

[0036] Comparative Example 2 Comparative Example 2 provides a preparation method of a polyphenol-based tissue adhesive for promoting wound healing. Compared with Example 1, the difference is that the concentration of the recombinant mussel adhesive protein in the solution in Step 1 is 0.1 mg / mL, and the other conditions are the same.

[0037] Comparative Example 3 Comparative Example 3 provides a preparation method of a polyphenol-based tissue adhesive for promoting wound healing. Compared with Example 1, the difference is that the concentration of the recombinant mussel adhesive protein in the solution in Step 1 is 55 mg / mL, and the other conditions are the same.

[0038] Comparative Example 4 Comparative Example 4 provides a preparation method of a polyphenol-based tissue adhesive for promoting wound healing. Compared with Example 1, the difference is that the concentration of the polyphenol compound in the solution in Step 1 is 5 mg / mL, and the other conditions are the same.

[0039] Comparative Example 5 Comparative Example 5 provides a preparation method of a polyphenol-based tissue adhesive for promoting wound healing. Compared with Example 1, the difference is that the concentration of the temperature-responsive polymer material in the solution in Step 2 is 1 mg / mL, and the other conditions are the same.

[0040] Comparative Example 6 Comparative Example 6 provides a method for preparing a polyphenol-based tissue adhesive for promoting wound healing. Compared with Example 1, the difference lies in replacing the temperature-responsive polymer with polyethylene glycol, that is, the tissue adhesive does not contain a temperature-responsive polymer, and the other conditions are the same.

[0041] Performance test: I. Temperature sensitivity test To study the temperature-sensitive properties of the tissue adhesive, the prepared polyphenol-based tissue adhesive for promoting wound healing was subjected to rheological tests. The tissue adhesive (150 μL) was placed on the rheometer plate, and the frequency range was set from 0.1 to 10 Hz. Frequency sweep tests were carried out at room temperature and 37 °C respectively. The storage modulus (solid-like property, G') and loss modulus (liquid-like property, G'') were recorded. The temperature-sensitive shrinkage characteristics of the adhesive were reflected by the change of modulus at different temperatures; II. Animal wound repair performance To study the repair effect of the tissue adhesive on wounds, a skin incision closure model was constructed using BALB / C mice. After anesthesia with isoflurane, an incision with a length of 1 cm was made on the back skin of each mouse. Those without subsequent treatment were used as the control group, and the other experimental groups were treated with the tissue adhesives obtained from the examples or comparative examples for wound closure. Photos were taken at 0 (before and after treatment), 3, 5, and 7 days to record the wound healing effect.

[0042] The temperature sensitivity test was carried out on the polyphenol-based tissue adhesive for promoting wound healing prepared by the method of Example 1, and the test results are as Figure 1 shown. Figure 1 This is the modulus change diagram of the polyphenol-based tissue adhesive for promoting wound healing prepared in Example 1 of the present invention. As Figure 1 shown, as the temperature changes from room temperature to body temperature (37 °C), both the storage modulus and loss modulus of the tissue adhesive increase significantly, indicating that the tissue adhesive of the embodiment of the present invention can respond to temperature changes. At 37 °C, the mechanical properties of the gel improve, the modulus increases, and further the colloid shrinks.

[0043] The temperature sensitivity test was carried out on the polyphenol-based tissue adhesive for promoting wound healing prepared by the method of Comparative Example 6, and the test results are as Figure 2 shown. Figure 2 This is the modulus change diagram of the polyphenol-based tissue adhesive for promoting wound healing prepared in Comparative Example 6 of the present invention. As Figure 2As shown, as the temperature changes from room temperature to human body temperature (37 °C), both the storage modulus and loss modulus of the tissue adhesive hardly change, indicating that the tissue adhesive of the present invention endows the tissue adhesive with the characteristic of temperature-sensitive shrinkage through temperature-responsive materials, and the tissue adhesive lacking temperature-responsive materials cannot respond to temperature changes.

[0044] The polyphenol-based tissue adhesives for promoting wound healing prepared by the preparation methods of Examples 1-3 and Comparative Example 1 were tested for their animal wound repair performance, and the test results are as Figure 3 shown. Figure 3 It is the wound repair effect diagram of the polyphenol-based tissue adhesives for promoting wound healing prepared by Examples 1-3 and Comparative Example 1 of the present invention. As Figure 3 shown, compared with the control group, the tissue adhesive-added groups (Examples 1, 2, 3 and Comparative Example 1) can all adhere to and close the wound in time. Compared with the control group (untreated) and the group without added recombinant mussel adhesive protein (Comparative Example 1), with the introduction of recombinant mussel adhesive protein (Examples 1, 2 and 3), the repair effect of the tissue adhesive on the wound is gradually improved. The effect of Example 1 is the best, and the wound has basically healed after 7 days.

