CN114668887A

CN114668887A - Biological material and preparation method and application thereof

Info

Publication number: CN114668887A
Application number: CN202210212225.8A
Authority: CN
Inventors: 田勇; 吴明一; 邓拓
Original assignee: Shanghai Zhenchen Cosmetics Co Ltd
Current assignee: Shanghai Zhenchen Cosmetics Co Ltd
Priority date: 2022-03-04
Filing date: 2022-03-04
Publication date: 2022-06-28

Abstract

The application relates to the technical field of biology, in particular to a biological material and a preparation method and application thereof. The biomaterial comprises a composition obtained by drying a viscous substance derived from gastropoda. The biomaterial has good moisturizing effect and remarkable tissue adhesion, and can promote the closure, healing and repair of acute wounds and chronic wounds including diabetic feet.

Description

Biological material and preparation method and application thereof

Technical Field

The application relates to the technical field of biology, in particular to a biological material and a preparation method and application thereof.

Background

Tens of millions of people suffer from tissue damage each year, including accidental injuries, surgical injuries, chronic ulcers, etc., and serious ones can cause amputation and even be life threatening. Sutures and staples are currently the first method of clinical wound closure. However, suturing a wound is a time-consuming step in the surgical procedure, and the removal of the suture causes secondary damage to the tissue, which not only increases the number of medical visits of the patient, but also easily causes the risks of scar hyperplasia, contracture and infection. The invention of tissue adhesives that do not require a stitch removal brings a great technical advance to the surgical procedure, which is more convenient to operate, greatly shortens the operation time, and significantly reduces the risk of secondary damage and infection of the tissue, compared to wound suturing methods.

Currently, the tissue adhesives in wide clinical use are mainly synthetic cyanoacrylates (e.g., medical 504 and 508 glues) and fibrin adhesives (e.g., porcine fibrin adhesive and lyophilized human fibrin adhesive). However, cyanoacrylate rapidly polymerizes to generate heat during the process of adhering and curing with tissues, and after curing, it presents a glass-like rigid solid, which has poor mechanical properties matching with soft tissues, very poor biodegradability, and poor biocompatibility limiting its external use only. The porcine fibrin adhesive can be used for stopping bleeding, sealing wound surface, promoting healing, preventing adhesion, and releasing drug. The freeze-dried human fibrin adhesive is a local hemostatic and is used for assisting in treating bleeding of burn wounds, abdominal incisions of general surgery, liver operation wounds and vascular surgery wounds. Because the fibrin adhesive has poor mechanical strength after being gelatinized, the fibrin adhesive is easy to tear and generate secondary damage to the wound surface and the longitudinal cut wound of the moving part of a body due to insufficient adhesive force. Both types of fibrin adhesives are blood products and are subject to the risk of allergic reactions and potential viral transmission. Therefore, there is a clinical need for tissue adhesives that are biocompatible, have high adhesion, and have low potential risks.

Disclosure of Invention

The embodiment of the application provides a biological material and a preparation method and application thereof.

In a first aspect, embodiments provide a biomaterial comprising a composition obtained by drying a viscous material derived from gastropods; alternatively, the gastropoda animal comprises an animal of the order gastropoda; further optionally, the gastropoda includes one or more of the families of stone sulphonidae, agate spirochaetaceae, costaphylspirochaceae, slug, mucoid slug and barnacidae.

In some embodiments, the composition comprises an acidic polysaccharide, wherein the acidic polysaccharide has a content a of more than or equal to 5% by weight of the composition, and optionally the acidic polysaccharide has a content a of more than or equal to 5% and less than or equal to 25% by weight of the composition.

In some embodiments, the acidic polysaccharide comprises a compound of formula (I),

in the formula (I), the compound is shown in the specification,

the A ring is alpha-D-2-amino-2-deoxy-glucosyl alpha-D-GlcN,

R₁is acetyl-COCH₃Or sulfonic acid group-SO₃ ^-；

The B ring is alpha-L-iduronate alpha-L-IdoA or alpha-D-glucuronate alpha-D-GlcA,

R₂is hydroxy-OH or sulphate-OSO₃ ^-，

R₃Is carboxy-COO^-And inorganic salts thereof;

n is an integer greater than 100;

the weight-average molecular weight Mw of the acidic polysaccharide is more than or equal to 400kDa and less than or equal to 1600kDa, and the polydispersity PDI value of the acidic polysaccharide is less than 5.0,

Wherein, based on the mass of the acidic polysaccharide, the sulfate ester group-OSO₃ ^-The mass percentage content c is that c is more than or equal to 8 percent.

In some embodiments, the acidic polysaccharide is formed by alternating between (1 → 4) glycosidic linkages of α -D-2-amino-2-deoxy-glucosyl α -D-GlcN in the A ring and α -L-iduronate α -L-IdoA or α -D-glucuronate α -D-GlcA in the B ring.

In some embodiments, the composition further comprises a polypeptide, and the mass percentage content b of the polypeptide is more than or equal to 10% of the mass of the composition, and the mass percentage content a + b is more than or equal to 50%.

In some embodiments, the polypeptide comprises greater than or equal to 5% of the first amino acid group and greater than or equal to 10% of the second amino acid group and greater than or equal to 30% of the third amino acid group by mass of the polypeptide. The first set of amino acids includes tyrosine and phenylalanine. The second group of amino acids includes lysine, arginine, and histidine. The third amino acid group includes leucine, isoleucine, valine, proline, methionine, and alanine.

In some embodiments, the composition further comprises allantoin and glycolic acid.

In a second aspect, embodiments also provide a method of preparing a biomaterial according to any of the embodiments of the first aspect, the method comprising providing a gastropod; stimulating the epidermis of the gastropod to cause the gastropod to secrete a viscous substance; drying the viscous mass to obtain a composition; optionally, the drying is reduced pressure drying or freeze drying.

In a third aspect, embodiments also provide a product for tissue adhesion, repair, haemostasis, tissue fluid penetration sealing, cosmetic or as a cell culture medium, protein or pharmaceutical carrier, the product comprising a biomaterial as defined in any one of the embodiments of the first aspect of the present application; optionally, the product is in the form of a powder, a tablet, a sponge, a gel, or a paste.

In some embodiments, the product is a medical adhesive.

The natural biomaterial is a medical adhesive prepared based on unmodified snail mucus, has the function of strongly adhering to human soft tissues such as skin, muscle, viscera and the like, can be used for tissue adhesion and wound healing and repair, and has comprehensive performance superior to the prior medical adhesives such as cyanoacrylate, fibrin glue and the like.

The natural biomaterial has the advantages of no need of adding other reagents during use, convenient operation, easy modification, good biocompatibility and the like, and provides an innovative selection which has strong practicability and can be adhered without sewing for wounds of skin, fragile organs and inaccessible internal tissues.

The natural biomaterial has biomechanical properties close to those of human soft tissues, has proper rigidity, elasticity, deformability and strong adhesion, has good biocompatibility and biodegradability, and overcomes the defects and limitations of the existing products.

The natural biomaterial has the functions of remarkably promoting the revascularization and the epidermal regeneration of acute and chronic wound tissues, can continuously and effectively heal various wounds, and avoids secondary injury caused by changing medicaments along with gradual degradation in the wound healing period.

The preparation method of the natural biological material has the remarkable technical advantages of simple process, simplicity and convenience in operation, environment-friendly process, easiness in amplification, low production cost and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for a person skilled in the art to obtain other drawings based on the drawings without any creative effort.

FIG. 1 illustrates the physicochemical properties of snail mucus provided by some embodiments of the present application; wherein, fig. 1A is white jade snail; FIG. 1B shows a bright snail; FIG. 1C shows fresh mucus of white jade snail; FIG. 1D shows the fresh mucus of a clear snail; FIG. 1E shows the lyophilized product of snail mucus; FIG. 1F shows the appearance of a freeze-dried snail product; FIG. 1G is an electron microscope scanning image of a freeze-dried white jade snail product; FIG. 1H is an electron microscope scan of a freeze-dried product of snail slime; figure 1I is a demonstration of the effect of two freeze-dried snail mucus products on wet skin adhesion, left: white jade snail mucus freeze-dried product, right: freeze-drying snail mucus; FIGS. 1J and 1K are the rheological curves of aqueous gel of snail mucus and its lyophilized product, respectively; FIG. 1L shows the main chemical components and contents of lyophilized product of snail mucus.

FIG. 2 is an electrophoretogram of snail mucus polypeptide provided by some embodiments of the present application; wherein, FIG. 2A shows the electrophoresis result of polypeptide from mucus of white jade snail; FIG. 2B shows the electrophoresis results of the polypeptide of snail slime.

FIG. 3 is a structural analysis of snail mucus acid polysaccharide provided by some embodiments of the present application; wherein, FIG. 3A is the 1H NMR spectrum of snail mucus acidic polysaccharide; FIG. 3B is a 13C NMR spectrum of snail slime acid polysaccharide; FIG. 3C is the 1H-1H COSY spectrum of snail mucus acid polysaccharide; FIG. 3D is the HSQC spectrum of snail slime acid polysaccharide.

FIG. 4 is a tissue adhesion performance test of snail mucus and its lyophilized products provided by some embodiments of the present application; wherein, fig. 4A is the adhesion performance display of the fresh snail mucus and the freeze-dried product to the visceral tissues; FIG. 4B is a graph of lap shear adhesion test, wherein i is a schematic of the test method, ii is a shear force-time curve, and iii is a histogram of shear adhesion strength for each group; FIG. 4C is a graph of the tensile adhesion test, wherein i is a schematic of the test method, ii is a tensile force-time curve, and iii is a histogram of the tensile adhesion strength of each group; fig. 4D is a 180 ° peel test, where i is a schematic of the test method, ii is a peel force-time curve, and iii is a histogram of interfacial adhesion energy for each group (P <0.05, P <0.01, P < 0.001).

FIG. 5 is an in vitro and in vivo hemostatic evaluation of freeze-dried snail mucus product provided by some embodiments of the present application; wherein FIG. 5A shows the trend of hemagglutination index of each group with time; FIG. 5B is a scanning electron micrograph of the adhesion state of red blood cells and platelets to the surface of various materials (scale bar: 10 μm and 2 μm); FIG. 5C shows the hemagglutination status of each group at 150 s; FIG. D is a schematic diagram of the evaluation of hemostasis of rat liver; fig. 5E shows the statistics of liver bleeding for each group (— P <0.05, — P <0.01, — P < 0.001;); fig. 5F is a display of the amount of liver bleeding collected with filter paper for each group.

