WO2021169075A1 - 一种集可注射与抗菌于一体的双功能水凝胶及其制备方法和用途 - Google Patents

一种集可注射与抗菌于一体的双功能水凝胶及其制备方法和用途 Download PDF

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WO2021169075A1
WO2021169075A1 PCT/CN2020/093461 CN2020093461W WO2021169075A1 WO 2021169075 A1 WO2021169075 A1 WO 2021169075A1 CN 2020093461 W CN2020093461 W CN 2020093461W WO 2021169075 A1 WO2021169075 A1 WO 2021169075A1
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hydrogel
bifunctional
antibacterial
nucleoside analog
present
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French (fr)
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赵行
刘江
张亚楠
韩向龙
陈谦明
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四川大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0063—Periodont
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/23—Carbohydrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • the invention belongs to biomedical materials, and specifically relates to a bifunctional hydrogel integrating injectable and antibacterial, and a preparation method and application thereof.
  • Periodontal disease is a chronic infectious disease, and studies have shown that its main pathogenic factor is bacteria. Periodontitis is treated with curettage and root leveling to remove plaque and tartar. However, due to the complex anatomical structure of the tooth and the deep periodontal pocket, some plaque and calculus are not easy to remove, and auxiliary antibacterial treatment is required. Adjuvant antibacterial therapy is mainly divided into two ways: oral antibiotics and topical medication. Oral antibiotics for the treatment of periodontitis have the following disadvantages: insufficient antibiotic concentration in the periodontal pocket, rapid decline of plasma antibiotic concentration to sub-therapeutic levels, prone to bacterial resistance, frequent medications for patients, and high compliance requirements for patients.
  • topical medication for antibacterial therapy which uses a local drug delivery system for delivery.
  • the local drug delivery system directly reaches the periodontal pocket.
  • This treatment has the following advantages: direct contact with the diseased site, improving patient compliance, avoiding gastrointestinal reactions caused by oral drugs, avoiding first-pass metabolism in the liver, and enhancing drug delivery
  • Therapeutic efficacy reduce the cost of treatment, provide a reliable drug delivery route for diseases that cannot swallow, a safer and more convenient route of administration, prolong the action time, and a non-invasive, painless and simple application method. Therefore, the current clinical application mainly uses topical drugs, such as minocycline, metronidazole gel, and biodegradable chlorhexidine gluconate tablets. The drugs are directly placed in the periodontal pocket to achieve antibacterial effects and have become the main auxiliary Antibacterial treatment method.
  • hydrogel As a carrier material for local drug delivery, hydrogel has attracted wide attention because of its unique structure and composition similar to natural extracellular matrix.
  • the preparation of polymers into hydrogels can expand its application in local drug-carrying carrier materials.
  • the local drug delivery system delivered into the periodontal pocket by injection has become the main clinical local drug delivery method due to the advantages of minimally invasive or non-invasive, simple operation, and reduced medication frequency. Therefore, injectable hydrogel drug delivery systems have gradually become a research hotspot in recent years.
  • thermosensitive hydrogels which are characterized by being a solution before injection, and turning into a gel when injected into the target part of the body.
  • CS chitosan
  • HTCC quaternized chitosan
  • ⁇ , ⁇ -glycerophosphate ⁇ , ⁇ -GP
  • Glue CS-HTCC/ ⁇ -GP
  • this hydrogel has both antibacterial and injectable properties, the sol-gel transition time is 3 minutes (37°C).
  • the solution After the solution is injected into the target site, it may penetrate into the surrounding tissues or be affected by the surrounding environment, limiting the water The formation of gel, which in turn causes toxicity problems.
  • the hydrogels that are physically cross-linked although some of the hydrogels can ignore the influence of the surrounding environment on the cross-linking process, and have fast self-repairing speed, they can quickly perform the function of the carrier, but most of them do not have antibacterial properties, such as Polo The mixture of Sham 407 (PX) and polyacrylic acid (PAA) behaves as a low-viscosity liquid at room temperature, with shear thinning behavior, which makes it injectable and gels rapidly at body temperature, but the water condenses The glue does not have antibacterial properties.
  • hydrogel carriers without antibacterial properties need to add antibacterial drugs to achieve antibacterial effects, but there may be the following problems: the wrapped drugs may affect the gel formation, the drugs are not easy to be wrapped, the drugs are inactivated, and they are wrapped after being injected into the body. The leakage of the drug causes toxic and side effects on local cell tissues, and it cannot achieve the purpose of sustained release of drugs in the drug-carrying system. There are few reports on injectable antibacterial local drug delivery systems, and there is no report that a hydrogel with antibacterial properties and shear thinning injectable ability may be used for local injection antibacterial treatment of periodontitis.
  • Natural nucleosides are one of the building blocks of supramolecular hydrogels. Under the action of reversible non-covalent bonds, they self-assemble to form hydrogels with a three-dimensional network structure. Compatibility has been extensively studied. Guanosine (G), as one of the simplest supramolecular hydrogel building units, has poor stability and requires a higher ionic solution, which hinders its further application in the field of biology. A large number of related studies are aimed at improving the performance of hydrogels. Nucleoside analogs are a class of small molecules that have been widely discussed. Seela et al.
  • isoguanosine an isomer of G
  • isoG an isomer of G
  • G and isoG are mixed and dissolved in the K + solution at a ratio of 1:1 to form a supramolecular hydrogel with good stability, short self-repair time, and injectability, but it also does not have antibacterial properties.
  • 2'-deoxy-2'-fluoroguanosine forms a poorly stable hydrogel in KCl solution, and can form a stable antibacterial hydrogel in an Ag+ aqueous solution, but the hydrogel No injectable properties.
  • the purpose of the present invention is to provide a bifunctional hydrogel integrating injectable and antibacterial, and a preparation method and application thereof.
  • the present invention provides a bifunctional hydrogel integrating injectability and antibacterial, which is a hydrogel prepared by dissolving nucleoside analogues in a solvent;
  • the concentration of the nucleoside analog is 1.67w/v% ⁇ 5w/v%;
  • the concentration of the nucleoside analog in the hydrogel is 2.5w/v% to 5w/v%.
  • the solvent is water or phosphate buffer.
  • the preparation method of the hydrogel is: heating and dissolving the nucleoside analog in a solvent, and cooling to room temperature to obtain it.
  • the present invention also provides a method for preparing the aforementioned bifunctional hydrogel, which includes the following steps:
  • the nucleoside analog is heated and dissolved in a solvent and cooled to room temperature to obtain it.
  • the concentration of the nucleoside analog is 1.67 w/v% to 5 w/v%;
  • the concentration of the nucleoside analog is 2.5w/v% to 5w/v%.
  • the present invention also provides the use of the aforementioned bifunctional hydrogel in the preparation of tissue repair materials and/or local injection of therapeutic drugs for periodontal disease.
  • tissue repair material is a craniofacial bone tissue repair material; and/or, the periodontal disease is periodontitis.
  • the present invention also provides a local injection treatment drug for periodontal disease, which is prepared by using the aforementioned bifunctional hydrogel as an active substance, plus pharmaceutically acceptable excipients or auxiliary components;
  • the periodontal disease is periodontitis.
  • the present invention also provides the use of the nucleoside analogue of the structure shown in formula I in the preparation of antibacterial drugs and/or the preparation of biomedical materials with antibacterial effects:
  • the biomedical material is a tissue repair material.
  • room temperature means 25 ⁇ 5°C.
  • nucleoside analogue of 2-amino-2'-fluoro-2'-deoxyadenosine
  • the analogue prepares a hydrogel with both injectability and antibacterial properties.
  • the present invention uses the nucleoside analogue to successfully construct a hydrogel with both injectability and antibacterial properties, which is the first such hydrogel reported so far.
  • the hydrogel has ultra-high strength, and its excellent mechanical properties are manifested in its MPa-level storage modulus and compressive strength: 5.0% hydrogel has a storage modulus of up to 1MPa, which is a low molecular weight gelling agent so far The highest value of injectable supramolecular hydrogel formed by self-assembly.
