WO2021109276A1 - 复合体、组织修复材料及其制备方法和应用 - Google Patents

复合体、组织修复材料及其制备方法和应用 Download PDF

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WO2021109276A1
WO2021109276A1 PCT/CN2019/127253 CN2019127253W WO2021109276A1 WO 2021109276 A1 WO2021109276 A1 WO 2021109276A1 CN 2019127253 W CN2019127253 W CN 2019127253W WO 2021109276 A1 WO2021109276 A1 WO 2021109276A1
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aptamer
exosomes
tissue
tissue repair
repair material
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PCT/CN2019/127253
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English (en)
French (fr)
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胡成虎
赵娜
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陕西佰傲再生医学有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33303—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group
    • C08G65/33306—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group acyclic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33331—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing imide group
    • C08G65/33337—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing imide group cyclic

Definitions

  • the invention belongs to the technical field of biological products, and particularly relates to a composite body, a tissue repair material, and a preparation method and application thereof.
  • tissue and organs The damage or dysfunction of tissues and organs is one of the main hazards facing human health, and it is also the main cause of human disease and death.
  • allogeneic tissue transplantation but there are problems of histocompatibility and serious shortage of donor sources; autologous tissue transplantation, which is at the expense of normal tissues and heterotopic tissues Often leads to dysfunction; the use of artificial synthetic material substitutes can easily lead to foreign body reactions, secondary infections and atrophy of surrounding tissues.
  • Mesenchymal stem cells are important members of the stem cell family. They are derived from the mesoderm in the early stage of development. They have the characteristics of multi-directional differentiation potential, hematopoietic support and promotion of stem cell implantation and immune regulation. They can regulate tissue regeneration and wound healing. Extracellular vesicles are one of the key secreted products of mesenchymal stem cells mediating intercellular communication to enhance tissue repair. Exosomes are a type of extracellular vesicles, which are round with a diameter of 40-100nm. The single-layer membrane structure is formed by the budding method of multiple cell inner membranes of the body and is widely distributed in different body fluids.
  • the targeted modification of exosomes is mainly through the method of transfection and then separation, that is, the cells express the targeted protein through transfection, and then the exosomes expressing the targeted protein are separated from the cell.
  • the modification efficiency is It is low, and the operation is cumbersome, so it is urgent to find a targeted modification method for exosomes with high modification efficiency and simple operation.
  • Aptamers are artificially synthesized single-stranded oligonucleotides that can be screened, amplified and enriched in vitro through the systematic evolution of exponentially enriched ligands (SELEX), often forming secondary structures such as hairpins, stem loops, and three-dimensional
  • SELEX exponentially enriched ligands
  • the three-dimensional structure can bind to the target through molecular conformation matching, and has the characteristics of high affinity, strong specificity, and low immunogenicity.
  • the present invention provides a complex that has stable pharmacodynamics, high targeting, high delivery efficiency, good tolerance in the body, and can be exerted by endogenous substances. A variety of biological functions, while avoiding the immune response in the body.
  • a further object of the present invention is to provide a tissue repair material that has a stable therapeutic effect and targeting in the treatment of skin diseases, and particularly has a significant effect in the treatment of chronic diabetic complications.
  • the first aspect of the present invention provides a complex formed by combining a targeting group and a medicinal vesicle, the targeting group is selected from an aptamer, and the medicinal vesicle is selected from outside Exosomes.
  • the present invention utilizes the targeting effect of aptamers on specific cells, enables exosomes to migrate and act on target cells, improves the bioavailability of exosomes, and achieves the purpose of promoting tissue repair.
  • Aptamer-modified exosomes can accelerate the growth and migration of fibroblasts from chronic non-healing wounds, and can effectively activate growth factor signaling pathways, increase the expression of angiogenesis-related genes and matrix proteins, and induce angiogenesis Therefore, the complex provided by the present invention can be used as a safe and effective product to promote the healing of chronic hard-to-heal wounds or bone defects.
  • the ratio of the number of aptamers and exosomes contained in each complex is: n:1, n ⁇ 1, and n is an integer.
  • the aptamer is selected from vascular endothelial cell targeting aptamers.
  • the vascular endothelial cell targeting aptamer has a targeting effect on vascular endothelial cells, allows exosomes to migrate and act on vascular endothelial cells, improves the bioavailability of exosomes, and promotes the proliferation of vascular endothelial cells. The purpose of vascular repair.
  • the aptamer is selected from osteoblast targeting aptamers.
  • the osteoblast targeting aptamer has a targeting effect on osteoblasts, allows exosomes to migrate and act on osteoblasts, improves the bioavailability of exosomes, and promotes the proliferation of osteoblasts. The purpose of increasing bone density and repairing bone defects.
  • sequence of the aptamer is:
  • the 3'or 5'end of the aptamer is modified with an amino group.
  • Ordinary aptamers are difficult to bind to exosomes. Using amide condensation reaction, the amino-modified aptamers can effectively bind to exosomes.
  • the modified aptamers are: aptamer-polyethylene glycol -Distearoylphosphatidylethanolamine, the chemical structure is:
  • the method for modifying the aptamer includes the following steps:
  • the present invention uses amide condensation reaction to modify the group of the aptamer, so that the aptamer obtains the lipid molecular structure so that it can be fused with exosomes, and the operation process is simple.
  • the obtained aptamer-polyethylene glycol- Distearoylphosphatidylethanolamine has high purity.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethyl carbon described in step 1) The ratio of the amount of diimine substances is 1: (10-30): (100-300).
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethyl carbon described in step 1) The ratio of the amount of diimine substances is 1: (15-20): (150-200).
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethyl carbon described in step 1) The ratio of the amount of diimine substance is 1:10:100.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethyl carbon described in step 1) The ratio of the amount of diimine substance is 1:30:300.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethyl carbon described in step 1) The ratio of the amount of diimine substance is 1:18:160.
  • the dialysis process in step 2) is specifically:
  • the ultrafiltration process is specifically:
  • the exosomes are mesenchymal stem cell exosomes.
  • the mesenchymal stem cell exosomes are selected from bone marrow mesenchymal stem cell exosomes, cord blood mesenchymal stem cell exosomes, umbilical cord tissue mesenchymal stem cell exosomes, placental tissue mesenchymal stem cells Exosomes or adipose tissue mesenchymal stem cell exosomes.
  • the second aspect of the present invention provides a composition, the active ingredient of the composition is the above-mentioned complex.
  • the composition of the present invention can accelerate the growth and migration of fibroblasts from chronic non-healing wounds, and can effectively activate growth factor signal pathways, increase the expression of angiogenesis-related genes and matrix proteins, and induce the formation of new blood vessels or osteoblasts Therefore, the composition provided by the present invention can be used as a safe and effective product to promote the healing of chronic difficult-to-heal wounds or bone defects.
  • the third aspect of the present invention provides the use of the above-mentioned complex or the above-mentioned composition in promoting angiogenesis.
  • the blood vessels include arterial blood vessels, venous blood vessels and capillaries.
  • the capillaries include capillaries existing in the dermis of the skin, bone tissue, and cardiomyocytes.
  • the fourth aspect of the present invention provides the use of the above-mentioned complex or the above-mentioned composition in the preparation of a drug for the treatment of skin diseases.
  • the skin diseases include skin injuries, skin infections, bedsores or diabetic ulcers.
  • a tissue repair material in a fifth aspect of the present invention, includes a composite body and a gel material, and the composite body is formed by a combination of aptamers and exosomes.
  • hydrogels are used to embed active drugs, proteins, cells, etc., which can achieve long-term sustained release of active substances through diffusion and degradation of the gel.
  • active drugs proteins, cells, etc.
  • hydrogels are used to embed active drugs, proteins, cells, etc., which can achieve long-term sustained release of active substances through diffusion and degradation of the gel.
  • due to the lack of vascularization and synthesis in the treatment The key link of bone and other tissue repair, the therapeutic effect is greatly reduced.
  • the tissue repair material constructed by the present invention using the amide condensation reaction, the principle of similar compatibility and the suspension method has been confirmed by experiments that the tissue repair material provided by the present invention can not only make the active substance achieve long-term effects through diffusion and gel degradation.
  • the effect of slow release, and the tissue repair material of the present invention has obvious ability to promote angiogenesis and tissue repair, can accelerate the healing of difficult-to-heal wounds, promote the deposition of collagen at the tissue damage site, and promote the maturation of granulation tissue and the regeneration of the epidermis.
  • the ratio of the number of aptamers and exosomes contained in each complex is: n:1, n ⁇ 1, and n is an integer.
  • the aptamer is selected from vascular endothelial cell targeting aptamers.
  • sequence of the aptamer is:
  • the 3'or 5'end of the aptamer is modified with -NH 2 and the modified aptamer is: aptamer-polyethylene glycol-distearoylphosphatidylethanolamine, and the chemical structural formula is :
  • the method for modifying the aptamer includes the following steps:
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethyl carbon described in step 1) The ratio of the amount of diimine substances is 1: (10-30): (100-300).
  • the dialysis process in step 2) is specifically:
  • the ultrafiltration process is specifically:
  • the exosomes are mesenchymal stem cell exosomes.
  • the mesenchymal stem cell exosomes are selected from bone marrow mesenchymal stem cell exosomes, cord blood mesenchymal stem cell exosomes, umbilical cord tissue mesenchymal stem cell exosomes, placental tissue mesenchymal stem cells Exosomes or adipose tissue mesenchymal stem cell exosomes.
  • the gel material is selected from at least one of collagen, alginate and sodium hyaluronate.
  • a method for preparing tissue repair materials which includes the following steps:
  • the tissue repair material obtained by using the preparation method of the tissue repair material of the present invention constructs a treatment system with strong targeting ability and low immunogenicity, and the preparation method is simple to operate, which solves the cumbersome synthesis of tissue repair materials in the prior art And the problem of low production efficiency.
  • the gel material is selected from at least one of collagen, alginate and sodium hyaluronate.
  • the aptamer-polyethylene glycol-distearoylphosphatidylethanolamine is prepared by the following method:
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethyl carbon described in step 1) The ratio of the amount of diimine substances is 1: (10-30): (100-300).
  • the dialysis process in step 2) is specifically:
  • the ultrafiltration process is specifically:
  • the seventh aspect of the present invention provides the use of the above-mentioned tissue repair material in the preparation of skin disease treatment products.
  • the skin diseases include skin injuries, skin infections, bedsores or diabetic ulcers.
  • a skin damage repair gel is provided, the components of the skin damage repair gel include the above-mentioned tissue repair material.
  • the tissue repair material constructed by the present invention using the amide condensation reaction, the principle of similar compatibility and the suspension method has been confirmed by experiments that the tissue repair material provided by the present invention can not only make the active substance achieve long-term effects through diffusion and gel degradation.
  • the effect of slow release, and the tissue repair material of the present invention has obvious ability to promote angiogenesis and tissue repair, can accelerate the healing of difficult-to-heal wounds, promote the deposition of collagen at the tissue damage site, and promote the maturation of granulation tissue and the regeneration of the epidermis.
  • a dressing for diabetic ulcer repair comprises the above-mentioned tissue repair material.
  • the diabetic ulcer repair dressing provided by the present invention has obvious ability to promote angiogenesis and tissue repair, can accelerate the wound healing of chronic difficult-to-heal ulcers caused by diabetes, promote the deposition of collagen in the ulcer, and promote the maturation of granulation tissue and the ulcer. Epidermal regeneration.
  • a foot patch comprising the above-mentioned tissue repair material and an adhesive patch, and the tissue repair material is pasted on the adhesive patch.
  • the shape of the foot patch provided by the present invention is designed for the foot, can be effectively fixed on the foot ulcer, does not affect daily walking, and can effectively promote the healing of foot wounds, especially chronic, difficult-to-heal diabetic foot ulcers.
  • the adhesive patch of the present invention is selected from non-woven fabric, spunbonded fabric, polyethylene film or medical PU film.
  • the ddH 2 O mentioned in the present invention refers to the water obtained by distilling once and then distilling again, also called double distilled water or double distilled water.