FIG. 6 is a graph showing the evaluation of the adhesion of freeze-dried snail mucus product to skin incisions, according to some embodiments of the present application; wherein, FIG. 6A is a diagram of skin adhesion effect or incision healing effect of each group at 0, 3, 5, 7 after operation (scale bar: 0.5 cm); FIG. 6B is a photograph of H & E staining of each group of tissue sections at day 7 after the operation (scale bar: 1mm, 200 μm); FIG. 6C is a heat map of the change in wound size over time during the healing of each set of incisions; fig. 6D is a statistics of the healing rate of the incisions for each group (— P <0.05, — P <0.01, —) P < 0.001.

Fig. 7 is an evaluation of the effect of freeze-dried snail mucus product on acute wound healing provided by some embodiments of the present application; wherein, FIG. 7A shows the healing effect of each group of wounds; fig. 7B is a statistics of the wound healing rates for each group (, indicates P <0.05, indicates P <0.01, indicates P < 0.001;); FIG. 7C shows H & E staining results (scale bar: 1mm and 100 μm) of tissue sections at 5 days and 15 days after operation.

Fig. 8 is a diagram illustrating the effect of freeze-dried snail mucus on healing of chronic wounds in diabetic rats, according to some embodiments of the present application, wherein fig. 8A is a schematic diagram illustrating the experimental design of animals; FIG. 8B is a representation of the healing of various groups of wounds; figure 8C is a summary of the healing rates of the groups (, indicates the significant difference between the freeze-dried snail mucus and the saline group;

the significant difference between the snail mucus acidic polysaccharide and the normal saline group is shown; to indicate alginate dressing^TMSignificant differences between groups and saline groups; wherein a single represents P<0.05, two represent P<0.01, three represent P<0.001); FIG. 8D is a photograph showing wound tissue sections H at day 7 and day 14 after surgery of each group&E staining results (scale bar: 1mm and 300 μm); panel E is granulation tissue thickness statistics at 3/7/14 days post-surgery for each group; FIG. F shows the results of Masson staining of wound tissue sections at day 7 and day 14 after surgery for each group (scale bar: 1mm and 300 μm); FIG. 8G shows the statistics of the thickness of granulation tissue in each group (P is indicated by<0.05, represents P<0.01, represents P<0.001)。

Fig. 9 is a biocompatibility evaluation of a freeze-dried snail mucus product provided by some embodiments of the present application; wherein, fig. 9A is a cell viability test statistical chart; FIG. 9B shows SMG subcutaneous degradation; fig. 9C is a graph of H & E staining of tissue sections where d-SMG was subcutaneously embedded) (. indicates P <0.05,. indicates P <0.01,. indicates P < 0.001; scale bar: 1 mm).

Detailed Description

In order to make the application purpose, technical solution and beneficial technical effects of the present application clearer, the present application is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are only for explaining the present application and are not intended to limit the present application.

For the sake of brevity, only a few numerical ranges are explicitly disclosed herein. However, any lower limit may be combined with any upper limit to form ranges not explicitly recited; and any lower limit may be combined with any other lower limit to form a range not explicitly recited, and similarly any upper limit may be combined with any other upper limit to form a range not explicitly recited. Also, although not explicitly recited, each point or individual value between endpoints of a range is encompassed within the range. Thus, each point or individual value can form a range not explicitly recited as its own lower or upper limit in combination with any other point or individual value or in combination with other lower or upper limits.

In the description herein, it is noted that, unless otherwise specified, "a plurality" means one or more than one; "several" means "plural" means two or more; the terms "above" and "below" are inclusive; the terms "upper", "lower", "inner", "outer", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the referred devices or elements must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present disclosure.

As used herein, the terms "optionally" and "optionally as" refer to embodiments of the present application that may provide certain benefits under certain circumstances. However, other embodiments may also be optional, under the same or other circumstances. Furthermore, the recitation of one or more alternative embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure.

The above summary of the present application is not intended to describe each disclosed embodiment or every implementation of the present application. The following description more particularly exemplifies illustrative embodiments. At various points throughout this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the list is provided only as a representative group and should not be construed as exhaustive.

In nature, mollusks such as mussels can firmly adhere to the surfaces of rocks, ships, sharks and the like in seawater, and the substances with adhesive property are mucin components which are secreted by the mollusks and are rich in dopamine modification. In recent years, mussel mucin extracted by modern biotechnology can be used as medical adhesive for ophthalmic surgery, skin tissue adhesion, bone adhesion, etc.; can also be used as trauma spray, and has antibacterial, analgesic, antipruritic, and healing promoting effects.

Inspired by mollusks such as mussel, scientists have made active progress in the research and development of various biomimetic medical adhesive materials by chemical synthesis.

The inventor finds that the catechol group of dopamine or other polyphenols in the mussel mucin and derivatives, whether of natural origin or biomimetic synthetic modification, is oxidized under alkaline conditions to form a covalent bond, which is the key of strong adhesion. Therefore, the involvement of an oxidizing agent, an alkaline agent or an enzyme is required for specific applications, which limits the range of clinical applications thereof.

In order to find a more suitable viscous substance, the inventors studied viscous substances secreted from terrestrial gastropods as a study object, found that the viscous substances secreted from gastropods are completely different in chemical composition from viscous substances secreted from mollusks such as mussels, and found that the viscous substances secreted from gastropods have unexpected effects in the fields of medical treatment, skin care and the like.

In view of this, the inventors propose a biomaterial comprising a composition obtained by drying a viscous substance derived from gastropods. The inventors found that mechanical stimulation of the gastropod portion of gastropods can cause a large amount of viscous material to be secreted, and that the composition obtained by drying the viscous material has excellent tissue adhesion and repair functions.

Gastropods are a class of terrestrial mollusks whose feet can protrude from the abdomen of the body and crawl in contact with the ground. Optionally, the gastropoda animals include animals of the order stemmatophora and/or the order basomophora. Further optionally, the gastropoda includes one or more of the families of the stone-sulphonidae (Onchididae), agate spirochaceae (Achatinidae), costaphrodisiae (pleuroodontidae), slug (Limacidae), myxophagous slug (phiomotidae) and barnacidae (Bradybaenidae).

Illustratively, the gastropoda includes one or more of a brown cloud agate snail, a white jade snail, a bright big snail, a south red snail and a french countryside snail; the foregoing is illustrative only and is not intended to limit the scope of the embodiments herein.

[ acidic polysaccharide ]

In some embodiments, the compositions of the embodiments include an acidic polysaccharide, wherein a is greater than or equal to 5% by weight of the composition; optionally, the mass content a of the acidic polysaccharide is more than or equal to 5% and less than or equal to 25%. The acidic polysaccharide is used as snail glycosaminoglycan, is a water-soluble macromolecular substance and has strong water absorption and moisture retention capacity; and within the above mass content range, the water absorbing and moisturizing ability is more excellent.

Alternatively, the acidic polysaccharide comprises a homologous compound of the structure shown in formula (I),

in the formula (I), the compound is shown in the specification,

the A ring is alpha-D-2-amino-2-deoxy-glucosyl alpha-D-GlcN,

R₁is acetyl-COCH₃Or sulfonic acid group-SO₃ ^-；

R₂is hydroxy-OH or sulphate-OSO₃ ^-，

R₃Is carboxy-COO^-And inorganic salts thereof;

n is an integer greater than 100;

wherein the sulfate-based OSO is based on the mass of the acidic polysaccharide₃ ^-The mass percentage content c is that c is more than or equal to 8 percent.

Acidic polysaccharides are carboxyl-and sulfate-rich glycosaminoglycans consisting of monosaccharides of α -D-2-amino-2-deoxy-glucose (α -D-GlcN, GlcN) and hexuronic acids including α -L-iduronic acid (α -L-IdoA, IdoA) and α -D-glucuronic acid (α -D-GlcA, GlcA). The acidic polysaccharide can be macromolecular polysaccharide formed by alternatively connecting monosaccharide alpha-D-GlcN and alpha-L-IdoA or alpha-D-GlcA by (1 → 4) glycosidic bonds.

The carboxyl, hydroxyl and other groups contained in the acidic polysaccharide have hydrophilicity, hydrogen bonds are easily formed between molecules and in the molecules, and molecules of the acidic polysaccharide can be crossed and wound, so that the water absorption and moisture retention performance and the tissue adhesion performance of the acidic polysaccharide can be further improved.

[ polypeptide ]

In some embodiments, the compositions of the embodiments herein can further include a polypeptide. Based on the mass of the composition, the mass percentage content b of the polypeptide is more than or equal to 10 percent, and a + b is more than or equal to 50 percent.

The polypeptide includes aspartic acid (Asp), threonine (Thr), serine (Ser), glutamic acid (Glu), glycine (Gly), alanine (Ala), cystine ((Cys)2), valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), histidine (His), lysine (Lys), arginine (Arg) and proline (Pro).

Optionally, the mass percentage content of the first amino acid group is greater than or equal to 5% based on the mass of the polypeptide. The first amino acid includes tyrosine and phenylalanine.

Optionally, the mass percentage content of the second amino acid group is greater than or equal to 10% based on the mass of the polypeptide. The second amino acid group includes lysine, arginine, and histidine.

Optionally, the third amino acid group is present in an amount of greater than or equal to 30% by mass of the polypeptide. The third amino acid group includes leucine, isoleucine, valine, proline, methionine, and alanine.

The polypeptide is water-soluble macromolecular substance, and also has water-absorbing and moisture-keeping capacity. The acidic polysaccharide and the polypeptide have synergistic effect, carboxyl and sulfate radical anions contained in the acidic polysaccharide can form stronger ionic bonds with amino and guanidyl and the like contained in the polypeptide, a large number of hydroxyl groups of the acidic polysaccharide and the polypeptide form hydrogen bonds, the acidic polysaccharide and the polypeptide can be crossed and wound mutually, the same molecules or the same molecules can be crossed and wound mutually, and the like, and a porous structure such as a sponge-shaped structure or a gel-shaped structure can be formed under the action of various chemical bonds and micromechanics.

In some embodiments, the compositions of the embodiments of the present application may also include allantoin and glycolic acid. Allantoin has effects of promoting cell growth, promoting wound healing, and softening keratin. Glycolic acid contains carboxyl and hydroxyl groups and readily forms intermolecular forces with other molecules. Optionally, the compositions of the embodiments of the present application may also include minerals and the like.

