WO2023231046A1 - 一种具有自适应性的促组织再生材料 - Google Patents

一种具有自适应性的促组织再生材料 Download PDF

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WO2023231046A1
WO2023231046A1 PCT/CN2022/097157 CN2022097157W WO2023231046A1 WO 2023231046 A1 WO2023231046 A1 WO 2023231046A1 CN 2022097157 W CN2022097157 W CN 2022097157W WO 2023231046 A1 WO2023231046 A1 WO 2023231046A1
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tissue regeneration
promoting
tissue
polyurethane
adaptive
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French (fr)
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高长有
李世分
胡欣蔓
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浙江大学
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    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • 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
    • 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
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • 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
    • A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, e.g. heart valves
    • 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
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction

Definitions

  • the invention relates to an adaptive material that promotes tissue regeneration and belongs to the technical field of biomedical materials.
  • tissue regenerative therapies aim to supply or repair organs or tissues by combining biomaterials, biochemical signals and cells.
  • the biggest challenge is to create and regulate the appropriate implantable or injectable macroenvironment and microenvironment according to the microenvironmental needs of the tissue to allow cell-induced tissue formation in vitro or in vivo.
  • a large number of studies have made great progress in the past few decades, it is still a challenge to construct biomaterials with native tissue-like structures and functions in vitro or in vivo, including spatiotemporal control of biological functional domains and mechanical properties.
  • tissue have varying degrees of complexity, from relatively simple two-dimensional structures (skin, cornea) to tubular tissues (blood vessels, urethra), hollow organs (e.g., bladder), and complex bulky organs (liver, heart).
  • Regenerative material strategies need to consider not only the biological function and mechanical properties of the implant, but also the specific material-level design of the tissue structure. Since each level can be divided into sub-hierarchies with the ability to rearrange in time, individual objects require dynamic characteristics to enable organizational remodeling and maturation.
  • tissue regeneration engineering has local heterogeneity, and the same regenerative therapy only targets a single level, resulting in limited application. Adaptive biomaterials have emerged as the times require.
  • Such materials are adaptive to tissue environments and can interact with signaling molecules in cells or extracellular matrices in different microenvironments.
  • Cells "communicate" with neighboring cells and the extracellular matrix through the transduction of signal molecules, cellular responses and other behaviors, thereby sensing their environment and inducing highly complex signaling networks, triggering downstream cellular processes and causing physiological changes, such as bone sclerosis. or wound healing.
  • Adaptable biomaterials can appropriately participate in the communication process. As long as they function at the appropriate location, they can adjust downstream cell behavior to promote tissue regeneration.
  • Protein is the main component of human cells. After protein in food enters the human body, it is first decomposed into amino acids after digestion. The human body then uses these amino acids to synthesize new human proteins, such as immune antibodies, digestive enzymes, plasma proteins, growth hormone, etc. It is a synthesized human protein. Among the various amino acids used to synthesize proteins, L-lysine is the most important; without it, other amino acids will be limited or unutilized. Scientists call it the first essential amino acid for the human body. Scientists have also discovered that L-lysine plays an important role in the human central nervous system and peripheral nervous system, and has been used in the fields of feed additives, medicine, cosmetics and polymer materials. Additionally, it is essential for proper cell growth and facilitates collagen formation.
  • Lysine has been recognized to accelerate the ability of lactate to form glucose in isolated rat hepatocytes. At the same time, lysine can promote cell adhesion and proliferation as well as tissue regeneration at the biomaterial interface.
  • Polyvinyl alcohol (PVA) fiber membranes chemically modified with L-lysine for extraction of nanoscale pollutants from aqueous environments.
  • the combination of L-lysine and PVA can improve the biological properties of electrospun membranes, while using different drug incorporation methods in order to tailor the effects of each biomolecule to a specific time frame. Therefore, hyperbranched polylysine (HBPL), as a polylysine with a wide molecular weight distribution and a randomly branched structure, is more effective than linear polylysine and dendritic polylysine.
  • HBPL hyperbranched polylysine
  • Amino acids have highly branched structures and abundant terminal amines. Since it has both ⁇ -PL and PLL units, in biological media, the available amine groups on the lysine molecule are protonated, thereby promoting its interaction with negatively charged cell membranes. This combination is conducive to regulating cell adhesion, cell proliferation and other cell behaviors, thereby promoting tissue repair and regeneration.
  • hyperbranched polylysine with different biomaterials can create a series of modular regenerative treatments.
  • composite myocardial patches of HBPL and polyurethane can be used in disease models of myocardial infarction; combinations of HBPL and hydrogels can be used in most injectable disease models; HBPL-loaded nanoparticles can be injected intravenously in the pneumonia model.
  • this type of material can be applied to but not limited to cartilage, bone, nerves, muscles, heart, and vascular tissues. This effectively solves the limitation of single therapeutic materials for regenerative therapy.
  • the application inclusiveness of hyperbranched polylysine allows the structure of the carrier material, the mode of loading, and the form of application to be designed.
  • the purpose of the present invention is to solve the problem of cell necrosis and apoptosis in different disease models, thereby promoting tissue necrosis, and to provide an adaptive hyperbranched polylysine that can be loaded on different materials to promote Tissue regeneration.