  • the diethyl pyrocarbonate treated water in the present invention refers to high-purity water that has been treated with diethyl pyrocarbonate and sterilized by high temperature and high pressure, and does not contain impurity RNA, DNA and protein.
  • the diethyl pyrocarbonate treated water of the present invention was purchased from Shenggong Bioengineering (Shanghai) Co., Ltd., number: B501005-0500.
  • the diethyl pyrocarbonate-treated PBS buffer solution of the present invention refers to a PBS buffer solution that has been treated with diethyl pyrocarbonate, and does not contain impurity RNA, DNA and protein.
  • the diethyl pyrocarbonate treated PBS buffer solution of the present invention was purchased from Shenggong Bioengineering (Shanghai) Co., Ltd., number: B501005-0500.
  • the PBS buffer in the present invention refers to a phosphate buffered saline solution.
  • TAE buffer refers to a buffer composed of Tris base, acetic acid and ethylenediaminetetraacetic acid (EDTA).
  • Figure 1A is a transmission electron microscopy result diagram of the exosomes described in an embodiment of the present invention.
  • FIG. 1B is a transmission electron microscope result diagram of the composite in the embodiment of the present invention.
  • Figure 2A is the particle size distribution of the exosomes in the embodiment of the present invention.
  • 2B is the particle size distribution of the composite in the embodiment of the present invention.
  • Figure 3 is a diagram showing the result of agarose gel electrophoresis identification of the complex according to the embodiment of the present invention.
  • Fig. 4A is a target fluorescence map of the aptamer used in the embodiment of the present invention to the vascular endothelial cell group;
  • Fig. 4B is a target fluorescence map of the aptamer used in the embodiment of the present invention to the mesenchymal stem cell group;
  • Fig. 5A is a Hochest staining image of skin wound tissue in the aptamer-carboxyfluorescein-hyaluronic acid group administered for 2 days in the embodiment of the present invention
  • Fig. 5B is the FAM fluorescence image of the skin wound tissue after the administration of the aptamer-carboxyfluorescein-hyaluronic acid group in the embodiment of the present invention for 2 days;
  • Fig. 5C is a CD31-stained fluorescence image of the skin wound in the aptamer-carboxyfluorescein-hyaluronic acid group administered for 2 days in the embodiment of the present invention
  • Fig. 5D is a merge fluorescence image of the skin wound in the aptamer-carboxyfluorescein-hyaluronic acid group administered for 2 days in the embodiment of the present invention
  • Fig. 6A is a Hochest staining image of skin wound tissue in the hyaluronic acid group 2 days after administration of the hyaluronic acid group in the embodiment of the present invention
  • Fig. 6B is a FAM fluorescence image of skin wound tissue in the hyaluronic acid group 2 days after administration of the hyaluronic acid group in the embodiment of the present invention
  • Fig. 6C is a fluorescence image of CD31 staining of skin wound tissue in the hyaluronic acid group 2 days after administration of the hyaluronic acid group in the embodiment of the present invention
  • Fig. 6D is a fluorescence chart of merge in the hyaluronic acid group 2 days after administration of the embodiment of the present invention.
  • Fig. 7A is a Hochest staining diagram of skin wound tissue in the PBS group after 2 days of instillation in the embodiment of the present invention.
  • Fig. 7B is a FAM fluorescence image of skin wound tissue in the PBS group after 2 days of instillation in the embodiment of the present invention.
  • Fig. 7C is a fluorescence image of the skin wound tissue stained by CD31 after 2 days of instillation in the PBS group in the embodiment of the present invention.
  • Fig. 7D is the merge fluorescence image of the skin wound tissue after 2 days of instillation in the PBS group in the embodiment of the present invention.
  • FIG. 8A is a fluorescence image of harness staining of skin wound tissue 4 days after administration of the tissue repair material group in the embodiment of the present invention.
  • Fig. 8B is an immunofluorescence image of the skin wound tissue stained by CD31 after administration of the tissue repair material group in the embodiment of the present invention for 4 days;
  • Figure 8C is an EMCN staining immunofluorescence image of skin wound tissue in the tissue repair material group 4 days after administration of the tissue repair material group in the embodiment of the present invention
  • Fig. 8D is a merge fluorescence image of skin wound tissue 4 days after administration of the tissue repair material group in the embodiment of the present invention.
  • Fig. 8E is a blood vessel diagram of the skin wound tissue 4 days after administration of the tissue repair material group in the embodiment of the present invention.
  • Fig. 9A is a fluorescence image of Hochest staining of skin wound tissue 4 days after administration of the exosomal gel group in the embodiment of the present invention.
  • Figure 9B is an immunofluorescence image of skin wound tissue stained with CD31 after administration of the exosomal gel group in the embodiment of the present invention for 4 days;
  • Fig. 9C is an immunofluorescence image of the skin wound tissue after being stained with EMCN in the exosomal gel group 4 days after administration of the embodiment of the present invention.
  • Fig. 9D is a merge fluorescence image of skin wound tissue 4 days after administration of the exosomal gel group in the embodiment of the present invention.
  • Figure 9E is a blood vessel diagram of skin wound tissue 4 days after administration of the exosomal gel group in the embodiment of the present invention.
  • Figure 10A is a fluorescence image of Hochest staining of skin wound tissue in the hyaluronic acid group 4 days after administration of the hyaluronic acid group in the embodiment of the present invention
  • Figure 10B is an immunofluorescence image of skin wound tissue stained by CD31 in the hyaluronic acid group 4 days after the administration of the hyaluronic acid in the embodiment of the present invention
  • FIG. 10C is an immunofluorescence image of the skin wound tissue after EMCN staining in the hyaluronic acid group 4 days after administration of the hyaluronic acid group in the embodiment of the present invention.
  • Fig. 10D is a merge fluorescence image of skin wound tissue in the hyaluronic acid group 4 days after administration of the hyaluronic acid group in the embodiment of the present invention
  • Figure 10E is a blood vessel diagram of the skin wound tissue 4 days after administration of the hyaluronic acid group in the embodiment of the present invention.
  • Figure 11A is a Hochest staining fluorescence image of skin wound tissue in the PBS group 4 days after instillation in the embodiment of the present invention.
  • Fig. 11B is an immunofluorescence image of skin wound tissue stained with CD31 after 4 days of instillation in the PBS group in the example of the present invention.
  • Fig. 11C is an immunofluorescence image of the skin wound tissue after EMCN staining in the PBS group 4 days after instillation in the embodiment of the present invention.
  • Fig. 11D is a merge fluorescence image of skin wound tissue after 4 days of instillation in the PBS group in the embodiment of the present invention.
  • Figure 11E is a blood vessel diagram of the skin wound tissue after 4 days of instillation in the PBS group in the embodiment of the present invention.
  • Figure 12A is a diagram of skin damage before applying the tissue repair material of the embodiment of the present invention.
  • Figure 12B is a diagram of skin damage before applying exosomal gel
  • Figure 12C is a diagram of skin damage before applying hyaluronic acid
  • Fig. 13A is a diagram of skin damage after applying the tissue repair material of the embodiment of the present invention for 4 days;
  • Fig. 13B is a picture of skin damage 4 days after applying exosomal gel
  • Figure 13C is a picture of skin damage 4 days after applying hyaluronic acid
  • Figure 14A is a diagram of skin damage 8 days after applying the tissue repair material of the embodiment of the present invention.
  • Figure 14B is a picture of skin damage 8 days after applying exosomal gel
  • Figure 14C is a diagram of skin damage after applying hyaluronic acid 8.
  • 15A is a diagram of skin damage after applying the tissue repair material of the embodiment of the present invention for 12 days;
  • Figure 15B is a picture of skin damage after applying exosomal gel for 12 days;
  • Figure 15C is a diagram of skin damage after applying hyaluronic acid for 12 days;
  • Figure 16A shows a hematoxylin-eosin staining image of the tissue repair material of an embodiment of the present invention after repairing damaged skin;
  • Figure 16B shows the hematoxylin-eosin staining image after the exosomal gel repairs damaged skin
  • Figure 16C shows a hematoxylin-eosin staining image after hyaluronic acid repairs damaged skin
  • FIG. 17A is a Masson staining diagram of the tissue repair material after treating damaged skin according to an embodiment of the present invention.
  • Figure 17B is a Masson staining image after exosomal gel treatment of damaged skin
  • Figure 17C is a Masson staining image after hyaluronic acid treatment of injured skin.
  • Distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl purchased from Avanti Polar Lipids, number 880135P;
  • N-Hydroxysuccinimide purchased from Aladdin Reagent (Shanghai) Co., Ltd., number H109330;
  • 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide purchased from Shanghai Macleans Biochemical Technology Co., Ltd., number N807578;
  • Vascular targeting aptamer Procured from SSBio Engineering (Shanghai) Co., Ltd., CCCACGTCTGCGCTTAGCTCCTGGGCCTGGATGGGC;
  • Triethylamine purchased from Aladdin Reagent (Shanghai) Co., Ltd., number T103289;
  • Diethyl pyrocarbonate treated water Procured from Zishenggong Bioengineering (Shanghai) Co., Ltd., number B501005-0500;
  • Hyaluronic acid purchased from Shanghai Macleans Biochemical Technology Co., Ltd., number H823435;
  • CD31 fluorescent primary antibody purchased from R&D company, number FAB3628G;
  • CD31 primary antibody purchased from Abcam, number AB28364;
  • EMCN primary antibody purchased from Santa Cruz Biotechnology company, number SC-6549;
  • Dialysis bag purchased from Shanghai Yuanye Biotechnology Co., Ltd., number SP131084;
  • 5kD ultrafiltration centrifuge tube purchased from Sartorius company, number VS0112;
  • 30kD ultrafiltration centrifuge tube purchased from Millipore company, number UFC203024.
  • the embodiment of the present invention provides a complex formed by combining a targeting group and a medicinal vesicle, the targeting group is selected from an aptamer, and the medicinal vesicle is selected from exocrine body.
  • the embodiment of the present invention utilizes the targeting effect of aptamers on specific cells to enable exosomes to migrate and act on target cells, thereby improving the bioavailability of exosomes and achieving the purpose of promoting tissue repair.
  • Aptamer-modified exosomes can accelerate the growth and migration of fibroblasts from chronic non-healing wounds, and can effectively activate growth factor signaling pathways, increase the expression of angiogenesis-related genes and matrix proteins, and induce angiogenesis Therefore, the complex provided by the present invention can be used as a safe and effective product to promote the healing of chronic hard-to-heal wounds or bone defects.
  • the ratio of the number of aptamers and exosomes contained in each complex is: n:1, n ⁇ 1, and n is an integer.
  • the aptamer is selected from vascular endothelial cell targeting aptamers.
  • the vascular endothelial cell targeting aptamer has a targeting effect on vascular endothelial cells, allows exosomes to migrate and act on vascular endothelial cells, improves the bioavailability of exosomes, and promotes the proliferation of vascular endothelial cells. The purpose of vascular repair.
  • the aptamer is selected from osteoblast targeting aptamers.
  • the osteoblast targeting aptamer has a targeting effect on osteoblasts, allows exosomes to migrate and act on osteoblasts, improves the bioavailability of exosomes, and promotes the proliferation of osteoblasts. The purpose of increasing bone density and repairing bone defects.
  • the sequence of the aptamer is:
  • the 3'or 5'end of the aptamer is modified with an amino group.
  • Ordinary aptamers are difficult to bind to exosomes. Utilizing amide condensation reaction, aptamers modified with amino groups can effectively bind to exosomes.
  • the modified aptamer is: aptamer-polyethylene glycol-distearoylphosphatidylethanolamine, and the chemical structural formula is:
  • the method for modifying the aptamer includes the following steps:
  • the present invention uses amide condensation reaction to modify the group of the aptamer, so that the aptamer obtains the lipid molecular structure so that it can be fused with exosomes, and the operation process is simple.
  • the obtained aptamer-polyethylene glycol- Distearoylphosphatidylethanolamine has high purity.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethyl as described in step 1) The ratio of the amount of carbodiimide is 1:(10-30):(100-300).