  • the hydrogel has good shear thinning performance and self-healing performance, and good injection performance. It is in a gel state before injection, and gels immediately after injection.
  • the hydrogel of the present invention is an injectable hydrogel with both high strength and shear thinning properties, which is used in the minimally invasive treatment of load-bearing tissues. Important role.
  • the hydrogel of the present invention has a potential broad-spectrum antibacterial effect.
  • the hydrogel has a good antibacterial effect on G(+) bacteria and G(-) bacteria model strains, and at the same time has a good antibacterial effect on oral pathogenic bacteria G(+).
  • the bacterium Streptococcus mutans and G(-) bacterium Porphyromonas gingivalis also showed good antibacterial effects, which solved the current existing periodontal local non-invasive antibacterial drug system.
  • the carrier has no antibacterial or poor antibacterial properties, and the efficiency of embedding drugs Low leakage and rapid release are easy to occur.
  • the hydrogel of the invention also has good biocompatibility, good stability at room temperature, is stable when injected into the human body, is degradable in the human body, and is convenient to use. Moreover, the preparation process of the hydrogel of the present invention is simple, convenient, and environmentally friendly: it can be obtained by heating, dissolving and cooling in dH 2 O and PBS.
  • the present invention prepares a bifunctional hydrogel through a simple and green method.
  • the hydrogel has good shear thinning ability and self-healing performance, good injectability, fast gel forming speed, and convenient use.
  • the hydrogel also has excellent mechanical properties, with super high strength, storage modulus and compressive strength up to MPa level.
  • the hydrogel has a good antibacterial effect, and has a good inhibitory effect on the growth of model strains and oral pathogenic bacteria.
  • the hydrogel has good stability at room temperature, can be stored for a long time, and can be quickly degraded after being implanted in the human body, and has good biocompatibility.
  • the hydrogel of the present invention is expected to be a candidate drug for local injection treatment of periodontal disease, and it also has potential application prospects in the minimally invasive treatment of cranio-maxillofacial bone tissue defects.
  • Figure 1 is an inversion test diagram of the bifunctional hydrogel prepared in Examples 1 to 3 of the present invention: a) is a small tube inversion test diagram of hydrogels with different concentrations of nucleoside analogs, Figure 1 is the preparation 1.67 in Example 1 % Hydrogel, 2 is the 2.5% hydrogel prepared in Example 2, and 3 is the 5.0% hydrogel prepared in Example 3; b) is the stable time of the 2.5% hydrogel at room temperature, 1.5M in the figure It means 1.5 months; c) is the stable time of 5.0% hydrogel at room temperature, 3M means 3 months in the figure, and 7M means 7 months.
  • Figure 3 shows the mechanical properties of the bifunctional hydrogel of the present invention: a) ⁇ d) are the results of frequency scanning of hydrogels of different concentrations, where a) is a 2.5% hydrogel with dH 2 O as a solvent, and b) is The solvent is a 2.5% hydrogel of PBS, c) is a 5.0% hydrogel of dH 2 O, d) is a 5.0% hydrogel of PBS; e) is a small amount of Thioflavin T (ThT) Or methyl blue 5.0% hydrogels are made into different shapes; f) is the puncture test diagram of the hydrogel sample; g) is the mechanical test results of 2.5% hydrogel and 5.0% hydrogel.
  • a) ⁇ d) are the results of frequency scanning of hydrogels of different concentrations, where a) is a 2.5% hydrogel with dH 2 O as a solvent, and b) is The solvent is a 2.5% hydrogel of PBS, c) is a 5.0% hydrogel of dH 2
  • Figure 4 shows the inhibitory effect of the 5.0% hydrogel of the present invention on Staphylococcus aureus and Escherichia coli:
  • A is the colony morphology of each group of Staphylococcus aureus and Escherichia coli. The left picture is Staphylococcus aureus and the right picture is Escherichia coli;
  • B is the count of Staphylococcus aureus colonies in each group;
  • C is the count of E. coli colonies in each group; in the figure, a represents the control group, b represents the PBS group, and c represents the hydrogel group .
  • Figure 5 shows the identification results of Streptococcus mutans and Porphyromonas gingivalis: a is the colony morphology of Streptococcus mutans, b is the SEM image of Streptococcus mutans, c is the colony morphology of Porphyromonas gingivalis, and d is the colony morphology of Porphyromonas gingivalis. SEM image of Phromonas spp.
  • Figure 6 shows the inhibitory effect of the bifunctional hydrogel of the present invention on Streptococcus mutans and Porphyromonas gingivalis: (a) is the inhibitory effect on Streptococcus mutans; (b) is the inhibitory effect on Porphyromonas gingivalis Effect; in the figure, C is the control group, P is the PBS group.
  • Figure 7 shows the toxicity of the hydrogel of the present invention to the NOK-SI cell line.
  • Figure 8 shows the degradation of the hydrogel of the present invention in mice at 0h, 3h and 6h.
  • (a) is the PBS group
  • (b) is the 1.67% hydrogel group
  • (c) is the 2.5% hydrogel Glue group.
  • Figure 9 shows the HE staining of mouse heart, liver, spleen, lung, and kidney after the hydrogel of the present invention is injected into mice.
  • (a) is the PBS group
  • (b) is the 1.67% hydrogel group.
  • (c) is the 2.5% hydrogel group.
  • FIG 10 shows the complete blood count and serum biochemical test biocompatibility results: (a) is the white blood cell (WBC) count; (b) is the red blood cell (RBC) count; (c) is the platelet (PLT) count; (d) is Hemoglobin (HGB) concentration; (e) is the average red blood cell (MCV) volume; (f) is the alanine aminotransferase (ALT, liver function index) content; (g) is the aspartate aminotransferase (AST, liver function index) content; (h ) Is the content of uric acid (UA, renal function index), (i) is the content of urea nitrogen (BUN, renal function index).
  • WBC white blood cell
  • RBC red blood cell
  • PHT platelet
  • HGB Hemoglobin
  • HGB Hemoglobin
  • HGB Hemoglobin
  • e is the average red blood cell (MCV) volume
  • (f) is the alanine aminotransferase
  • the raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products.
  • nucleoside analogs used in the specific embodiments are all 2-amino-2'-fluoro-2'-dexoyadenosine (2-amino-2'-fluoro-2'-dexoyadenosine), and the molecular formula is C 10 H 13 FN 6 O 3 , the molecular weight is 284.25, and the specific structural formula is as follows:
  • the "one-step method” is used to construct a bifunctional hydrogel, and the specific preparation method is as follows:
  • the "one-step method” is used to construct a bifunctional hydrogel, and the specific preparation method is as follows:
  • the nucleoside analogue is heated in 100 mL of phosphate buffered saline (PBS) to completely dissolve, and then cooled to room temperature to obtain the bifunctional hydrogel (2.5% hydrogel) of the present invention.
  • PBS phosphate buffered saline
  • the concentration of the nucleoside analog in the bifunctional hydrogel is 2.5w/v%
  • the transition temperature of the bifunctional hydrogel is 37°C (transition temperature refers to the temperature at which the hydrogel starts to transform from a gel state to a liquid state) .
  • the "one-step method” is used to construct a bifunctional hydrogel, and the specific preparation method is as follows:
  • the 5.0 g nucleoside analog is heated in 100 mL of phosphate buffered saline (PBS) to completely dissolve, and then cooled to room temperature to obtain the bifunctional hydrogel (5.0% hydrogel) of the present invention.
  • PBS phosphate buffered saline
  • the concentration of the nucleoside analog in the bifunctional hydrogel is 5.0w/v%, and the transition temperature of the bifunctional hydrogel is 47°C (transition temperature refers to the temperature at which the hydrogel begins to transform from a gel state to a liquid state) .
  • the "one-step method” is used to construct a bifunctional hydrogel, and the specific preparation method is as follows:
  • the "one-step method” is used to construct a bifunctional hydrogel, and the specific preparation method is as follows:
  • the concentration of the nucleoside analog in the bifunctional hydrogel is 2.5w/v%, and the transition temperature of the bifunctional hydrogel is 37°C (transition temperature refers to the temperature at which the hydrogel starts to transform from a gel state to a liquid state) .