The composition of the embodiment of the application can be prepared into a powdery substance after drying treatment, and also can be prepared into a sponge shape, a sheet shape, a gel shape or a paste shape according to different application scenes. The product in the form of sponge, sheet, gel or paste can be further prepared from the powder. Alternatively, the drying treatment may be drying under reduced pressure or freeze-drying.

When the biological material is applied to biological tissue repair, the powdery biological material in the same form can rapidly absorb water and gel after contacting and wetting the biological tissue, and the excellent adhesion performance is embodied; and the biological material has the biomechanical property close to that of biological tissues, particularly human soft tissues, and has good matching property with the biological tissues. In this context, biological tissue includes skin, muscle, or viscera, among others.

Based on the same application concept, the embodiment of the application also provides a method for manufacturing the biological material, which comprises the following steps:

providing an animal of the gastropoda class;

stimulating the epidermis of the gastropod to cause the gastropod to secrete a viscous substance;

drying the viscous mass to obtain a composition;

optionally, the drying is reduced pressure drying or freeze drying.

The method provided by the embodiment of the application has the advantages of simple process, simplicity and convenience in operation, low production cost and easiness in industrialization. The prepared biological material has excellent water absorption, moisture retention and adhesion performances as the biological materials in the embodiments.

The embodiments of the present application also provide a product for tissue adhesion, repair, hemostasis, tissue fluid permeation sealing, cosmetology, or as a cell culture medium, protein, or drug carrier, comprising the biomaterial of each of the above embodiments. Optionally, the product is in the form of a powder, a tablet, a sponge, a gel, or a paste.

The product of the embodiment of the application can be used for tissue adhesion and repair; illustratively, the product is a medical adhesive, i.e., the embodiments of the present application provide for the use of a biomaterial in the preparation of an adhesive for surgery, dermal tissue adhesion, bone adhesion, and the like.

When the medical adhesive is adhered to biological tissues, the adhesion force of the medical adhesive is basically not influenced by water and blood. The medical adhesive can instantly absorb interfacial water when contacting wet biological tissues, so that the interference of the interfacial water is avoided, and the spatial distance between the medical adhesive and the biological tissues is shortened, so as to achieve the degree of easily forming intermolecular acting force; the medical adhesive contains acidic polysaccharide which is rich in carboxyl and sulfate radical anions and can form stronger ionic bonds with amino groups, guanidyl groups and the like in proteins of biological tissues, the medical adhesive contains polypeptide, amino groups, guanidyl groups or carboxyl groups of the polypeptide and can form ionic bonds with carboxyl groups, amino groups, guanidyl groups and the like in proteins of the biological tissues, and abundant hydrogen bonds and the like can be formed between a large number of hydroxyl groups of the acidic polysaccharide and the polypeptide and the biological tissues, so that the adhesion performance of the medical adhesive is improved.

The product of the embodiment of the application also has good performances of sealing the wound surface, stopping bleeding, promoting wound healing and repairing, and can be used as a trauma spray for repairing skin and mucosa, repairing nerves and the like caused by burns, scalds, operations and the like. Namely, the embodiment of the application provides an application of the biological material in wound tissue repair-skin wound repair.

Because the biological material comprises the biological material with better biocompatibility with biological tissues and has lower blood coagulation index, when the biological material is applied to hemostasis, red blood cells and platelets in blood can be adhered to the surface of the biological material or embedded in tiny holes of the biological material to form an aggregation state, thereby being capable of rapidly stanching, reducing the bleeding amount and shortening the bleeding time. Therefore, the biological material has good functions of adhering wounds and stopping bleeding.

The biomaterial of the embodiments of the present application can also be used for the closure and repair of chronic wounds, including diabetic feet. The diabetic skin tissue ulcer including diabetic foot is a common high-risk complication in diabetic patients, and the diabetic patients are affected by foot ulcers with different degrees, and can amputate the limbs of the patients in serious conditions, so that serious health threat is brought to the diabetic patients, and heavy social and economic burden is caused. The product provided by the embodiment of the application can be used as an external dressing, and can obviously promote the healing of skin wounds and/or wound surfaces and/or ulcers of diabetic feet and the like, so that the dressing has an important application value for preventing and/or treating diabetic skin tissue ulcers. Based on the application, the embodiment also provides the application of the biological material in healing of wounds of tissues which are difficult to heal, such as diabetic feet and/or pressure sores, bedsores, burns and the like.

The main components of the biomaterial in the embodiment of the application are acidic polysaccharide and polypeptide, the freeze-dried patch and the freeze-dried sponge are porous sponge-shaped or honeycomb-shaped, and the biomaterial can absorb tissue fluid permeation and is further used for preparing a tissue fluid permeation plugging product. Therefore, the application also provides an application of the biological material in preparing tissue fluid permeation plugging products.

The biomaterial of the embodiment of the application comprises the acidic polysaccharide, and the acidic polysaccharide can form hydrogen bonds, ionic bonds and the like with small molecule drugs, proteins, nucleic acids and cells due to the rich functional groups such as hydroxyl, carboxyl, sulfate and the like, so that the biomaterial in the product can be used as a carrier to load functional components such as drugs, proteins, nucleic acids and cells, and the chemical bonds can play a role in slow release due to reversibility. Therefore, the application also provides a biological material which is applied to wound surface sealing and used as a carrier of medicines, proteins and cells in tissue engineering and regenerative medicine.

The biological material of the embodiment of the application can be used as a functional raw material of beauty cosmetics and the like, and can be added into the cosmetics to be further prepared into various liquid, solid or semisolid masks, creams, water aqua and the like. Therefore, the application also provides an application of the biological material in preparing a cosmetic product.

Examples

The present disclosure is more particularly described in the following examples that are intended as illustrations only, since various modifications and changes within the scope of the present disclosure will be apparent to those skilled in the art. Unless otherwise indicated, all parts, percentages, and ratios reported in the following examples are on a weight basis, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further treatment, and the equipment used in the examples is commercially available.

Example 1

Preparation of freeze-dried snail mucus and chemical composition analysis thereof

1.1 Experimental materials

Raw materials: white jade snails (Achatina furica "white") and bright big snails (Helix lucorum) are commercially available.

Reagent: bicinchoninic Acid protein detection kit, beijing solibao science and technology ltd; triton X-100, 1, 9-dimethyl methylene blue, alkaline protease, glycine, tris, sodium dodecyl sulfate, ammonium persulfate, N, N, N ', N' -tetramethyl ethylenediamine, allantoin, glycolic acid, NaCl, NaOH and other reagents are all commercially available analytical pure reagents.

1.2 Snail mucus Collection and Freeze-dried product preparation

Cleaning snail, placing in clean culture dish, stimulating the ventral foot of snail with sterile wood stick, allowing the snail to secrete mucus (snail mucous gel, abbreviated as SMG), freezing the obtained fresh mucus in liquid nitrogen for 15min or at-20 deg.C in refrigerator, and drying under reduced pressure by freeze dryer to obtain freeze-dried product of snail mucus (abbreviated as d-SMG).

1.3 analysis of the polypeptide content, amino acid composition and gel electrophoresis of the freeze-dried snail mucus

(1) Polypeptide content analysis: weighing 25mg of freeze-dried snail mucus, dissolving in 20mL of PBS buffer solution containing 0.5% triton X-100, and diluting to 25.00mL to obtain a test solution. And (3) taking bovine serum albumin as a protein standard substance, and drawing a standard curve by adopting a Bicinchoninic Acid protein detection kit. And operating the test solution according to the instruction, substituting the measured absorbance value into a standard curve equation to calculate the corresponding protein concentration, and calculating the polypeptide content in the freeze-dried snail mucus product. Samples were taken in triplicate.

(2) Amino acid composition analysis: weighing 25mg of freeze-dried snail mucus, adding 10mL of 6mol/L hydrochloric acid, heating at 110 ℃ for 20h under the protection of nitrogen, cooling to room temperature, filtering, and metering the volume of the filtrate to 25 mL. Deacidifying 1.00mL of solution at 60 ℃ for 2h, adding 1.00mL of sample diluent to obtain a test solution, separating by adopting cation chromatography, and analyzing the standard sample and the test sample in sequence by a method of detecting by a ninhydrin post-column derivatization method. Samples were run in triplicate.

The chromatographic conditions were as follows:

a chromatograph: amino acid analyzer A300

A chromatographic column: sulfonic acid type cationic resin, 4.0X 125mm

Sample introduction amount: 20 μ L column temperature: 40-70 DEG C

Mobile phase: flow rate of sodium salt buffer: 0.24mL/min

A detector: mini diode photometer (570nm, 440nm two detection wavelength)

(3) Electrophoretic analysis: 4.5375g of tris (hydroxymethyl) aminomethane was weighed, dissolved in water, adjusted to pH 8.8 with hydrochloric acid, and diluted to 25mL with water to obtain a separation gel buffer. 1.5125g of tris (hydroxymethyl) aminomethane is weighed, dissolved in water, the pH value is adjusted to 6.8 by hydrochloric acid, and the solution is diluted to 25mL by water to obtain concentrated gel buffer solution. 0.2g of sodium dodecyl sulfate is weighed, and is dissolved and diluted to 2mL by water, thus obtaining 10 percent SDS solution. 0.2g of ammonium persulfate is weighed and dissolved in water to be diluted to 2mL, and then 10 percent ammonium persulfate solution is obtained. 0.2g of N, N, N ', N' -tetramethylethylenediamine is weighed and dissolved in water to be diluted to 2mL, and then 10% TEMED solution is obtained. Taking 4mL of 30% Acr-bis, 2.5mL of separation gel buffer solution, 0.1mL of 10% SDS solution, 0.1mL of 10% ammonium persulfate solution, 0.004mL of TEMED solution and 3.3mL of water in a beaker, homogenizing, pouring into a mold, adding water, sealing, and standing for 30 min; 5.8mL of deionized water, 1.5mL of 30% Acr-bis, 2.5mL of concentrated gel buffer solution, 0.1mL of 10% SDS solution, 0.1mL of 10% ammonium persulfate solution and 0.01mL of TEMED solution are put in a beaker, after the polymerization of the separation gel is finished, the upper water is sucked off, and the concentrated gel solution is poured into the beaker and inserted into a sample comb. Taking 2.5mg of the freeze-dried snail mucus, adding 1mL of phosphate buffer saline solution, ultrasonically dissolving for 30min at 37 ℃, centrifuging (15000rpm multiplied by 10min), and taking the supernatant to obtain a sample solution to be detected. And respectively loading the protein standard substance and the sample for gel electrophoresis, taking out the gel after the protein band electrophoresis is carried out to a proper position, and carrying out dyeing analysis. Samples were run in triplicate.