  • An adaptive material that promotes tissue regeneration has both amide bonds that can provide hydrogen bonding and reactive amino functional groups.
  • the material contains hyperbranched polylysine, and the number of amino groups contained in each hyperbranched polylysine is 22 to 36, and the tissue is at least one of cardiac tissue, nervous tissue, and vascular tissue.
  • the material is extremely adaptable and can promote tissue regeneration in various forms and under various conditions. And the material can function in an environment with a pH of 5-10. Compared with the application environment of amino acids, HBPL will not be inactivated in acidic or alkaline environments.
  • the material can be dissolved in a solution and directly used as a drug to promote tissue regeneration.
  • the drug can be loaded on the carrier material through physical and/or chemical effects to form a drug that promotes tissue regeneration.
  • the physical effect is one or more of blending, adsorption, penetration, self-assembly, ionic complexation, and chain entanglement;
  • the chemical effect is bulk grafting, surface grafting, or copolymerization. of one or more.
  • the carrier material may be a polymer, and may exist in the form of one or more of hydrogel, microgel, polyurethane, and nanoparticles.
  • the carrier material may also be a metal or non-metal material.
  • tissue regeneration-promoting material is a polyurethane material loaded with hyperbranched polylysine, with diol and diamine as soft segments, diisocyanate and chain extender as hard segments, and HBPL by physical blending. load into it;
  • polyurethane fiber membrane loaded with hyperbranched polylysine and resistant to adhesion which is obtained by grafting hydrophilic polyethylene glycol on the outer surface of the polyurethane elastomer material layer.
  • the polyurethane elastomer includes polyurethane elastomer.
  • the soft segment consists of propylene glycol fumarate (PPF) and poly ⁇ -caprolactone diol (PCL) and the hard segment consists of saturated aliphatic diisocyanate and L-lysine dimethyl ester hydrochloride, which is porous. structure, in which HBPL is adsorbed;
  • the inventive principle of the present invention is:
  • the HBPL of the present invention which has the effect of promoting tissue regeneration, can be loaded on different materials through physical and/or chemical effects, and the application form of the materials is not limited.
  • the material form can be selected according to different application scenarios. For example, polyurethane patches loaded with HBPL can be used for myocardial infarction, hydrogels can be used for intrapericardial injection, and microgels can be used for the treatment of disease models such as pneumonia.
  • the different functional responses of the carrier material can synergize with the tissue regeneration promoted by HBPL, and then interact with the tissue microenvironment to promote the regeneration of different tissues.
  • the HBPL that promotes tissue regeneration can provide amide bonds with strong hydrogen bonding and very active reactive amino functional groups, which greatly broadens its application.
  • the tissue regeneration-promoting effect of hyperbranched polylysine can act on different tissues, such as cardiac tissue, nervous tissue, vascular tissue, etc.
  • HBPL can be loaded on different materials through physical (blending, adsorption, penetration, self-assembly, ionic complexation, chain entanglement, etc.) or chemical effects.
  • HBPL is loaded on different materials through physical and chemical effects, and the forms of materials are diversified, such as polyurethane patches, injectable hydrogels, nanoparticles, microgels, metal surfaces, etc.
  • the corresponding material functional response can be customized according to different disease models, and then play a synergistic effect with HBPL in promoting tissue regeneration.
  • the tissue regeneration-promoting material loaded with hyperbranched polylysine has universal applicability.
  • Figure 1 is a diagram showing the effect of hyperbranched polylysine on promoting cardiomyocyte proliferation in Example 1;
  • Figure 2 is a synthesis route diagram of the tissue regeneration-promoting polyurethane material loaded with hyperbranched polylysine in Example 2;
  • Figure 3 shows the effect of promoting fibroblast proliferation of the tissue regeneration-promoting polyurethane material loaded with hyperbranched polylysine in Example 2;
  • Figure 4 shows the cytotoxicity of the tissue regeneration-promoting hydrogel loaded with hyperbranched polylysine in Example 3.
  • the invention provides an adaptive material that promotes tissue repair and regeneration and has both amide bonds that can provide hydrogen bonding and reactive amino functional groups.
  • the material contains hyperbranched polylysine, and each hyperbranched polylysine contains 22 to 36 amino groups.
  • the material is extremely adaptable and can be in various forms and states. Promote tissue regeneration, and the tissue is at least one of cardiac tissue, nervous tissue, and vascular tissue.
  • the material can be dissolved in a solution and directly used as a drug to promote tissue regeneration. Or it can be loaded on the carrier material through physical action and/or chemical action to form a tissue regeneration-promoting drug.
  • the physical action can be one or more of blending, adsorption, penetration, self-assembly, ion complexation, and chain entanglement.
  • species, the chemical reaction may be one or more of grafting, coupling, click, cyclization, etc.
  • the carrier material may be a polymer, and may exist in the form of one or more of hydrogel, microgel, polyurethane, and nanoparticles.
  • the carrier material can also be a metal or non-metal material.
