WO2023088415A1 - Andrias davidianus skin secretion hydrolysate, and preparation method therefor and application thereof - Google Patents

Abstract

An Andrias davidianus skin secretion hydrolysate, and a preparation method therefor and an application thereof The preparation method comprises: S1: mixing a disulfide bond reducing agent and a first solvent to obtain a second solvent; S2: mixing Andrias davidianus skin secretion freeze-dried powder with the second solvent to obtain a first mixed solution; S3: fully hydrolyzing the first mixed solution to obtain a second mixed solution; S4: centrifuging the second mixed solution and collecting a supernatant; and S5: putting the supernatant into a dialysis bag having a molecular weight cut-off of less than or equal to 1000 and placing the dialysis bag in the first solvent for full dialysis to obtain the Andrias davidianus skin secretion hydrolysate, wherein the disulfide bond reducing agent is tris(2-carboxyethyl)phosphine hydrochloride. The Andrias davidianus skin secretion hydrolysate has adjustable viscosity, good biocompatibility, and regeneration promotion and coarse proliferation promotion capabilities, and is used for preparing adhesives, a wound dressings and photosensitive hydrogels.

Description

A kind of hydrolyzate of salamander skin secretion and its preparation method and application

This application claims the priority of the Chinese invention patent application [CN2021113742137] filed on November 19, 2021, titled "A Preparation Method for Hydrolyzate of Salamander Skin Exudation", which is incorporated by reference in its entirety .

technical field

The invention relates to the field of biological materials, in particular to a hydrolyzate of salamander skin secretions and a preparation method and application thereof.

Background technique

Wound closure is critical in surgical procedures. Clinically, 60% of wounds are fixed by sutures or staples after surgery. Due to soft tissue fragility and local stress, mechanical closure may cause secondary injury and require removal of non-degradable sutures or staples. When suturing external injuries, hypertrophic scars are often caused due to poor alignment, excessive skin treatment, and deep skin abrasions.

To date, most of the adhesives available in the art for wound closure have limitations. For example, cyanoacrylate (CA), the most widely used commercial chemically synthesized adhesive, despite its excellent bond strength, cannot be ignored due to its cytotoxicity and non-biodegradability. In addition, cyanoacrylate has poor elasticity and cannot adapt to the adhesion of soft tissues with large mobility such as joints and skin. Compared with cyanoacrylate, adhesives of biological origin (such as fibrin glue, gelatin, hyaluronic acid, etc.) have good biocompatibility, but their adhesive strength is low, and they can only be used as sutures when used It cannot be used alone, which limits its application.

Contents of the invention

In a first aspect, the present invention provides a method for preparing a hydrolyzate of salamander skin secretion, comprising the following steps:

S1 mixes the disulfide bond reducing agent and the first solvent to obtain the second solvent;

S2 mixing the lyophilized powder of salamander skin secretion with the second solvent to obtain a first mixed solution;

S3 fully hydrolyzing the first mixed solution to obtain a second mixed solution;

S4 centrifuging the second mixed solution and collecting the supernatant;

S5 put the supernatant into a dialysis bag with a molecular weight cut-off ≤ 1000 and place it in the first solvent for sufficient dialysis to obtain a hydrolyzate of the salamander skin secretion;

Wherein the disulfide bond reducing agent includes a specific disulfide bond reducing agent, the specific disulfide bond reducing agent is tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl), and the first solvent Including one or more of ultrapure water, deionized water, water for injection, and pure water.

In some embodiments, the mass ratio of the first solvent to the lyophilized powder of salamander skin secretion is at least 4:1.

In some embodiments, the mass ratio of the lyophilized salamander skin secretion powder to the disulfide bond reducing agent is at least 3:1.

In some embodiments, the hydrolysis time of the hydrolysis comprises 2-168 hours.

In some embodiments, the hydrolysis time of the hydrolysis is 12-48 hours.

In some embodiments, the temperature conditions of S3, S4 and S5 include 0-8°C.

In some embodiments, the temperature conditions of S3, S4 and S5 are 0-4°C.

In some embodiments, the S3 is performed in a vacuum reactor, and the vacuum degree of the vacuum reactor is less than 5kPa.

In some embodiments, the disulfide bond reducing agent further includes vitamin C.

In some embodiments, the dialysis time of the dialysis includes 48 hours.

In some embodiments, the obtained concentration of the salamander skin secretion hydrolyzate includes 3%-7%.

In some embodiments, the method further includes S6 concentrating the hydrolyzate of salamander skin secretion to obtain a concentrated hydrolyzate of salamander skin secretion.

In some embodiments, the concentration method includes putting the hydrolyzate of the salamander skin secretion into a concentrated centrifuge tube with a molecular weight cut-off ≤ 3K for centrifugation or evaporating and concentrating the hydrolyzate of the salamander skin secretion.

In the second aspect, the present invention also provides a hydrolyzate of salamander skin secretion, which is characterized in that the hydrolyzate of salamander skin secretion is prepared by the above method.

In the third aspect, the present invention also provides the use of the hydrolyzate of salamander skin secretion in the preparation of bioadhesive, characterized in that the hydrolyzate of salamander skin secretion is prepared by the above method.

In some embodiments, the concentration of the salamander skin secretion hydrolyzate includes 5%-7%.

In some embodiments, after the bioadhesive is applied to the wound, the bioadhesive penetrates into the tissue of the wound by itself or through external pressure to form a microgel structure.

In some embodiments, the wound comprises a skin wound.

In some embodiments, the bioadhesive is used to promote healing of the wound.

In the fourth aspect, the present invention also provides the use of a hydrolyzate of salamander skin secretion in preparing a wound dressing, which is characterized in that the hydrolyzate of salamander skin secretion is prepared by the above method.

In the fifth aspect, the present invention also provides a use of a hydrolyzate of salamander skin secretion in the preparation of a photosensitive hydrogel, characterized in that the photosensitive hydrogel includes a concentrated hydrolyzate of salamander skin secretion and a photoinitiator. Under the irradiation of visible light, the concentrated salamander skin secretion hydrolyzate is solidified to form the photosensitive hydrogel; the concentrated salamander skin secretion hydrolyzate is prepared by the above method, and the photoinitiator includes Ru/SPS system .

In some embodiments, the intensity of the visible light comprises 7000W.

In some embodiments, the photosensitive hydrogel is used as a hemostatic agent.

In some embodiments, the photosensitive hydrogel is used as a bioadhesive.

In some embodiments, the photosensitive hydrogel is used as a wound dressing.

As used herein, the term "bioadhesive" refers to a biocompatible substance that can adhere to and remain in biological tissue. In some embodiments, the bioadhesive is bioglue. In some embodiments, the bioadhesive can replace sutures/staples to seal wounds and stop bleeding. In some embodiments, the bioadhesive also has the functions of anti-infection, promoting healing, and inhibiting scar hyperplasia. The salamander skin secretion hydrolyzate prepared by the preparation method provided by the present invention can be used as a bioadhesive to achieve effects such as adhesion, wound closure (even hemostasis, antibacterial) and promotion of wound healing, which can reduce the time required for suturing and technical sensitivity, reduce the difficulty of postoperative care, avoid secondary damage to patients caused by the removal of sutures/staples, and reduce the number and time of patient visits.

As used herein, the term "hydrogel" refers to a natural or synthetic polymer network that is highly absorbent (eg, can absorb and/or retain large volumes of water).

As used in the present invention, the term "microgel" refers to hydrogel particles with an intramolecular cross-linked structure and a micron-scale particle size.

As used in the present invention, the term "wound dressing" refers to a material used to cover the surface of a wound and to treat or protect the wound. In some embodiments, the wound dressings are divided into: (1) passive dressings (i.e. traditional dressings), which passively cover the wound surface and absorb exudate, and provide limited protection for the wound surface; (2) interactive dressings, There are many forms of interaction between the dressing and the wound surface, such as absorbing exudate and toxic substances, allowing gas exchange, thereby creating an ideal environment for healing; providing a barrier outer structure to prevent the intrusion of microorganisms in the environment, preventing Wound cross-infection, etc. The salamander skin secretion hydrolyzate and the photosensitive hydrogel formed by the invention belong to the interactive dressing.