[0045] The polyphenol-based tissue adhesives for promoting wound healing prepared by the preparation methods of Comparative Example 1 and Comparative Example 2 were tested for their animal wound repair performance, and the test results are as Figure 4 shown. As Figure 4 shown, compared with the control group, the tissue adhesive-added group (Comparative Example 2) can adhere to and close the wound in time. Compared with Comparative Example 1 (without added recombinant mussel protein), it has a certain wound healing promoting effect. Since the concentration of recombinant mussel adhesive protein in the preparation method of Comparative Example 2 is not within the range defined by the present invention, the repair effect of the tissue adhesive prepared thereby on the wound is not as good as that of the tissue adhesive of Example 1, but better than that of Comparative Example 1 without added recombinant mussel adhesive protein. Therefore, the concentration of recombinant mussel adhesive protein in the present invention is preferably 1-10 mg / mL.

[0046] In addition, since the adhesion effect of the polyphenol-based tissue adhesives for promoting wound healing prepared by the preparation methods of Comparative Examples 3-5 is poor, the animal wound repair performance test cannot be carried out. It can be seen that the present invention forms a condensate with certain adhesion performance through polyphenol compounds and temperature-responsive polymer materials, and stimulates collagen regeneration at the wound site and remodels the wound microenvironment by introducing recombinant mussel adhesive protein to achieve the purpose of promoting wound healing. The adhesion effect of the polyphenol-based tissue adhesives for promoting wound healing prepared by using recombinant mussel adhesive protein, polyphenol compounds or temperature-responsive polymer materials not within the range defined by the present invention is poor. Therefore, the concentration of the temperature-responsive polymer material in the present invention is preferably 5-200 mg / mL. The concentration of the polyphenol compound in the present invention is preferably 10-500 mg / mL.

[0047] In summary, the present invention provides a polyphenol-based tissue adhesive for promoting wound healing and a preparation method thereof. Through the synergistic effect of polyphenol compounds, temperature-responsive polymer materials and recombinant mussel adhesive protein, the tissue adhesive has a certain ability to adhere to and close wounds. At the same time, the temperature-responsive material endows the tissue adhesive with the property of thermosensitive contraction. When acting on the wound site, it further contracts the wound site on the basis of adhesion to help close the wound. On the other hand, the introduction of recombinant mussel adhesive protein stimulates tissue regeneration at the wound site and activates the immune mechanism, accelerating the wound repair process.

[0048] As described above, the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of various equivalent modifications or substitutions, and these modifications or substitutions should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A preparation method of a polyphenol-based tissue adhesive for promoting wound healing, characterized in that, It includes the following steps: Step 1: Prepare a solution by configuring polyphenolic compounds and recombinant mussel adhesive protein, stir evenly, and set aside, which is called solution A; Step 2: Prepare a solution by configuring a temperature-responsive polymer material, and set aside, which is called solution B; Step 3: Mix the solution A prepared in Step 1 and the solution B prepared in Step 2, mix well, centrifuge by shaking, and discard the supernatant to obtain a polyphenol-based tissue adhesive for promoting wound healing.

2. The preparation method of the polyphenol-based tissue adhesive for promoting wound healing according to claim 1, wherein, In Step 1, the polyphenolic compounds include at least one of catechol, dopamine, 1-(3,4-dihydroxyphenyl)-2-aminoethanol, flavonol, ellagic acid, chrysin, hesperetin, kaempferol, morin, catechin, luteolin, quercetin, pyrogallol, gallic acid, baicalin, 1,2,4-benzenetriol, tannic acid, epigallocatechin gallate, epicatechin gallate, epigallocatechin.

3. The preparation method of the polyphenol-based tissue adhesive for promoting wound healing according to claim 1, characterized in that, In Step 2, the temperature-responsive polymer material includes at least one of poly(N-isopropylacrylamide), poly(N,N-diethylacrylamide), poly(dimethylaminoethyl methacrylate), Pluronic F127, poly(N-vinylcaprolactam), poly(oligoethylene glycol monomethyl ether (meth)acrylate).

4. The preparation method of the polyphenol-based tissue adhesive for promoting wound healing according to claim 1, characterized in that, In Step 1, the recombinant mussel adhesive protein is type V.

5. The preparation method of the polyphenol-based tissue adhesive for promoting wound healing according to claim 1, characterized in that, The concentration of the polyphenolic compounds in the solution of Step 1 is 10 - 500 mg / mL.

6. The preparation method of the polyphenol-based tissue adhesive for promoting wound healing according to claim 1, characterized in that, The concentration of the recombinant mussel adhesive protein in the solution of Step 1 is 1 - 50 mg / mL.

7. The preparation method of the polyphenol-based tissue adhesive for promoting wound healing according to claim 1, characterized in that, In Step 1, the molecular weight of the recombinant mussel adhesive protein is 8 - 10 kDa.

8. The preparation method of the polyphenol-based tissue adhesive for promoting wound healing according to claim 1, characterized in that, The concentration of the temperature-responsive polymer material in the solution of Step 2 is 5 - 200 mg / mL.

9. The preparation method of the polyphenol-based tissue adhesive for promoting wound healing according to claim 6, characterized in that, The concentration of the recombinant mussel adhesive protein in the solution of Step 1 is 1 - 10 mg / mL.

10. A polyphenol-based tissue adhesive for promoting wound healing, characterized in that, It is prepared by the preparation method of the polyphenol-based tissue adhesive for promoting wound healing according to any one of claims 1 - 9.

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