  • the tissue regeneration-promoting material is a polyurethane material loaded with hyperbranched polylysine, with macromolecular diol and diamine as soft segments, and diisocyanate and small molecule chain extender as hard segments. Sections and HBPL are loaded in it by means of physical blending, and the polyurethane material has good mechanical properties.
  • the polyurethane material contains poly ⁇ -caprolactone diol, 1,3-bis(4-hydroxybutyl)tetramethyldisiloxane soft segment, and diisocyanate , 1,4-butanediamine is a hard segment. Its structure is as follows:
  • the number average molecular weight of poly ⁇ -caprolactone diol is 2.0 to 4.0 kDa
  • the number average molecular weight of 1,3-bis(4-hydroxybutyl)tetramethyldisiloxane is The molecular weight is 0.2 ⁇ 0.5kDa.
  • the diisocyanates are aliphatic diisocyanates (isophorone diisocyanate, hexamethylene diisocyanate, 4,4'-dicyclohexylmethane diisocyanate) and aromatic diisocyanates. At least one of isocyanates.
  • the polyurethane material is made of poly ⁇ -caprolactone diol and 1,3-bis(4-hydroxybutyl)tetramethyldisiloxane as macromolecular diols and Saturated aliphatic diisocyanate is used as raw material and is produced using a two-step process, including the following steps:
  • step 2) Add 1,3-bis(4-hydroxybutyl)tetramethyldisiloxane to the polyurethane prepolymer solution obtained in step 1), and stir for 2 hours under the protection of nitrogen atmosphere.
  • 1,3-bis(4-hydroxybutyl)tetramethyldisiloxane is added as the first component chain extender of the system, and the molar ratio of unreacted poly ⁇ -caprolactone diol in step 1) is 0.5:1.
  • the reaction temperature is lowered to 50°C, the stirring speed is reduced, and the anhydrous dioxane solution of the second component chain extender 1,4-butanediamine is slowly added dropwise.
  • 1,4-butanediamine is completely added, resume the stirring speed until all the isocyanate and chain extender react completely to obtain a clear and transparent polyurethane solution.
  • the molar ratio of the two chain extenders is 1:1;
  • step 3 Pour the polyurethane solution prepared in step 2) into ice-cold absolute ethanol to precipitate, centrifuge and collect. Dissolve the collected polyurethane solid in dioxane solvent again, and pour it into ice-cold absolute ethanol again to precipitate. Repeat several times. Finally, place the obtained polyurethane solid in absolute ethanol and stir for more than 4 hours to fully replace the solvent. . Then put it into deionized water to fully replace the absolute ethanol and freeze-dry. Finally, polyurethane with good mechanical properties is obtained;
  • step 4) Take a certain amount of the polyurethane prepared in step 3) and dissolve it in the solvent hexafluoroisopropyl alcohol. Dissolve HBPL in a certain amount of hexafluoroisopropanol, and then mix it evenly with the hexafluoroisopropanol solution of polyurethane. Pour it into a polytetrafluoroethylene mold, place it in a fume hood to fully evaporate the solvent, and obtain a polyurethane film loaded with HBPL, which has the function of promoting tissue regeneration.
  • the tissue regeneration-promoting material is an anti-adhesion polyurethane fiber membrane loaded with hyperbranched polylysine, which is grafted with hydrophilic polyethylene on the outer surface of the polyurethane elastomer material layer. Obtained from diol.
  • the polyurethane elastomer includes a soft segment composed of polypropylene fumarate (PPF) and poly ⁇ -caprolactone diol (PCL) and a saturated aliphatic diisocyanate and L-lysine dimethyl ester hydrochloride. Hard segment composed of salt.
  • the polyurethane fiber membrane has a porous structure and can effectively adsorb HBPL to achieve loading.
  • the preparation method of the polyurethane fiber membrane includes the following steps:
  • PCL poly ⁇ -caprolactone diol
  • step 1) Mix the polyurethane solutions obtained in step 1) and step 2) together under nitrogen protection, slowly add the supernatant obtained in step 3) for chain extension, and continue the reaction at 60-80°C for at least 6 hours to obtain a polyurethane solution;
  • step 5 Pour the polyurethane solution obtained in step 4) into deionized water to precipitate, centrifuge and collect. Dissolve the collected polyurethane solid in N,N-dimethylformamide solvent again, and pour it into deionized water again to precipitate. , repeated several times, and finally put the obtained polyurethane solid into a vacuum oven to dry for more than 24 hours to obtain a polyurethane elastomer material;
  • step 6) Take a certain amount of the polyurethane elastomer material prepared in step 5) and dissolve it in the solvent hexafluoroisopropyl alcohol.
  • the dissolved polyurethane solution is used for electrospinning to obtain a fiber membrane with a nanofiber structure. After drying the fiber membrane for 12 hours, it was immersed in a solution of polyethylene glycol containing a photoinitiator and irradiated under ultraviolet light for 10 to 15 minutes. Then wash with deionized water more than 5 times and freeze-dry. Obtain fiber membrane;
  • the tissue regeneration-promoting material is an injectable hydrogel loaded with hyperbranched polylysine.
  • HBPL can penetrate into the interior of the hydrogel driven by a concentration gradient.