  • the "one-step method” is used to construct a bifunctional hydrogel, and the specific preparation method is as follows:
  • the bifunctional hydrogel (5.0% hydrogel) of the present invention.
  • concentration of the nucleoside analog in the bifunctional hydrogel is 5.0w/v%
  • transition temperature of the bifunctional hydrogel is 47°C (transition temperature refers to the temperature at which the hydrogel begins to transform from a gel state to a liquid state) .
  • Figure 1 is an inverted test diagram of the hydrogels prepared in Examples 1 to 3. It can be seen from a) in Figure 1 that as the concentration of the nucleoside analog increases, the appearance of the hydrogel changes from transparent to translucent.
  • Figure 1 b) and c) are the stable time of 2.5% hydrogel and 5.0% hydrogel at room temperature respectively. It can be seen that the stable time of 2.5% hydrogel at room temperature is 1.5 months, 5.0% The stability time of the hydrogel at room temperature can be up to 7 months. It shows that the bifunctional hydrogel prepared by the present invention is very stable, which is beneficial to the storage of the later hydrogel at room temperature, and the stability performance of the 5.0% hydrogel is the best.
  • Table 1 shows the gelation properties of the bifunctional hydrogels prepared in Example 2, Example 3, Example 5 and Example 6 of the present invention. It can be seen from Table 1 that nucleoside analogs can self-assemble to form hydrogels in both dH 2 O and PBS.
  • the gelation performance of hydrogels is mainly affected by the concentration of nucleoside analogs, and the type of solvent has basically no effect on its performance. Influence.
  • the concentration of nucleoside analogues increases, the gelation time of the hydrogel decreases, the stabilization time increases, and the transition temperature (transition temperature refers to the temperature at which the hydrogel begins to transform from the gel state to the liquid state) also increases. When the concentration exceeds 2.5w/v%, the transition temperature of the hydrogel is higher than 38°C. This indicates that as the concentration of nucleoside analogs increases, it is easier to gel, and the resulting hydrogel will be more stable in vivo.
  • the bifunctional hydrogel of the present invention is completely thermoreversible.
  • the hydrogel of the present invention has a short gelation time, has good stability, and can exist stably in the body.
  • 5.0% hydrogel has a faster gelation time and better stability, which is the best hydrogel.
  • Test Example 2 Detection of shear thinning performance, self-healing performance and injectable performance of the dual-functional hydrogel of the present invention
  • Example 2 Take the 2.5% hydrogel and 5.0% hydrogel prepared in Example 2, Example 3, Example 5 and Example 6, and use a rheometer cycle strain time scan to detect the self-healing performance of the hydrogel; use a syringe to inject
  • the experiment explored the injectability of the hydrogels of Example 2 and Example 3.
  • the conditions for scanning the cycle strain time of the rheometer are as follows: take the hydrogel, preheat the parallel plate at 80°C and apply the sample, and add a thin layer of silicone oil to the exposed surface of the sample around the parallel plate after the sample is added to prevent the test process The water evaporates. All tests are performed at 25°C.
  • a) and b) are the shear viscosity test results of the 2.5% hydrogel of Example 2 and the 5.0% hydrogel of Example 3. It can be seen from a) and b) of Figure 2 that the viscosity of both 2.5% hydrogel and 5.0% hydrogel decreases with the increase of shear rate, and the shear stress drops sharply, which shows that the hydraulic gel prepared by the present invention Glue has shear thinning properties and can be used for injection.
  • the bifunctional hydrogel of the present invention is not only injectable, but can be changed from gel to fluid when pressure is applied, and has good self-repair performance. After the pressure is removed, it can change from fluid to gel immediately.
  • Figure 2 g) and h) show the shear thinning injectability of the hydrogel.
  • Figure 2 g) in Figure 2 when a shear force is applied, the 2.5% hydrogel is transformed into a fluid material.
  • Figure 2 h) shows: use a syringe to inject 5.0% hydrogel into an empty bottle at room temperature or 37°C, the hydrogel will turn into a solution state when the sample is injected, and the vial will be inverted immediately after injection, and the solution will quickly return to gel State, the transition between solution and gel takes only a few seconds.
  • the results of the syringe injection experiment are consistent with the results of c) to f) in Figure 2. Once the injection shear force is removed, the hydrogel can quickly return to a gel state at the injection site.
  • the above test results indicate that the hydrogel of the present invention has good shear thinning performance and self-healing performance.
  • the hydrogel has good injection performance and can quickly change between solution and gel states.
  • Example 2 Take the 2.5% hydrogels and 5.0% hydrogels prepared in Example 2, Example 3, Example 5 and Example 6 for mechanical performance testing.
  • the test conditions are: Anton Paar is used for the rheological test of the hydrogel Modular intelligent rotary rheometer (MCR302) to detect.
  • MCR302 hydrogel Modular intelligent rotary rheometer
  • the prepared hydrogel solution was preheated on the parallel plate at 80°C for sample application. After the sample was added, a thin layer of silicone oil was added to the exposed surface of the sample around the parallel plate to prevent moisture from evaporating during the test. All measurements are performed at 25°C, and the detection frequency range is 0.1-100rad/s.
  • the hydrogel of the present invention has MPa-level storage modulus and compressive strength: the storage modulus of the 5.0% hydrogel is as high as 1 MPa, and the compressive strength is as high as 0.2 MPa.
  • Example 3 Using G(+) bacterium Staphylococcus aureus (S. aureus) and G(-) bacterium Escherichia coli (E. coli) as the representative strains of the model strains, the 5.0% hydrogel prepared in Example 3 was initially checked by the bacterial colony counting method Antibacterial activity of glue.
  • the specific test method is as follows:
  • the antibacterial performance of the bifunctional hydrogel of the present invention against Staphylococcus aureus and Escherichia coli is shown in FIG. 4. It can be seen from Figure 4 that the number of bacterial colonies in the control group is greater than that in the PBS group, but there is no significant difference between the control group and the PBS group (P>0.05), indicating that PBS does not affect the growth of bacterial cultures, but only shows Dilution effect on bacterial suspension. Compared with the PBS group, the 5.0% hydrogel group had fewer bacterial colonies (P ⁇ 0.05), indicating that the bifunctional hydrogel of the present invention has significant antibacterial properties against both Staphylococcus aureus and Escherichia coli.
  • Example 2 Using G(+) bacterium Streptococcus mutans (S.mutans) and G(-) bacterium Porphyromonas gingivalis as representative strains of oral pathogens, the example 2 was explored by bacterial colony counting method and scanning electron microscope And the antibacterial properties of the bifunctional hydrogel prepared in Example 3.
  • the specific test method is as follows:
  • Each group took 100 ⁇ L of bacterial solution at the time point, and after performing the same gradient dilution, the same volume of the dilution was inoculated on the agar solid plate. Streptococcus mutans was cultured for 24 hours, and Porphyromonas gingivalis was cultured for 7 days. The in vitro bactericidal activity was determined by counting bacterial colonies.
  • Scanning electron microscopy Place the cleaned round slides in a 24-well plate, and take S. mutans bacterial liquid (Streptococcus mutans bacterial liquid) cultured for 12 hours or P.gingivalis bacterial liquid cultured for 24 hours (gingival porphyrin Spore bacteria liquid) onto a round glass slide, fixed with the same amount of 5% glutaraldehyde, gradient elution with different concentrations of ethanol (50%, 70%, 95%, 100%) for 10 min, take out the slide to dry naturally, and after labeling Observe under the scanning electron microscope.
  • S. mutans bacterial liquid Streptococcus mutans bacterial liquid
  • P.gingivalis bacterial liquid cultured for 24 hours gingival porphyrin Spore bacteria liquid
  • strain identification observation of colony morphology and scanning electron microscopy results (a to d in Figure 5) showed that the strains were Streptococcus mutans and Porphyromonas gingivalis.
  • a single colony of Streptococcus mutans is milky white with the size of a millet grain, uplifted, rounded, with neat edges.