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:10:100.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:10:150.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:10:200.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:10:300.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:20:100.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:20:150.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:20:200.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:20:300.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:30:100.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:30:150.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:30:200.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-carboxy, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3- The ratio of the amount of ethylcarbodiimide is 1:30:300.
  • step 2) The dialysis process described in step 2) is specifically:
  • step 5 The ultrafiltration process described in step 5) is specifically:
  • the exosomes are mesenchymal stem cell exosomes, for example: bone marrow mesenchymal stem cell exosomes, cord blood mesenchymal stem cell exosomes, umbilical cord tissue mesenchymal stem cell exosomes , Placental tissue mesenchymal stem cell exosomes or adipose tissue mesenchymal stem cell exosomes.
  • compositions and the active ingredient of the composition is the complex described in Example 1.
  • the composition of the present invention can accelerate the growth and migration of fibroblasts from chronic non-healing wounds, and can effectively activate growth factor signal pathways, increase the expression of angiogenesis-related genes and matrix proteins, and induce the formation of new blood vessels or osteoblasts Therefore, the composition provided by the present invention can be used as a safe and effective product to promote the healing of chronic difficult-to-heal wounds or bone defects.
  • the blood vessels include arterial blood vessels, venous blood vessels and capillaries.
  • the capillaries include capillaries existing in the dermis of the skin, bone tissue, and cardiomyocytes.
  • This example provides the use of the complex in Example 1 or the composition in Example 2 in the preparation of a medicament for the treatment of skin diseases.
  • the skin disease includes skin damage, skin infection, bedsores, or diabetic ulcers.
  • tissue repair material This embodiment provides a tissue repair material.
  • the components of the tissue repair material include a composite body and a gel material, and the composite body is formed by a combination of an aptamer and an exosome.
  • hydrogels are used to embed active drugs, proteins, cells, etc., which can achieve long-term sustained release of active substances through diffusion and degradation of the gel.
  • active drugs proteins, cells, etc.
  • hydrogels are used to embed active drugs, proteins, cells, etc., which can achieve long-term sustained release of active substances through diffusion and degradation of the gel.
  • due to the lack of vascularization and synthesis in the treatment The key link of bone and other tissue repair, the therapeutic effect is greatly reduced.
  • the tissue repair material constructed by the present invention using the amide condensation reaction, the principle of similar compatibility and the suspension method has been confirmed by experiments that the tissue repair material provided by the present invention can not only make the active substance achieve long-term effects through diffusion and gel degradation.
  • the effect of slow release, and the tissue repair material of the present invention has obvious ability to promote angiogenesis and tissue repair, can accelerate the healing of difficult-to-heal wounds, promote the deposition of collagen at the tissue damage site, and promote the maturation of granulation tissue and the regeneration of the epidermis.
  • the ratio of the number of aptamers and exosomes contained in each complex is: n:1, n ⁇ 1, and n is an integer.
  • the aptamer is selected from vascular endothelial cell targeting aptamers.
  • the sequence of the aptamer is:
  • the 3'or 5'end of the aptamer is modified with -NH 2 and the modified aptamer is: aptamer-polyethylene glycol-distearoylphosphatidylethanolamine,
  • the chemical structural formula is:
  • the method for modifying the aptamer includes the following steps:
  • Step 1) Distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide described in step 1) The ratio of the amount of the substance is 1:(10 ⁇ 30):(100 ⁇ 300).
  • step 2) The dialysis process described in step 2) is specifically:
  • step 5 the ultrafiltration process is specifically:
  • the exosomes are mesenchymal stem cell exosomes, for example: bone marrow mesenchymal stem cell exosomes, cord blood mesenchymal stem cell exosomes, umbilical cord tissue mesenchymal stem cell exosomes , Placental tissue mesenchymal stem cell exosomes or adipose tissue mesenchymal stem cell exosomes.
  • the gel material is selected from at least one of collagen, alginate and sodium hyaluronate.
  • This embodiment provides a method for preparing a tissue repair material, including the following steps:
  • the tissue repair material obtained by using the preparation method of the tissue repair material of the present invention constructs a treatment system with strong targeting ability and low immunogenicity, and the preparation method is simple to operate, which solves the cumbersome synthesis of tissue repair materials in the prior art And the problem of low production efficiency.
  • the aptamer-polyethylene glycol-distearoylphosphatidylethanolamine is prepared by the following method:
  • Step 1) Distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide described in step 1) The ratio of the amount of the substance is 1:(10 ⁇ 30):(100 ⁇ 300).
  • step 2) The dialysis process described in step 2) is specifically:
  • step 5 the ultrafiltration process is specifically:
  • This embodiment provides the use of the above-mentioned tissue repair material in the preparation of a skin disease treatment product.
  • the skin disease includes skin damage, skin infection, bedsores, or diabetic ulcers.
  • This embodiment provides a skin damage repair gel, and the components of the skin damage repair gel include the tissue repair material in embodiment 5.
  • the tissue repair material constructed by the present invention using the amide condensation reaction, the principle of similar compatibility and the suspension method has been confirmed by experiments that the tissue repair material provided by the present invention can not only make the active substance achieve long-term effects through diffusion and gel degradation.
  • the effect of slow release, and the tissue repair material of the present invention has obvious ability to promote angiogenesis and tissue repair, can accelerate the healing of difficult-to-heal wounds, promote the deposition of collagen at the tissue damage site, and promote the maturation of granulation tissue and the regeneration of the epidermis.
  • This embodiment provides a dressing for repairing diabetic ulcers, and the dressing for repairing diabetic ulcers includes the tissue repairing material in embodiment 5.
  • the diabetic ulcer repair dressing provided by the present invention has obvious ability to promote angiogenesis and tissue repair, can accelerate the wound healing of chronic difficult-to-heal ulcers caused by diabetes, promote the deposition of collagen in the ulcer, and promote the maturation of granulation tissue and the ulcer. Epidermal regeneration.
  • This embodiment provides a foot patch comprising the tissue repair material and an adhesive patch, and the tissue repair material is pasted on the adhesive patch.
  • the shape of the foot patch provided by the present invention is a shape that fits the foot structure, which can be square or round, and can be arbitrarily cut into the required shape.
  • the foot patch provided by the present invention can be fixed on the ulcer of the foot without affecting daily life. Walking can effectively promote the healing of foot wounds, especially chronic, difficult-to-heal diabetic foot ulcers.
  • the material of the adhesive sticker is selected from non-woven fabric, spunbonded fabric, polyethylene film or medical PU film.
  • Figure 1A is an electron microscope image of exosomes.
  • Figure 1A shows a cup-shaped membrane bubble with a clear membrane structure with a diameter of about 100 nm.
  • Figure 1B is an electron microscope image of the complex. It can be seen that the aptamer is successfully attached to the surface of the exosomal membrane and is in the form of microspheres;
  • Figure 2A shows the particle size distribution of the exosomes;
  • Figure 2B shows the particle size distribution of the complex; The particle size is mostly distributed around 90nm, and the particle size of the composite is mostly around 300nm.
  • band 1 is the identification result of aptamer agarose gel electrophoresis
  • band 2 is the agarose gel electrophoresis identification of aptamer-polyethylene glycol-distearoylphosphatidylethanolamine
  • band 3 is the result of agarose gel electrophoresis identification of the complex.
  • the band As the molecular weights of the aptamer, aptamer-polyethylene glycol-distearoylphosphatidylethanolamine and the complex increase in sequence, the band’s The electrophoresis speed also slowed down successively, proving that the intermediate product aptamer-polyethylene glycol-distearoylphosphatidylethanolamine and the final product complex were successfully synthesized.
  • the cell slides are placed in a 24-well plate, and vascular endothelial cells and mesenchymal stem cells are respectively seeded on the 24-well plate at an appropriate concentration.
  • the 5OD aptamer-carboxyfluorescein (Apt- FAM) Dissolve in 100 ⁇ l of diethyl pyrocarbonate-treated water to prepare a stock solution, place it in a water bath at 85°C for 10 minutes, then quickly ice bath for 10 minutes, dilute the Apt-FAM stock solution to 10 ⁇ M with DMEM medium, treat the cells for 2h, and wash with PBS Several times, the nuclei were stained with 5 ⁇ M Hochest for 5min, fixed, mounted, and observed and imaged with a fluorescence microscope.
  • Figure 4A shows the targeted fluorescence of the vascular endothelial cell group
  • Figure 4B shows the targeted fluorescence of the mesenchymal stem cell group
  • the fluorescence intensity of the vascular endothelial cell group is higher than that of the mesenchymal stem cell group.
  • the plasmonic stem cell group indicates that the aptamer used has good targeting ability to vascular endothelial cells.
  • mice After the mice were anesthetized, the middle back hair was shaved, and a round wound with a diameter of 1.0cm was cut with a special puncher, deep into the skin, to form an animal model of mechanical injury.
  • the mice On the second day after modeling, the mice were randomly divided into 3 groups.
  • the PBS group, the hyaluronic acid group and the aptamer-carboxyfluorescein-hyaluronic acid group were administered respectively.
  • the materials were taken on the second day after the administration, and immediately Put it in 4% paraformaldehyde and fix for 4-6 hours, then transfer to 30% sucrose solution to the bottom of the tissue, embed the frozen tissue block, slice 30 ⁇ m in the cryostat, put it at room temperature for more than 30 minutes to prevent peeling off, wash 3 with 1 ⁇ PBS Times, 5 minutes each time, circled by immunohistochemistry, 2% BSA at room temperature for 1 hour, 1 ⁇ PBS washing, CD31 primary antibody 4°C incubation overnight, 1 ⁇ PBS washing, secondary antibody room temperature and dark incubation for 1h, 1 ⁇ PBS washing, Hochest stains the nucleus at room temperature for 20 minutes, washes with 1 ⁇ PBS, drops anti-quenching agent, mounts, and the results of fluorescence microscope observation are shown in Figure 5A-7D.
  • Figure 5A shows the aptamer-carboxyfluorescein-hyaluronan Hochest staining image of skin wound tissue in the acid group 2 days after administration
  • Figure 5B shows the FAM fluorescence image of skin wound tissue in the aptamer-carboxyfluorescein-hyaluronic acid group 2 days after administration
  • Figure 5C shows the aptamer- Fluorescence image of skin wound tissue stained by CD31 after 2 days of administration in the carboxyfluorescein-hyaluronic acid group
  • Figure 5D shows the merge fluorescence image of skin wound tissue after 2 days of administration in the aptamer-carboxyfluorescein-hyaluronic acid group
  • Figure 6A shows the Hochest staining image of skin wound tissue in the hyaluronic acid group 2 days after administration
  • Figure 6B shows the FAM fluorescence image of skin wound tissue in the hyaluronic acid group 2 days after administration
  • Figure 6C shows the hyaluronic acid group administration The fluor
  • CD31 also known as platelet-endothelial cell adhesion molecule
  • CD31 is usually located in vascular endothelial cells, platelets, macrophages, etc., and can be used to assess angiogenesis.
  • Figure 5A-7D the aptamer-carboxyfluorescein- The aptamer with FAM fluorophore in the hyaluronic acid group partially overlapped with CD31 in the skin wound tissue after co-staining, while the hyaluronic acid group and the PBS group had no FAM coloration, indicating the aptamer in this example
  • the son also has good blood vessel targeting ability in the body.
  • mice After the mice were anesthetized, the middle back hair was shaved, and a round wound with a diameter of 1.0cm was cut with a special puncher, deep into the skin, to form an animal model of mechanical injury. After modeling, the mice were randomly divided into 4 groups.
  • the tissue repair material group, the exosomal gel group, the hyaluronic acid group and the PBS group were administered respectively.
  • the materials were taken on the 4th day after the administration and immediately put in 4% Fix in paraformaldehyde for 4-6 hours, then transfer to 30% sucrose solution to the bottom of the tissue, embed the frozen tissue block, slice 30 ⁇ m on the cryostat, put it at room temperature for more than 30 minutes to prevent peeling off, wash 3 times with 1 ⁇ PBS, each time 5min, immunohistochemical stroke circle, 2% BSA room temperature blocking for 1h, 1 ⁇ PBS washing, CD31 fluorescent primary antibody and EMCN primary antibody incubated overnight at 4°C, 1 ⁇ PBS washing, secondary antibody incubating at room temperature for 1h, 1 ⁇ PBS washing , Hochest stained the nucleus at room temperature for 20 minutes, washed with 1 ⁇ PBS, dripped anti-quenching agent, and mounted.