As used herein, the term "biological tissue" or "tissue" refers to any tissue of or derived from a living or dead organism. A biological tissue can include any single tissue (eg, a collection of cells that can be interconnected) or a group of tissues that make up an organ or part or region of an organism. The biological tissue includes one or more animal tissues among mucosal tissue, epidermal tissue, dermal tissue and subcutaneous tissue. Different biological tissues can form organs with specific functions. Biological Tissue The organism associated or derived (ie, subject or individual) can be any animal, including mammals and non-mammals (eg, invertebrates). Biological tissue may be intact, or may have one or more cuts, tears, defects, or other types of wounds. In some embodiments, the biological tissue is mammalian tissue.

As used herein, "wound" refers to a physical disruption of the continuity or integrity of a biological tissue structure. Wounds may result from cuts, abrasions, avulsions, lacerations, punctures, cancer, diabetic ulcers or lesions, burns, surgery, or other injuries. In some embodiments, the wound may result in bleeding.

As used herein, "wound healing" refers to partial or complete restoration of tissue integrity. In some embodiments, the tissue is skin, ie the wound is a skin wound, such as a dermal or epidermal wound. In some embodiments, wound healing includes temporal and spatial healing programs that include wound closure and processes involving wound closure. "Promoting wound healing" is understood as restoring tissue from a break in continuity or integrity. In some embodiments, promoting wound healing includes achieving the same degree of healing with less healing time required. In some embodiments, promoting wound healing includes achieving a greater degree of healing within the same healing time. Specifically, one or more of: neovascularization; migration of fibroblasts, endothelial cells, and epithelial cells; extracellular matrix deposition; epidermal replantation; and remodeling may be involved.

As used herein, the term "wound closure" refers to the closure of a wound, including closing the wound without exposing the internal tissues. In some embodiments, the wound closure includes rejoining the sides of the wound to form a continuous barrier (eg, intact skin). In some embodiments, the wound closure further comprises hemostasis.

As used herein, "full-thickness wound" refers to a full-thickness disruption of the dermis (and epidermis).

As used herein, "scar" refers to the appearance, morphology and histopathological changes of tissues caused by trauma, which is an inevitable product of the body's wound repair process. When the scar grows beyond a certain limit, various complications may occur (such as the destruction of appearance and functional disturbance, etc.), which will bring huge physical pain and mental pain to the patient (especially burns, scalds, and scars left after severe trauma) scar). "Scarless healing" means less, significantly less or no scarring that would result from natural wound healing.

As used herein, the term "biocompatible" refers to a material that is substantially non-toxic in its intended in vivo environment and is not substantially rejected by an individual's physiological system. The evaluation of biocompatibility of biomaterials should follow the two principles of biosafety and biofunctionality, which require that biomaterials have very low toxicity, and at the same time require that biomaterials can properly stimulate the corresponding functions of the body in specific applications (such as , the material interacts with the body environment). In some embodiments, the biocompatibility meets FDA requirements. The immune response and tissue repair process in the body are very complex, and it is often not enough to determine the biocompatibility of a certain material through a cell or tissue. The present invention refers to the requirements of the International Standards Organization (International Standards Organization, ISO) 10993 and the national standard GB/T16886, through a series of in vitro and in vivo experiments, it is verified that the hydrolyzate of salamander skin secretion has a higher effect on organisms (especially the wound environment). biocompatibility.

As used herein, "regeneration" is a repair process in which a part of a tissue or organ is lost due to external effects, and a structure identical in shape and/or function to the missing part grows on the basis of the remaining part.

As used herein, "biogenic" means derived from or taken from naturally occurring organisms and parts of organisms. In other words, the freeze-dried powder of salamander skin secretion and the hydrolyzate of salamander skin secretion involved in the present invention are not obtained through gene recombination technology.

As used herein, the term "substantially free of" or "substantially free of" means that the referred substance is absent or present in a certain substance in an amount that cannot be detected analytically.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the description of the embodiments or the prior art. Apparently, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can obtain other drawings according to these drawings without any creative effort.

Fig. 1 is the representative physical picture of the hydrogel formed by the lyophilized powder of salamander skin secretion and water;

Figure 2a is a representative image of salamander skin secretion hydrolyzate before hydrolysis, after hydrolysis and the obtained viscous supernatant;

Figure 2b is a representative image of the supernatant in Figure 2a before, during and after dialysis;

Fig. 2c is a representative physical figure of viscous salamander skin secretion hydrolyzate provided by the present invention;

Fig. 3a and Fig. 3c are the experimental result diagrams of detecting the cytotoxicity of salamander skin secretion hydrolyzate;

Figure 3b shows the high cytocompatibility of the hydrolyzate of the TCEP group of the present invention;

Figure 3d and Figure 3e are the experimental results of the expression of angiogenesis-related genes in cells promoted by the hydrolyzate of salamander skin secretions on the 7th day and 14th day of culture respectively;

Figure 3f shows the scratch test results of vitamin C group and TCEP group;

Fig. 3 g shows the Transwell experiment result of vitamin C group and TCEP group;

Figure 3h shows the permeability of salamander skin secretion hydrolyzate;

Figure 3i shows a representative SEM image of the bonding interface of salamander skin secretion hydrolyzate to pig skin;

Fig. 4 a is the macroscopic experimental result figure of the skin incision healing experiment of each experimental group (blank control group, hydrolyzate group, fibrin adhesive group, suturing group, cyanoacrylate group);

Fig. 4 b is the experimental result figure of the HE staining of the skin wound healing experiment of each experimental group (blank control group, hydrolyzate group, fibrin adhesive group, cyanoacrylate group);

Figure 4c is the macroscopic experimental results of the skin incision healing experiment of each experimental group (blank control group, vitamin C group, TCEP group, fibrin adhesive (FB) group, cyanoacrylate (CA) group and suture group) ;

Figure 4d is the HE staining experiment of the skin wound healing experiment of each experimental group (blank control group, vitamin C group, TCEP group, fibrin adhesive (FB) group, cyanoacrylate (CA) group and suture group) Result graph;

Figure 5a is a diagram of the experimental results of the co-culture of salamander skin secretion hydrolyzate + Ru/SPS photosensitizer and cells;

Figure 5b shows the hemostatic effect of the photosensitive hydrogel formed by curing salamander skin secretion hydrolyzate + photosensitizer Ru/SPS (abbreviated as photosensitive hydrogel) as a photosensitive hemostatic agent;

Figure 5c shows the effect of photosensitive hydrogel as a wound dressing;

Figure 6a shows the macroscopic effect diagram of salamander skin secretion hydrolyzate used as a liquid wound dressing to promote wound healing of full-thickness skin defects;

Fig. 6 b shows the experimental result figure of the HE staining of the wound healing experiment of skin full-thickness defect promoting the salamander skin secretion hydrolyzate as liquid wound dressing;

Fig. 6c shows the macroscopic experimental result figure of the skin incision healing experiment of each experimental group (blank control group, hydrolyzate group, fibrin adhesive (FG) group, cyanoacrylate (CA) group and suturing group);

Fig. 7 is the summary diagram of the performance and the specific use of the salamander skin secretion hydrolyzate provided by the present invention;

Figure 8a shows representative SEM images and viscosities of unconcentrated and concentrated salamander skin exudate hydrolyzates cured by visible light;

Figure 8b shows the maximum bond strength of CA, the concentrated hydrolyzate, the unconcentrated hydrolyzate and the photosensitive hydrogel and the schematic diagram of the detection device;

Figure 8c shows the in vivo biodegradable properties of photosensitive hydrogels;

Figure 8d shows the operation process of the liver hemorrhage model constructed by the present invention;

Figure 8e shows the SEM image of the photosensitive hydrogel tightly bound to the liver after hemostasis on the bleeding liver;

Figure 8f shows the hemostatic effect of each experimental group (blank group, hydrolyzate group, fibrin glue (FB) group and photosensitive hydrogel group) on the liver;

Figure 9a shows the Raman spectra of different samples (lyophilized powder of salamander skin secretion, hydrogel of salamander skin secretion, TCEP group and vitamin C group);

Fig. 9b shows the infrared spectrum analysis of different samples (lyophilized powder of salamander skin secretion, hydrogel of salamander skin secretion, TCEP group and vitamin C group).