  • the hydrogel can The glue can be injected through minimally invasive implantation, reducing the cost of surgery and secondary injuries.
  • the preparation method of the hydrogel includes the following steps:
  • step 2) Place the hydrogel obtained in step 1) in an aqueous solution containing HBPL for more than 12 hours to obtain an injectable hydrogel loaded with hyperbranched polylysine, which has the function of promoting tissue regeneration.
  • the tissue regeneration-promoting material is a self-assembled nanoparticle loaded with hyperbranched polylysine, which is made by using chitosan as the core and hyaluronic acid and HBPL as two shells in sequence. It is obtained by superimposing layers by utilizing the electrostatic adsorption of materials.
  • the nanoparticles can be used as regeneration-promoting drugs to quickly reach the diseased site through intravenous injection, in situ injection and other means, and provide corresponding treatment.
  • the preparation method of nanoparticles includes the following steps:
  • step 2) Disperse the chitosan particles obtained in step 2) in the hyaluronic acid solution for more than 12 hours, mix and stir, then centrifuge and discard the supernatant, then centrifuge and wash with deionized water, and finally use deionized water to disperse the centrifuged precipitate.
  • chitosan-hyaluronic acid nanoparticles with core-shell structure can be obtained;
  • step 4) Disperse the nanoparticles obtained in step 3) in the HBPL solution for more than 12 hours, mix and stir, then centrifuge to discard the supernatant, then centrifuge and wash with deionized water, and finally use deionized water to disperse the centrifugal precipitate to obtain three-dimensional Self-assembled nanoparticles with a layered structure have the function of promoting tissue regeneration.
  • the material that promotes tissue regeneration is a metal material loaded with hyperbranched polylysine, which is achieved by treating aluminum metal with plasma and then loading HBPL that can promote regeneration.
  • the preparation method of the metal material includes the following steps:
  • the tissue regeneration-promoting material is a microgel loaded with hyperbranched polylysine, which is achieved through the reaction of the amino functional group of HBPL and the double bond.
  • the preparation method of the tissue regeneration-promoting microgel is as follows:
  • This type of reaction can be extended to other monomers containing double bonds.
  • the tissue regeneration-promoting material is a nanoparticle loaded with hyperbranched polylysine, which is obtained by using polyurethane as a base material.
  • the preparation method of the nanoparticles is as follows:
  • the tissue regeneration-promoting material can be hyperbranched polylysine through physical effects (blending, adsorption, penetration, self-assembly, ion complexation, etc.) and chemical effects (click reactions of amino groups and double bonds, etc.)
  • At least one of the methods is loaded on a carrier, and the carrier form includes but is not limited to materials such as metals, polymers (hydrogel, microgel, polyurethane, nanoparticles, etc.), inorganic non-metals, etc.
  • the preparation process of HBPL for promoting tissue regeneration of the present invention is as follows:
  • HBPL was prepared into a solution of 25 ⁇ g/mL.
  • the CCK-8 method was used to test the proliferation of cells in HBPL solution. Take the H9C2 cells in good growth status and divide them into two groups. One group is added with ordinary culture medium, and the other group is added with medium mixed with HBPL. 5 ⁇ 10 3 cells are added to each well and cultured for 1-2 days respectively. At each time point, aspirate the culture medium inside, rinse it three times with sterile phosphate buffer saline (PBS), add 100 ⁇ L of the prepared CCK-8 solution to each well, and incubate in the dark for 2 hours, then take out 80 ⁇ L from each well. , test its absorbance value at a wavelength of 450nm ( Figure 1). As shown in the figure, the group mixed with HBPL solution promoted cell proliferation, proving the ability of HBPL to promote cardiomyocyte proliferation.
  • PBS sterile phosphate buffer saline
  • tissue regeneration-promoting polyurethane material loaded with hyperbranched polylysine of the present invention is obtained by physical blending of HBPL and polyurethane materials.
  • the synthesis route is shown in Figure 2.
  • Tissue regeneration-promoting polyurethane material loaded with hyperbranched polylysine composed of poly ⁇ -caprolactone diol, 1,3-bis(4-hydroxybutyl)tetramethyldisiloxane soft segment, and hexamethylene HDI and 1,4-butanediamine are hard segments.
  • the number average molecular weight of poly ⁇ -caprolactone diol is 2.0kDa
  • the number average molecular weight of 1,3-bis(4-hydroxybutyl)tetramethyldisiloxane is 0.278kDa.
  • 1,3-bis(4-hydroxybutyl)tetramethyldisiloxane (0.167g, 0.6mmol) to the obtained polyurethane prepolymer solution, and stir for 2 hours under the protection of nitrogen atmosphere.
  • 1,3-bis(4-hydroxybutyl)tetramethyldisiloxane is added as the first component chain extender of the system, and the molar ratio of unreacted poly ⁇ -caprolactone diol in step 1) is 0.5:1.
  • the reaction temperature lower the reaction temperature to 50°C, reduce the rotation speed, and slowly add 1 mL of anhydrous dioxane solution of the second component chain extender 1,4-butanediamine (0.0576g, 0.6mmol) dropwise. After the 1,4-butanediamine is completely added, resume the stirring speed until all the isocyanate and chain extender react completely to obtain a clear and transparent polyurethane solution.