  • Figures 8A-11E show the immunofluorescence imaging and blood vessel images after administration of the tissue repair material group, exosomal gel group, hyaluronic acid group and PBS group.
  • Figure 8A shows the administration of the tissue repair material group The sharkest stained immunofluorescence image of the skin wound tissue after 4 days
  • Figure 8B shows the immunofluorescence image of the skin wound tissue stained by CD31 after 4 days of administration of the tissue repair material group
  • Figure 8C shows the skin wound surface of the tissue repair material group 4 days after the administration
  • Figure 8D shows the merge fluorescence image of the skin wound tissue in the tissue repair material group 4 days after administration
  • Figure 8E shows the blood vessel image of the skin wound tissue in the tissue repair material group 4 days after administration
  • 9A shows the harnessest stained immunofluorescence image of the skin wound tissue in the exosomal gel group 4 days after administration
  • Figure 9B shows the immunofluorescence image of the skin wound surface after administration of the exosom
  • tissue repair material in Example 5 has significantly enhanced fluorescence and a significant increase in the number of blood vessels compared with the exosomal gel group, hyaluronic acid group and PBS group, indicating that the tissue repair material has Obviously promote angiogenesis.
  • mice After the mice were anesthetized, the middle back hair was shaved, and a round wound with a diameter of 1.0cm was cut with a special puncher, deep into the skin, to form an animal model of mechanical injury. After modeling, they were randomly divided into 3 groups. The tissue repair materials, exosomal gel and hyaluronic acid of the examples of the present invention were applied respectively. The wounds were photographed on the 0th, 4th, 8th, and 12th days after administration, and the wound surface was calculated. Healing rate.
  • the tissue repair material of Example 5 was used to treat skin defects in mice. Refer to Figures 12A-15C for the treatment effect. On the 12th day, the wound healing rate of the tissue repair material group was 99.04%, while the control group exosomal gel and hyaluronic acid The healing rates were 88.92% and 87.20%, respectively, showing that tissue repair materials have good repairing ability and can accelerate wound healing.
  • 12A is a diagram of skin damage before applying the tissue repair material of an embodiment of the present invention
  • FIG. 12B is a diagram of skin damage before applying exosomal gel
  • FIG. 12C is a diagram of skin damage before applying hyaluronic acid
  • FIG. 13B is a picture of skin damage after applying the tissue repair material of an embodiment of the present invention for 4 days; Fig. 13B is a diagram of skin damage after applying exosomal gel for 4 days; Fig. 13C is a diagram of skin damage after applying hyaluronic acid for 4 days; 14A is a diagram of skin damage after 8 days of applying the tissue repair material of the embodiment of the present invention; FIG. 14B is a diagram of skin damage after applying exosomal gel for 8 days; FIG. 14C is a diagram of skin damage after applying hyaluronic acid 8; Fig. 15A is a diagram of skin damage after applying the tissue repair material of the embodiment of the present invention for 12 days; Fig. 15B is a diagram of skin damage after applying exosomal gel for 12 days; Fig. 15C is a diagram of skin damage after applying hyaluronic acid for 12 days.
  • Figure 16A shows the hematoxylin-eosin staining image of the tissue repair material of the embodiment of the present invention after repairing the damaged skin
  • Figure 16B shows the hematoxylin-eosin staining image of the exosomal gel after repairing the damaged skin
  • Figure 16C shows the hematoxylin-eosin staining image after hyaluronic acid repairs damaged skin.
  • tissue repair materials Take tissue repair materials, exosomal gel and tissue treated with hyaluronic acid, slice, dehydrate, embed, and dewax; stain with Weigert iron hematoxylin (mixed with liquid A and B in equal proportions) for 10 minutes, wash with water; 1% hydrochloric acid Differentiate in alcohol solution for a few seconds and wash with water; dye with Ponceau acid fuchsin solution for 7-8 minutes and wash quickly; treat slices with phosphomolybdic acid for 1 minute and pour off the excess liquid; re-stain with aniline blue dye solution for 5 minutes and wash quickly; use after dehydration Sealing with sex gum.
  • FIG. 17A-17C Compared with the exosomal gel group and the hyaluronic acid group, the tissue repair material group significantly improves Figure 17A is a Masson staining image of damaged skin treated with tissue repair materials; 17B is a Masson staining image of damaged skin treated with exosomal gel; 17C is a Masson staining image of damaged skin treated with hyaluronic acid .

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Abstract

一种复合体、组织修复材料及其制备方法和应用，所述复合体的药效稳定、靶向性高、传递效率高，在体内有良好的耐受性，并可以通过内源物发挥多种生物学功能，同时避免了体内的免疫反应。所述组织修复材料在皮肤疾病治疗方面具有稳定的治疗效果和靶向性，特别在久治不愈的糖尿病并发症治疗方面具有效果。

Description

复合体、组织修复材料及其制备方法和应用 技术领域
本发明属于生物制品技术领域，尤其涉及一种复合体、组织修复材料及其制备方法和应用。
背景技术
组织、器官的损伤或功能障碍是人类健康所面临的主要危害之一，也是人类疾病和死亡的最主要原因。目前临床常见的组织器官缺损修复技术主要有三种：异体组织移植，但存在组织相容性及供体来源严重不足的问题；自体组织移植，这种移植以牺牲正常组织为代价，且组织异位常导致功能紊乱；应用人工合成材料代用品，易导致异物反应、继发感染及周围组织萎缩等。
间充质干细胞是干细胞家族的重要成员，来源于发育早期的中胚层，具有多向分化潜能、造血支持和促进干细胞植入和免疫调控等特点，可以调控组织再生和伤口愈合。细胞外囊泡是间充质干细胞介导细胞间通讯以增强组织修复的关键分泌产物之一，外泌体就属于细胞外囊泡的一种，它是一种直径在40～100nm的圆形单层膜结构，由机体多种细胞内膜以出芽方式形成，并广泛分布于不同体液当中，携带多种蛋白质、mRNA和microRNA等，可通过生物合成与内吞作用参与到精确而复杂调控的细胞通讯、细胞迁移、细胞增殖与分化、调节免疫等过程。但未做任何改变的外泌体会在肝、肾、脾等器官集聚，并迅速被胆管系统、泌尿系统排泄，或被网状内皮系统吞噬，不能有效地到达目标组织器官。
外泌体的靶向修饰主要是通过先转染后分离的方式，即先通过转染的方式使细胞表达靶向蛋白，然后从细胞中分离出表达靶向蛋白的外泌体，其修饰效率低，操作较繁琐，所以寻找修饰效率高、操作简便的外泌体靶向修饰方法迫在眉睫。适配子是人工合成的单链寡核苷酸，可通过指数富集配基的系统进化技术(SELEX)进行体外筛选、扩增和富集，常形成发卡、茎环等二级结构及三 维立体空间结构，可通过分子构象匹配与靶标结合，具有亲和力高、特异性强、免疫原性低的特点。已有研究将核酸适配体通过化学键连接到人工合成的脂质体上，以制备适配体靶向脂质体载药系统，以增强药物的稳定性、靶向性和传递效率。然而人工合成的脂质体虽然便于修饰功能基团，但成本大幅增加，且合成过程也更加繁琐，而且脂质体来源于外来物质，人体耐受性有限，容易在体内产生免疫反应。