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Apparently, the described embodiments are some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

It should be noted that, in this document, the term "comprising", "comprising" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements, It also includes other elements not expressly listed, or elements inherent in the process, method, article, or device. Without further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus comprising that element.

As used in this specification, the term "about" typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 4% of the stated value /-3%, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, even more typically +/-0.5% of the stated value.

In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description "within a certain range" is merely for convenience and brevity, and should not be construed as an inflexible limitation on the disclosed scope. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, range

The description should be read as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and within this range Individual numbers such as 1, 2, 3, 4, 5 and 6. The above rules apply regardless of the breadth of the scope.

The preparation method of the salamander skin secretion hydrolyzate provided by the invention comprises the following steps:

S1 Mixing the disulfide bond reducing agent and the first solvent to obtain the second solvent.

A suitable first solvent should substantially not contain ions, such as ultrapure water, deionized water, water for injection, pure water and the like.

S2 Mixing the lyophilized powder of salamander skin secretion with the second solvent to obtain a first mixed solution.

Actually, the order of mixing the disulfide bond reducing agent, the first solvent and the lyophilized salamander skin secretion powder (that is, the step of obtaining the first mixed solution) is not specifically limited. For example, the first mixed solution can be obtained by adding and mixing the disulfide bond reducing agent, the first solvent and the lyophilized salamander skin secretion, or by first removing the disulfide bond The reducing agent is mixed with the first solvent, and then the lyophilized powder of the skin secretion of the salamander is added therein and mixed, or the lyophilized powder of the skin secretion of the salamander is first mixed with the first solvent, and then It can be obtained by adding the disulfide bond reducing agent into it and mixing it, or it can also be obtained by first mixing the disulfide bond reducing agent with the lyophilized powder of salamander skin secretion, and then adding the first solvent into it and mixing it. . The proportions of the disulfide bond reducing agent, the first solvent and the lyophilized powder of salamander skin secretion can be adjusted according to actual needs, which is not limited in the present invention. As an example, the mass ratio of the lyophilized powder of the salamander skin secretion to the disulfide bond reducing agent can be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4 :1 or 3:1. The mass ratio of the first solvent to the lyophilized powder of salamander skin secretions can be 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5: 1 or 4:1. The concentration of the second solvent can be about 1%-5% (the specific value can be selected as about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, w/w%). The lower limit (minimum value) of the mass ratio of the salamander skin secretion freeze-dried powder to the disulfide bond reducing agent may be 3:1. The lower limit (minimum value) of the mass ratio of the first solvent to the lyophilized salamander skin secretion may be 4:1.

The preparation method of exemplary salamander skin secretion freeze-dried powder is as follows (taking Chinese giant salamander as an example): adult healthy male Chinese giant salamander (3-6 years old, body length 65-110cm, body weight 2.8 ± 0.7kg) was washed with clean water cleaning. Under mild mechanical stimulation, mucus secretion from the dorsal skin of Chinese giant salamanders was induced and collected in clean tubes. After collection, wash the skin secretion mucus of Chinese giant salamander with sterilized PBS or other suitable solvents, shake, centrifuge and collect the supernatant. The supernatant was freeze-dried for 24 hours, and ground into a powder (ie freeze-dried powder of giant salamander skin secretion, SSAD for short) by a freeze ball mill, as shown in the left figure of FIG. 1 . The lyophilized powder of salamander skin secretions was stored at -20°C until further use. The SSAD is preferably freshly prepared. In the present invention, giant salamander skin secretion freeze-dried powder, SSAD, SSAD freeze-dried powder and SSAD powder have the same meaning unless otherwise specified. The skin is an important respiratory organ of salamanders, with mucus glands and granular glands distributed. The reason why the present invention selects the skin secretion of the Chinese giant salamander is that it can represent the mucus secreted by stimulated amphibians such as the Chinese giant salamander. Electrical stimulation and skin scraping can promote mucus secretion.

S3 fully hydrolyzing the first mixed solution to obtain a second mixed solution.

The temperature conditions for the hydrolysis include 0-8°C (preferably 0-4°C). The hydrolysis time is related to factors such as the specific hydrolysis temperature, the concentration of the second solvent, the concentration of the lyophilized powder of salamander skin secretions, and the like. Within the temperature range of 0-8°C, the higher the temperature, the shorter the hydrolysis time; the higher the concentration of the second solvent, the shorter the hydrolysis time; the higher the concentration of the salamander skin secretion freeze-dried powder, the shorter the hydrolysis time. Relatively longer. The hydrolysis time may also be related to the specific disulfide bond reducing agent used. Therefore, the hydrolysis time can be adjusted accordingly according to actual needs, which is not limited in the present invention. Exemplary hydrolysis times include 2-168h, preferably 12-48h. Mixing can be carried out by a common mixing method in the art, such as vibrating and vortex mixing. Preferably, S3 can be carried out in a low-temperature vacuum reactor, and the vacuum degree of the vacuum reactor is less than 5kPa.

S4 Centrifuge the second mixture and collect the supernatant.

The specific conditions of centrifugation can be adjusted accordingly according to actual needs, which is not limited in the present invention. Exemplary centrifugation conditions include centrifugation at 4500 rpm for 10 min at 4°C.

S5 Put the supernatant into a dialysis bag with a molecular weight cut-off ≤ 1000, place it in the first solvent and fully dialyze it to obtain the hydrolyzate of the salamander skin secretion.

The temperature conditions of the dialysis include 0-8°C (preferably 0-4°C). Preferably a dialysis bag with a molecular weight cut off of 500 or 1000 is used. The dialysis time is actually related to the treatment method during the dialysis period. For example, the dialysis time can be shortened by increasing the frequency of changing the first solvent in which the dialysis bag is placed. Therefore, the dialysis time can be adjusted accordingly according to actual needs, which is not limited in the present invention.

Wherein, the disulfide bond reducing agent includes a specific disulfide bond reducing agent. In the present invention, "specific disulfide bond reducing agent" refers to a disulfide bond reducing agent capable of reducing intermolecular disulfide bonds in the protein or polypeptide to be reduced while keeping intramolecular disulfide bonds from being reduced as much as possible. The specific disulfide bond reducing agent includes tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl). Of course, in the method for preparing the hydrolyzate of salamander skin secretion provided by the present invention, it can also be used together with common protein denaturants/organic solvents known in the art, such as vitamin C, acetic acid, hydrochloric acid, β-mercaptoethanol, di Thiothreitol, EDTA, urea, ethanol, acetone, guanidine hydrochloride, sodium lauryl sulfate, and sodium hydroxide, and proteases such as pepsin, trypsin, papain, and subtilisin.

Further, the method for preparing the hydrolyzate of salamander skin secretion provided by the present invention may also include S6 concentrating the hydrolyzate of salamander skin secretion to obtain the concentrated hydrolyzate of salamander skin secretion.

An exemplary concentration method includes putting the hydrolyzate of salamander skin secretion into a concentrated centrifuge tube with a molecular weight cut-off ≤ 3K for centrifugation or evaporating and concentrating the hydrolyzate of salamander skin secretion.

Preferably, any of the above steps are carried out under vacuum or inert atmosphere.