1.4 content determination of acidic polysaccharide in freeze-dried snail mucus product

Putting 8mg of 1, 9-dimethylmethylene blue, 1.52g of glycine and 1.185g of NaCl in a beaker, adding deionized water to dissolve the materials, fixing the volume in a 500ml volumetric flask, and shaking up to obtain the dye stock solution. White jade snail acidic polysaccharide reference substance is weighed, and water is added to prepare solution containing 0.1mg of glycosaminoglycan per 1 ml. The control solutions 0.2, 0.4, 0.6, 0.8 and 1.0ml were measured separately and placed in 1.5ml test tubes, water was added to 1.0ml and shaken up. And (3) weighing 0.1ml of each solution, putting the solution into a cuvette, and adding 3ml of dye stock solution to obtain a reference substance determination solution. The absorbance was measured at a wavelength of 592nm by UV-visible spectrophotometry. And drawing a standard curve by taking the absorbance as the ordinate and the acid polysaccharide concentration as the abscissa.

Weighing white jade snail mucus freeze-dried product 100mg, adding 5ml deionized water, and performing ultrasonic treatment until no particles exist. After the pH value of 6M NaOH solution is adjusted to 9, 2mg of alkaline protease is added, the mixture is stirred for 36 hours in water bath at the temperature of 60 ℃, and the pH value is controlled to 9 in the enzymolysis process. The enzymatic hydrolysate was centrifuged (4000 rpm. times.20 min) to obtain a supernatant, which was then diluted 10-fold with 100. mu.l of a volumetric flask of 25 ml. Weighing 500 μ l, placing in 1.5ml test tube, adding water to 1ml, and shaking to obtain test solution. Repeating the above operations, measuring absorbance value of the sample, substituting into a standard curve equation to calculate corresponding concentration, and calculating polysaccharide content in the freeze-dried snail mucus product. Samples were taken in 6 portions for parallel testing.

1.5 content determination of allantoin in Snail mucus lyophilized products

The allantoin content of the freeze-dried snail mucus product was determined by High Performance Liquid Chromatography (HPLC), and analyzed by HILIC hydrophilic column (3 μm, 150X 2mm, Phenomenex, USA) on high performance liquid chromatography (Agilent 1260, USA).

The chromatographic conditions were as follows: mobile phase system: acetonitrile 10mM ammonium formate solution (pH 2.70) 90: 10; flow rate: 0.2 mL/min; detection wavelength: 200 nm; column temperature: 30 ℃; sample introduction amount: 0.5. mu.L.

Allantoin standard solutions were prepared at concentrations of 0.04, 0.06, 0.08, 0.12 and 0.16mg/mL, respectively. Weighing freeze-dried snail mucus 15mg, adding 10mL mobile phase, mixing with ultrasound for 10min, heating and dissolving at 60 deg.C for 1h, centrifuging (15000rpm × 15min), and collecting supernatant as sample. Samples were taken in triplicate for testing.

Substituting the integral areas of the standard sample and the test sample into a standard curve equation to calculate the allantoin concentration in the test sample, and further calculating the content of the allantoin in the freeze-dried snail mucus product.

1.6 content determination of glycolic acid in lyophilized product of snail mucus

And (3) measuring the content of glycolic acid in the snail mucus freeze-dried product by adopting ion chromatography. Weighing 100mg of freeze-dried snail mucus, adding into 40mL of deionized water, ultrasonic bath for dissolving for 30min, diluting to 50.0mL of solution, centrifuging (3000g × 15min), and collecting supernatant to obtain test solution. The sample and the standard were analyzed and measured by an ICS-1100 ion chromatograph (Thermo Dionex, USA).

1.7 results of the experiment

The results of the experiment are shown in FIGS. 1 and 2, and see Table 1 together.

The inventor has observed that the ventral foot part of the snail secretes mucus, adhering itself to the surface of other objects (branches, leaves, etc.), a reversible adhesion mode, which is clearly different from the adhesion of mussel foot silk to the surface of submarine rocks, ships and sharks, etc.

The fresh white jade snail mucus is in a semitransparent gel state, and the bright snail mucus is in a milk white gel state, and has good elasticity and ductility; the freeze-dried product is porous sponge-shaped, can form sticky jelly when mixed with interfacial water on the surface of wet skin, and has strong adhesiveness.

According to the method for preparing snail mucus and freeze-dried product thereof, about 20g of fresh mucus can be taken by 10 snails on average; the solid content of the white jade snail mucus is 3.2 percent and the solid content of the bright snail mucus is 5.3 percent calculated by the dry basis of the freeze-dried snail mucus.

The content of acidic polysaccharide in the mucus frozen product of white jade snail and bright snail is 15.91 + -0.23% and 9.26 + -0.17% respectively.

The content of snail mucus polypeptide in the mucus frozen products of white jade snail and bright snail is 33.27 + -1.78% and 35.71 + -1.45% respectively on a dry basis; electrophoresis results show that the molecular weight of the white jade snail mucus polypeptide is distributed in three regions of 45kDa, 60-100 kDa and >100 kDa; the snail slime polypeptide is distributed in the range of 15-35 kDa, 45kDa, 60kDa, 75kDa and >100 kDa.

The amino acid composition analysis results show that the snail mucus polypeptide consists of dozens of amino acids such as aspartic acid (Asp), threonine (Thr), serine (Ser), glutamic acid (Glu), glycine (Gly), alanine (Ala), cystine ((Cys)2), valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), histidine (His), lysine (Lys), arginine (Arg) and proline (Pro) (Table 1), wherein the aromatic amino acid accounts for 10.38% and 10.65% of the two snail mucus polypeptides, the basic amino acid accounts for 16.98% and 15.29% of the polypeptides, and the nonpolar amino acid accounts for 38.34% and 36.80% of the polypeptides.

TABLE 1 Snail mucus polypeptide amino acid content (mg/g)

On a dry basis, the freeze-dried mucus products of white jade snails and clear big snails contain 2.74 +/-0.07% of allantoin and 1.03 +/-0.04% of allantoin respectively, and 7.13 +/-0.41 mg/g and 0.98 +/-0.18 mg/g of glycolic acid respectively.

Example 2

Chemical structure analysis of acidic polysaccharide in freeze-dried snail mucus product

2.1 reagent:

d-2-acetamido-glucose (D-GlcNAc) is an Alfa Aesar product; l-iduronic acid (L-IdoA), product of Carbosynth, UK; glucose (Glc) is a product of Chinese drug inspection; 3-methyl-1-phenyl-2-pyrazolin-5-one (PMP), an alatin product; dextran standards D1(2500Da), D2(4600Da), D3(7100Da), D4(10000Da), D5(21400Da), D6(41100Da), D7(84400Da), D8(133800Da) and D2000(2000000Da) China drug biological product accreditation offices; 3000Da dialysis bag, Spectrum Laboratories, USA; the reagents such as alkaline protease, benzethonium chloride, potassium acetate, silver nitrate, trifluoroacetic acid, NaCl, NaOH and the like are all commercially available analytical pure reagents.

2.2 extraction and purification of acidic polysaccharide in freeze-dried snail mucus product

20g of freeze-dried snail mucus obtained in example 1 and 400mg of alkaline protease are weighed and placed in a flask, 200mL of water and 2M NaOH are added to adjust the pH value to 9, the mixture is subjected to enzymolysis reaction and extraction for 36h under the condition of continuous stirring in a water bath at 60 ℃, cooled to room temperature, kept stand overnight at 4 ℃, and centrifuged (4000rpm × 15min) to obtain a supernatant. 11.6g of potassium acetate and 95% ethanol were added to the supernatant to a final concentration of 75%, and the mixture was centrifuged (4000 rpm. times.15 min) to obtain a precipitate. After the precipitate was redissolved in water, 4% benzethonium chloride solution was added dropwise with stirring until no white precipitate was formed, and the mixture was centrifuged (4000 rpm. times.15 min) to obtain a precipitate. Washing the precipitate with deionized water for 3 times, adding saturated salt water of the same volume, stirring, and adding anhydrous ethanolThe final concentration was 80% (v/v), and the precipitate was obtained by centrifugation (4000 rpm. times.15 min), and the salt exchange step was repeated 3 times. Dissolving the precipitate with appropriate amount of water, dialyzing with 3000Da dialysis bag, and detecting with 0.1M silver nitrate until no Cl is present in the dialyzed water^-Concentrating, and lyophilizing to obtain snail mucus acidic polysaccharide.

2.3 structural analysis of acidic polysaccharide in freeze-dried snail mucus product

(1) Analysis of monosaccharide composition: according to the technical personnel familiar with the column PMP derivatization method for monosaccharide composition analysis, acid polysaccharide is completely hydrolyzed with trifluoroacetic acid, then reacts with PMP under alkaline condition, after extraction, the high performance liquid chromatograph C18 column analysis is carried out, the chromatographic condition is ZORBAX SB-C18(4.6mm x 150mm x 5 μ M) chromatographic column, the mobile phase is 0.1M ammonium acetate (pH5.5) buffer solution-acetonitrile (83:17), the column temperature is 25 ℃, the flow rate is 1 mL/min.

(2)-OSO₃-/-COO^-And (3) analysis of molar ratio: analyzing the molar ratio of sulfate groups to carboxylic acid groups contained in the snail acidic polysaccharide by adopting a conductivity titration method, and calculating sulfate groups (-OSO)₃ ^-) Accounts for the content of the acidic polysaccharide. Samples of 5mg each were weighed and dissolved in 1mL of deionized water to prepare 5mg/mL aqueous solutions. Exchange the solution with Dowex 50w × 850-100H strong acid cation exchange resin to be hydrogen type, the flow rate is 3s/d, and 40mL of eluent is collected. Titrations with 0.02M NaOH titrant, 50 μ L each time, and recording the titration volume and conductivity values. Calculation of-OSO from the consumption volume of NaOH₃ ^-/-COO^-Molar ratio and-OSO₃ ^-Mass fraction of (c).