发明内容
针对现有技术存在的问题，本发明提供一种复合体，所述复合体的药效稳定、靶向性高、传递效率高，在体内有良好的耐受性，并可以通过内源物发挥多种生物学功能，同时避免了体内的免疫反应。
本发明的进一步目的是提供一种组织修复材料，所述组织修复材料在皮肤疾病治疗方面具有稳定的治疗效果和靶向性，特别在久治不愈的糖尿病并发症治疗方面具有显著的效果。
本发明的目的是通过以下技术方案来实现的：
本发明第一方面提供一种复合体，所述复合体由靶向基团和药用囊泡结合而成，所述靶向基团选自适配子，所述药用囊泡选自外泌体。
本发明利用适配子对特定细胞的靶向作用，使外泌体迁移并作用于目标细胞，提高了外泌体的生物利用率，达到促进组织修复的目的。经过适配子修饰的外泌体可以加快来自慢性不愈合伤口的成纤维细胞的生长和迁移，并能有效激活生长因子信号通路，提高血管生成相关基因和基质蛋白的表达，诱导新生血管的生成，因此，本发明提供的复合体可作为安全有效的产品促进慢性难愈合伤口或骨缺损伤口的愈合。
优选地，每个复合体包含所述适配子和外泌体的数量比为：n:1，n≧1，n为整数。
优选地，所述适配子选自血管内皮细胞靶向适配子。所述血管内皮细胞靶向适配子对血管内皮细胞具有靶向作用，可使外泌体迁移并作用于血管内皮细 胞，提高了外泌体的生物利用率，达到促进血管内皮细胞增殖，组织血管修复的目的。
优选地，所述适配子选自成骨细胞靶向适配子。所述成骨细胞靶向适配子对成骨细胞具有靶向作用，可使外泌体迁移并作用于成骨细胞，提高了外泌体的生物利用率，达到促进成骨细胞增殖，促进骨密度增加、修复骨缺损的目的。
优选地，所述适配子的序列为：
5′-CCCACGTCTGCGCTTAGCTCCTGGGCCTGGATGGGC-3′。
优选地，所述适配子的3′或5′端进行了氨基修饰。普通的适配子难以和外泌体结合，利用酰胺缩合反应，进行氨基修饰后的适配子能与外泌体进行有效结合，修饰后的适配子为：适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺，化学结构式为：
Figure PCTCN2019127253-appb-000001
。使用二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基对适配子进行修饰，得到具有脂质分子结构的适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺，能与外泌体进行有效结合。
优选地，所述适配子的修饰方法包括以下步骤：
1)将二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺溶于适量ddH 2O中，调节反应体系的pH值为5～6，室温搅拌40～60min，获得第一溶液；
2)将所述第一溶液用焦碳酸二乙酯处理水透析后，得到二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺；
3)取未经修饰的适配子，溶于焦碳酸二乙酯处理水，置于85℃水浴10min，然后迅速冰浴10min，获得活化适配子的焦炭酸二乙酯处理水溶液；
4)将所述二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺、活化适配子的焦炭酸二乙酯处理水溶液和三乙胺混合，室温搅拌8-12h，获得第二溶液；
5)将所述第二溶液利用焦碳酸二乙酯处理水超滤后，得到适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺。
本发明利用酰胺缩合反应对适配子进行基团修饰，使适配子获得脂质分子结构，以便能够与外泌体进行融合，并且操作过程简单，获得的适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺纯度高。
优选地，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:(10～30):(100～300)。
优选地，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:(15～20):(150～200)。
优选地，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:10:100。
优选地，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:30:300。
优选地，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:18:160。
优选地，步骤2)中所述透析过程具体为：
1)反应结束后，将所述第一溶液装入透析袋，将透析袋浸入焦碳酸二乙酯处理水中，缓慢搅拌，直至透析袋内外物质浓度达到平衡；
2)透析袋内外物质浓度达到平衡后，更换新的焦碳酸二乙酯处理水，重复步骤1)3次，每次透析2-4h。
优选地，步骤5)中，所述超滤过程具体为：
1)将所述第二溶液装入超滤管内管，10000rpm离心30-40min；
2)当超滤管内管液体达到最小体积后，加焦碳酸二乙酯处理水稀释样品，重复步骤1)3次。
优选地，所述外泌体为间充质干细胞外泌体。
更优选地，所述间充质干细胞外泌体选自骨髓间充质干细胞外泌体、脐带血间充质干细胞外泌体、脐带组织间充质干细胞外泌体、胎盘组织间充质干细胞外泌体或脂肪组织间充质干细胞外泌体。
本发明第二方面，提供一种组合物，所述组合物的活性成分为上述的复合体。本发明的组合物可以加快来自慢性不愈合伤口的成纤维细胞的生长和迁移，并能有效激活生长因子信号通路，提高血管生成相关基因和基质蛋白的表达，诱导新生血管的生成或成骨细胞的生成，因此，本发明提供的组合物可作为安全有效的产品促进慢性难愈合伤口或骨缺损伤口的愈合。
本发明第三方面，提供上述复合体或上述组合物在促进血管新生方面的用途。所述血管包括动脉血管、静脉血管和毛细血管。所述毛细血管包括存在于皮肤真皮层、骨组织、心肌细胞中的毛细血管。
本发明第四方面，提供上述的复合体或上述的组合物在制备皮肤疾病治疗药物中的用途。
优选地，所述皮肤疾病包括皮肤损伤、皮肤感染、褥疮或糖尿病溃疡。
本发明第五方面，提供一种组织修复材料，所述组织修复材料的组分包括复合体和凝胶材料，所述复合体由适配子和外泌体结合而成。
现有技术中，利用水凝胶包埋活性药物、蛋白质和细胞等，可以通过扩散作用和凝胶的降解作用使活性物质达到长效缓释的效果，但在治疗中由于缺少成血管和成骨等组织修复的关键环节，治疗效果大打折扣。本发明利用酰胺缩合反应、相似相溶原理和混悬的方法构建的组织修复材料，通过实验证实，本发明提供的组织修复材料不但可以通过扩散作用和凝胶的降解作用使活性物质达到长效缓释的效果，而且本发明的组织修复材料具有明显的促进血管生成的能力和组织修复能力，可以加速难愈合伤口愈合，促进胶原在组织损伤部位的沉积，促肉芽组织成熟和表皮再生。
优选地，每个复合体包含所述适配子和外泌体的数量比为：n:1，n≧1，n为整数。
优选地，所述适配子选自血管内皮细胞靶向适配子。
优选地，所述适配子的序列为：
5′-CCCACGTCTGCGCTTAGCTCCTGGGCCTGGATGGGC-3′。
优选地，所述适配子的3′或5′端进行了-NH 2修饰，修饰后的适配子为：适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺，化学结构式为：
Figure PCTCN2019127253-appb-000002
优选地，所述适配子的修饰方法包括以下步骤：
1)将二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺溶于适量ddH 2O中，调节反应体系的pH值为5～6，室温搅拌40～60min，获得第一溶液；
2)将所述第一溶液用焦碳酸二乙酯处理水透析后，得到二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺；
3)取未经修饰的适配子，溶于焦碳酸二乙酯处理水，置于85℃水浴10min，然后迅速冰浴10min，获得活化适配子的焦炭酸二乙酯处理水溶液；
4)将所述二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺、活化适配子的焦炭酸二乙酯处理水溶液和三乙胺混合，室温搅拌8-12h，获得第二溶液；
5)将所述第二溶液利用焦碳酸二乙酯处理水超滤后，得到适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺。
优选地，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:(10～30):(100～300)。
优选地，步骤2)中所述透析过程具体为：
1)反应结束后，将所述第一溶液装入透析袋，将透析袋浸入焦碳酸二乙酯处理水中，缓慢搅拌，直至透析袋内外物质浓度达到平衡；
2)透析袋内外物质浓度达到平衡后，更换新的焦碳酸二乙酯处理水，重复步骤1)3次，每次透析2-4h。
优选地，步骤5)中，所述超滤过程具体为：
1)将所述第二溶液装入超滤管内管，10000rpm离心30-40min；
2)当超滤管内管液体达到最小体积后，加焦碳酸二乙酯处理水稀释样品，重复步骤1)3次。
优选地，所述外泌体为间充质干细胞外泌体。
更优选地，所述间充质干细胞外泌体选自骨髓间充质干细胞外泌体、脐带血间充质干细胞外泌体、脐带组织间充质干细胞外泌体、胎盘组织间充质干细胞外泌体或脂肪组织间充质干细胞外泌体。
优选地，所述凝胶材料选自胶原蛋白、海藻酸盐和透明质酸钠中的至少一种。
本发明第六方面，提供一种组织修复材料的制备方法，包括以下步骤：
1)常规培养间充质干细胞，利用超速离心法提取间充质干细胞来源的外泌体；
2)用焦碳酸二乙酯处理PBS缓冲液作为溶剂，将适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺与所述外泌体37℃搅拌4～6h；
3)利用焦碳酸二乙酯处理PBS缓冲液超滤后，得到适配子修饰的间充质干细胞外泌体悬液；
4)向上述适配子修饰的间充质干细胞外泌体悬液中加入适量凝胶材料，充分混匀，即得到所述组织修复材料。
使用本发明组织修复材料的制备方法获得的组织修复材料构造了一种具有强靶向能力和低免疫原性的治疗体系，并且该制备方法操作简单，解决了现有技术中组织修复材料合成繁琐和制备效率低下的问题。
优选地，所述凝胶材料选自胶原蛋白、海藻酸盐和透明质酸钠中的至少一种。
优选地，所述适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺由以下方法制备而成：
1)将二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺溶于适量ddH 2O中，调节反应体系的pH值为5～6，室温搅拌40～60min，获得第一溶液；
2)将所述第一溶液用焦碳酸二乙酯处理水透析后，得到二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺；
3)取未经修饰的适配子，溶于焦碳酸二乙酯处理水，置于85℃水浴10min，然后迅速冰浴10min，获得活化适配子的焦炭酸二乙酯处理水溶液；
4)将所述二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺、活化适配子的焦炭酸二乙酯处理水溶液和三乙胺混合，室温搅拌8-12h，获得第二溶液；
5)将所述第二溶液利用焦碳酸二乙酯处理水超滤后，得到适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺。
优选地，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:(10～30):(100～300)。
优选地，步骤2)中所述透析过程具体为：
1)反应结束后，将所述第一溶液装入透析袋，将透析袋浸入焦碳酸二乙酯处理水中，缓慢搅拌，直至透析袋内外物质浓度达到平衡；
2)透析袋内外物质浓度达到平衡后，更换新的焦碳酸二乙酯处理水，重复步骤1)3次，每次透析2-4h。
优选地，步骤5)中，所述超滤过程具体为：
1)将所述第二溶液装入超滤管内管，10000rpm离心30-40min；
2)当超滤管内管液体达到最小体积后，加焦碳酸二乙酯处理水稀释样品， 重复步骤1)3次。
本发明第七方面，提供上述组织修复材料在制备皮肤疾病治疗产品中的用途。
优选地，所述皮肤疾病包括皮肤损伤、皮肤感染、褥疮或糖尿病溃疡。
本发明第八方面，提供一种皮肤损伤修复凝胶，所述皮肤损伤修复凝胶的组分包含上述的组织修复材料。
本发明利用酰胺缩合反应、相似相溶原理和混悬的方法构建的组织修复材料，通过实验证实，本发明提供的组织修复材料不但可以通过扩散作用和凝胶的降解作用使活性物质达到长效缓释的效果，而且本发明的组织修复材料具有明显的促进血管生成的能力和组织修复能力，可以加速难愈合伤口愈合，促进胶原在组织损伤部位的沉积，促肉芽组织成熟和表皮再生。
本发明第九方面，提供一种糖尿病溃疡修复敷料，所述糖尿病溃疡修复敷料包含上述的组织修复材料。本发明提供的糖尿病溃疡修复敷料，具有明显的促进血管生成的能力和组织修复能力，可以加速糖尿病导致的慢性难愈合溃疡的伤口愈合，促进胶原在溃疡部位的沉积，促进肉芽组织成熟和溃疡部位表皮再生。