Preparation Example 1: Preparation of SSAD Hydrolysis Dialysate Using TCEP-HCl ("TCEP" for short)

Weigh 100mg TCEP and dissolve it in 5mL deionized water, and add 500mg SSAD powder into it, shake and mix to obtain a mixed solution. The mixed solution was hydrolyzed at 4°C for 48 hours, and the mixed solution after hydrolysis for 48 hours was centrifuged at 4°C and 4000 rpm for 20 minutes to obtain about 3.5 mL of supernatant. The SSAD hydrolysis dialysate was obtained by dialysis in deionized water at low temperature (4° C.) for 48 h with a dialysis bag with a molecular weight cut-off of 1000.

The SSAD powder was hydrolyzed with the mass ratio of SSAD powder and TCEP as 10:1, 5:1, and 3:1. It was found that after 1 day of hydrolysis, the colloid was dispersed in a flocculent form, and the obtained supernatant maintained the viscosity of SSAD itself, while maintaining the activity of the protein and polypeptide contained in SSAD, and the obtained lower precipitate was non-viscous. Preferably, the mass ratio of SSAD powder to TCEP can be 5:1, and the mass ratio of SSAD powder to deionized water can be 10:1.

The representative images of SSAD hydrolyzate before hydrolysis, after hydrolysis and the obtained viscous supernatant (taking the mass ratio of SSAD powder to TCEP as 5:1 as an example) are shown in Fig. 2a. The mixed solution obtained after hydrolysis is centrifuged, and the obtained supernatant has obvious viscosity. Representative images of the supernatant before, during and after dialysis are shown in Figure 2b. It was found that the clear and transparent liquid before dialysis turned pale milky white after dialysis. The pale milky color may be caused by oxidation during dialysis. Therefore, if conditions permit, any step in the preparation method provided by the present invention is preferably carried out under vacuum or inert atmosphere. Figure 2c is a representative image of the prepared SSAD hydrolyzate, in which, the left picture is the prepared SSAD hydrolyzate, which is uniform and translucent; the middle upper picture is the SSAD hydrolyzate loaded in the syringe, and the middle lower picture is A drop of SSAD hydrolyzate dripped on the finger, which shows that SSAD hydrolyzate has good fluidity and can be injected; and the right picture: After the SSAD hydrolyzate is dropped on the finger, the two fingers can be pinched for less than 1min, and the upper and lower fingers can be stuck , showing obvious stickiness. In the present invention, SSAD hydrolysis dialysate, SSAD hydrolyzate and hydrolyzate have the same meaning unless otherwise specified.

Preparation Example 2: Preparation of SSAD Hydrolyzed Dialysate Using Vitamin C

Weigh 66mg vitamin C and dissolve it in 5mL deionized water, and add 500mg SSAD powder into it, shake and mix to obtain a mixed solution. The mixture was hydrolyzed at 4°C for 48h. The mixture after hydrolysis for 48 hours was centrifuged at 4°C and 4000 rpm for 20 minutes to obtain about 3.5 mL of supernatant. The SSAD hydrolysis dialysate was obtained by dialysis in deionized water at low temperature (4° C.) for 48 h with a dialysis bag with a molecular weight cut-off of 1000.

The concentration of the salamander skin secretion hydrolyzate prepared by the preparation method provided by the invention is usually between 3% and 7%. In this paper, the concentration of salamander skin secretion hydrolyzate (such as SSAD hydrolyzate) was calculated by weighing after freeze-drying, unless otherwise stated. Unless otherwise specified (for example, vitamin C group), "salamander skin secretion hydrolyzate" and "hydrolyzate" in the present invention both refer to the salamander skin secretion hydrolyzate prepared using TCEP.

In fact, the present invention also attempts to use other common protein denaturants/organic solvents in the art to hydrolyze the lyophilized powder of salamander skin secretions, including 25%, 50%, 75% and 99.5% acetic acid at a concentration of 21.6% and 36% hydrochloric acid, concentrations of 25%, 50% and 57% urea in water, and β-mercaptoethanol, dithiothreitol (DTT), EDTA, sodium hydroxide, ethanol, acetone, guanidine hydrochloride, ten Sodium dialkyl sulfate (SDS) and proteases, etc. (pepsin, trypsin, papain, subtilisin, etc.). The experimental results show that even if a strong base is added to adjust the pH, the supernatant obtained by hydrolysis with acetic acid or hydrochloric acid is still too acidic and has extremely low viscosity; while the colloid swells after hydrolysis with urea aqueous solution for 5 hours, the viscosity of the supernatant obtained is medium to low, The resulting lower precipitate was instead moderately viscous.

Embodiment one: the characteristic of the hydrolyzate of salamander skin secretion

1.1 Viscosity of salamander skin secretion hydrolyzate

The lyophilized powder of salamander skin secretion (such as powdered SSAD) will immediately form a highly viscous hydrogel when it meets water, but it is difficult to disperse (that is, insoluble) in aqueous solution, as shown in the right figure of Figure 1.

Compared with the previously prepared lyophilized salamander skin secretion hydrolyzate (taking SSAD hydrolyzate) provided by the present invention, it has better biocompatibility and similar viscosity (Table 1 ) and the viscosity is adjustable. Different from the viscosity result (that is, the viscosity of the salamander skin secretion hydrolyzate increases with the increase of the concentration) according to the pre-experiment speculation, the present invention unexpectedly finds that the lower concentration of the salamander skin secretion hydrolyzate (such as the concentration below 5%, 7% hydrolyzate), the adhesion strength is higher than the higher concentration salamander skin secretion hydrolyzate (for example, the following concentration is 10%, 15%, 20% hydrolyzate) . In other words, the viscosity and concentration-dependent degree of the hydrolyzate of salamander skin secretion present a downward-opening parabola: when the concentration is 5-7%, the viscosity of the hydrolyzate of salamander skin secretion is the highest; The viscosity of skin secretion hydrolyzate increased with the increase of concentration; when the concentration was >7%, the viscosity of salamander skin secretion hydrolyzate decreased with the increase of concentration.

Table 1: Viscosity of SSAD hydrolyzate

Concentration of SSAD hydrolyzate	sticky skin
3%	10.8kPa±2.3kPa
5%	26.5kPa±3.9kPa
7%	27.6kPa±3.2kPa
10%	21.8kPa±2.7kPa
15%	17.7kPa±3.6kPa
20%	16.3kPa±2.1kPa

1.2 Biocompatibility of hydrolyzate of salamander skin secretion

The biocompatibility of the salamander skin secretion hydrolyzate was assessed using the Cell Viability Assay Kit (CCK-8) following the manufacturer's instructions. As shown in Fig. 3a and Fig. 3c (Fig. 3a and Fig. 3c are different manifestations of the same data), detect different concentrations respectively (1/100, 1/1000, 1/10000 in the figure are w/w% ) powder extract, dialyzed supernatant and non-dialyzed supernatant on skin fibroblast cytotoxicity. The results prove that the dialyzed supernatant (i.e. SSAD hydrolyzate) has good biocompatibility, not only has no obvious cytotoxicity, but also has the effect of promoting cell proliferation.

Using polymerase chain reaction (PCR) technology, the expression of angiogenesis-related genes in human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells, HUVEC) co-cultured with different concentrations of SSAD hydrolyzate was detected. Figure 3d and Figure 3e show the changes in the expression levels of angiogenesis-related genes in HUVECs. As shown in Figure 3d (co-cultivation for 7 days) and Figure 3e (co-cultivation for 14 days), the 1/50 group (the concentration of SSAD hydrolyzate was about 5%, mixed with the volume ratio of cell culture medium 1:49) and 1 In the /100 group (the concentration of SSAD hydrolyzate is about 5%, mixed with the cell culture medium at a volume ratio of 1:99), CD31, VEGF, EphrinB2 and other angiogenesis-related genes were significantly highly expressed in a dose-dependent manner. In other words, as the concentration of SSAD hydrolyzate increased, the expression of angiogenesis-related genes in cells increased in a concentration-dependent effect, indicating that SSAD hydrolyzate could promote angiogenesis.