  • the molar ratio of the two chain extenders is 1:1;
  • the obtained polyurethane film was made into a disc with a diameter of 8 mm using a hole punch.
  • the CCK-8 method was used to test the value-added status of cells on the material surface. Sterilize the polyurethane membrane and put it into a 48-well plate. Take the fibroblasts in good growth status, add 2 ⁇ 10 4 cells to each well, and culture them for 1-3 days. At each time point, aspirate the culture medium inside, rinse it three times with sterile PBS, add 200 ⁇ L of the prepared CCK-8 solution to each well, and incubate for 2 hours in the dark, take out 100 ⁇ L from each well, and test it at a wavelength of 450 nm. The absorbance value is below (Figure 3). As shown in the figure, polyurethane materials loaded with HBPL can promote cell proliferation, proving the ability of HBPL to promote the proliferation of fibroblasts.
  • the tissue regeneration-promoting hydrogel loaded with hyperbranched polylysine of the present invention is obtained through a chemical reaction between the epoxy group of the active oxygen-responsive hydrogel and the amino group of HBPL.
  • the preparation steps are as follows:
  • the hydrogel pre-solution was injected into a mold with a diameter of 1.1 cm, sealed, placed in a 37°C constant temperature water bath, and reacted for 24 hours to obtain a hydrogel. Then put it in a -20°C refrigerator to freeze for 24 hours, then take it out and put it in a 4°C refrigerator to thaw for 12 hours. Repeat three times.
  • the prepared hydrogel was cut into 1 mm thick slices and freeze-dried to obtain dry hydrogel sheets. Then it is placed in a 0.5 mg/mL hyperbranched polylysine solution for swelling equilibrium to obtain a tissue regeneration-promoting hydrogel with reactive oxygen species responsiveness.
  • the obtained hydrogel was soaked in serum-free culture medium for 24 hours, and serum was added after sterilization for later use.