本发明第十方面，提供一种足贴，所述足贴包含上述述的组织修复材料和胶贴，所述组织修复材料黏贴在所述胶贴上。本发明提供的足贴的形状针对足部设计，能有效固定于足部溃疡部位，不影响日常行走，并能有效促进足部伤口特别是足部慢性难愈合糖尿病足溃疡的愈合。优选地，本发明所述胶贴选自无纺布、纺粘布、聚乙烯薄膜或医用PU膜。
术语解释：
本发明所述的ddH 2O指将经过一次蒸馏后，再次蒸馏所得到的水，也称双蒸水或重蒸水。
本发明所述的焦碳酸二乙酯处理水是指经焦碳酸二乙酯处理过并经高温高压灭菌的高纯水，不含杂质RNA、DNA和蛋白质。本发明所述的焦碳酸二乙酯处理水采购自生工生物工程(上海)股份有限公司，编号：B501005-0500。
本发明所述的焦碳酸二乙酯处理PBS缓存液是指经过焦碳酸二乙酯处理的PBS缓存液，不含杂质RNA、DNA和蛋白质。本发明所述的焦碳酸二乙酯处理PBS缓存液采购自生工生物工程(上海)股份有限公司，编号：B501005-0500。
本发明所述的PBS缓冲液是指磷酸缓冲盐溶液。
TAE缓冲液是指由三羟甲基氨基甲烷(Tris base)、乙酸(acetic acid)和乙二胺四乙酸(EDTA)组成的缓冲液。
附图说明
图1A为本发明实施例中所述外泌体的透射电镜结果图；
图1B为本发明实施例中所述复合体的透射电镜结果图；
图2A为本发明实施例中所述外泌体的粒径分布；
图2B为本发明实施例中所述复合体的粒径分布；
图3为本发明实施例所述的复合体的琼脂糖凝胶电泳鉴定结果图；
图4A为本发明实施例中所用适配子对血管内皮细胞组的靶向荧光图；
图4B为本发明实施例中所用适配子对间充质干细胞组的靶向荧光图；
图5A为本发明实施例中适配子-羧基荧光素-透明质酸组给药2天后皮肤创面组织的Hochest染色图；
图5B为本发明实施例中适配子-羧基荧光素-透明质酸组给药2天后皮肤创面组织的FAM荧光图；
图5C为本发明实施例中适配子-羧基荧光素-透明质酸组给药2天后皮肤创面的经CD31染色后的荧光图；
图5D为本发明实施例中适配子-羧基荧光素-透明质酸组给药2天后皮肤创面的的merge荧光图；
图6A为本发明实施例中透明质酸组给药2天后皮肤创面组织的Hochest染色图；
图6B为本发明实施例中透明质酸组给药2天后皮肤创面组织的FAM荧光图；
图6C为本发明实施例中透明质酸组给药2天后皮肤创面组织经CD31染色后的荧光图；
图6D为本发明实施例中透明质酸组给药2天后的merge荧光图；
图7A为本发明实施例中PBS组滴注2天后皮肤创面组织的Hochest染色图；
图7B为本发明实施例中PBS组滴注2天后皮肤创面组织的FAM荧光图；
图7C为本发明实施例中PBS组滴注2天后皮肤创面组织经CD31染色后的荧光图；
图7D为本发明实施例中PBS组滴注2天后皮肤创面组织的merge荧光图；
图8A为本发明实施例中组织修复材料组给药4天后皮肤创面组织的hochest染色荧光图；
图8B为本发明实施例中组织修复材料组给药4天后皮肤创面组织的经CD31染色后的免疫荧光图；
图8C为本发明实施例中组织修复材料组给药4天后皮肤创面组织的经EMCN染色后的免疫荧光图；
图8D为本发明实施例中组织修复材料组给药4天后皮肤创面组织的merge荧光图；
图8E为本发明实施例中组织修复材料组给药4天后皮肤创面组织的血管图；
图9A为本发明实施例中外泌体凝胶组给药4天后皮肤创面组织的Hochest染色荧光图；
图9B为本发明实施例中外泌体凝胶组给药4天后皮肤创面组织经CD31染色后的免疫荧光图；
图9C为本发明实施例中外泌体凝胶组给药4天后皮肤创面组织经EMCN染色后的免疫荧光图；
图9D为本发明实施例中外泌体凝胶组给药4天后皮肤创面组织的merge荧光图；
图9E为本发明实施例中外泌体凝胶组给药4天后皮肤创面组织的血管图；
图10A为本发明实施例中透明质酸组给药4天后皮肤创面组织的Hochest染色荧光图；
图10B为本发明实施例中透明质酸组给药4天后皮肤创面组织经CD31染色后的免疫荧光图；
图10C为本发明实施例中透明质酸组给药4天后皮肤创面组织经EMCN染色后的免疫荧光图；
图10D为本发明实施例中透明质酸组给药4天后皮肤创面组织的merge荧光图；
图10E为本发明实施例中透明质酸组给药4天后皮肤创面组织的血管图；
图11A为本发明实施例中PBS组滴注4天后皮肤创面组织的Hochest染色荧光图；
图11B为本发明实施例中PBS组滴注4天后皮肤创面组织经CD31染色后的免疫荧光图；
图11C为本发明实施例中PBS组滴注4天后皮肤创面组织经EMCN染色后的免疫荧光图；
图11D为本发明实施例中PBS组滴注4天后皮肤创面组织的merge荧光图；
图11E为本发明实施例中PBS组滴注4天后皮肤创面组织的血管图；
图12A为涂抹本发明实施例的组织修复材料前的皮肤损伤图；
图12B为涂抹外泌体凝胶前的皮肤损伤图；
图12C为涂抹透明质酸前的皮肤损伤图；
图13A为涂抹本发明实施例的组织修复材料4天后的皮肤损伤图；
图13B为涂抹外泌体凝胶4天后后的皮肤损伤图；
图13C为涂抹透明质酸4天后后的皮肤损伤图；
图14A为涂抹本发明实施例的组织修复材料8天后的皮肤损伤图；
图14B为涂抹外泌体凝胶8天后后的皮肤损伤图；
图14C为涂抹透明质酸8后后的皮肤损伤图；
图15A为涂抹本发明实施例的组织修复材料12天后的皮肤损伤图；
图15B为涂抹外泌体凝胶12天后的皮肤损伤图；
图15C为涂抹透明质酸12天后的皮肤损伤图；
图16A显示了本发明实施例的组织修复材料修复损伤皮肤后的苏木精—伊红染色图；
图16B显示了外泌体凝胶修复损伤皮肤后的苏木精-伊红染色图；
图16C显示了透明质酸修复损伤皮肤后的苏木精-伊红染色图；
图17A为本发明实施例所述组织修复材料治疗损伤皮肤后的Masson染色图；
图17B为外泌体凝胶治疗损伤皮肤后的Masson染色图；
图17C为透明质酸治疗损伤皮肤后的Masson染色图。
具体实施例
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
一、试剂耗材
二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基：采购自Avanti Polar Lipids公司，编号880135P；
N-羟基琥珀酰亚胺：采购自阿拉丁试剂(上海)有限公司，编号H109330；
1-(3-二甲氨基丙基)-3-乙基碳二亚胺：采购自上海麦克林生化科技有限公司，编号N807578；
血管靶向适配子：采购自生工生物工程(上海)股份有限公司，CCCACGTCTGCGCTTAGCTCCTGGGCCTGGATGGGC；
三乙胺：采购自阿拉丁试剂(上海)有限公司，编号T103289；
焦碳酸二乙酯处理水：采购自生工生物工程(上海)股份有限公司，编号B501005-0500；
焦碳酸二乙酯处理PBS：采购自生工生物工程(上海)股份有限公司，编号B540627-0500；
透明质酸：采购自上海麦克林生化科技有限公司，编号H823435；
CD31荧光一抗：采购自R&D公司，编号FAB3628G；
CD31一抗：采购自Abcam公司，编号AB28364；
EMCN一抗：采购自Santa Cruz Biotechnology公司，编号SC-6549；
二抗：采购自上海翊圣生物科技有限公司，编号33308ES60；
透析袋：采购自上海源叶生物科技有限公司，编号SP131084；
5kD超滤离心管：采购自Sartorius公司，编号VS0112；
30kD超滤离心管：采购自Millipore公司，编号UFC203024。
实施例1：
本发明实施例提供一种复合体，所述复合体由靶向基团和药用囊泡结合而成，所述靶向基团选自适配子，所述药用囊泡选自外泌体。
本发明实施例利用适配子对特定细胞的靶向作用，使外泌体迁移并作用于目标细胞，提高了外泌体的生物利用率，达到促进组织修复的目的。经过适配子修饰的外泌体可以加快来自慢性不愈合伤口的成纤维细胞的生长和迁移，并能有效激活生长因子信号通路，提高血管生成相关基因和基质蛋白的表达，诱导新生血管的生成，因此，本发明提供的复合体可作为安全有效的产品促进慢性难愈合伤口或骨缺损伤口的愈合。
在一些实施例中，每个复合体包含所述适配子和外泌体的数量比为：n:1，n≧1，n为整数。
在一些实施例中，所述适配子选自血管内皮细胞靶向适配子。所述血管内皮细胞靶向适配子对血管内皮细胞具有靶向作用，可使外泌体迁移并作用于血管内皮细胞，提高了外泌体的生物利用率，达到促进血管内皮细胞增殖，组织血管修复的目的。
在一些实施例中，所述适配子选自成骨细胞靶向适配子。所述成骨细胞靶向适配子对成骨细胞具有靶向作用，可使外泌体迁移并作用于成骨细胞，提高了外泌体的生物利用率，达到促进成骨细胞增殖，促进骨密度增加、修复骨缺损的目的。
在一些实施例中，所述适配子的序列为：
5′-CCCACGTCTGCGCTTAGCTCCTGGGCCTGGATGGGC-3′。
在一些实施例中，所述适配子的3′或5′端进行了氨基修饰。普通的适配子难以和外泌体结合，利用酰胺缩合反应，进行氨基修饰后的适配子能与外泌体进行有效结合。
在一些实施例中，修饰后的适配子为：适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺，化学结构式为：
Figure PCTCN2019127253-appb-000003
。使用二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基对适配子进行修饰，得到具有脂质分子结构的适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺，能与外泌体进行有效结合。
在一些实施例中，所述适配子的修饰方法包括以下步骤：
1)将二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺溶于适量ddH 2O中，调节反应体系的pH值为5～6，室温搅拌40～60min，获得第一溶液；
2)将所述第一溶液用焦碳酸二乙酯处理水透析后，得到二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺；
3)取未经修饰的适配子，溶于焦碳酸二乙酯处理水，置于85℃水浴10min，然后迅速冰浴10min，获得活化适配子的焦炭酸二乙酯处理水溶液；
4)将所述二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺、活化适配子的焦炭酸二乙酯处理水溶液和三乙胺混合，室温搅拌8-12h，获得第二溶液；
5)将所述第二溶液利用焦碳酸二乙酯处理水超滤后，得到适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺。
本发明利用酰胺缩合反应对适配子进行基团修饰，使适配子获得脂质分子结构，以便能够与外泌体进行融合，并且操作过程简单，获得的适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺纯度高。
本实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:(10～30):(100～300)。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:10:100。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:10:150。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:10:200。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:10:300。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:20:100。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:20:150。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:20:200。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:20:300。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:30:100。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:30:150。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:30:200。
在一些实施例中，步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:30:300。
步骤2)中所述透析过程具体为：
1)反应结束后，将所述第一溶液装入透析袋，将透析袋浸入焦碳酸二乙酯处理水中，缓慢搅拌，直至透析袋内外物质浓度达到平衡；
2)透析袋内外物质浓度达到平衡后，更换新的焦碳酸二乙酯处理水，重复步骤1)3次，每次透析2-4h。
步骤5)中所述超滤过程具体为：
1)将所述第二溶液装入超滤管内管，10000rpm离心30-40min；
2)当超滤管内管液体达到最小体积后，加焦碳酸二乙酯处理水稀释样品，重复步骤1)3次。
在一些实施例中，所述外泌体为间充质干细胞外泌体，例如：骨髓间充质干细胞外泌体、脐带血间充质干细胞外泌体、脐带组织间充质干细胞外泌体、胎盘组织间充质干细胞外泌体或脂肪组织间充质干细胞外泌体。
实施例2