Vitamin C, also known as ascorbic acid, is a strong reducing agent. Meanwhile, vitamin C is an essential nutrient for living organisms, especially humans, and thus it is considered to be high in biosafety. Using the Cell Viability Detection Kit (CCK-8), the present invention further compared the hydrolyzate prepared in Preparation Example 1 (abbreviated as TCEP group, the same below) and the hydrolyzate prepared in Preparation Example 2 (abbreviated as vitamin C group, the same below) ) biocompatibility. Surprisingly, however, as shown in Figure 3b, the biocompatibility of the TCEP group was higher than that of the vitamin C group on day 1, day 3, and day 5. Scratch test (Figure 3f) and Transwell test (Figure 3g) also further confirmed that the biocompatibility of TCEP group was higher than that of vitamin C group.

The exemplary experimental method of the scratch test includes: seeding about ^5X105 cells in each well of a 6-well plate, and culturing for 24 hours under suitable conditions; after 24 hours, use a pipette tip perpendicular to the bottom of the culture well to draw horizontal and vertical intersecting lines 5 strips each; the cells were washed 3 times with PBS to remove the scratched cells, and then serum-free medium was added; the 6-well plate was placed in a 37°C, 5% CO2 incubator for cultivation, and 0, 12, 24, 48 hours sampling and photo recording. An exemplary experimental method of the Transwell experiment includes: taking cells in the logarithmic growth phase, digesting them routinely, resuspending the cells in medium containing 1% FBS and adjusting the cell density to 5×10 ⁵ /mL. 100 μL of cell suspension was inoculated in the upper chamber of the Transwell (24-well plate), and 600 μL of medium containing 10% FBS was added to the lower chamber, and 1% SSAD hydrolyzate was added to the control group. After culturing the cells for 24h or 48h, the small chamber was taken out and rinsed in a beaker, and then methanol was added to the small chamber at 800 μL/well. The liquid in the upper chamber was blotted dry, and fixed with 4% paraformaldehyde at room temperature for 10-30 min. After absorbing the fixative in the upper chamber, add 0.1% crystal violet staining solution and stain at room temperature for 20 min. The cells were gently rinsed with running water and soaked 3 times for depigmentation staining. Aspirate the liquid in the upper chamber and gently wipe off the unmigrated cells on the membrane surface at the bottom of the upper chamber with a cotton swab. Cells in 5 fields of view were randomly observed under a microscope and photographed and recorded (images were processed using ImageJ software).

In this embodiment, Raman spectroscopy and infrared spectroscopy are used to further analyze the hydrolyzate of TCEP group and vitamin C group. Raman spectroscopy and infrared spectroscopy are based on different physical principles to detect the vibrational properties of molecules, and have the advantages of simple measurement methods and no damage to samples. Infrared spectroscopy is based on the absorption properties of the sample, where the signal intensity follows Beer's law, while Raman spectroscopy relies on the photons scattered inelastically by the sample being detected, and the intensity of the Raman shift is proportional to the molecular concentration.

Figure 9a shows that, compared with the lyophilized powder of salamander skin secretion and the hydrogel of salamander skin secretion, the TCEP group and the vitamin C group have no peak at the wavelength corresponding to the disulfide bond. Specifically, as shown in Figure 9a, the lyophilized powder of salamander skin secretion (referred to as "powder") has a corresponding SS vibration mode at 499 cm ^-1 , and the hydrogel of salamander skin secretion (referred to as "hydrogel") The corresponding SS vibration mode appeared at 492cm ^-1 , but the corresponding SS vibration mode did not appear in the TCEP group and the vitamin C (referred to as VC) group, which indicated that the disulfide bonds in the hydrolyzate of the TCEP group and the vitamin C group may be mostly is restored. Based on this, this embodiment further analyzes the disulfide bond (SS) sub-conformation of different samples.

Disulfide bond (SS) will undergo stretching vibration, and the Raman spectrum near 500-550cm ^-1 belongs to the characteristic band of disulfide bond. The Raman peak here can be divided into three sub-conformations of SS stretching vibration. Among them, the Raman peak around 508cm ^-1 corresponds to the SS stretching vibration of the gauche-gauche-gauche (ggg) conformation, the Raman peak around 525cm ^-1 corresponds to the SS stretching vibration of the gauche-gauche-trans (ggt) conformation, and around 545cm ^-1 The Raman peak corresponds to the SS stretching vibration of the trans-gauche-trans(tgt) conformation. The tgt and ggg conformations belong to the intermolecular and intramolecular disulfide bond configurations, respectively. The results are shown in Table 3. Compared with the powder alone, the three sub-conformations of the SS of the hydrogel were significantly changed. Compared with the hydrogel, the vitamin C group had a small change, only a slight change in the tgt sub-conformation; while the TCEP group had a large change, and there were large changes in the ggt and tgt. It can be seen that compared with vitamin C treatment, TCEP treatment may have a greater impact on the change of protein spatial structure. Further analysis showed that compared with powder, the content of ggg and tgt conformation in the vitamin C group decreased, while the content of ggg conformation decreased and the content of tgt conformation increased in the TCEP group, which indicated that vitamin C treatment damaged some of the intermolecular disulfide bonds , which may lead to a decrease in the intermolecular force, and ultimately lead to a decrease in the stability of the protein structure after vitamin C treatment. This also shows that TCEP treatment increases the content of intermolecular disulfide bonds, which may lead to an increase in the force between molecules, and ultimately leads to an increase in the stability of the protein structure after TCEP treatment.

Table 3 S-S subconformation (Raman spectrum) of different samples

the	ggg	ggt	tgt
Hydrogels	45.47%	48.62%	5.91%
pink	55.84%	31.04%	13.12%
Vitamin C	44.20%	47.13%	8.67%
TCEP	47.29%	34.00%	18.71%

The Raman spectrum also shows that the TCEP group has the amide I band vibration at 1667cm ^-1 and the CC stretching vibration at 1453cm ^-1 . In the vitamin C group, the absorption peak of the amide I band appeared at 1659cm ^-1 , and the CC stretching vibration appeared at 1445cm ^-1 . The powder has an amide I band absorption peak at 1670cm ^-1 , CC stretching vibration at 1448cm ^-1 , CC stretching vibration at 1003cm ^-1 , and SS vibration mode at 499cm ^-1 . The hydrogel exhibited amide I band vibrations at 1674 cm ^-1 , amide II band vibrations at 1585 cm ^-1 , CC stretching vibrations at 1454 cm ^-1 , and amide III band vibrations at 1307 cm ^-1 , The CC stretching vibration appeared at 1007 cm ⁻¹ and the SS stretching vibration at 492 cm ⁻¹ (shown in Fig. 9a and Table 4).

Table 4 Amide Ⅰ band, amide Ⅱ band, amide Ⅲ band and C-C conformation of different samples (Raman spectrum)

the	Amide band I	Amide II band	Amide III band	C-C
Hydrogels	1674cm -1	1585cm -1	1307cm -1	1007, 1454cm -1
pink	1670cm -1	-	-	1003, 1448cm -1
Vitamin C	1659cm -1	-	-	1445cm -1
TCEP	1667cm -1	-	-	1453cm -1

Figure 9b and Table 5 show the FTIR analysis of different samples. Disulfide bonds play an important role in stabilizing the tertiary structure of proteins. During the preparation of the hydrolyzate, the disulfide bonds are destroyed, and the protein needs to form a new tertiary structure, so that all the atoms in the peptide chain can be rearranged in space. The appearance of the aromatic CH structure in the infrared spectrum shows that the protein molecules in the hydrolyzate have aromatic amino acids such as tyrosine and tryptophan inside, which further indicates that the preparation process of the hydrolyzate will change the conformation of the protein, thereby making the internal structure Exposure occurs. Compared with the powder, the amide II band of the TCEP group gradually weakened, and the amide II band even disappeared in the hydrogel, which indicated that the sample generation process of the TCEP group and the hydrogel had a certain influence on the protein content and structure. However, CH vibration peaks appeared at 931 and 3080 cm ^-1 in the vitamin C group, which indicated that the protein structure and composition changed significantly, and a large number of aromatic proteins (tyrosine, tryptophan, etc.) appeared. An increase in the relative content of aromatic proteins may change the hydrophobicity and structural stability of the hydrolyzate treated with vitamin C, resulting in an increase in hydrophobicity and a decrease in structural stability of the hydrolyzate treated with vitamin C. This indicates that vitamin C treatment will not only reduce the disulfide bonds between molecules and intramolecules, but also reduce oxidized groups such as aldehyde groups. The obvious increase of methyl and methylene intensity indicates that the content of aliphatic hydrocarbons in TCEP group and hydrogel is higher, which may be mainly derived from the structure of peptidoglycan.