  • scanning electron microscopy it can be seen that a single bacterial cell is spherical, or multiple bacteria are connected in a chain; a single colony of Porphyromonas gingivalis It is round with neat edges, black with metallic luster; under the scanning electron microscope, the bacteria are oval or round with smooth edges. All are consistent with the characteristics of the standard strain.
  • the test results show that the bifunctional hydrogel of the present invention can effectively inhibit the growth of bacteria, especially the growth of the main pathogenic bacteria Streptococcus mutans and Porphyromonas gingivalis. Among them, 2.5% has a good inhibitory effect on both Streptococcus mutans and Porphyromonas gingivalis, and 5.0% hydrogel has a good inhibitory effect on Porphyromonas gingivalis.
  • Example 2 The cytotoxicity of the bifunctional hydrogel prepared in Example 1 and Example 2 was tested in vitro.
  • CCK8 cytotoxicity test detects the toxicity to NOK-SI cell line.
  • the specific test method is as follows:
  • Example 1 and Example 2 Collect well-growing NOK-SI cells, prepare them as cell suspensions, adjust their cell density and inoculate them in 24-well plates; wait for 4-6 hours for the cells to adhere to the wall, and add 5 ⁇ L of the hydrogel prepared in Example 1 and Example 2 respectively.
  • Glue experimental group
  • 5 ⁇ L PBS as PBS experimental group
  • put it in the upper chamber of the transwell chamber, place it in a 24-well plate, and incubate at 37°C for 24h; then add 10% CCK8 reagent, incubate for 3h and use the blank Adjust the hole to zero and use a microplate reader to detect the absorbance value (OD) at 450nm wavelength.
  • Each group is designed with ⁇ 3 auxiliary holes, the results are averaged, and the experiment is repeated 3 times independently.
  • a cell control group was set up.
  • the CCK8 kit was used to evaluate the toxicity of the hydrogel of the present invention to the NOK-SI cell line.
  • the results are shown in Figure 7 (the cell activity data in Figure 7 is not multiplied by 100%, it is directly the experimental group (OD)/control group (OD)).
  • the results in Figure 7 show: 1.67% hydrogel and 2.5% hydrogel
  • the cell activity of the gel is as high as 95% and 90%, respectively, which indicates that the hydrogel material of the present invention is non-toxic or low-toxic to cells and has good cell compatibility.
  • the hydrogel of the present invention has no acute toxicity in vitro and has potential applications as biological materials.
  • Example 1 After subcutaneous injection of the bifunctional hydrogel prepared in Example 1 and Example 2 into the skin and mucous membrane of the back of female BALB/c mice, the degradation was detected. And at a certain time point (0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h), the blood was divided into 2 parts, and the blood was subjected to complete blood count and serum biochemical test for biocompatibility. The mice were sacrificed and the heart, liver, spleen, lung, and kidney were processed for HE staining; the organs were observed for damage. An equal amount of PBS was injected into the skin and mucous membrane of the back of BALB/c mice as the PBS group for control.
  • FIG. 8 shows the degradation of the hydrogel of the present invention in mice. The results of Figure 8 show that the hydrogel degrades within 6 hours, and the degradation rate of 1.67% of the hydrogel is faster than that of 2.5% of the hydrogel.
  • Figure 9 shows the results of hematoxylin and eosin (H&E) staining. The staining results show that the heart, liver, spleen, lung and kidney of the 1.67% hydrogel and 2.5% hydrogel treatment groups are all compared with the control group. No obvious signs of organ damage were found.