(3) Determination of molecular weight and distribution thereof: taking about 10mg of sample, adding mobile phase to prepare 10mg/mL solution, filtering with 0.22 μm microporous membrane, and performing liquid-phase analysis on the filtrate; weighing dextran reference substances with known molecular weights, and preparing into a solution of 10 mg/mL; and processing the data by adopting GPC software, drawing a standard curve, and calibrating the molecular weight of the snail mucus acidic polysaccharide by using the curve. The chromatographic conditions were as follows:

chromatograph: agilent Technologies 1260series high performance liquid chromatograph

A chromatographic column: shodex SB-804HQ (8.0X 300mm)

Sample introduction amount: 10 μ L column temperature: 40 deg.C

Mobile phase: flow rate of 0.1M NaCl: 0.5mL/min

A detector: differential Refractometer (RID) and Diode Array Detector (DAD)

(3) Nuclear magnetic resonance spectroscopy: about 10mg of sample was taken after 3 times of D₂Exchange of O, D₂Dissolving O to 20mg/mL, detecting 1D by 800MHz nuclear magnetism instrument¹H、¹³C NMR and 2D¹H-¹H (COSY, TOCSY, ROESY) and¹H-¹³C(HSQC,HMBC,HSQC-TOCSY)。

2.4 results of the experiment

The results of the experiment are shown in fig. 3, table 2 and table 3.

Monosaccharide composition analysis results show that the acidic polysaccharide of the white jade snail mucus consists of D-2-amino-2-deoxy-glucose (D-GlcN, GlcN) and L-iduronic acid group (L-IdoA, IdoA), and the acidic polysaccharide of the bright big snail consists of N-acetylglucosamine (GlcNAc), D-glucuronic acid (GlcA), iduronic acid (IdoA) and the like.

The acidic polysaccharide in the white jade snail mucus has weight average molecular weight (Mw) and number average molecular weight (Mn) of 696.7kDa and 540.7kDa, respectively, and polydispersity index (PDI) of 1.29; the acidic polysaccharides in the clear snail mucus have Mw and Mn of 900.0kDa and 725.8kDa polydispersity numbers (PDI) of 1.24, respectively.

In the white jade snail mucus and the bright snail mucus, the molar ratio of the sulfuric acid and the carboxylic acid of the acidic polysaccharide is 1:1.02 and 1:1.25 respectively; with sulfate (-OSO)₃ ^-) Accounting for the mass fraction of the acidic polysaccharide, the content of the acidic polysaccharide is 15.4 percent and 13.6 percent respectively.

TABLE 2 physicochemical Properties of Snail mucus acidic polysaccharide

As shown in figure 3 of the drawings,¹H、¹³analysis of the C and 2D NNR spectrogram shows that the acidic polysaccharide of the mucus of the two snails is formed by alternately connecting D-GlcN and L-IdoA and/or alpha-D-GlcA by alpha (1 → 4) glycosidic bonds.

Taking white jade snail mucus acidic polysaccharide as an example, the complete assignment of NMR spectra of H and C of GlcNAc and IdoA of the polysaccharide can be carried out, and the results are shown in Table 3.

TABLE 3 chemical shift tables for H and C of acidic polysaccharides in white jade snail mucus

Example 3

Scanning electron microscope observation of freeze-dried snail mucus product

Placing snail mucus hydrogel in a sample tank, freezing the sample tank in liquid nitrogen for 10min, taking out the sample tank, placing in a freezing vacuum chamber at (-90 deg.C) for sublimation for 10min, plating platinum on the surface of the sample, and scanning with an electron microscope. And selecting three magnification factors of 800X, 1200X and 1600X, and randomly searching a flat area for image recording.

The results are shown in fig. 1G and 1H, the interior of the white jade snail mucus freeze-dried product is in a honeycomb-like or spongy three-dimensional space structure, and the bright snail mucus freeze-dried product is similar to the white jade snail mucus freeze-dried product, but the pore size is slightly different.

Example 4

Rheological property test of snail mucus freeze-dried product hydrogel

The rheological tests were carried out using a rheometer type MCR302 (germany).

And (3) uniformly paving 0.5g of snail mucus hydrogel on a test platform, setting the amplitude range to be 5% and the frequency range to be 0.1-10 Hz, and carrying out test by using a test bench at a constant temperature of 25 ℃.

The results of storage modulus (G ') and loss modulus (G') at different solids contents for the two snail fresh mucus and mucus lyophilisate hydrogels are shown in FIGS. 1J and 1K and Table 4. The modulus values of the two kinds of fresh snail mucus show that the two kinds of snail mucus are gel substances with better elasticity; after the freeze-dried product of the snail mucus is hydrated, the freeze-dried product of the snail mucus has viscoelasticity similar to and stronger than that of fresh mucus. Along with the increase of solid content, the storage modulus of the gelled snail mucus freeze-dried product is increased by multiple times, and the storage modulus is matched with the elastic modulus range of various organs and tissues of a human body.

TABLE 4 storage and loss moduli of hydrated gels of fresh snail mucilage and its lyophilized products

Remarking: the table shows the data at a frequency of 0.631 Hz.

Example 5

In-vitro adhesion performance test of snail mucus freeze-dried powder

5.1 Experimental materials

Freeze-dried snail mucus powder: prepared from example 1.

Rat whole blood and fresh ex vivo viscera were collected from SD rats that were about to be euthanized in other examples; porcine fibrin adhesive (fibrin glue for short), medical grade, Guangzhou Beixiu biotechnology limited; cutting fresh pigskin into a rectangle of 1.0cm multiplied by 7.0cm when in use; the pig intestine casing is cut into 1.0 × 3.0cm and 1.0 × 1.0cm shapes when in use, and is adhered to a plastic tape or a glass sheet.

5.2 tissue adhesion test

Fresh mucus group: approximately 200mg of fresh mucus was taken, placed on fresh isolated rat visceral tissue, pulled with forceps, and the mucus adhesion performance was observed.

Mucus freeze-dried powder group: the lyophilized mucus product of 5mg was placed on the fresh isolated rat visceral tissue, pulled with forceps, and the mucus adhesion performance was observed.

The experimental result is shown in fig. 4A, fresh mucus has certain adhesive capacity, a freeze-dried mucus product has strong adhesive capacity, and a forceps is used for clamping one corner of the mucus to lift off body tissues. The results show that the freeze-dried product of the fresh snail mucus is remarkably improved in the ability of adhering to various tissues after freeze-drying.

5.3 shear adhesion Strength test

Mucus freeze-dried powder group: weighing 5mg snail mucus lyophilized powder, spreading the lyophilized powder in a square region of casing 1.0cm × 1.0cm, dripping 10 μ L deionized water or fresh whole blood, overlapping two casings as shown in figure, and fixing for 1 min.

Fibrin glue group: 20 μ L fibrin glue is dropped into a square area of 1.0cm × 1.0cm of casing, and is coated uniformly and fixed for 1 min.

Fixing the two ends of each group of sausage casings on a clamp of a universal tensile machine, carrying out a shearing test at the speed of 0.5mm/s, and recording the maximum tensile value and a time-stress curve.

The results are shown in FIG. 4B, wherein the tissue adhesion strength of the fresh white jade snail mucus and the bright snail mucus are respectively 4.62 + -0.56 kPa and 10.30 + -0.78 kPa; the tissue adhesion strength of the two freeze-dried products is 23.64 +/-2.00 kPa and 35.67 +/-2.09 kPa respectively, which is close to or better than fibrin glue (20.79 +/-0.51 kPa); the adhesion strength of the tissue surface covered by blood in the two mucus groups is 33.85 +/-3.09 kPa and 40.5 +/-3.86 kPa respectively, and is obviously superior to that of fibrin glue (P < 0.01).

5.4 tensile adhesion Strength test

Mucus freeze-dried powder group: weighing 5mg snail mucus lyophilized powder, uniformly spreading in a square region of casing 1.0cm × 1.0cm, dripping 10 μ L deionized water or whole blood, overlapping two casings as shown in figure, and fixing for 1 min.

Fibrin glue group: dripping 20 μ L fibrin glue into square area of casing 1.0cm × 1.0cm, coating, and fixing for 1 min.

After the fixation is finished, the two ends of each group of sausage casings are fixed on a clamp of a universal tensile machine, the tensile test is carried out at the speed of 0.1mm/s, and the maximum tensile value and a time-stress curve are recorded.

The experimental results are shown in figure 4C, wherein the tissue adhesion strength of the fresh white jade snail mucus and the bright snail mucus are respectively 2.86 + -0.13 kPa and 2.20 + -0.39 kPa; the tissue adhesion strength of the two freeze-dried products is 51.38 +/-3.18 kPa and 82.59 +/-7.39 kPa respectively, and the tissue adhesion strength is obviously superior to that of fibrin glue (0.57 +/-0.10 kPa) (P is less than 0.001); while the blood-covered tissue surfaces of the two mucus groups produced adhesive strengths of 60.51 + -2.18 kPa and 52.31 + -4.62 kPa, respectively, which were significantly better than those of the fibrin glue group (P < 0.001).

5.5 Peel test

Mucus freeze-dried powder group: weighing 20mg snail mucus lyophilized powder, uniformly spreading in rectangular area of pigskin 1.0cm × 3.0cm, dripping 40 μ L deionized water or whole blood, overlapping two sheets of pigskin as shown in figure, and fixing for 1 min.

Fibrin glue group: dripping fibrin glue 80 μ L into pigskin 1.0cm × 3.0cm, coating, and fixing for 1 min.

After the fixation is finished, the two free ends of the pigskins of each group are fixed on a clamp of a universal tensile machine, a 180-degree peeling test is carried out at the speed of 0.5mm/s, and a time-stress curve is recorded.

The experimental results are shown in FIG. 4D, and the interfacial adhesion energy of the fresh white jade snail and the bright snail mucus of the bright snail are respectively 7.37 +/-0.58J/m²And 7.07. + -. 0.62J/m²(ii) a The interfacial adhesion energy of the two freeze-dried products is respectively 24.27 +/-1.37J/m²And 33.70. + -. 4.46J/m²Are all obviously superior to fibrin glue (6.77 +/-0.67J/m)²)(P<0.001，P<0.01); the interfacial adhesion energy generated by the tissue surface covered by blood of the two freeze-dried product groups is respectively 30.13 +/-7.74J/m²And 16.87. + -. 4.52J/m²Is superior to fibrin glue (P)<0.05)。

In summary, the biomaterial of the embodiments of the present application is exemplified by adhesion of lyophilized product to pig intestine or pig skin, and mechanical parameters such as lap shear, tensile adhesion and 180 ° peel are tested in the presence of water or plasma or blood. The results show that the material can strongly adhere to biological tissues including skin, muscle, viscera and the like, and the adhesion is not influenced in the presence of water, blood and the like. According to various indexes of the adhesion mechanical test, the biological material of the embodiment of the application is obviously superior to the porcine fibrin glue used clinically.