Infrared spectrum analysis of different samples in table 5

the	Aromatic C-H	Amide II band	Amide III band	methyl and methylene
Hydrogels	877cm -1	-	-	2924cm -1
pink	-	1544cm -1	-	2927cm -1
Vitamin C	931, 3080cm -1	1533cm -1	1235cm -1	2967, 2927, 2870cm -1
TCEP	868cm -1	1544cm -1	-	2924cm -1

In addition, as shown by semi-quantitative analysis of infrared spectroscopy (Table 6), compared with samples from other groups, the secondary structure of vitamin C group changed significantly, and the relative content of β-turn and Random decreased and transformed into β-sheet structure.

Infrared spectrum semi-quantitative analysis of different samples in table 6

the	α-helix	β-sheet	β-turn	Random
pink	14.78%	27.92%	24.36%	32.94%
TCEP	15.23%	26.51%	25.32%	32.94%
Hydrogels	16.81%	26.09%	23.23%	33.87%
Vitamin C	14.24%	32.02%	22.57%	31.17%

According to reports, the tertiary structure models of regular proteins can be divided into four categories: (1) all-alpha type: dominated by α-helical structure, its content is greater than 40%, while the content of β-sheet is less than 5%; (2) Full β type: mainly β-sheet structure, its content is more than 40%, while the content of α-helix is less than 5%; (3) α+β type: the content of both α-helix and β-sheet is more than 15%, These two structures are separated in space, and more than 60% of the folded chains are antiparallel; (4) α/β type: the content of α-helix and β-sheet is more than 15%, and these two structures are in Spatially alternate, and more than 60% of the folded chains are aligned in parallel. As shown by the circular dichroism spectrum analysis (Table 7), the protein sample in the powder extract group is an α+β protein with a relatively stable structure. The protein conformation of the vitamin C group changed dramatically, and its secondary structural features basically disappeared, mainly showing a Random (random coil) conformation with a relative content of more than 90%, which further proved that vitamin C significantly denatured the protein. Powder (acetic acid) group (SSAD freeze-dried powder was treated with 0.75mol/L acetic acid + 500mg SSAD freeze-dried powder + 5mL ddH2O) and TCEP group samples, the protein secondary structure changed to β-sheet (β-sheet) and Random Mainly, it may be that acetic acid or TCEP combines with the amino group and side chain group of the amino acid in the α-helix (α-helix) structure of the protein, so that the protein molecule unfolds to change its spatial conformation.

Table 7 Circular dichroism analysis of different samples

the	α-helix	β-sheet	β-turn	Random
powder (acetic acid)	20.7%	24.1%	6.9%	48.3%
TCEP	20.7%	24.1%	19.0%	36.2%
powder extract	31.7%	21.6%	26.4%	20.3%
Vitamin C	4%	4%	2%	90%

In addition, compared with SSAD lyophilized powder, SSAD hydrolyzate has a lighter odor. This may be due to the fact that TCEP weakened or decomposed dimethyl disulfide (which may be the source of odor and low toxicity of SSAD powder) during the hydrolysis of SSAD powder, which further reduced the odor and toxicity of SSAD hydrolyzate.

In summary, the present invention uses a specific disulfide bond reducing agent (such as TCEP-HCl), which can reduce the intermolecular disulfide bonds in the lyophilized powder of salamander skin secretions, while retaining intramolecular disulfide bonds and oxidized groups. groups are not reduced (even a slight excess of this specific disulfide reducing agent will not significantly affect intramolecular disulfide bonds). Furthermore, during the hydrolysis process of this specific disulfide bond reducing agent, the protein structure of the original lyophilized salamander skin secretion powder is rearranged, so that the intramolecular disulfide bonds of the obtained salamander skin secretion hydrolyzate The increase of the content of , in turn leads to the improvement of the stability of the hydrolyzate of salamander skin secretion. In addition, this specific disulfide bond reducing agent can also reduce the toxic and odorous substances (for example, dimethyl disulfide) in the lyophilized powder of the skin secretion of the salamander, and reduce the concentration of the hydrolyzate of the skin secretion of the salamander. odor and toxicity. Therefore, the present invention uses a specific disulfide bond reducing agent to hydrolyze the lyophilized powder of salamander skin secretions to prepare a water-soluble, injectable hydrolyzate that maintains viscosity and improves biological safety (with Compared with the hydrogel formed by the lyophilized powder of salamander skin secretion meeting water).

Using the three-dimensional gel formed by rat tail collagen capable of simulating extracellular matrix as an in vitro tissue model, the present invention evaluates the tissue permeability of SSAD hydrolyzate. Add 100 μL of rat tail collagen solution dropwise to a clean glass slide and cover the slide with a cover glass, then place it on a shaker at 37° C. and wait for it to solidify (about 3 min). Fluorescein-labeled hydrolyzate was added dropwise around the glass slide to allow it to permeate naturally (the whole process was observed under an immunofluorescence microscope (magnification 4×) and photographed and recorded). Figure 3h shows the penetration of SSAD hydrolyzate into rat tail glue at 0 min, 3 min, 5 min and 7 min, in which the upper layer is rat tail glue and the lower layer is fluorescein-labeled hydrolyzate. The results showed that the gradual penetration of SSAD hydrolyzate into the rat tail gum was observed, which indicated that the appropriate concentration of SSAD hydrolyzate has good permeability as a bioadhesive and can enter the extracellular matrix-like environment over time.

An incision with a length of 1.5 cm was made on the dorsal skin of SD rats. As shown in the macroscopic image of Figure 6c, the adhesive performance of the photosensitive hydrogel group (see below for the preparation method of the photosensitive hydrogel) is better than that of the blank group, cyanoacrylate glue (CA) group, and fibrin glue (FB) group , which exhibited potential as an adhesive. Among these 6 groups, SSAD hydrolyzate had better healing speed and healing effect, and could even promote scarless wound healing. The incision bonded with SSAD hydrolyzed solution was sliced, and placed under a scanning electron microscope (SEM) to observe the incision bonding interface. As shown in the scanning electron microscope image of Figure 3i, after the SSAD hydrolyzate was bonded to the skin tissue, the gap of the incision was closed well, and the margin of the incision was hardly visible.

As summarized in Figure 7, when the hydrolyzed salamander skin exudate in solution form is applied to the wound at a low concentration (e.g., 5%-7%), it can cover the wound surface in an injectable, thinner, and uniform form, compared with The degree of adhesion to the wound is higher, only slight pressure (for example, pressing with fingers) can achieve stable adhesion to the wound, and the whole operation process is more convenient and controllable.