  • H&E hematoxylin and eosin
  • serum biochemical indicators compared with the PBS treatment group, in the 1.67% hydrogel group, blood urea nitrogen decreased after 3 hours (BUN, *P ⁇ 0.05), and aspartate aminotransferase increased 3 hours after injection (AST, *P ⁇ 0.05) , Other serum biochemical indicators were not significantly different (P>0.05); the 2.5% hydrogel group except for the decrease of plasma BUN (*P ⁇ 0.05) after 3 hours of injection and the increase of uric acid (UA, *P ⁇ 0.05) 24 hours after injection There is no difference in other test indicators. But all the detected values are within the normal range. The test results show that the bifunctional hydrogel of the present invention has no acute toxicity in vivo.
  • the present invention prepares a bifunctional hydrogel through a simple and green method.
  • the hydrogel has good shear thinning ability and self-healing performance, good injectability, fast gel forming speed, and convenient use.
  • the hydrogel also has excellent mechanical properties, with super high strength, storage modulus and compressive strength up to MPa level.
  • the hydrogel has a good antibacterial effect, and has a good inhibitory effect on the growth of model strains and oral pathogenic bacteria.
  • the hydrogel has good stability at room temperature, can be stored for a long time, and can be quickly degraded after being implanted in the human body, and has good biocompatibility.
  • the hydrogel of the present invention is expected to be a candidate drug for local injection treatment of periodontal disease, and it also has potential application prospects in the minimally invasive treatment of cranio-maxillofacial bone tissue defects.

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Abstract

本发明提供了一种集可注射与抗菌于一体的双功能水凝胶及其制备方法和用途，属于生物医用材料。该双功能水凝胶是由核苷类似物溶于溶剂后制备而得的水凝胶；所述核苷类似物的结构如式I所示。该水凝胶制备简单、环保，具有良好的剪切稀化能力和自修复性能，可注射性能良好，成胶速度快，使用方便。该水凝胶也具备出色的机械性能。同时，该水凝胶具有良好的抗菌作用。此外，该水凝胶室温下具有良好的稳定性，可长期保存，而植入人体后可快速降解，且具有良好的生物相容性。这些突出的特征使本发明水凝胶有望成为用于牙周病局部注射治疗的候选药物，同时在颅颌面骨组织缺损的微创疗法中也有潜在的应用前景。

Description

一种集可注射与抗菌于一体的双功能水凝胶及其制备方法和用途 技术领域
本发明属于生物医用材料，具体涉及一种集可注射与抗菌于一体的双功能水凝胶及其制备方法和用途。
背景技术
牙周疾病是慢性感染性疾病，研究表明其主要致病因素是细菌。牙周炎采用以刮治和根面平整为主的治疗来去除菌斑牙石。但由于牙齿解剖结构复杂和牙周袋较深，部分菌斑牙石不易清除，需要进行辅助抗菌治疗。辅助抗菌治疗主要分为口服抗生素和局部用药两种方式。口服抗生素治疗牙周炎存在以下缺点：牙周袋部位的抗生素浓度不足、血浆抗生素浓度迅速下降至亚治疗水平、易产生细菌耐药性、患者需频繁服药、对患者的依从性要求高。鉴于以上不足，已经有大量的研究聚焦于局部用药进行抗菌治疗，这种用药方式利用局部载药系统给药。局部载药系统直接到达牙周袋内，这种治疗方式有以下优势：与疾病部位直接接触，改善患者依从性，避免口服药物引起的胃肠道反应，避免肝脏的首过代谢，增强药物的治疗功效，降低治疗成本，为不能吞咽的患病提供可靠的药物输送途径，更安全，更方便的给药途径，延长作用时间，无创、无痛且简单的应用方式。因此，目前临床上主要采用局部用药，如米诺环素、甲硝唑凝胶、可降解葡萄糖酸洗必泰片，直接将药物放置至牙周袋内，达到抗菌效果，已成为主要的辅助抗菌治疗方式。
作为局部载药的载体材料，水凝胶因为与天然细胞外基质的独特结构和组成相似，广受关注。将聚合物制备成水凝胶，可拓展其在局部载药的载体材料中的应用。此外，通过注射方式将局部药物释放系统送至牙周袋内，由于微创或无创、操作简单、降低用药频率等优点，已成为临床上主要的局部给药方式。因此，可注射水凝胶药物释放系统近年来逐渐成为研究热点。
目前，关于牙周治疗的可注射水凝胶研究多为聚合物热敏性水凝胶，其特征是：注射前是溶液，注射到体内目标部位变为凝胶。比如有研究使用壳聚糖(CS)、季铵化壳聚糖(HTCC)和α，β-甘油磷酸酯(α，β-GP)成功设计并制备了具有抗菌性能的可注射热敏水凝胶(CS–HTCC/αβ-GP)。但此水凝胶虽然兼具抗菌性和可注射能力，但是溶胶-凝胶转变时间为3分钟(37℃)，溶液注射到目标部位后可能深入周围组织或者受到周围环境的影响，限制了水凝胶的形成，进而产生毒性问题。在物理交联的水凝胶中，虽然有一部分水凝胶可忽略周围环境对交联过程的影响，并且自修复速度快， 能够迅速发挥载体的功能，但大多不具备抗菌性能，如泊洛沙姆407(PX)与聚丙烯酸(PAA)混合物在室温下表现为低粘度液体，具有剪切稀化行为，从而使其具有可注射性，并在体温下迅速凝胶化，但该水凝胶不具备抗菌性能。这些不具备抗菌性能的水凝胶载体在使用时需要加抗菌药物达到抗菌效果，但可能存在以下问题：包裹的药物可能影响凝胶形成，药物不易被包裹，药物失活，注射到体内后包裹的药物渗漏从而对局部细胞组织产生毒副作用，不能达到载药系统缓释药物的目的。关于可注射抗菌局部载药系统的报道较少，尚无兼具抗菌性和剪切稀化可注射能力的水凝胶可能用于牙周炎局部注射抗菌治疗的的报道。
天然核苷，一种内源性小分子，是超分子水凝胶构建单元之一，在可逆的非共价键的作用下自组装形成具有三维网络结构的水凝胶，由于其良好的生物相容性被广泛研究。鸟苷(G)作为一种最简单超分子水凝胶构建单元，其稳定性较差，且需要较高的离子溶液，这阻碍了其在生物学领域的进一步应用。大量相关研究都旨在提高水凝胶的性能。核苷类似物是其中被广泛探讨的一类小分子。Seela等最近发现，G的异构体-异鸟苷(isoG)可以自组装成稳定性更好的水凝胶，但其不可注射且无抗菌性能。G和isoG以1:1的比例混合溶解在K +溶液中，可以形成稳定性好、自修复时间短、可注射性的超分子水凝胶，但其也不具有抗菌性能。也有研究报道，2’-脱氧-2’-氟鸟苷在KCl溶液中形成稳定性较差的水凝胶，在含Ag+水溶液中可以形成稳定性好的抗菌水凝胶，但该水凝胶没有可注射性能。