Example 6

Evaluation of hemostatic efficacy of freeze-dried snail mucus product

6.1 materials and instruments

SD rat, male, 180-: 2019 and 0004; fresh whole blood (containing sodium citrate anticoagulant) was collected from healthy SD rats.

0.2mol/L CaCl₂Solution, Phosphate Buffered Saline (PBS), 3% glutaraldehyde solution,Sterile gauze, round filter paper, surgical instruments and the like are all of analytical pure or medical grade sold in the market.

Microplate reader (Flex Station 3), Thermo Scientific, USA; scanning electron microscopy (Sigma 300) zeiss, germany; small animal anesthesia machine (VOR76X-6), Lenbell instruments manufacture, Haiman.

6.2 in vitro hemostasis test

(1) Blood coagulation index determination

Grouping experiments:

positive control: medical gauze;

negative control: blank processing;

experimental groups: white jade snail mucus freeze-dried product group and bright snail mucus freeze-dried product group.

The experimental method comprises the following steps: weigh 44.4mg CaCl₂Dissolving the solid in 2.0mL of water to obtain 0.2M CaCl₂A solution; 0.2M CaCl₂Adding 50 mu L of the solution into 1.0mL of rat whole blood, and uniformly mixing to prepare the calcium-enriched whole blood solution.

Adding 10 μ L of the calcium-enriched whole blood solution into the sample or the control, incubating at 37 deg.C for 60, 90, 120 and 150s, slowly adding 1.0mL of deionized water at corresponding time points, washing out free red blood cells, centrifuging the solution (116g × 10min), obtaining supernatant, and measuring the absorbance value As at 540 nm.

mu.L of the calcium-enriched whole blood solution was added to 1.0mL of deionized water, and centrifuged (116 g.times.10 min) to obtain a supernatant, and the absorbance value thereof was measured as a reference value Ar.

Blood Clotting Index (BCI) was calculated as follows: BCI (%). As/Ar. times.100%

(2) Observation of blood cell adhesion State

Grouping experiments:

positive control: medical gauze;

negative control: blank processing;

The experimental method comprises the following steps: 2.5mL of rat whole blood is taken and centrifuged (116g multiplied by 10min) to obtain supernatant, namely platelet rich plasma. Freeze-drying 2 pieces of snail mucus with diameter of 8mm and thickness of 2mm, and placing in a culture dish. Taking a square gauze with the length and the width of 8mm and the thickness of 4 layers, and placing the square gauze in a culture dish for later use.

mu.L of whole blood was taken, dropped on each group of materials, and incubated at 37 ℃ for 5 min. 10 μ L of platelet-rich plasma was dropped onto each group of materials and incubated at 37 ℃ for 1.0 h. After the incubation is finished, 5ml PBS is added to wash free blood cells, and the blood cells are washed for three times; adding 5.0mL of 3% glutaraldehyde solution, fixing for 4.0h, soaking in 50%, 70%, 90% and 100% gradient ethanol for 10min, gradually dehydrating, and freeze drying. After the freeze-dried samples are sprayed with gold, the freeze-dried samples are photographed and recorded under an electron microscope, and the adhesion state of blood cells of each group is observed.

(3) Results of the experiment

The results of the in vitro coagulation experiments are shown in figures 5A, 5B and 5C, and the freeze-dried products of the two snail mucus can remarkably accelerate the blood coagulation, and are superior to those of a gauze group and a blank group. Scanning electron microscope observation shows that a large amount of red blood cells are adhered and gathered on the surface of the snail mucus freeze-dried product, and only a small amount of red blood cells are gathered in gauze fibers, but a firm adhesion state is not observed.

6.3 rat liver hemostasis experiment

(1) Laboratory animal

The whole process of the animal experiment strictly complies with the regulations of experimental animal management regulations (revised 2017) issued by State institutes. The SPF grade adult male SD rat is 20, 180-220 g. Rats are raised under constant temperature conditions, and water and food are freely drunk and eaten in light and dark circulation every 12 hours, and padding is replaced every day. After 7 days of acclimatized feeding of rats under laboratory conditions, the markers were weighed.

(2) Experiment grouping and method

The adaptively fed rats were divided into 4 groups of 5 rats each according to the weight and size balance.

Negative control: blank processing;

positive control: treating gauze;

The experimental method comprises the following steps: rat isoflurane is inhaled for anesthesia, abdominal hair is removed, the abdominal cavity is opened to expose the left lobe of the liver, tissue fluid around the liver is carefully wiped off by a cotton swab, circular filter paper is placed below the liver, tissue with the length of 2cm below the lower edge of the liver is cut off to cause bleeding, each group is correspondingly treated, each group of filter paper is taken down after 2min for weighing, and the area of blood on the filter paper is photographed and recorded.

(3) Results of the experiment

The results of the rat liver hemostasis experiments are shown in figures 5D, 5E and 5F, the bleeding amount of the negative control group is 238.2 +/-24.90 mg, the bleeding amounts of the white jade snail and the bright snail mucus freeze-dried product group are 113.0 +/-17.16 mg and 114.2 +/-9.60 mg respectively, and are obviously lower than that of the negative control group; the amount of bleeding from gauze treatment was 213.0 + -46.06 mg, which is slightly better than that of the negative group.

And (3) comprehensive analysis: the experimental result of a rat liver bleeding model shows that compared with medical hemostatic gauze, the freeze-dried powder can obviously reduce the liver bleeding amount of a rat and shorten the bleeding time, and can also seal the liver wound surface to realize strong hemostasis. Therefore, the biomaterial has good wound-adhering and hemostatic functions.

Example 7

Snail mucus lyophilized product used as adhesive for adhering and repairing skin incision

7.1 Instrument

Rapid histopathological section staining system (including paraffin embedding machine, microtome and bakc machine, etc.), Thermo Scientific corporation, usa; small animal anesthesia machine (VOR76X-6), Lenbell instruments manufacture, Haiman.

7.2 reagents and controls

The main reagents are as follows: hematoxylin-eosin staining solution, Masson trichrome staining solution, neutral gum, anti-run glass slide, and the like, the american company Auragene; the reagents such as isoflurane, iodophor, chloral hydrate, xylene, absolute ethyl alcohol and the like are commercially available, biological-grade or medical-grade reagents.

Positive control: absorbable suture, Shanghai Pudong gold ring medical supplies GmbH; medical 504 glue (main component: α -n-butyl cyanoacrylate), Beijing Kangpi medical instruments GmbH; porcine fibrin adhesive (abbreviated as fibrin glue), medical grade, Guangdong Beixiu Biotechnology Co., Ltd.

Experimental groups: a lyophilized product of white jade snail mucus.

7.3 animal experiments

(1) Condition of animal

The whole process of the animal experiment strictly complies with the regulations of experimental animal management regulations (revised 2017) issued by State institutes. 45 adult male SD rats of SPF grade, 200-220 g. Rats were kept at constant temperature, with light-dark cycles every 12h, water was freely drunk, food was taken, and bedding was changed every day. After 7 days of acclimatization, the rats were weighed and labeled.

(2) Experiment grouping

The adaptively fed rats were divided into 5 groups of 9 animals per group according to the weight and size balance.

Negative control group: saline group, 50 μ Ι _ per wound;

positive control group: absorbable suture lines, 3-4 needles are sutured on each wound, and the drug is administered once; medical 504 groups: administering 30 μ L of the drug to each wound, and fixing the wound for 10s after administration;

fibrin glue group: administering 200 μ L of the medicine to each wound, and fixing the wound for 1min after administration;

Experimental groups: white jade snail mucus lyophilized product (d-SMG), 15mg per wound, and wound fixing for 1min after administration.

(3) Animal skin incision molding

Rat isoflurane is inhaled for anesthesia, back hair is removed, a 2 cm-long linear incision is cut at a position 3.5cm away from the rear edge of an ear of a rat, iodophor is used for disinfection, and after adhesive closing treatment, photographing and recording are carried out. Rats were raised in a single cage after surgery with free access to water and food.

(4) Data recording

Observing for 1 time every day from the date of establishing the skin incision model, and recording animal behaviors and physiological conditions;

observing and recording the healing of the wound and the surrounding inflammation of the rat on the 0 th, 1 th, 3 th, 5 th and 7 th days after operation, calculating and measuring the length of the incision which is not healed by using Image J software, and calculating the healing rate of the incision:

percent healing (%) - (initial incision length-length of incision for n days of treatment)/initial incision area x 100%

And thirdly, randomly selecting 3 rats for anesthesia in each group on

days

5 and 7 after the operation, fixing wound granulation tissue samples by 4 percent paraformaldehyde solution, embedding the fixed tissue samples by conventional paraffin, slicing the fixed tissue samples, dyeing the fixed tissue samples by H & E, and observing the regeneration of epidermis and dermis, the thickness of granulation tissue, the formation of new blood vessels and inflammatory infiltration conditions under a light microscope.

(5) Statistical analysis

Statistical data were analyzed for one-way anova using SPSS17.0 software, and data were expressed as mean ± standard error (x ± s), with differences of P <0.05 being statistically significant.

7.4 results of the experiment

Animal behavior changes: from the day of establishment of the skin incision model of the normal and healthy rat, observation every day does not find that the skin incision model rat has obvious indexes of ethology (activity frequency, behavior state and the like) and physiology (indexes such as body weight, drinking water, diet, excrement and the like) which are abnormal.

Peri-incision inflammation and incision healing: figure 6A shows the healing of each group of skin incisions. No obvious suppurative infection was observed in each group of skin incisions on the 3 rd day after the operation. On the 7 th day after the operation, the skin incision of the snail mucus freeze-dried product group is almost completely healed, and no scar or mucus residue is generated; obvious scabbing was seen in the saline group, and the skin was not completely closed; the absorbable suture set skin incision was nearly healed, but the suture was not completely degraded; the medical fibrin glue group is not completely healed and has scars; the medical 504 glue group has better incision bonding effect, but the adhesive is not degraded, so that the normal healing and growth of the skin incision are hindered.