Surprisingly, the present invention finds that the actual bonding effect of the salamander skin secretion hydrolyzate is not the higher the concentration, the higher the viscosity, and the better the bonding effect as generally believed; but it is at a relatively low concentration. When the state (for example 5%-7%), the viscous effect of the salamander skin secretion hydrolyzate is better. Before application to the wound, a relatively low concentration (eg, 5%-7%) of the hydrolyzed salamander skin secretion is in a "saturated" state (ie, a "solution" state); before reaching the "saturated" state, the salamander skin The viscosity of secretion hydrolyzate will increase with the increase of concentration; after reaching the "saturation" state, the viscosity of salamander skin secretion hydrolyzate will decrease with the increase of concentration. When it is applied to a wound, under slight pressure, it will adhere to the wound and can penetrate deeper into the interior of the biological tissue along with the solvent (such as water in the hydrolyzed solution). As the solvent enters the tissue, the local concentration of salamander skin secretion hydrolyzate increases (for example, to a concentration >7%) and reaches a "supersaturated" state (ie, a "microgel" state), and is able to interact with biological tissue The proteins in the tissue react to form a cross-linked network between tissues. Eventually, the microgels can form a gel inside the tissue. In other words, after being applied to the wound and subjected to a certain pressure, the solvent (for example, water) in the hydrolyzed solution of salamander skin secretion provided by the present invention can penetrate deeper into the tissue, so that the hydrolyzed solution of salamander skin secretion The local concentration increases and forms a microgel structure with a cross-linked network between tissues. Microgel particles with a smaller particle size can self-assemble at the wound tissue, so the formed cross-linked network (ie microgel structure) is denser and more adaptable to the wound environment. Therefore, the microgel structure can bond the tissues at the wound more tightly, strengthen the connection between the tissues at the wound, improve the continuity and integrity of the tissue at the wound, and finally promote the healing of the wound. The salamander skin secretion hydrolyzate provided by the invention can change in concentration and state at the wound, which provides a suitable healing environment for the wound. In addition, compared with the lyophilized powder of salamander skin secretion, the hydrolyzed salamander skin secretion provided by the present invention requires less amount of adhesion to a wound of the same area, has lower inflammatory response, and has shorter wound recovery time, and has the advantages of Better wound healing properties.

1.3 Wound-healing properties of SSAD hydrolyzate

Previous studies have shown that SSAD powder can promote scarless wound healing, and the healing effect is the best among the groups tested (fibrin glue group, hemostatic control group, cyanoacrylate group and suture group); No signs of infection or inflammation were seen, hair regrowth was observed without scarring, and SSAD promoted overall wound healing. Figure 4a shows the SSAD hydrolyzate (which can be understood as a bioadhesive) with other 4 wound closure methods (negative control group (blank), suture (suture), cyanoacrylate adhesive (commercial Glue), fibrin glue (biological glue)) in vivo effect comparison. The 0, 1, 3, and 5 days after the operation of rat back incision (incision length is 2cm) images show that SSAD hydrolyzate maintains the ability to promote cell proliferation, migration, wound regeneration, and angiogenesis, which is similar to that of the above-mentioned SSAD powder. The ability to promote wound healing is similar. Figure 4b HE staining images of damaged tissue (epidermal (E), dermis (D), scab (SC), incision position (★)) showed that the remaining crack width of the SSAD hydrolyzed group was significantly narrower than that of the blank group and fibrin glue group and cyanoacrylate group, the number of proliferating epithelial cells was significantly higher than the other groups, and the granulation tissue was less.

Figure 4c is the macroscopic image of rat back skin incision (incision length is 2cm) model shows that on the 5th day after operation, the wounds of vitamin C group and TCEP group have healed and scab, and the healing speed and effect are significantly better than cyano Acrylate glue (CA) group, fibrin glue (FB) group and suture group were better than blank control group. The histological section further confirmed that on the 5th day after operation, the healing effect of the TCEP group and the vitamin C group was better, and the inflammatory reaction of the incision margin in the TCEP group was the lightest (Fig. 4d, E represents the epidermis, D represents the dermis, CA group "CA" in the lower figure means that CA cannot be degraded).

This embodiment further compares the characteristics of SSAD hydrolyzate and SSAD freeze-dried powder, as shown in Table 2.

Table 2 Comparison of SSAD hydrolyzate and SSAD freeze-dried powder

In conclusion, the hydrolyzate of salamander skin secretion not only has excellent adhesion properties, good biocompatibility, biodegradability, and promotes tissue regeneration, but also has the characteristics of injectability, low inflammatory response, and deeper penetration depth in tissues, etc. Features, can be applied to the wound site in an easy way.

Example 2: Salamander skin secretion hydrolyzate as bioadhesive and liquid dressing

The above experiments showed that the hydrolyzate of salamander skin secretion can be used as a bioadhesive (Fig. 6b). After the salamander skin secretion hydrolyzate was used to bond the skin tissue, the incision gap was closed better, the incision margin was almost invisible, the inflammatory response was lower, and the wound recovery time was shorter. In addition, the rat skin full-thickness defect model (a circle with a diameter of 1.5 cm) shown in Figure 6a shows that the SSAD hydrolyzate can also be used as a liquid dressing to promote wound healing and regeneration.

Example 3: Salamander skin secretion hydrolyzate as photosensitive hydrogel

Concentration method can be used to further concentrate the hydrolyzate of salamander skin secretion. SEM images and viscosities of unconcentrated and concentrated hydrolyzates after visible light curing are shown in Figure 8a. The concentrated hydrolyzate (one-fold concentration means that the volume of the concentrated hydrolyzate is reduced to half of that before concentration) has a denser pore size and higher viscosity than the unconcentrated hydrolyzate. A suitable concentrating method includes putting the hydrolyzate of salamander skin secretion into a centrifuge tube with a molecular weight cut-off ≤ 3K for centrifugation or evaporating and concentrating the hydrolyzate of salamander skin secretion.

The preparation method of the exemplary photosensitive hydrogel includes: in a clean EP tube (full tinfoil is protected from light), 500 μ L of concentrated SSAD hydrolyzate (concentration is about 15%) and photoinitiator Ru/SPS (ruthenium (ruthenium ( II) compound and sodium persulfate) were mixed according to the ratio of 50:1 (that is, adding 10 μL RU+10 μL SPS, the concentration of Ru/SPS was 1/25mM). Then, the mixture was added to a 96-well plate (50ul/well), and repeated for 8 wells. Then use 7000w visible light to cure and check whether it is glued after 30s (the stronger the intensity of visible light, the shorter the curing time, the intensity and curing time of visible light can be adjusted according to the actual situation). The macroscopic images of the photosensitive hydrogel before and after illumination are shown in the lower part of Figure 7. The photosensitive hydrogel before illumination is in a liquid state, while the photosensitive hydrogel after illumination is in a solid state.

Co-culture of cells and photosensitive hydrogels. Preparation of medium: Add 5mL FBS and 500ul double antibody to a 50mL centrifuge tube, and dilute to 50mL with basic DMEM medium to obtain 2 tubes of 50mL complete medium (1% double antibody + 10% FBS + basic DMEM medium). Add 2.5mL FBS and 1.25mL double antibody to a 50mL centrifuge tube, and dilute to 25mL with basic DMEM medium to obtain 1 tube of 25mL complete medium (5% double antibody + 10% FBS + basic DMEM medium). Digest adherent cells: Take the 10cm culture dish out of the incubator, observe the growth of the cells to 70%-80% under the microscope, transfer to the biological safety cabinet; discard the old medium, rinse once with PBS, aspirate, add 1ml Trypsinize for 2-3 minutes, when the cells become round under the microscope, add 3ml of complete medium to stop the digestion; pipette the cells, collect the cell suspension into a 15ml centrifuge tube, centrifuge at 1000rpm for 3min, discard the supernatant, add 3ml of complete culture base (5% double antibody + 10% FBS + basic DMEM medium) for resuspension. Cell counting: draw 200ul of the cell suspension into an EP tube, add 600ul of 5% double antibody medium, dilute the original solution 4 times, draw 10ul of the cell suspension into a hemocytometer for cell counting. The cell suspension (ie 6000cells/well) was added to the above-mentioned 96-well plate containing the photosensitive hydrogel at 3ul/well, and then 100ul of 5% double-antibody medium was added. Then, the 96-well plate was transferred to an incubator and cultured at 37° C., 5% CO 2 .