本实施例提供一种组合物，所述组合物的活性成分为实施例1中所述的复合体。本发明的组合物可以加快来自慢性不愈合伤口的成纤维细胞的生长和迁移，并能有效激活生长因子信号通路，提高血管生成相关基因和基质蛋白的表达，诱导新生血管的生成或成骨细胞的生成，因此，本发明提供的组合物可作为安全有效的产品促进慢性难愈合伤口或骨缺损伤口的愈合。
实施例3
本实施例提供实施例1中的复合体或实施例2中的组合物在促进血管新生方面的用途。所述血管包括动脉血管、静脉血管和毛细血管。所述毛细血管包括存在于皮肤真皮层、骨组织、心肌细胞中的毛细血管。
实施例4
本实施例提供实施例1中的复合体或实施例2中的组合物在制备皮肤疾病治疗药物中的用途。
在一些实施例中，所述皮肤疾病包括皮肤损伤、皮肤感染、褥疮或糖尿病溃疡。
实施例5
本实施例提供一种组织修复材料，所述组织修复材料的组分包括复合体和凝胶材料，所述复合体由适配子和外泌体结合而成。
现有技术中，利用水凝胶包埋活性药物、蛋白质和细胞等，可以通过扩散作用和凝胶的降解作用使活性物质达到长效缓释的效果，但在治疗中由于缺少成血管和成骨等组织修复的关键环节，治疗效果大打折扣。本发明利用酰胺缩合反应、相似相溶原理和混悬的方法构建的组织修复材料，通过实验证实，本发明提供的组织修复材料不但可以通过扩散作用和凝胶的降解作用使活性物质达到长效缓释的效果，而且本发明的组织修复材料具有明显的促进血管生成的能力和组织修复能力，可以加速难愈合伤口愈合，促进胶原在组织损伤部位的沉积，促肉芽组织成熟和表皮再生。
在一些实施例中，每个复合体包含所述适配子和外泌体的数量比为：n:1，n≧1，n为整数。
在一些实施例中，所述适配子选自血管内皮细胞靶向适配子。
在一些实施例中，所述适配子的序列为：
5′-CCCACGTCTGCGCTTAGCTCCTGGGCCTGGATGGGC-3′。
在一些实施例中，所述适配子的3′或5′端进行了-NH 2修饰，修饰后的适配子为：适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺，化学结构式为：
Figure PCTCN2019127253-appb-000004
在一些实施例中，所述适配子的修饰方法包括以下步骤：
1)将二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺溶于适量ddH 2O中，调节反应体系的pH值为5～6，室温搅拌40～60min，获得第一溶液；
2)将所述第一溶液用焦碳酸二乙酯处理水透析后，得到二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺；
3)取未经修饰的适配子，溶于焦碳酸二乙酯处理水，置于85℃水浴10min，然后迅速冰浴10min，获得活化适配子的焦炭酸二乙酯处理水溶液；
4)将所述二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺、活化适配子的焦炭酸二乙酯处理水溶液和三乙胺混合，室温搅拌8-12h，获得第二溶液；
5)将所述第二溶液利用焦碳酸二乙酯处理水超滤后，得到适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺。
步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:(10～30):(100～300)。
步骤2)中所述透析过程具体为：
1)反应结束后，将所述第一溶液装入透析袋，将透析袋浸入焦碳酸二乙酯处理水中，缓慢搅拌，直至透析袋内外物质浓度达到平衡；
2)透析袋内外物质浓度达到平衡后，更换新的焦碳酸二乙酯处理水，重复步骤1)3次，每次透析2-4h。
步骤5)中，所述超滤过程具体为：
1)将所述第二溶液装入超滤管内管，10000rpm离心30-40min；
2)当超滤管内管液体达到最小体积后，加焦碳酸二乙酯处理水稀释样品，重复步骤1)3次。
在一些实施例中，所述外泌体为间充质干细胞外泌体，例如：骨髓间充质干细胞外泌体、脐带血间充质干细胞外泌体、脐带组织间充质干细胞外泌体、胎盘组织间充质干细胞外泌体或脂肪组织间充质干细胞外泌体。
在一些实施例中，所述凝胶材料选自胶原蛋白、海藻酸盐和透明质酸钠中的至少一种。
实施例6
本实施例提供一种组织修复材料的制备方法，包括以下步骤：
1)常规培养间充质干细胞，利用超速离心法提取间充质干细胞来源的外泌体；
2)用焦碳酸二乙酯处理PBS缓冲液作为溶剂，将适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺与所述外泌体37℃搅拌4～6h；
3)利用焦碳酸二乙酯处理PBS缓冲液超滤后，得到适配子修饰的间充质干细胞外泌体悬液；
4)向上述适配子修饰的间充质干细胞外泌体悬液中加入适量凝胶材料，充分混匀，即得到所述组织修复材料。
使用本发明组织修复材料的制备方法获得的组织修复材料构造了一种具有强靶向能力和低免疫原性的治疗体系，并且该制备方法操作简单，解决了现有技术中组织修复材料合成繁琐和制备效率低下的问题。
在一些实施例中，所述适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺由以下方法制备而成：
1)将二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺溶于适量ddH 2O中，调节反应体系的pH值为5～6，室温搅拌40～60min，获得第一溶液；
2)将所述第一溶液用焦碳酸二乙酯处理水透析后，得到二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺；
3)取未经修饰的适配子，溶于焦碳酸二乙酯处理水，置于85℃水浴10min，然后迅速冰浴10min，获得活化适配子的焦炭酸二乙酯处理水溶液；
4)将所述二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺、活化适配子的焦炭酸二乙酯处理水溶液和三乙胺混合，室温搅拌8-12h，获得第二溶液；
5)将所述第二溶液利用焦碳酸二乙酯处理水超滤后，得到适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺。
步骤1)中所述的二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺的物质的量之比为1:(10～30):(100～300)。
步骤2)中所述透析过程具体为：
1)反应结束后，将所述第一溶液装入透析袋，将透析袋浸入焦碳酸二乙酯处理水中，缓慢搅拌，直至透析袋内外物质浓度达到平衡；
2)透析袋内外物质浓度达到平衡后，更换新的焦碳酸二乙酯处理水，重复步骤1)3次，每次透析2-4h。
步骤5)中，所述超滤过程具体为：
1)将所述第二溶液装入超滤管内管，10000rpm离心30-40min；
2)当超滤管内管液体达到最小体积后，加焦碳酸二乙酯处理水稀释样品，重复步骤1)3次。
实施例7
本实施例提供上述组织修复材料在制备皮肤疾病治疗产品中的用途。
在一些实施例中，所述皮肤疾病包括皮肤损伤、皮肤感染、褥疮或糖尿病溃疡。
实施例8
本实施例提供一种皮肤损伤修复凝胶，所述皮肤损伤修复凝胶的组分包含实施例5中的组织修复材料。
本发明利用酰胺缩合反应、相似相溶原理和混悬的方法构建的组织修复材料，通过实验证实，本发明提供的组织修复材料不但可以通过扩散作用和凝胶的降解作用使活性物质达到长效缓释的效果，而且本发明的组织修复材料具有明显的促进血管生成的能力和组织修复能力，可以加速难愈合伤口愈合，促进胶原在组织损伤部位的沉积，促肉芽组织成熟和表皮再生。
实施例9
本实施例提供一种糖尿病溃疡修复敷料，所述糖尿病溃疡修复敷料包含实施例5中的组织修复材料。本发明提供的糖尿病溃疡修复敷料，具有明显的促进血管生成的能力和组织修复能力，可以加速糖尿病导致的慢性难愈合溃疡的伤口愈合，促进胶原在溃疡部位的沉积，促进肉芽组织成熟和溃疡部位表皮再生。
实施例10
本实施例提供一种足贴，所述足贴包含所述的组织修复材料和胶贴，所述组织修复材料黏贴在所述胶贴上。本发明提供的足贴的形状为贴合足部结构的形状，可以是方形、圆形，并可任意裁剪成需要的形状，本发明提供的足贴可固定于足部溃疡部位，不影响日常行走，并能有效促进足部伤口特别是足部慢性难愈合糖尿病足溃疡的愈合。
在一些实施例中，所述胶贴的材料选自无纺布、纺粘布、聚乙烯薄膜或医用PU膜。
效果验证：
一、实施例1所述的复合体的透射电子显微镜(TEM)及粒径分析
将30μg的外泌体和复合体分别用PBS稀释至适当浓度，滴在200/300目F/C的TEM铜网上，自然吸附样品至3分钟后将过量样品吸出，随后将吸附的样品用10μL 3％的磷钨酸染色30秒，再将多余的染剂用滤纸吸出，自然风干后检测。同时，取30μg的外泌体和复合体，分别用PBS稀释至适当浓度，使用纳米激光粒度分析仪测量粒径分布。
参考图1A-图2B，图1A为外泌体的电镜图，图1A可以看到直径约为100nm的具有清晰膜结构的杯状膜泡；图1B为复合体的电镜图，从图1B可以看到，适配子成功连接到外泌体膜表面，呈微球状；图2A显示了外泌体的粒径分布；图2B显示了复合体的粒径分布；可以看出，外泌体的粒径尺寸大多分布在90nm左右，复合体的粒径尺寸大多分布在300nm左右。
二、复合体的琼脂糖凝胶电泳鉴定
将有机玻璃的电泳凝胶床洗净，晾干，放在水平的工作台上，插上样品梳；称取0.6g琼脂糖置于锥形瓶中，加入30ml TAE缓冲溶液，微波炉加热至全部融化，加入3μl核酸染料后摇匀，待琼脂糖溶液冷却到50～60℃，缓慢倒入安装好的电泳槽水平板上；待琼脂糖溶液凝固后，在电泳槽内加入电泳缓冲液，然后拔出梳子，将适配子、适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺及复合体与电泳缓冲液按10：1混匀后，用微量移液器将混合液加到样品槽中，每槽加10μl；安装好电极导线，点样孔一端接负极，另一端接正极，打开电源，调电压至30-40V/cm，当溴酚蓝移到距凝胶前沿1-2cm时，停止电泳，采用凝胶成像系统拍照保存。
电泳结果如图3所示，条带1为适配子琼脂糖凝胶电泳鉴定结果，条带2为适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺的琼脂糖凝胶电泳鉴定结果，条带3为复合体的琼脂糖凝胶电泳鉴定结果，由于适配子、适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺及复合体的分子量依次增大，条带的电泳速度也依次减慢，证明中间产物适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺及最终产物复合体成功合成。
三、适配子特异性靶向血管内皮细胞能力检测
细胞爬片放于24孔板中，将血管内皮细胞和间充质干细胞分别以适宜浓度接种于24孔板，待细胞长至对数生长期，将5OD适配子-羧基荧光素(Apt-FAM) 溶于100μl焦碳酸二乙酯处理水配制贮存液，置于85℃水浴10min，然后迅速冰浴10min，用DMEM培养基将Apt-FAM贮存液稀释至10μM，处理细胞2h后，PBS清洗数次，用5μM的Hochest染核5min，固定，封片，用荧光显微镜进行观察成像。
图4A显示了血管内皮细胞组的靶向荧光图；图4B显示了间充质干细胞组的靶向荧光图；从图4A和图4B可以看出，血管内皮细胞组的荧光强度高于间充质干细胞组，说明所用的适配子对血管内皮细胞具有较好的靶向能力。
四、适配子在体内特异性靶向血管能力检测
将小鼠麻醉后剃去背中部毛，用特制打孔器切割一个直径为1.0cm的圆形创面，深至皮下，形成机械损伤动物模型。造模后第2天将小鼠随机分为3组，分别设置PBS组、透明质酸组和适配子-羧基荧光素-透明质酸组给药，于给药后第2天取材，立即放入4％多聚甲醛中固定4～6h，后转至30％蔗糖溶液至组织沉底，包埋冰冻组织块，冰冻切片机切片30μm，室温放置30min以上防脱片，1×PBS洗3次，每次5min，免疫组化笔画圈，2％BSA室温封闭1h，1×PBS清洗，CD31一抗4℃孵育过夜，1×PBS清洗，二抗室温避光孵育1h，1×PBS清洗，Hochest室温染核20min，1×PBS清洗，滴加防淬灭剂，封片，荧光显微镜观察结果如图5A-图7D所示，其中，图5A显示了适配子-羧基荧光素-透明质酸组给药2天后皮肤创面组织的Hochest染色图；图5B显示了适配子-羧基荧光素-透明质酸组给药2天后皮肤创面组织的FAM荧光图；图5C显示了适配子-羧基荧光素-透明质酸组给药2天后皮肤创面组织经CD31染色后的荧光图；图5D显示了适配子-羧基荧光素-透明质酸组给药2天后皮肤创面组织的merge荧光图；图6A显示了透明质酸组给药2天后皮肤创面组织的Hochest染色图；图6B显示了透明质酸组给药2天后的皮肤创面组织FAM荧光图；图6C显示了透明质酸组给药2天后皮肤创面组织经CD31染色后的荧光图；图6D显示了透明质酸组给药2天后皮肤创面组织的merge荧光图；图7A显示了PBS组滴注2天后皮肤创面组织的Hochest染色图；图7B显示了PBS组滴注2天后皮肤创面组织的FAM荧光图；图7C显示了PBS组滴注2天后皮肤创面组织经CD31染色后的荧光图；图7D显示了PBS组滴注2天后皮肤创面组织的merge荧光图。
CD31又称为血小板-内皮细胞粘附分子，通常位于血管内皮细胞、血小板、 巨噬细胞等，可以用于评估血管生成，结合图5A-图7D可以看出，适配子-羧基荧光素-透明质酸组中带有FAM荧光基团的适配子与皮肤创面组织中的CD31共染后有部分重叠，而透明质酸组和PBS组无FAM显色，表明该实施例中的适配子在体内也具有良好的血管靶向能力。
五、本实施例5所述的组织修复材料促进血管生成能力检测