  • the CCK-8 method was used to test the cytotoxicity of cells in hydrogel extract. Fibroblasts in good growth status were taken and divided into three groups, and 200 ⁇ L of ordinary culture medium, the extract of reactive oxygen-responsive hydrogel, and the extract of HBPL-loaded hydrogel were added respectively. Add 5 ⁇ 10 3 cells to each well. After co-culture for 1 day, suck out the medium inside and rinse it three times with sterile PBS. Add 100 ⁇ L of the prepared CCK-8 solution to each well.

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Abstract

一种具有自适应性的促组织再生材料，所述材料兼具有可提供氢键作用的酰胺键和可反应的氨基官能团。所述材料中含有超支化聚赖氨酸，且每个超支化聚赖氨酸含有的氨基数量为22～36个，所述的组织为心肌组织、神经组织、血管组织中的至少一种。所述材料可以溶于溶液中直接作为促组织再生药物，也可以通过物理作用和/或化学作用负载于载体材料上形成促组织再生药物。所述材料可以在不同的组织微环境中，与环境内的各种信号相互作用，促进组织修复和再生。

Description

一种具有自适应性的促组织再生材料 技术领域
本发明涉及一种具有自适应性的促组织再生材料，属于生物医用材料技术领域。
背景技术
全球器官移植的短缺推动了组织再生疗法的进步。组织工程和再生医学旨在通过结合生物材料、生化信号和细胞来供应或修复器官或组织。最大的挑战便是根据组织所在的微环境需要，创建并调控合适的可植入或可注射的宏观环境和微环境，以允许离体或体内细胞诱导的组织形成。尽管在过去几十年中，大量的研究取得了巨大进展，但在体外或体内构建具有类似天然组织结构和功能的生物材料仍然是一个挑战，包括对生物功能域和机械性能的时空控制。同时由于组织具有不同程度的复杂性，从相对简单的二维结构(皮肤、角膜)到管状组织(血管、尿道)、中空器官(例如，膀胱)和复杂的大块器官(肝脏、心脏)，再生材料策略不仅需要考虑植入物的生物功能、力学性能，重要的是组织结构的特定材料层次设计。由于每个级别都可以分为具有及时重新排列能力的子层次结构，因此单个对象需要动态特征来实现组织重塑和成熟。通常情况下，组织再生工程的设计都具有局部异质化，同一种再生疗法只针对单一的级别层次，导致其应用受到限制。具有自适应性的生物材料应运而生，这类材料具有组织环境自适应性，可以在不同的微环境中与细胞内或者细胞外基质的信号分子产生相互作用。细胞与邻近细胞和细胞外基质之间通过信号分子的转导、细胞反应等行为进行“交流”，从而感知其环境并诱导高度复杂的信号网络，触发下游细胞过程，引起生理变化，例如骨骼硬化或伤口愈合。自适应性的生物材料便可以适当地参与其中的交流过程，只要在适当的位置发挥作用，便可以通过调整下游的细胞行为，进而促进组织再生。
蛋白质是构成人体细胞的主要成份，食物中的蛋白质进入人体后经过消化先分解成氨基酸，然后人体又利用这些氨基酸再合成新的人体蛋白质，如免疫抗体、 消化酶、血浆蛋白、生长激素等都是合成后的人体蛋白质。在合成蛋白质的各种氨基酸中，L-赖氨酸是最重要的一种；少了它，其它氨基酸就受到限制或得不到利用，科学家称它为人体第一必需氨基酸。科学家还发现，L-赖氨酸对人的中枢神经和周围神经系统都起着重要作用，已在饲料添加剂、医药、化妆品和高分子材料领域得到应用。此外，它对于适当的细胞生长至关重要，并且有利于胶原蛋白的形成。赖氨酸已被公认为能够加速分离的大鼠肝细胞中乳酸形成葡萄糖的能力。同时赖氨酸可以在生物材料界面促进细胞粘附和增殖以及组织再生。用L-赖氨酸进行化学改性的聚乙烯醇(PVA)纤维膜，用于从水环境中提取纳米级污染物。L-赖氨酸和PVA的结合可以改善静电纺丝膜的生物学特性，同时使用不同的药物掺入方法，以便将每个生物分子的作用调整到特定的时间范围。因此，超支化聚赖氨酸(HBPL)作为一种分子量分布较宽、结构无规支化的聚赖氨酸，与线性聚赖氨酸和树枝状聚赖氨酸相比，超支化聚赖氨酸具有高度支化的结构和丰富的末端胺。由于其同时具有ε-PL和PLL单元，在生物介质中，赖氨酸分子上可用的胺基被质子化，从而促进其与带负电荷的细胞膜的相互作用。这样的结合有利于调控细胞黏附、细胞增殖等细胞行为，从而促进组织修复与再生。
将超支化聚赖氨酸与不同的生物材料进行结合，便可以形成一系列模块化的再生治疗方法。例如，HBPL和聚氨酯的复合的心肌补片可应用于心肌梗死的疾病模型中；HBPL和水凝胶的组合可以应用于大多数可注射的疾病模型中；负载HBPL的纳米粒子可以通过静脉注射作用于肺炎模型。通过设计生物载体材料的自适应性，与组织的微环境进行响应并调控其细胞行为，可以将这类材料应用但不限于软骨、骨骼、神经、肌肉、心脏和血管组织等。如此便很好地解决了再生疗法的材料具有单一治疗性的限制。超支化聚赖氨酸的应用包容性允许载体材料的结构、负载的方式以及应用的形式都可以进行设计。
发明内容