Then, after 24 and 48 hours of co-cultivation, whether there was cell growth in the photosensitive hydrogel was observed by live-death staining. Observation under a fluorescence microscope showed (Fig. 5a) that L929 cells adhered to the hydrogel, and the photosensitive hydrogel had good cytocompatibility in vitro. Exemplary experimental methods include: first prepare the living death staining working solution: thaw the living death staining storage solution in advance in the dark, take a clean EP tube, keep tinfoil away from light, add 800ul PBS+1.6ul Ca+2.4ul PI, mix well and set aside Then, take the 96-well plate out of the incubator, centrifuge and discard the supernatant. Rinse the cell pellet once with PBS, after aspiration, add the staining working solution at 100ul/well, and incubate in the dark for 15min; finally, observe and take pictures under a fluorescent microscope.

As shown in the setup in Figure 8b, after the pigskin was bonded in place, a shear force was applied at a rate of 0.1 N/s using a universal mechanical tester to measure CA (cyanoacrylate), concentrated hydrolyzate, unconcentrated The maximum bond strength (maximum shear stress) of hydrolyzate and photosensitive hydrogel. The results showed that the maximum bond strength of concentrated hydrolyzate and photosensitive hydrogel was not as good as that of unconcentrated hydrolyzate.

Figure 8c shows the staining results of the sample sections after 7 days and 14 days after the subcutaneous implantation of 100 mg of photosensitive hydrogel at the back incision of SD rats. Both HE staining and Masson staining indicated that the photosensitive hydrogel can be degraded in vivo (it can be completely degraded at 21 days after implantation, experimental data not shown), and has good biocompatibility.

Figure 8d shows the surgical procedure of the rat liver hemorrhage model: first, an 8-mm defect was made on the liver, and then the liver was excised. Then, gauze is used to stop bleeding from the liver. Finally, the photosensitive hydrogel is injected and exposed to visible light. As shown in Figure 5b, the prepared SSAD photosensitive hydrogel can be used as a photosensitive hemostatic agent, and compared with the control group and the fibrin glue (FB) group, the photosensitive hydrogel group significantly reduced the bleeding volume and Clotting time. The SEM images of Figure 8e (350× magnification on the left and 1000× on the right) show that the light-sensitive hydrogel can bind very tightly to tissues that require hemostasis (eg, the liver). Figure 8f further shows the hemostatic effect of each experimental group (blank group, hydrolyzate group, fibrin glue (FB) group and photosensitive hydrogel group) on the liver at 0min, 2min, 4min, and 6min, and the results show that photosensitive hydrogel Glue can stop the massive hemorrhage of the liver in 1 minute, while the other groups can't control the bleeding in 6 minutes.

The rat skin full-thickness defect model (circle with a diameter of 1.5 cm) shown in Figure 5c shows that the SSAD photosensitive hydrogel is also effective in promoting wound healing (as a liquid dressing), and can promote wound healing and regeneration.

In addition, the SSAD hydrolyzate added with a photoinitiator (such as Ru/SPS) can also be used alone or in combination with other methacrylic bioink substrates (such as GelMA, HAMA, FibMA, CSMA, Col1MA, HepMA, AlgMA, DexMA, ChsMA, CHIMA, PCLMA, ElaMA, etc.) are used as bioinks for 3D printing. This also shows that the hydrolyzate of salamander skin secretion is an excellent new biomaterial, which can be used in many fields with stronger and adjustable viscosity, higher biocompatibility and excellent ability to promote regeneration and proliferation.

Embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementations, and the above-mentioned specific implementations are only illustrative, rather than restrictive, and those of ordinary skill in the art will Under the enlightenment of the present invention, many forms can also be made without departing from the gist of the present invention and the protection scope of the claims, and these all belong to the protection of the present invention.

Claims (23)

1. A preparation method of salamander skin secretion hydrolyzate, comprising the following steps:

   S1 mixes the disulfide bond reducing agent and the first solvent to obtain the second solvent;

   S2 mixing the lyophilized powder of salamander skin secretion with the second solvent to obtain a first mixed solution;

   S3 fully hydrolyzing the first mixed solution to obtain a second mixed solution;

   S4 centrifuging the second mixed solution and collecting the supernatant;

   S5 put the supernatant into a dialysis bag with a molecular weight cut-off ≤ 1000 and place it in the first solvent for sufficient dialysis to obtain a hydrolyzate of the salamander skin secretion;

   Wherein the disulfide bond reducing agent includes a specific disulfide bond reducing agent, the specific disulfide bond reducing agent is tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl), and the first solvent Including one or more of ultrapure water, deionized water, water for injection, and pure water.
2. The method according to claim 1, characterized in that the mass ratio of the first solvent to the lyophilized powder of salamander skin secretion is at least 4:1.
3. The method according to claim 1, wherein the mass ratio of the lyophilized salamander skin secretion powder to the disulfide bond reducing agent is at least 3:1.
4. The method according to claim 1, characterized in that, the hydrolysis time of the hydrolysis comprises 2-168 hours, optionally 12-48 hours.
5. The method according to claim 1, characterized in that the temperature conditions of said S3, said S4 and said S5 include 0-8°C.
6. The method according to claim 1, characterized in that said S3 is carried out in a vacuum reactor, and the vacuum degree of said vacuum reactor is <5kPa.
7. The method of claim 1, wherein the disulfide bond reducing agent further comprises vitamin C.
8. The method according to claim 1, wherein the dialysis time of the dialysis includes 48 hours.
9. The method according to claim 1, characterized in that the concentration of the obtained hydrolyzate of the salamander skin secretion comprises 3%-7%.
10. The method according to any one of claims 1-9, characterized in that, the method further comprises S6 concentrating the hydrolyzate of salamander skin secretion to obtain the concentrated hydrolyzate of salamander skin secretion.
11. The method according to claim 10, characterized in that, the concentrated method comprises putting the hydrolyzate of the salamander skin secretion into a concentrated centrifuge tube with a molecular weight cut-off≤3K or centrifuging the hydrolyzate of the skin secretion of the salamander Concentrated by evaporation.
12. A hydrolyzate of salamander skin secretion, characterized in that the hydrolyzate of salamander skin secretion is prepared by the method described in any one of claims 1-11.
13. A use of the hydrolyzate of salamander skin secretion in the preparation of bioadhesive, characterized in that the hydrolyzate of salamander skin secretion is prepared by the method described in any one of claims 1-9.
14. The use according to claim 13, characterized in that the concentration of the salamander skin secretion hydrolyzate comprises 5%-7%.
15. The use according to claim 13, characterized in that, after the bioadhesive is applied to the wound, the bioadhesive penetrates into the tissue of the wound by itself or through external pressure to form a microgel structure.
16. The use according to claim 15, wherein the wound comprises a skin wound.
17. The use according to claim 15, wherein the bioadhesive is used to promote the healing of the wound.
18. A use of a hydrolyzate of salamander skin secretion in preparing a wound dressing, characterized in that the hydrolyzate of salamander skin secretion is prepared by the method described in any one of claims 1-11.
19. A use of the hydrolyzate of salamander skin secretion in the preparation of photosensitive hydrogel, characterized in that the photosensitive hydrogel includes concentrated salamander skin secretion hydrolyzate and a photoinitiator, and under the irradiation of visible light, the concentrated salamander The skin secretion hydrolyzate is solidified to form the photosensitive hydrogel; the concentrated salamander skin secretion hydrolyzate is prepared by the method described in claim 10, and the photoinitiator includes Ru/SPS system.
20. The use according to claim 19, the intensity of the visible light comprises 7000W.
21. The use according to claim 19, the photosensitive hydrogel is used as a hemostatic agent.
22. The use according to claim 19, the photosensitive hydrogel is used as a bioadhesive.
23. The use according to claim 19, the photosensitive hydrogel is used as a wound dressing.

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