将小鼠麻醉后剃去背中部毛，用特制打孔器切割一个直径为1.0cm的圆形创面，深至皮下，形成机械损伤动物模型。造模后将小鼠随机分为4组，分别设置组织修复材料组、外泌体凝胶组、透明质酸组和PBS组给药，于给药后第4天取材，立即放入4％多聚甲醛中固定4～6h，后转至30％蔗糖溶液至组织沉底，包埋冰冻组织块，冰冻切片机切片30μm，室温放置30min以上防脱片，1×PBS洗3次，每次5min，免疫组化笔画圈，2％BSA室温封闭1h，1×PBS清洗，CD31荧光一抗和EMCN一抗4℃孵育过夜，1×PBS清洗，二抗室温避光孵育1h，1×PBS清洗，Hochest室温染核20min，1×PBS清洗，滴加防淬灭剂，封片。
图8A-图11E显示了组织修复材料组、外泌体凝胶组、透明质酸组和PBS组给药后的免疫荧光成像图和血管图，其中，图8A显示了组织修复材料组给药4天后皮肤创面组织的hochest染色免疫荧光图；图8B显示了组织修复材料组给药4天后皮肤创面组织经CD31染色后的免疫荧光图；图8C显示了组织修复材料组给药4天后皮肤创面组织经EMCN染色后的免疫荧光图；图8D显示了组织修复材料组给药4天后皮肤创面组织的merge荧光图；图8E显示了组织修复材料组给药4天后皮肤创面组织的血管图；图9A显示了外泌体凝胶组给药4天后皮肤创面组织的hochest染色免疫荧光图；图9B显示了外泌体凝胶组给药4天后皮肤创面组织经CD31染色后的免疫荧光图；图9C显示了外泌体凝胶组给药4天后皮肤创面组织经EMCN染色后的免疫荧光图；图9D显示了外泌体凝胶组给药4天后皮肤创面组织的merge荧光图；图9E显示了外泌体凝胶组给药4天后皮肤创面组织的血管图；图10A显示了透明质酸组给药4天后皮肤创面组织的hochest染色免疫荧光图；图10B显示了透明质酸组给药4天后皮肤创面组织经CD31染色后的免疫荧光图；图10C显示了透明质酸组给药4天后皮肤创面组织经EMCN染色后的免疫荧光图；图10D显示了透明质酸组给药4天后皮肤创面组织的merge荧光图；图10E显示了透明质酸组给药4天后皮肤创面组织的血管图；图11A显示了PBS组滴注4天后皮肤创面组织的hochest染色免疫 荧光图；图11B显示了PBS组滴注4天后皮肤创面组织CD31染色后的免疫荧光图；图11C显示了PBS组滴注4天后皮肤创面组织经EMCN染色后的免疫荧光图；图11D显示了PBS组滴注4天后皮肤创面组织的merge荧光图；图11E显示了PBS组滴注4天后皮肤创面组织的血管图。
从图8A-图11E可以看出，实施例5中的组织修复材料与外泌体凝胶组、透明质酸组和PBS组相比荧光显著增强，血管数目也明显增多，说明组织修复材料具有明显的促进血管生成的能力。
六、本实施例5所述的组织修复材料治疗皮肤损伤效果检测
将小鼠麻醉后剃去背中部毛，用特制打孔器切割一个直径为1.0cm的圆形创面，深至皮下，形成机械损伤动物模型。造模后随机分为3组，分别涂抹本发明实施例的组织修复材料、外泌体凝胶以及透明质酸，于给药后第0、4、8、12天对创伤进行拍照，计算创面愈合率。
愈合率＝(原创伤面积－现创伤面积)/原创伤面积×100％
利用实施例5的组织修复材料治疗小鼠皮肤缺损，治疗效果参考图12A-15C，在第12天，组织修复材料组伤口愈合率为99.04％，而对照组外泌体凝胶和透明质酸的愈合率分别为88.92％和87.20％，显示组织修复材料具有良好的修复能力，可以加速伤口愈合。其中，图12A为涂抹本发明实施例的组织修复材料前的皮肤损伤图；图12B为涂抹外泌体凝胶前的皮肤损伤图；图12C为涂抹透明质酸前的皮肤损伤图；图13A为涂抹本发明实施例的组织修复材料4天后的皮肤损伤图；图13B为涂抹外泌体凝胶4天后后的皮肤损伤图；图13C为涂抹透明质酸4天后后的皮肤损伤图；图14A为涂抹本发明实施例的组织修复材料8天后的皮肤损伤图；图14B为涂抹外泌体凝胶8天后后的皮肤损伤图；图14C为涂抹透明质酸8后后的皮肤损伤图；图15A为涂抹本发明实施例的组织修复材料12天后的皮肤损伤图；图15B为涂抹外泌体凝胶12天后的皮肤损伤图；图15C为涂抹透明质酸12天后的皮肤损伤图。
七、本实施例5所述的组织修复材料治疗皮肤损伤后的苏木精-伊红染色检测
用无菌手术器械取上述组织修复材料组、外泌体凝胶组以及透明质酸组的创面新生皮肤及创周部分正常皮肤，深达肌层；立即放入4％多聚甲醛中固定过夜；将固定的组织依次置于下列溶剂中脱水：于50％酒精中脱水0.5h→70％酒 精脱水0.5h→80％酒精脱水0.5h→95％酒精脱水0.5h→100％酒精脱水0.5h，然后进行透明处理：1/2二甲苯+1/2酒精处理2h→二甲苯Ⅰ处理1.5h→二甲苯Ⅱ处理1.5h；将组织置于溶化的石蜡中，待石蜡完全浸入组织块后进行包埋，冷却凝固成块；将包埋好的蜡块固定于切片机上，切成3～5μm的薄片，贴到载玻片上，自然风干；将切片依次放入下列溶剂中脱蜡：放入二甲苯Ⅰ中10min→二甲苯Ⅱ中5min→1/2二甲苯+1/2酒精中5min→100％乙醇中2min→95％的乙醇中1min→80％乙醇中1min→75％乙醇中1min→蒸馏水中1min；将脱蜡的切片放入苏木精溶液中染色约10min，然后用自来水冲洗；放入1％盐酸酒精溶液中分化数秒，用自来水漂洗；将切片依次置于下列溶剂中脱水：在70％酒精中脱水2h→80％酒精中脱水2h→95％酒精中脱水2h，然后置于伊红液中复染2min；对切片进行二次脱水：在80％酒精中脱水10min→95％酒精中脱水5min→100％酒精中脱水5min，然后进行透明处理：3/4二甲苯+1/4石碳酸中脱水1min→二甲苯Ⅰ中脱水3min→二甲苯Ⅱ中脱水5min；给已透明的组织切片滴加少量中性树脂，盖上盖玻片封固。
参考图16A-16C，苏木精-伊红染色检测结果显示，组织修复材料组具有更小的肉芽组织间隙宽度，表明其具有优良的促肉芽组织成熟能力和表皮再生能力。其中，图16A显示了本发明实施例的组织修复材料修复损伤皮肤后的苏木精—伊红染色图；图16B显示了外泌体凝胶修复损伤皮肤后的苏木精-伊红染色图；图16C显示了透明质酸修复损伤皮肤后的苏木精—伊红染色图。
八.本实施例5所述的组织修复材料治疗皮肤损伤的Masson染色
取组织修复材料、外泌体凝胶以及透明质酸治疗后的组织，进行切片，脱水，包埋，脱蜡；Weigert铁苏木素(A、B液等比例混合)染色10min，水洗；1％盐酸酒精溶液中分化数秒，水洗；丽春红酸性品红溶液染7～8min，快速水洗；磷钼酸处理切片1min，倾去多余液体；苯胺蓝染液复染5min，快速水洗；脱水后用中性树胶封固。
胶原的合成和沉积也是伤口愈合的关键过程，Masson染色是胶原纤维染色经典的技术方法，参考图17A-17C，相比于外泌体凝胶组以及透明质酸组，组织修复材料组显著促进了胶原沉积，其中，图17A为组织修复材料治疗损伤皮肤后的Masson染色图；17B为外泌体凝胶治疗损伤皮肤后的Masson染色图；17C为透明质酸治疗损伤皮肤后的Masson染色图。
尽管以上结合附图对本发明的实施方案进行了描述，但是本发明并不局限于上述的具体实施方案和应用领域，上述的具体实施方案仅仅是示意性的、指导性的，而不是限制性的。本领域的普通技术人员在说明书的启示下，在不脱离本发明权利要求所保护的范围的情况下，还可以做出多种形式的变化，这些均属于本发明保护之列。

Claims (27)

  1. 复合体，其特征在于，所述复合体由靶向基团和药用囊泡结合而成，所述靶向基团选自适配子，所述药用囊泡选自外泌体。
  2. 根据权利要求1所述的复合体，其特征在于，每个复合体包含所述适配子和外泌体的数量比为：n:1，n≧1，n为整数。
  3. 根据权利要求1所述的复合体，其特征在于，所述适配子选自血管内皮细胞靶向适配子。
  4. 根据权利要求1所述的复合体，其特征在于，所述适配子的序列为：5′-CCCACGTCTGCGCTTAGCTCCTGGGCCTGGATGGGC-3′。
  5. 根据权利要求1所述的复合体，其特征在于，所述适配子的3′或5′端进行了-NH 2修饰，修饰后的适配子为：适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺，化学结构式为：
    Figure PCTCN2019127253-appb-100001
  6. 根据权利要求5所述的复合体，其特征在于，所述适配子的修饰方法包括以下步骤：
    1)将二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧基、N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺溶于适量ddH 2O中，调节反应体系的pH值为5～6，室温搅拌40～60min，获得第一溶液；
    2)将所述第一溶液用焦碳酸二乙酯处理水透析后，得到二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺；
    3)取未经修饰的适配子，溶于焦碳酸二乙酯处理水，置于85℃水浴10min，然后迅速冰浴10min，获得活化适配子的焦炭酸二乙酯处理水溶液；
    4)将所述二硬脂酰磷脂酰乙醇胺-聚乙二醇-N-羟基琥珀酰亚胺、活化适配子的焦炭酸二乙酯处理水溶液和三乙胺混合，室温搅拌8-12h，获得第二溶液；
    5)将所述第二溶液利用焦碳酸二乙酯处理水超滤后，得到适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺。
  7. 根据权利要求1所述的复合体，其特征在于，所述外泌体为间充质干细胞外泌体。
  8. 根据权利要求1所述的复合体，其特征在于，所述间充质干细胞外泌体选自骨髓间充质干细胞外泌体、脐带血间充质干细胞外泌体、脐带组织间充质干细胞外泌体、胎盘组织间充质干细胞外泌体或脂肪组织间充质干细胞外泌体。
  9. 组合物，其特征在于，所述组合物的活性成分为根据权利要求1-8任一项所述的复合体。
  10. 根据权利要求1-8任一项所述的复合体或根据权利要求9所述的组合物在促进血管新生方面的用途。
  11. 根据权利要求1-8任一项所述的复合体或根据权利要求9所述的组合物在制备皮肤疾病治疗药物中的用途。
  12. 根据权利要求11所述的用途，其特征在于，所述皮肤疾病包括皮肤损伤、皮肤感染、褥疮或糖尿病溃疡。
  13. 组织修复材料，其特征在于，所述组织修复材料的组分包括所述复合体和凝胶材料，所述复合体由适配子和外泌体结合而成。
  14. 根据权利要求13所述的组织修复材料，其特征在于，每个复合体包含所述适配子和外泌体的数量比为：n:1，n≧1，n为整数。
  15. 根据权利要求13所述的组织修复材料，其特征在于，所述适配子选自血管内皮细胞靶向适配子。
  16. 根据权利要求15所述的组织修复材料，其特征在于，所述适配子的序列为：5′-CCCACGTCTGCGCTTAGCTCCTGGGCCTGGATGGGC-3′。
  17. 根据权利要求13所述的组织修复材料，其特征在于，所述适配子的3′或5′端进行了-NH 2修饰，修饰后的适配子为：适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺，化学结构式为：
    Figure PCTCN2019127253-appb-100002
  18. 根据权利要求13所述的组织修复材料，其特征在于，所述外泌体为间充质干细胞外泌体。
  19. 根据权利要求13所述的组织修复材料，其特征在于，所述间充质干细胞外泌体选自骨髓间充质干细胞外泌体、脐带血间充质干细胞外泌体、脐带组织间充质干细胞外泌体、胎盘组织间充质干细胞外泌体或脂肪组织间充质干细胞外泌体。
  20. 根据权利要求13所述的组织修复材料，其特征在于，所述凝胶材料选自胶原蛋白、海藻酸盐和透明质酸钠中的至少一种。
  21. 权利要求13-20任一项所述的组织修复材料的制备方法，其特征在于，包括以下步骤：
    1)常规培养间充质干细胞，利用超速离心法提取间充质干细胞来源的外泌体；
    2)用焦碳酸二乙酯处理PBS缓冲液作为溶剂，将适配子-聚乙二醇-二硬脂酰磷脂酰乙醇胺与所述外泌体37℃搅拌4～6h；
    3)利用焦碳酸二乙酯处理PBS缓冲液超滤后，得到适配子修饰的间充质干细胞外泌体悬液；
    4)向上述适配子修饰的间充质干细胞外泌体悬液中加入适量凝胶材料，充分混匀，即得到所述组织修复材料。
  22. 权利要求13～20任意一项所述组织修复材料在制备皮肤疾病治疗产品中的用途。
  23. 根据权利要求22所述的用途，其特征在于，所述皮肤疾病包括皮肤损伤、皮肤感染、褥疮或糖尿病溃疡。
  24. 皮肤损伤修复凝胶，其特征在于，所述皮肤损伤修复凝胶包含权利要求13～20任一项所述的组织修复材料。
  25. 糖尿病溃疡修复敷料，其特征在于，所述糖尿病溃疡修复敷料包含权利要求13～20任一项所述的组织修复材料。
  26. 足贴，其特征在于，所述足贴包含权利要求13～20任一项所述的组织修复材料和胶贴，所述组织修复材料黏贴在所述胶贴上。
  27. 根据权利要求26所述的足贴，其特征在于，所述胶贴选自无纺布、纺粘布、聚乙烯薄膜或医用PU膜。
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CN114904043B (zh) * 2022-04-28 2023-07-21 深圳市儿童医院 复合水凝胶及其制备方法和应用
CN115054678A (zh) * 2022-07-12 2022-09-16 康膝生物医疗(深圳)有限公司 一种用于子宫内膜修复的温敏型胶原外泌体复合水凝胶制剂的制备方法及其应用
CN116036310A (zh) * 2023-01-13 2023-05-02 广东医科大学附属医院 琥珀酰化壳聚糖修饰外泌体及其制备方法和应用

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