本发明的目的是为了解决在不同的疾病模型中细胞坏死和凋亡，进而促进组织坏死的问题，提供一种具有自适应性的超支化聚赖氨酸，可以负载在不同的材料上，促进组织再生。
本发明的目的是通过以下技术方案实现的：
一种具有自适应性的促组织再生材料，所述材料兼具有可提供氢键作用的酰 胺键和可反应的氨基官能团。
进一步的，所述材料中含有超支化聚赖氨酸，且每个超支化聚赖氨酸含有的氨基数量为22～36个，所述的组织为心肌组织、神经组织、血管组织中的至少一种。所述材料具有极强的自适应性，可以以多种形式多种状态下发挥促组织再生作用。且所述材料在pH为5-10的环境下均可发挥作用。与氨基酸的应用环境相比，HBPL在酸性或碱性的环境中也不会存在失活的情况。
进一步的，所述材料可以溶于溶液中，直接作为促组织再生药物。或者通过物理作用和/或化学作用负载于载体材料上形成促组织再生药物。
进一步的，所述的物理作用为共混、吸附、渗透、自组装、离子络合、链缠结中的一种或多种；所述的化学作用为本体接枝、表面接枝、共聚中的一种或多种。
进一步的，所述的载体材料可以为聚合物，存在形式可以为水凝胶、微凝胶、聚氨酯、纳米粒子中的一种或多种。
进一步的，所述的载体材料也可以为金属或非金属材料。
进一步的，所述促组织再生材料是一种负载超支化聚赖氨酸的聚氨酯材料，以二醇和二胺为软段，以二异氰酸酯和扩链剂为硬段、HBPL以物理共混的手段负载其中；
或者是一种负载超支化聚赖氨酸的可抗粘连的聚氨酯纤维膜，是在聚氨酯弹性体材料层的外表面接枝亲水性聚乙二醇得到的，所述聚氨酯弹性体包括由聚富马酸丙二醇酯(PPF)和聚ε-己内酯二醇(PCL)组成的软段和由饱和脂肪族二异氰酸酯及L-赖氨酸二甲酯盐酸盐组成的硬段，具有多孔结构，HBPL吸附于其中；
或者是一种负载超支化聚赖氨酸的可注射水凝胶，HBPL在浓度梯度的驱动下，渗透进入水凝胶内部；
或者是一种负载超支化聚赖氨酸的自组装纳米粒子，是通过以壳聚糖为核，透明质酸和HBPL依次为两层外壳，利用静电吸附作用，层层叠加得到；
本发明的发明原理为：
本发明的具有促组织再生作用的HBPL，可以通过物理和/或化学的作用负载在不同的材料上，且材料的应用形式没有限制。可以根据不用的应用场景来选择 材料形式，比如负载HBPL的聚氨酯补片可以用于心肌梗死、水凝胶可以用于心包内注射、微凝胶可以用于肺炎等疾病模型的治疗。此外，载体材料的不同功能性响应可以和HBPL的促组织再生发挥协同作用，进而和组织微环境进行相互的响应，促进不同组织的再生。
与现有的医用材料相比，本发明的有益效果是：
1)本发明中，所述的具有促组织再生的HBPL可以提供具有强氢键作用的酰胺键和非常活泼的可反应的氨基官能团，极大地拓宽了其应用。
2)本发明中，超支化聚赖氨酸的促组织再生作用，可以作用于不同的组织，如心肌组织、神经组织、血管组织等。
3)本发明中，通过物理(共混、吸附、渗透、自组装、离子络合、链缠结等)或化学作用可以将HBPL负载在不同的材料上。
4)本发明中，通过物理和化学作用将HBPL负载在不同的材料上，材料的形式多样化，如聚氨酯补片、可注射水凝胶、纳米粒子、微凝胶、金属表面等等。
5)本发明中，可以根据不同的疾病模型，来定制相应的材料功能响应，进而和HBPL的促组织再生发挥协同作用。
6)本发明中，负载超支化聚赖氨酸的促组织再生材料，具有普适性。
附图说明
图1为实施例1中超支化聚赖氨酸的促心肌细胞增殖的效果图；
图2为实施例2中负载超支化聚赖氨酸的促组织再生聚氨酯材料合成路线图；
图3为实施例2中负载超支化聚赖氨酸的促组织再生聚氨酯材料的促成纤维细胞增殖的效果；
图4为实施例3中负载超支化聚赖氨酸的促组织再生水凝胶的细胞毒性情况。
具体实施方式
本发明提供了一种具有自适应性的促组织修复与再生材料，兼具有可提供氢键作用的酰胺键和可反应的氨基官能团。
所述材料中含有超支化聚赖氨酸，且每个超支化聚赖氨酸含有的氨基数量为22～36个，所述材料具有极强的自适应性，可以以多种形式多种状态下发挥促组织再生作用，所述的组织为心肌组织、神经组织、血管组织中的至少一种。
在本发明中，所述材料可以溶于溶液中，直接作为促组织再生药物。或者通过物理作用和/或化学作用负载于载体材料上形成促组织再生药物，所述的物理作用可以为共混、吸附、渗透、自组装、离子络合、链缠结中的一种或多种，所述的化学作用可以为接枝、偶联、点击、环化等作用中的一种或多种。
在本发明中，所述的载体材料可以为聚合物，存在形式可以为水凝胶、微凝胶、聚氨酯、纳米粒子中的一种或多种。所述的载体材料也可以为金属或非金属材料。
在本发明具体实施例中，所述促组织再生材料是一种负载超支化聚赖氨酸的聚氨酯材料，以大分子二醇和二胺为软段，以二异氰酸酯和小分子扩链剂为硬段、HBPL以物理共混的手段负载其中，而且所述的聚氨酯材料具有良好的力学性能。
在所述具体实例中，进一步地，所述聚氨酯材料，含有聚ε-己内酯二醇、1,3-双(4-羟基丁基)四甲基二硅氧烷软段，以二异氰酸酯、1,4-丁二胺为硬段。其结构如下：
Figure PCTCN2022097157-appb-000001
在具体实施例中，进一步的，所述的聚ε-己内酯二醇数均分子量为2.0～4.0kDa，1,3-双(4-羟基丁基)四甲基二硅氧烷数均分子量为0.2～0.5kDa。