The statistical result of the healing rate is shown in fig. 6D, and on the 5 th day after the operation, the healing rate of the cut of the snail mucus freeze-dried product group is significantly different from that of the normal saline group, the fibrin glue group and the medical 504 glue group; on the 7 th day after the operation, the healing rate of the cut of the snail mucus freeze-dried product group is obviously different from that of the normal saline group, the fibrin glue group, the medical 504 glue group and the medical fibrin glue group.

H & E staining analysis results: as shown in fig. 6B, on day 7 after the operation, epidermis and dermis at skin incision of the snail mucus freeze-dried product group are regenerated, and clearly visible skin organ tissues such as sebaceous gland, hair follicle, pore and the like are regenerated and intact; the physiological saline group still has no obvious granulation tissue, and the epidermis is not completely regenerated; the fibrin glue group has complete epidermis regeneration and visible granulation tissues; the suture group has good epidermis regeneration and no obvious inflammation; significant adhesive residue was visible at the skin incision of the medical 504 gel group and tissue regeneration was hindered.

And (3) comprehensive analysis: when the freeze-dried snail mucus product is applied to skin incision adhesion, the freeze-dried snail mucus product has the effect of promoting tissue regeneration at the skin incision, and the overall effect is obviously superior to clinical common medical adhesives such as fibrin glue, 504 glue and the like.

Example 8

Evaluation of effect of freeze-dried snail mucus product in promoting healing and repair of acute skin wound

8.1 instruments

8.2 reagents

Hematoxylin-eosin staining solution (H & E), Masson trichrome staining solution, neutral gum, anti-run glass slide, and the like, Auragene corporation, usa; the reagents such as isoflurane, iodophor, chloral hydrate, xylene, absolute ethyl alcohol and the like are commercially available biological-grade or medical-grade reagents.

8.3 animal experiments

(1) Condition of animal

The whole process of the animal experiment strictly complies with the regulations of experimental animal management regulations (revised 2017) issued by State institutes. The adult male SD rat with SPF grade is 12, and the weight is about 200-220 g. Rats were kept at constant temperature, with light-dark cycles every 12h, water was freely drunk, food was taken, and bedding was changed every day. After 7 days of acclimatization, the rats were weighed and labeled.

(2) Experiment grouping

Normal healthy rats were divided into 2 groups of 6 rats each according to weight and size balance.

Blank control: saline group, 50 μ L per well, single administration.

Experimental groups: the freeze-dried snail mucus product is 5mg per hole and is administrated once.

(3) Skin injury model

Rat isoflurane is inhaled for anesthesia, back hair is removed, two symmetrical round wounds with the diameter of 10mm are made at the position 3.5cm away from the rear edge of an ear of a rat, iodophor is used for disinfection, a silica gel gasket (with the diameter of 1.5mm) is sewn at the wound for fixing the wound, and after the administration treatment of the wound surface part, photographing records are carried out. Rats were raised in a single cage after surgery, with free access to water and food.

(4) Data recording

Firstly, observing animal behaviors and conventional physiological indexes every day from the establishment of an acute skin injury animal model;

Observing and recording the healing of the wound of the rat and the surrounding inflammation conditions on the 0 th, 2 th, 5 th, 8 th and 11 th days after operation, and calculating and measuring the area of the ulcer wound by using Image J software to analyze the healing rate of the wound:

percent healing (%) (initial wound area-treated n days wound area)/initial wound area x 100%

And thirdly, randomly selecting 3 rats for anesthesia in each group on 5 th and 15 th days after operation, fixing wound granulation tissue samples by 4 percent paraformaldehyde solution, embedding the fixed tissue samples by conventional paraffin, slicing the fixed tissue samples, and observing regeneration of epidermis and dermis, granulation tissue thickness, neovascularization and inflammatory cell infiltration under a light microscope after H & E staining.

(5) Statistical analysis

Statistical data were analyzed for one-way anova using SPSS 17.0 software, and data were expressed as mean ± standard error (x ± s), with differences of P <0.05 being statistically significant.

8.4 results of the experiment

Animal behavior changes: from the establishment of the normal healthy rat full-skin injury model, no obvious behavioral (activity frequency, behavior state and the like) and physiological (indexes such as body weight, drinking water, diet, excrement and the like) index abnormality of the rat is observed every day.

Inflammatory conditions around the wound and the wound healing rate: as shown in fig. 7A, the wound surface of the snail mucus freeze-dried product group is obviously reduced after 5 days of administration treatment; the statistical result of the wound healing rate is shown in figure 7B, and the wound healing rate of the snail mucus freeze-dried product treatment group is obviously superior to that of the normal saline treatment group in

days

2, 5 and 8 (the corresponding P is less than 0.001, 0.01 and 0.05);

Tissue staining analysis: as shown in fig. 7C, 7D, 7E and 7F, the epidermal histology results were consistent with wound contraction observed in the photographs of wound healing. On the 5 th day after the operation, the epidermis of the snail mucus freeze-dried product group is obviously regenerated, rich new blood vessels can be seen in granulation tissues, no obvious edema exists, the epidermis of the normal saline group is slowly regenerated, and obvious edema exists under the skin; the granulation tissue thickness and the collagen deposition rate are both higher than those of the normal saline group; on the 15 th day after operation, the skin tissues of the two groups of wounds are completely healed, and the skin organs are obviously regenerated; granulation tissue thickness and collagen deposition rate were significantly greater than those of the saline group (P <0.01, 0.001, respectively);

comprehensive analysis: the experimental result of an acute full-skin wound model of a normal rat shows that the freeze-dried snail mucus product can obviously close the wound, and the wound healing rate is obviously superior to that of a normal saline treatment group at 2, 5 and 8 days. Histopathological staining analysis on the 5 th day after operation shows that the result of the epidermic histology is consistent with the wound contraction observed in the picture of wound healing, the epidermis of the snail mucus freeze-dried product group is obviously regenerated, abundant new vessels can be seen in granulation tissues, and no obvious edema exists; the normal saline group had slow regeneration of epidermis and marked edema under the skin. Therefore, when the freeze-dried snail mucus product is applied to acute full cortical wounds of normal rats, the freeze-dried snail mucus product has obvious effect of promoting wound healing, can relieve edema, relieve inflammation, promote epidermal regeneration, angiogenesis and the like.

Example 9

Evaluation of effect of freeze-dried snail mucus product on promoting healing of diabetic chronic wounds

9.1 Instrument

Rapid histopathological section staining system (including paraffin embedding machine, microtome and roast microtome, etc.), Thermo Scientific, USA; small animal anesthesia machine (VOR76X-6), Leibel instruments manufacturing, Inc. of Haiman.

9.2 reagents and controls

The main reagents are as follows: streptozotocin (STZ), Sigma company; hematoxylin-eosin staining solution (H & E), Masson trichrome staining solution, neutral gum, and anti-drop slides, etc., Auragene corporation; the reagents such as isoflurane, iodophor, chloral hydrate, xylene, absolute ethyl alcohol and the like are commercially available biological-grade or medical-grade reagents.

Negative control: physiological saline

Positive control: alginate dressing (3M)^TM Tegaderm^TMAlginate Dressing, Minnesota Mining and Manufacturing Company, 3M Company, USA).

Experimental groups: freeze-dried snail mucus product as described in example 1.2 of the present application; the snail mucus acid polysaccharide (GAG) of example 1.4.1 of the present application.

9.3 animal experiments

(1) Condition of animal

The whole process of the animal experiment strictly complies with the regulations of experimental animal management regulations (revised 2017) issued by State institutes. 60 SPF adult male SD rats weighing about 300-330 g are selected. Rats were kept at constant temperature, with light-dark cycles every 12h, water was freely drunk, food was taken, and bedding was changed every day. Before the start of the experiment, all rats were weighed after 7 days of acclimatization feeding under laboratory conditions.

(2) Diabetic rat model

All experimental animals were blood-sampled on fasting to test blood glucose and to record body weight, and the dose of STZ (10 mg/mL concentration) required for each experimental rat was calculated at a dose of 50mg/kg and subjected to intraperitoneal injection. After injection is completed for 5-7 days, all model rats are subjected to tail vein blood sampling and blood sugar is detected by a glucometer. If the random blood sugar is more than or equal to 16.7mmol/L, the model of the diabetes mellitus model is successfully modeled.

(3) Experiment grouping and method

The diabetic rats successfully modeled were divided into 4 groups of 15 rats each according to the weight and size balance.

Blank control: saline group, 50 μ L per well; positive control: alginate dressing, 8mm diameter size per hole; test drug groups: snail slime polysaccharide, 0.9mg per hole, is administered continuously for 5 days; the freeze-dried snail mucus product is 5mg per hole and is administrated once.

(4) Diabetic ulcer model

Isoflurane is inhaled to anesthetize a diabetic rat, back hair is removed, two symmetrical round wounds with the diameter of 10mm are made at the position 3.5cm away from the rear edge of an ear of the rat, iodophor is used for disinfection, a silica gel gasket (with the diameter of 1.5mm) is sewn and fixed on the wounds through an operation suture line, and after the wounds are administrated, photographing records are carried out. Rats were raised in a single cage after surgery, with free access to water and food.

(5) Data recording

Observing for 1 time every day from the date of establishing the diabetic ulcer model, and observing whether the behavior of the animal is abnormal compared with that of a normal animal;

observing and recording the wound healing and peripheral inflammation conditions of the rats on

days

0, 3, 7, 10, 14 and 21 after administration, and calculating and measuring the area of the ulcer wound by using Image J software to analyze the wound healing rate:

And thirdly, 3 rats are randomly selected from each group for anesthesia on 3, 7, 10 and 14 days after treatment, wound granulation tissue samples are taken and fixed by 4 percent paraformaldehyde solution, are embedded and sliced by conventional paraffin, are dyed by H & E, and then the regeneration of epidermis and dermis, the thickness of granulation tissue, the formation of new blood vessels and the infiltration of fibroblasts are observed under a light microscope.

(6) Statistical analysis

Wound healing rate, epidermal regeneration rate, granulation tissue thickness, collagen deposition area ratio and the like: data are expressed as mean ± standard error (x ± s), statistical data are analyzed for one-way variance using SPSS17.0 software, and differences of P <0.05 are statistically significant.