因此，有必要探究集剪切稀化可注射性抗菌性于一体的水凝胶，同时起到局部注射和抗菌作用，在未来牙周局部无创治疗中发挥潜在效应。
发明内容
本发明的目的是提供一种集可注射与抗菌于一体的双功能水凝胶及其制备方法和用途。
本发明提供了一种集可注射与抗菌于一体的双功能水凝胶，它是由核苷类似物溶于溶剂后制备而得的水凝胶；
所述核苷类似物的结构如式I所示：
Figure PCTCN2020093461-appb-000001
进一步地，所述水凝胶中，核苷类似物的浓度为1.67w/v％～5w/v％；
优选地，所述水凝胶中，核苷类似物的浓度为2.5w/v％～5w/v％。
进一步地，所述溶剂为水或磷酸盐缓冲液。
进一步地，所述水凝胶的制备方法为：将核苷类似物加热溶解于溶剂中，冷却至室温，即得。
本发明还提供了一种前述的双功能水凝胶的制备方法，它包括如下步骤：
将核苷类似物加热溶解于溶剂中，冷却至室温，即得。
进一步地，所述核苷类似物溶于溶剂后，核苷类似物的浓度为1.67w/v％～5w/v％；
优选地，所述核苷类似物的浓度为2.5w/v％～5w/v％。
本发明还提供了前述的双功能水凝胶在制备组织修复材料，和/或牙周病局部注射治疗药物中的用途。
进一步地，所述组织修复材料为颅颌面骨组织修复材料；和/或，所述牙周病为牙周炎。
本发明还提供了一种牙周病局部注射治疗药物，它是由前述的双功能水凝胶为活性物质，加上药物上可接受的辅料或辅助性成分制备而成；
优选地，所述牙周病为牙周炎。
本发明还提供了式I所示结构的核苷类似物在制备抗菌药物，和/或制备具有抗菌作用的生物医用材料中的用途：
Figure PCTCN2020093461-appb-000002
优选地，所述生物医用材料为组织修复材料。
本发明中，室温是指25±5℃。
现有技术目前关于2-氨基-2’-氟-2’-脱氧腺苷这种核苷类似物的研究较少，现有技术中尚未发现该核苷类似物具有抗菌作用，也未见利用该类似物制备兼具可注射性和抗菌性的水凝胶。
本发明利用该核苷类似物成功构建一种兼具可注射性和抗菌性的水凝胶，是迄今为止报道的第一个此类水凝胶。该水凝胶具有超高强度，其出色的机械性能表现在具有MPa级的储能模量和抗压强度：5.0％水凝胶的储能模量高达1MPa，是迄今为止低分子量胶凝剂自组装形成的可注射超分子水凝 胶的最高值。该水凝胶具有良好的剪切稀化性能和自修复性能，且注射性能良好，注射前是凝胶状态，注射后立即成胶，在37℃下用注射器注射后，从液体状物质到固体状凝胶的转变需要几秒钟。大多数研究报道中高强度和剪切稀化性能往往不可兼得，但本发明水凝胶是兼具高强度和剪切稀化性能的可注射水凝胶，其在承重组织微创治疗中发挥重要作用。
此外，本发明水凝胶具有潜在的广谱抗菌效应，该水凝胶对G(+)菌和G(-)菌模式菌株均有良好的抗菌作用，同时对口腔致病菌G(+)菌变异链球菌和G(-)菌牙龈卟啉单胞菌也表现出良好的抗菌效果，解决了目前存在的牙周局部无创抗菌药物系统存在的载体无抗菌性或抗菌性差，包埋药物效率低易出现渗漏及释放过快等问题。本发明水凝胶还具有良好的生物相容性，室温下稳定性好，其注入人体时稳定，在人体中又可降解，使用方便。而且，本发明水凝胶制作过程简单方便、绿色环保：在dH 2O和PBS中加热溶解冷却即得。
总之，本发明通过一种简单、绿色的方法制备得到一种双功能水凝胶。该水凝胶具有良好的剪切稀化能力和自修复性能，可注射性能良好，成胶速度快，使用方便。该水凝胶也具备出色的机械性能，其具有超高强度，储能模量和抗压强度可达MPa级。同时，该水凝胶具有良好的抗菌作用，对模式菌株和口腔致病菌的生长均有良好抑制作用。此外，该水凝胶室温下具有良好的稳定性，可长期保存，而植入人体后可快速降解，且具有良好的生物相容性。这些突出的特征使本发明水凝胶有望成为用于牙周病局部注射治疗的候选药物，同时在颅颌面骨组织缺损的微创疗法中也有潜在的应用前景。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为本发明实施例1～3制备的双功能水凝胶倒置试验图：a)为具有不同核苷类似物浓度的水凝胶的小管倒置试验图，图中1为实施例1制备1.67％水凝胶，2为实施例2制备的2.5％水凝胶，3为实施例3制备的5.0％水凝胶；b)为2.5％水凝胶在室温下的稳定时间，图中1.5M表示1.5个月；c)为5.0％水凝胶在室温下的稳定时间，图中3M表示3个月，7M表示7个月。
图2为本发明双功能水凝胶剪切稀化性能、自修复性能和可注射性能的结果图：a)～b)为实施例2的2.5％水凝胶和实施例3的5.0％水凝胶的剪切粘 度测试结果；c)～f)为在ω＝1rad·s -1时的循环应变时间扫描评估水凝胶自修复能力的结果，其中c)为溶剂为dH 2O的2.5％水凝胶，d)为溶剂为dH 2O的5.0％水凝胶，e)为溶剂为PBS的2.5％水凝胶，f)为溶剂为PBS的5.0％水凝胶；g)～h)为注射器在室温(25℃)下注射2.5％水凝胶和5.0％水凝胶，其中g)为2.5％水凝胶，h)为5.0％水凝胶。
图3为本发明双功能水凝胶的机械性能：a)～d)为不同浓度水凝胶频率扫描后的结果，其中a)为溶剂为dH 2O的2.5％水凝胶，b)为溶剂为PBS的2.5％水凝胶，c)为溶剂为dH 2O的5.0％水凝胶，d)为溶剂为PBS的5.0％水凝胶；e)为含少量的硫黄素T(ThT)或甲基蓝的5.0％水凝胶被制成不同形状；f)为水凝胶样品穿刺试验图；g)为2.5％水凝胶和5.0％水凝胶的力学检测结果。
图4为本发明5.0％水凝胶对金黄色葡萄球菌和大肠杆菌的抑制作用：(A)为各组金黄色葡萄球菌和大肠杆菌菌落形态，其中左图为金黄色葡萄球菌，右图为大肠杆菌；(B)为各组金黄色葡萄球菌菌落数统计；(C)为各组大肠杆菌菌落数统计；图中a均表示对照组，b均表示PBS组，c均表示水凝胶组。
图5为变异链球菌和牙龈卟啉单胞菌鉴定结果：a为变异链球菌的菌落形态，b为变异链球菌的SEM图，c为牙龈卟啉单胞菌的菌落形态，d为牙龈卟啉单胞菌的SEM图。
图6为本发明双功能水凝胶对变异链球菌和牙龈卟啉单胞菌的抑制作用：(a)为对变异链球菌的抑制作用；(b)为对牙龈卟啉单胞菌的抑制作用；图中，C均为对照组，P均为PBS组。
图7为本发明水凝胶对NOK-SI细胞系的毒性。
图8为本发明水凝胶在小鼠体内0h、3h和6h的降解情况，图中，(a)为PBS组，(b)为1.67％水凝胶组，(c)为2.5％水凝胶组。
图9为本发明水凝胶注入小鼠体内后，小鼠心、肝、脾、肺、肾的HE染色，图中，(a)为PBS组，(b)为1.67％水凝胶组，(c)为2.5％水凝胶组。
图10为全血细胞计数和血清生化检测生物相容性结果：(a)为白细胞(WBC)计数；(b)为红细胞(RBC)计数；(c)为血小板(PLT)计数；(d)为血红蛋白(HGB)浓度；(e)为平均红细胞(MCV)体积；(f)为谷丙转氨酶(ALT，肝功能指标)含量；(g)为谷草转氨酶(AST，肝功能指标)含量；(h)为尿酸(UA，肾功能指标)含量，(i)为尿素氮(BUN，肾功能指标)含量。
具体实施方式
本发明具体实施方式中使用的原料、设备均为已知产品，通过购买市售 产品获得。
其中，具体实施方式中使用的核苷类似物均为2-氨基-2’-氟-2’-脱氧腺苷(2-amino-2’-fluoro-2’-dexoyadenosine)，分子式为C 10H 13FN 6O 3，分子量为284.25，具体结构式如下：
Figure PCTCN2020093461-appb-000003
实施例1、本发明双功能水凝胶的制备
采用“一步法”构建双功能水凝胶，具体制备方法如下：
将1.67g核苷类似物在100mL磷酸盐缓冲液(PBS)中加热至完全溶解，然后冷却至室温，即得本发明双功能水凝胶(1.67％水凝胶)。该双功能水凝胶中核苷类似物的浓度为1.67w/v％，该双功能水凝胶的转变温度为35℃(转变温度是指水凝胶由凝胶态开始转变为液态的温度)。
实施例2、本发明双功能水凝胶的制备
采用“一步法”构建双功能水凝胶，具体制备方法如下：
将2.5g核苷类似物在100mL磷酸盐缓冲液(PBS)中加热至完全溶解，然后冷却至室温，即得本发明双功能水凝胶(2.5％水凝胶)。该双功能水凝胶中核苷类似物的浓度为2.5w/v％，该双功能水凝胶的转变温度为37℃(转变温度是指水凝胶由凝胶态开始转变为液态的温度)。
实施例3、本发明双功能水凝胶的制备
采用“一步法”构建双功能水凝胶，具体制备方法如下：