9.4 results of the experiment

Animal behavior changes: from the date of establishment of the diabetic ulcer model, no obvious behavioral (activity frequency, behavior state and the like) and physiological (indexes such as body weight, drinking water, diet, excrement and the like) index abnormality of the diabetic rat is observed every day.

Inflammatory conditions around the wound and the wound healing rate: as shown in fig. 8A, 8B and 8C, the hydrogel hydrated on the wound surface of the snail mucus freeze-dried product group was not visible to the naked eye at day 7 after the operation, and the alginate dressing group had significant hydrogel residue. The wound healing rate of the freeze-dried snail mucus product group is obviously superior to that of the normal saline group in 7 th and 10 th days; the healing rate was better than that of the normal saline group at day 7 after the operation in the alginate group, but there was no statistical difference. On the 14 th day after the operation, obvious new hair can be seen on the wound surface of the snail mucus freeze-dried product group, and scar tissues are hardly seen; the alginate group healed slower with the saline group and hair regrowth was rarely observed. The results show that the freeze-dried snail mucus product has obvious drug effect of promoting the healing of diabetic wounds, and the effect is superior to that of the alginate dressing for diabetic foot ulcers sold in the market.

Tissue staining analysis: as shown in fig. 8D, 8E, 8F and 8G, the epidermal histology results were consistent with wound contraction observed in the optical images of diabetic wounds. The freeze-dried snail mucus product group continuously promotes re-epithelialization, thickens granulation tissues, generates blood vessels and leads to early wound healing, and is remarkably superior to the normal saline solution group and the alginate dressing group on the whole. The results of the statistics of the thicknesses of the granulation tissues at the 3 rd, 7 th and 14 th days after the operation show that the thicknesses of the granulation tissues of the snail mucus freeze-dried gel group and the snail mucus acidic polysaccharide group are obviously greater than those of the normal saline group and the alginate dressing group at the 3 rd day after the operation; on day 7 post-surgery, the snail mucus acidic polysaccharide group was significantly larger than the alginate group; on the 14 th day after the operation, the thicknesses of the granulation tissues of the snail mucus freeze-dried product group and the snail mucus acidic polysaccharide group are both obviously larger than those of the normal saline group.

Comprehensive analysis: when the freeze-dried snail mucus product is applied to the skin wound surface of a diabetic rat, the freeze-dried snail mucus product is hydrated into gel on tissues to seal the wound surface, the wound surface is invisible by naked eyes after the 7 th day of operation, and the alginate dressing group has large hydrogel residues due to the undegradability. The statistical result of the wound healing rate shows that the freeze-dried snail mucus product group is remarkably superior to the control normal saline group and the alginate group on the 7 th day and the 10 th day. On the 14 th day after the operation, the wound surface part of the snail mucus freeze-dried product group has no scar and obvious new hair generation, while the alginate group and the normal saline group have not completely healed and no obvious hair regeneration is observed. Hematoxylin-eosin (H & E) staining and masson staining results show that compared with the normal saline solution group and the alginate dressing group, the snail mucus freeze-dried product group can continuously promote re-epithelialization of wound parts, thickening of granulation tissues, collagen deposition and angiogenesis, so that early healing of the wound is accelerated. The results show that the freeze-dried snail mucus product can remarkably accelerate the regeneration of epidermis and dermis and the deposition of collagen, thereby promoting the healing of the diabetic wound and the reconstruction and regeneration of skin organs such as hair follicle, sebaceous gland and the like, and the effect is remarkably superior to that of the alginate dressing for diabetic foot ulcer in the market. The freeze-dried snail mucus product can obviously promote the wound healing of diabetic rats, and the healing physiological indexes of re-epithelization, granulation tissue thickening, angiogenesis and the like are all obviously superior to those of an alginate dressing group.

Example 10

Biocompatibility test of freeze-dried snail mucus product

10.1 cell viability assay

(1) Experimental methods

Weighing 1.6mg of freeze-dried snail mucus, dissolving in 4.0mL of complete culture medium, and filtering with 0.22 μm filter membrane to obtain 400 μ g/mL snail mucus culture medium; l929 cells are inoculated in a 96-well plate at the density of 2000 cells per well, after 24h of incubation under the conventional condition, the original culture medium is replaced by a snail mucus freeze-dried culture medium, the original culture medium is discarded after 24h, 48h and 72h of incubation respectively, a culture medium containing MTT is added, the incubation is carried out for 4h at 37 ℃, 100 mu L DMSO is added after the culture medium is absorbed, and after 10min of low-speed shaking table oscillation, the absorbance value of each well is tested at 570 nm.

(2) Results of the experiment

After 24h of culture, the survival condition of the cells in the experimental group is not obviously different from that of the blank group; when the culture is carried out for 48 hours, the cell density of the experimental group is obviously higher than that of the blank group; at 72h, no obvious difference was observed between the two groups due to the fact that the cell growth entered the plateau phase. Cell statistics shows that the number of living cells of the freeze-dried snail mucus product is more than 80 percent in the presence of the freeze-dried snail mucus product, which indicates that the freeze-dried snail mucus product has no cytotoxicity.

And (3) comprehensive analysis: cell culture experimental observation is carried out after the snail mucus freeze-dried powder is mixed in a cell culture medium, the cell survival rate under different concentrations and different time points is not lower than 80 percent, and the snail mucus freeze-dried powder has no cytotoxicity.

10.2 in vivo degradation experiments

(1) Experimental method

And (4) performing a living body subcutaneous embedding experiment on the freeze-dried snail mucus product, and evaluating the biodegradability of the freeze-dried snail mucus product.

200-220 g of male SD rats are randomly divided into two groups, and each group has at least 5 animals.

Rats were anesthetized with isoflurane, a 2.0cm long skin incision was made 3.5cm behind the ear, 100mg of snail mucus lyophilizate was subcutaneously placed, and the wound was sutured with sutures. The normal saline was used as a control group for surgery, and the treatment was performed in the same manner. And (3) after operation, on

days

3, 7, 14 and 21, photographing and recording embedding positions of animals in each group, randomly selecting the animals, taking subcutaneous embedding tissue blocks to perform a pathological tissue section H & E staining experiment, and observing degradation conditions of the mucus freeze-dried products and inflammatory conditions of surrounding tissues.

(2) Results of the experiment

The experimental results are shown in figure 9. After 7 days of subcutaneous embedding, the freeze-dried snail mucus product group is not completely degraded, no bulge is formed at the embedded part on 14 days, the embedded part is completely healed after 21 days, and the skin surface is completely repaired without scars. H & E staining analysis results show that obvious embedded objects can be seen on day 3; on day 7, only a small amount of the inclusion remained; on day 14, the inclusion was not visible; after 21 days, the tissue of the embedded part is completely regenerated, which indicates that the freeze-dried product of the snail mucus is completely degraded; moreover, during this process, no significant inflammatory infiltration and any tissue proliferative fibrosis of the surrounding tissues was observed.

Comprehensive analysis: the snail mucus freeze-dried powder is embedded under the animal skin, the degradation process is observed by photographing, and the degradation process is analyzed by tissue H & E staining. The result shows that the snail mucus freeze-dried powder does not have side effects of causing inflammatory reaction of subcutaneous tissues, bleeding or thrombosis and the like, can be completely degraded in 7-21 days, and the degradation process is basically consistent with the time process of wound healing. However, after 21 days, the cyanoacrylate medical adhesive is still not degraded significantly, and the surrounding tissues have significant adverse reactions such as edema, inflammatory infiltration and the like. Therefore, the natural biomaterial disclosed by the application has good biocompatibility and degradability, and has significant technical advantages compared with cyanoacrylate used clinically (such as medical 504).

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent application shall be subject to the appended claims.

Claims

1. A biomaterial comprising a composition obtained by drying a viscous substance derived from gastropoda;

optionally, the gastropoda animal comprises an animal of the order gastropoda;

further optionally, the gastropoda includes one or more of the families of stone sulphonidae, agate spirochaetaceae, costaphylspirochaceae, slug, mucoid slug and barnacidae.

2. The biomaterial of claim 1, wherein the composition comprises an acidic polysaccharide,

based on the weight of the composition, the mass percent a of the acidic polysaccharide is more than or equal to 5%, and optionally, the mass percent a of the acidic polysaccharide is more than or equal to 5% and less than or equal to 25%.

3. The biomaterial according to claim 2, characterized in that the acidic polysaccharide comprises a compound of formula (I),

in the formula (I), the compound is shown in the specification,

the A ring is alpha-D-2-amino-2-deoxy-glucosyl alpha-D-GlcN,

R₁is acetyl-COCH₃Or sulfonic acid group-SO₃ ^-；

R₂is hydroxy-OH or sulphate-OSO₃ ^-，

R₃Is carboxy-COO^-And inorganic salts thereof;

n is an integer greater than 100;

4. The biomaterial according to claim 3, wherein the acidic polysaccharide is formed by alternating (1 → 4) glycosidic linkages of α -D-2-amino-2-deoxy-glucosyl α -D-GlcN in the A-ring and α -L-iduronate α -L-IdoA or α -D-glucuronate α -D-GlcA in the B-ring.

5. The biomaterial of claim 2, wherein the composition further comprises a polypeptide,

based on the mass of the composition, the mass percentage content b of the polypeptide is more than or equal to 10 percent, and a + b is more than or equal to 50 percent.

6. The biomaterial of claim 5, wherein the polypeptide comprises, by mass of the polypeptide:

greater than or equal to 5% of a first set of amino acids comprising tyrosine and phenylalanine;

a second group of amino acids greater than or equal to 10% comprising lysine, arginine, and histidine; and

and (c) 30% or more of a third amino acid group including leucine, isoleucine, valine, proline, methionine and alanine.

7. The biomaterial of claim 1,

The composition further comprises allantoin and glycolic acid.

8. A method of making the biomaterial of any one of claims 1 to 7, comprising:

providing a gastropod;

stimulating the epidermis of the gastropod to cause the gastropod to secrete viscous material;

drying said viscous mass to obtain a composition;

optionally, the drying is reduced pressure drying or freeze drying.

9. A product for tissue adhesion, repair, haemostasis, tissue fluid penetration occlusion, cosmetic or as a cell culture medium, protein or drug carrier, the product comprising a biomaterial as claimed in any one of claims 1 to 8;

optionally, the product is in the form of a powder, a tablet, a sponge, a gel, or a paste.

10. Biomaterial according to claim 9, characterized in that the product is a medical adhesive.