将5.0g核苷类似物在100mL磷酸盐缓冲液(PBS)中加热至完全溶解，然后冷却至室温，即得本发明双功能水凝胶(5.0％水凝胶)。该双功能水凝胶中核苷类似物的浓度为5.0w/v％，该双功能水凝胶的转变温度为47℃(转变温度是指水凝胶由凝胶态开始转变为液态的温度)。
实施例4、本发明双功能水凝胶的制备
采用“一步法”构建双功能水凝胶，具体制备方法如下：
将1.67g核苷类似物在100mL蒸馏水(dH 2O)中加热至完全溶解，然后冷却至室温，即得本发明双功能水凝胶(1.67％水凝胶)。该双功能水凝胶中核苷类似物的浓度为1.67w/v％，该双功能水凝胶的转变温度为35℃(转变温度是指水凝胶由凝胶态开始转变为液态的温度)。
实施例5、本发明双功能水凝胶的制备
采用“一步法”构建双功能水凝胶，具体制备方法如下：
将2.5g核苷类似物在100mL蒸馏水(dH 2O)中加热至完全溶解，然后冷却至室温，即得本发明双功能水凝胶(2.5％水凝胶)。该双功能水凝胶中核苷类似物的浓度为2.5w/v％，该双功能水凝胶的转变温度为37℃(转变温度是指水凝胶由凝胶态开始转变为液态的温度)。
实施例6、本发明双功能水凝胶的制备
采用“一步法”构建双功能水凝胶，具体制备方法如下：
将5.0g核苷类似物在100mL蒸馏水(dH 2O)中加热至完全溶解，然后冷却至室温，即得本发明双功能水凝胶(5.0％水凝胶)。该双功能水凝胶中核苷类似物的浓度为5.0w/v％，该双功能水凝胶的转变温度为47℃(转变温度是指水凝胶由凝胶态开始转变为液态的温度)。
以下通过具体的试验例证明本发明的有益效果。
试验例1、本发明双功能水凝胶的性质
1、试验方法
通过小管倒置试验检验实施例1～6制备的水凝胶构建是否成功。并利用常规方法检测实施例2、实施例3、实施例5和实施例6制备的水凝胶的凝胶化性质。
2、试验结果
通过小管倒置试验证明本发明实施例1～6制备的水凝胶均构建成功。图1为实施例1～3制备的水凝胶的倒置试验图。由图1中的a)可知：随着核苷类似物浓度的增加，水凝胶外观从透明变为半透明。图1中b)和c)分别为2.5％水凝胶和5.0％水凝胶的在室温下的稳定时间，可以看出2.5％水凝胶在室温下的稳定时间为1.5个月，5.0％水凝胶的在室温下的稳定时间可达7个月。说明本发明制备的双功能水凝胶非常稳定，有利于后期水凝胶的在室温下的保存，其中5.0％水凝胶的稳定性能最优。
表1为本发明实施例2、实施例3、实施例5和实施例6制备的双功能水凝胶的凝胶化性质。由表1可知：核苷类似物在dH 2O和PBS中都可以自组装形成水凝胶，水凝胶的凝胶化性能主要受核苷类似物浓度的影响，溶剂种类对其性能基本无影响。随着核苷类似物浓度增加，水凝胶的成胶时间减少，稳定时间增加，转变温度(转变温度是指水凝胶由凝胶态开始转变为液态的温度)也增加，当核苷类似物浓度超过2.5w/v％时，水凝胶的转变温度高于38℃。说明随着核苷类似物浓度增加，更容易成胶，且得到的水凝胶在体内会更稳定。同时本发明双功能水凝胶是完全热可逆的。
表1.本发明双功能水凝胶的凝胶化性质
Figure PCTCN2020093461-appb-000004
根据试验例1可知：本发明水凝胶成胶时间短，且具有良好的稳定性，在体内也能够稳定存在。其中5.0％水凝胶成胶速率具有更快的成胶时间，稳定性也更好，是最优的水凝胶。
试验例2、本发明双功能水凝胶剪切稀化性能、自修复性能和可注射性能的检测
1、试验方法
取实施例2、实施例3、实施例5和实施例6制备的2.5％水凝胶和5.0％水凝胶，利用流变仪循环应变时间扫描检测水凝胶的自修复性能；采用注射器注射实验探究实施例2和实施例3水凝胶的可注射性。流变仪循环应变时间扫描的条件为：取水凝胶，在80℃预热平行板上进行加样，加样后在平行板周围样品的裸露表面加一层薄薄的硅油，防止测试过程中水分蒸发。所有检测均在25℃下进行。
2、试验结果
(1)流变仪检测结果
图2中a)和b)为实施例2的2.5％水凝胶和实施例3的5.0％水凝胶的剪切粘度测试结果。由图2的a)和b)可知：2.5％水凝胶和5.0％水凝胶的粘度均随剪切速率的增加而降低，并且剪切应力的急剧下降，这说明本发明制备的水凝胶均具有剪切稀化特性，可以用于注射。
弹性和流体性质通过储能模量和损耗模量测量，分别表示为G’和G”。G’＜G”表明测试样品处于溶液状态；G’＞G”表明测试样品处于凝胶化状态。在稳定的粘弹性条件下，在ω＝1rad·s -1时的循环应变时间扫描评估水凝胶的自修复能力，结果如图2中的c)～f)所示。由图2中的c)～f)可知：各组水凝胶在1％的应变下均表现出凝胶特性。当应变直接增加到100％时(第2分钟)，G’和G”迅速下降，G’＜G”，表明凝胶被破坏，水凝胶状态由凝胶状转变为流体状。当应变恢复到1％时，样品在几秒内(溶剂为PBS时约为0.17秒，溶剂为dH 2O时约为0.155秒)完成流体-凝胶的转变，且G’和G”值完全恢复到与初始模量相等，重要的是，在几个循环应变变化周期之后，G’和G”值仍等于初始值。说明本发明双功能水凝胶不仅具有可注射性，施加压力即 可由凝胶状变为流体状，且自修复性能良好，撤去压力后可由流体状立即变为凝胶状。
(2)注射器注射实验结果
图2中的g)和h)显示了水凝胶的剪切稀化可注射能力。图2中的g)所示：当施加剪切力时，2.5％水凝胶转变为流体材料。图2中的h)所示：使用注射器将5.0％水凝胶在室温或者37℃注入空瓶中，样本注射时水凝胶转变为溶液状态，注射后立即倒置小瓶，溶液迅速恢复成凝胶状态，溶液-凝胶之间的转变仅几秒钟。注射器注射实验结果与图2中c)～f)的结果一致，一旦去除了注射剪切力，水凝胶可在注射部位迅速恢复至凝胶状态。
上述试验结果说明：本发明水凝胶具有良好的剪切稀化性能和自修复性能。该水凝胶注射性能良好，且能快速在溶液和凝胶状态间改变。
试验例3、本发明双功能水凝胶的机械性能研究
1、试验方法
取实施例2、实施例3、实施例5和实施例6的制备的2.5％水凝胶和5.0％水凝胶，进行机械性能检测，测试条件为：水凝胶流变测试使用安东帕模块化智能型旋转流变仪(MCR302)来检测。制备的水凝胶溶液在80℃预热平行板上进行加样，加样后在平行板周围样品的裸露表面加一层薄薄的硅油，防止测试过程中水分蒸发。在25℃进行所有测量，检测频率范围为0.1-100rad/s。
2、试验结果
流变学测试进一步探索水凝胶的机械性能。在对水凝胶施加的频率范围内，G'和G”几乎不发生变化，且G'始终大于G”(如图3a)～d)所示)，表明所有被测样本始终表现为凝胶状态。此外，水凝胶的储能模量极高：2.5％水凝胶的储能模量大于0.1MPa，5.0％水凝胶的储能模量大于1MPa。加入少量的硫黄素T(ThT)或甲基蓝的5.0％水凝胶可被成功制成厚度约为1厘米的各种形状并被拿在手中(如图3e)所示)。如图3中的f)所示：所有水凝胶样品都能够承受一定的压缩应力而不发生破坏，高浓度水凝胶能够承受更高的压缩应力。如图3中的g)所示：实施例2和实施例3的水凝胶在25℃下进行测量，应变连续增加，应变范围为0～100％，2.5％水凝胶可以抵抗超过70％的应变，抗压强度高达0.2MPa，5.0％水凝胶可以抵抗超过80％的应变，抗压强度高达0.2MPa。
试验结果说明：本发明水凝胶具有MPa级的储能模量和抗压强度：5.0％水凝胶的储能模量高达1MPa，抗压强度高达0.2MPa。
试验例4、本发明双功能水凝胶的抗菌性能研究
一、本发明双功能水凝胶对金黄色葡萄球菌和大肠杆菌的抗菌性能
1、试验方法
使用G(+)菌金黄色葡萄球菌(S.aureus)及G(-)菌大肠杆菌(E.coli)作为模式菌株代表菌株，通过细菌菌落计数法初检实施例3制备的5.0％水凝胶的抗菌活性。具体试验方法如下：
按照实施例3所述方法在1.5mL离心管中直接形成60μL 5.0wt％水凝胶，在超净台内紫外线灭菌30分钟，在新的1.5mL离心管中加入相同体积的PBS作为PBS对照组(PBS组)，未处理的1.5mL离心管作为对照组(各组n＝3)。用无菌LB培养基将细菌培养物稀释至10 5CFU/ml，然后每个离心管中加入100μL菌液。在37℃下孵育24小时后，分别将各1.5mL离心管中的细菌按照相同梯度稀释后，将相同体积的稀释液接种在Luria-Bertani培养基上，在37℃下培养，24h后通过计数细菌菌落来确定本发明水凝胶体外杀菌活性。
2、试验结果
16SRNA鉴定结果确认菌种为金黄色葡萄球菌和大肠杆菌。本发明双功能水凝胶对金黄色葡萄球菌和大肠杆菌的抗菌性能如图4所示。由图4可知细菌菌落计数的结果为：对照组细菌菌落数大于PBS组，但是对照组与PBS组之间无明显差异(P>0.05)，说明PBS不影响细菌培养物的生长，仅表现为对细菌悬浮液的稀释作用。与PBS组相比，5.0％水凝胶组的细菌菌落数更少(P<0.05)，说明本发明双功能水凝胶对金黄色葡萄球菌和大肠杆菌均具有显著的抗菌性。
二、本发明双功能水凝胶对口腔主要致病菌的抗菌性能
1、试验方法
使用G(+)菌变异链球菌(Streptococcus mutans,S.mutans)及G(-)菌牙龈卟啉单胞菌为口腔致病菌代表菌株，通过细菌菌落计数法和扫描电子显微镜探究实施例2和实施例3制备的双功能水凝胶的抗菌性能。具体试验方法如下：
按照实施例2和实施例3所述方法分别在15mL离心管中形成1mL 2.5％水凝胶和5.0％水凝胶，并在超净台内紫外线灭菌30分钟，在新的15mL离心管中加入相同体积的PBS作为PBS对照组(PBS组)，未处理的15mL离心管作为对照组(各组n＝4)。用无菌BHI培养基将细菌培养物稀释至10 7CFU/mL，然后每个离心管中加入2mL菌液。在37℃下培养，变异链球菌在以下时间点检测：6小时、12小时、24小时，牙龈卟啉单胞菌在48小时取菌液进行检测。
各组分别在时间点取100μL菌液，进行相同的梯度稀释后，将相同体积的稀释液接种至琼脂固体平板上。变异链球菌培养24小时，牙龈卟啉单胞菌 培养7天后，通过计数细菌菌落来确定体外杀菌活性。
扫描电镜：将洗涤干净的圆玻片置于24孔板内，取培养了12小时的S.mutans菌液(变异链球菌菌液)或培养了24h的P.gingivalis菌液(牙龈卟啉单胞菌菌液)至圆玻片上，等量5％戊二醛固定后，不同浓度乙醇(50％，70％，95％，100％)梯度洗脱10min，取出玻片自然晾干，标记后在扫描电镜下观察。
2、试验结果
菌种鉴定：菌落形态观察和扫描电镜结果(图5的a～d)显示菌种为变异链球菌和牙龈卟啉单胞菌。变异链球菌单个菌落呈小米粒大小的乳白色状，隆起，呈圆形、边缘整齐，扫描电镜下可见菌体单个菌体球状，或多个细菌连接呈链状；牙龈卟啉单胞菌单个菌落呈圆形、边缘整齐，黑色，有金属光泽；扫描电镜下菌体呈卵圆形或圆形，边缘光滑。均与标准菌株的特征一致。
细菌菌落计数结果如图6所示：变异链球菌和牙龈卟啉单胞菌的对照组均较PBS组菌落数多，但无明显差异(P>0.05)，说明PBS不影响细菌培养物的生长，仅表现为对细菌悬浮液的稀释作用。对于变异链球菌：2.5％水凝胶组较PBS组有显著性差异(**P<0.01)，说明2.5％水凝胶组能有效抑制变异链球菌生长。对于牙龈卟啉单胞菌：2.5％水凝胶组和5.0％水凝胶组均无菌落生长，说明均可以抑制牙龈卟啉单胞菌的生长。
试验结果说明本发明双功能水凝胶能够有效抑制细菌生长，特别是抑制口腔主要致病菌变异链球菌和牙龈卟啉单胞菌的生长。其中2.5％对于变异链球菌和牙龈卟啉单胞菌均具有良好抑制作用，5.0％水凝胶对于牙龈卟啉单胞菌具有良好抑制作用。
试验例5、本发明双功能水凝胶的生物相容性评价
一、本发明双功能水凝胶的体外毒性评价
1、试验方法
体外检测实施例1和实施例2制备的双功能水凝胶的细胞毒性。CCK8细胞毒性实验检测对NOK-SI细胞系的毒性。具体试验方法如下：
分别收集生长良好的NOK-SI细胞，配制成细胞悬液，调整其细胞密度后接种于24孔板中；等待4～6h细胞贴壁，分别加入5μL实施例1和实施例2制备的水凝胶(实验组)，以及5μL PBS(作为PBS实验组)，至transwell小室的上室内，将其放置在24孔板内，37℃共培养24h；然后加入10％CCK8试剂，孵育3h后使用空白孔调零，使用酶标仪检测450nm波长的吸光度值(OD)。每组设计≥3个副孔，结果取平均值，实验独立重复3次。试验中设置细胞对照组(对照组，Control)。
细胞活性百分率(％)＝实验组(OD)/对照组(OD)×100％。
2、试验结果
采用CCK8试剂盒评估本发明水凝胶对NOK-SI细胞系的毒性。结果如图7所示(图7中的细胞活性数据没有乘以100％，直接是实验组(OD)/对照组(OD))，图7结果显示：1.67％水凝胶和2.5％水凝胶的细胞活性分别高达95％和90％，这表明本发明水凝胶材料对细胞无毒或低毒，具有良好的细胞相容性。本发明水凝胶无体外急性毒性，作为生物材料有潜在的应用。
二、本发明双功能水凝胶的体内毒性评价
1、试验方法
在雌性BALB/c小鼠背部皮肤粘膜下注射实施例1和实施例2制备的双功能水凝胶后检测降解情况。并在一定的时间点(0h、3h、6h、9h、12h、15h、18h、21h和24h)取血液分成2份，血液分别进行全血细胞计数和血清生化检测生物相容性。处死小鼠取心、肝、脾、肺、肾处理后进行HE染色；观察器官是否有损伤。在BALB/c小鼠背部皮肤粘膜下注射等量PBS作为PBS组，进行对照。
2、试验结果
小鼠背部皮肤粘膜下注射水凝胶检测材料的降解以及生物安全性，评估指标包括全血细胞计数(CBC)、血清生化指标和组织学。图8为本发明水凝胶在小鼠体内降解情况，图8结果显示：水凝胶在6小时内降解，其中1.67％水凝胶降解速度快于2.5％水凝胶。图9为苏木精和伊红(H&E)染色结果，其染色结果显示：1.67％水凝胶和2.5％水凝胶处理组的心脏、肝脏、脾脏、肺和肾脏与对照组相比，均未发现明显的器官损伤迹象。
此外，如图10所示：与PBS组对比，处理组的各项CBC指标，除注射水凝胶后3小时白细胞(WBC)，其它均不存在差异(P>0.05)。注射水凝胶3h后的WBC，1.67％水凝胶组(p＝0.00，***P<0.001)和2.5％水凝胶组(P＝0.03，*P<0.05)均高于PBS组，但它们的值在正常范围内。这种差异可能是由于动物之间的个体差异所致。关于血清生化指标，与PBS治疗组相比，1.67％水凝胶组中3小时后血尿素氮减少(BUN，*P<0.05)，注射后3h谷草转氨酶升高(AST，*P<0.05)，其他血清生化指标没有显着差异(P>0.05)；2.5％水凝胶组除注射3小时后血浆BUN(*P<0.05)减少和注射后24小时尿酸(UA，*P<0.05)增加外无其它检测指标无差异。但是所有检测值都在正常范围内。试验结果说明本发明双功能水凝胶无体内的急性毒性。
上述试验结果表明本发明双功能水凝胶具有良好的降解性能，且在体内外均具有良好的生物相容性。
综上，本发明通过一种简单、绿色的方法制备得到一种双功能水凝胶。该水凝胶具有良好的剪切稀化能力和自修复性能，可注射性能良好，成胶速度快，使用方便。该水凝胶也具备出色的机械性能，其具有超高强度，储能模量和抗压强度可达MPa级。同时，该水凝胶具有良好的抗菌作用，对模式菌株和口腔致病菌的生长均有良好抑制作用。此外，该水凝胶室温下具有良好的稳定性，可长期保存，而植入人体后可快速降解，且具有良好的生物相容性。这些突出的特征使本发明水凝胶有望成为用于牙周病局部注射治疗的候选药物，同时在颅颌面骨组织缺损的微创疗法中也有潜在的应用前景。

Claims (10)

  1. 一种集可注射与抗菌于一体的双功能水凝胶，其特征在于：它是由核苷类似物溶于溶剂后制备而得的水凝胶；
    所述核苷类似物的结构如式I所示：
    Figure PCTCN2020093461-appb-100001
  2. 根据权利要求1所述的双功能水凝胶，其特征在于：所述水凝胶中，核苷类似物的浓度为1.67w/v％～5w/v％；
    优选地，所述水凝胶中，核苷类似物的浓度为2.5w/v％～5w/v％。
  3. 根据权利要求1所述的双功能水凝胶，其特征在于：所述溶剂为水或磷酸盐缓冲液。
  4. 根据权利要求1～3任一项所述的双功能水凝胶，其特征在于：所述水凝胶的制备方法为：将核苷类似物加热溶解于溶剂中，冷却至室温，即得。
  5. 一种权利要求1～4任一项所述的双功能水凝胶的制备方法，其特征在于：它包括如下步骤：
    将核苷类似物加热溶解于溶剂中，冷却至室温，即得。
  6. 根据权利要求5所述的制备方法，其特征在于：所述核苷类似物溶于溶剂后，核苷类似物的浓度为1.67w/v％～5w/v％；
    优选地，所述核苷类似物的浓度为2.5w/v％～5w/v％。
  7. 权利要求1～4任一项所述的双功能水凝胶在制备组织修复材料，和/或牙周病局部注射治疗药物中的用途。
  8. 根据权利要求7所述的用途，其特征在于：所述组织修复材料为颅颌面骨组织修复材料；和/或，所述牙周病为牙周炎。
  9. 一种牙周病局部注射治疗药物，其特征在于：它是由权利要求1～4任一项所述的双功能水凝胶为活性物质，加上药物上可接受的辅料或辅助性成分制备而成；
    优选地，所述牙周病为牙周炎。
  10. 式I所示结构的核苷类似物在制备抗菌药物，和/或制备具有抗菌作用的生物医用材料中的用途：
    Figure PCTCN2020093461-appb-100002
    优选地，所述生物医用材料为组织修复材料。
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