在具体实施例中，进一步地，所述的二异氰酸酯为脂肪族二异氰酸酯(异佛尔酮二异氰酸酯、六亚甲基二异氰酸酯、4,4'-二环己基甲烷二异氰酸酯)和芳香族二异氰酸酯其中的至少一种。
在具体实施例中，进一步地，所述的聚氨酯材料是以聚ε-己内酯二醇、1,3-双(4-羟基丁基)四甲基二硅氧烷为大分子二元醇和饱和脂肪族二异氰酸酯为原料，采用两步法制，包括以下步骤：
1)取一定量的聚ε-己内酯二醇放入真空烘箱，干燥4h以上。将干燥后的聚ε-己内酯二醇加入到干燥容器中，在110℃真空(0.1mbar)中脱气干燥1h。然后加入饱和脂肪族二异氰酸酯及催化剂辛酸亚锡，在氮气保护下80～100℃反应至少2h，再加入无水二氧六环溶解，得到固含量为12.5wt％的聚氨酯预聚物溶液。其中饱和脂肪族二异氰酸酯中异氰酸根基团与聚ε-己内酯二醇中羟基的摩尔比为1.5:1～2.0:1；
2)在步骤1)得到的聚氨酯预聚物溶液中，加入1,3-双(4-羟基丁基)四甲基二硅氧烷，在氮气氛围的保护下搅拌反应2h。1,3-双(4-羟基丁基)四甲基二硅氧烷为体系的第一组分扩链剂加入，和步骤1)未反应的聚ε-己内酯二醇的摩尔比为0.5:1。反应结束之后，将反应温度降至50℃，降低搅拌速度，缓慢滴加第二组分扩链剂1,4-丁二胺的无水二氧六环溶液。在完全加入1,4-丁二胺后，恢复搅拌速度直到所有异氰酸酯与扩链剂反应完全，得到澄清透明的聚氨酯溶液。两种扩链剂的摩尔比为1：1；
3)将步骤2)制得的聚氨酯溶液倒入冰无水乙醇中沉淀，离心、收集。将收集的聚氨酯固体再次溶于二氧六环溶剂中，并再次倒入冰无水乙醇中沉淀，反复数次，最后将所得的聚氨酯固体置于无水乙醇中搅拌4h以上，充分置换出溶剂。再放入去离子水中充分置换无水乙醇，冻干。最后得到具有良好力学性能的聚氨酯；
4)取一定量在步骤3)制得的聚氨酯，溶于溶剂六氟异丙醇中。将HBPL溶于一定量的六氟异丙醇中，然后与聚氨酯的六氟异丙醇溶液混合均匀。倒入聚四氟乙烯的模具中，放入通风橱中充分挥发溶剂，得到负载HBPL的聚氨酯薄膜，具有促组织再生功能。
在本发明具体实施例中，所述促组织再生材料是一种负载超支化聚赖氨酸的可抗粘连的聚氨酯纤维膜，是在聚氨酯弹性体材料层的外表面接枝亲水性聚乙二醇得到的。所述聚氨酯弹性体包括由聚富马酸丙二醇酯(PPF)和聚ε-己内酯二醇(PCL)组成的软段和由饱和脂肪族二异氰酸酯及L-赖氨酸二甲酯盐酸盐组成的硬段。
在具体实施例中，进一步地，聚氨酯纤维膜具有多孔结构，有效地吸附HBPL实现负载。
在具体实施例中，所述的聚氨酯纤维膜的制备方法，包括以下步骤：
1)将聚富马酸丙二醇酯(PPF)加入到干燥容器中，减压除去残余的水分，然后加入无水N,N-二甲基甲酰胺溶剂溶解，再加入饱和脂肪族二异氰酸酯及催化剂辛酸亚锡，在氮气保护下60～80℃反应至少3h，得到聚氨酯溶液；
2)同时将聚ε-己内酯二醇(PCL)加入到另一个干燥容器中，减压除去残余的水分，然后加入无水N,N-二甲基甲酰胺溶剂溶解，再加入饱和脂肪族二异 氰酸酯及催化剂辛酸亚锡，在氮气保护下60～80℃反应和步骤1)相同的时间，得到聚氨酯溶液；
3)称取一定量的L-赖氨酸甲酯二盐酸盐于干燥容器中，加入无水N,N-二甲基甲酰胺溶剂，摇匀，再加入三乙胺。振荡至少3h以后，离心，收集上清液；
4)在氮气保护下将步骤1)和步骤2)得到的聚氨酯溶液混合到一起，缓慢加入步骤3)得到的上清液进行扩链，60～80℃继续反应至少6h以上，得到聚氨酯溶液；
5)将步骤4)得到的聚氨酯溶液，倒入去离子水中沉淀，离心、收集，将收集的聚氨酯固体再次溶于N,N-二甲基甲酰胺溶剂中，并再次倒入去离子水中沉淀，反复数次，最后将所得的聚氨酯固体放入真空烘箱干燥24h以上，得到聚氨酯弹性体材料；
6)取一定量在步骤5)制得的聚氨酯弹性体材料，溶于溶剂六氟异丙醇中。将溶解后的聚氨酯溶液用于静电纺丝，得到具有纳米纤维结构的纤维膜。将纤维膜干燥12h以后，浸没在含有光引发剂的聚乙二醇的溶液中，在紫外光下照射10～15min。再用去离子水清洗5遍以上，冻干。得到纤维膜；
7)再将纤维膜浸没在HBPL的水溶液中，浸泡24h以上。然后冻干，得到负载超支化聚赖氨酸的可抗粘连的聚氨酯纤维膜，具有促组织再生功能。
在本发明具体实施例中，所述促组织再生材料是一种负载超支化聚赖氨酸的可注射水凝胶，HBPL可以在浓度梯度的驱动下，渗透进入水凝胶内部，该水凝胶可以通过微创植入的手段进行注射，降低手术的成本和二次伤害。
在具体实施例中，所述的水凝胶的制备方法，包括以下步骤：
1)将甲基丙烯酸羟乙酯、甲叉双丙烯酰胺、苯基-2,4,6-三甲基苯甲酰基亚磷酸锂分别按质量体积分数10％、0.5％、0.05％溶于水中，将其置于紫外光照下成胶。得到固含量为10％的水凝胶；更进一步地，甲基丙烯酸羟乙酯可以换成各种水溶性的丙烯酸酯类单体。
2)将步骤1)得到水凝胶，置于含有HBPL的水溶液中12h以上，得到具有负载超支化聚赖氨酸的可注射水凝胶，具有促组织再生功能。
在本发明具体实施例中，所述促组织再生材料是一种负载超支化聚赖氨酸的自组装纳米粒子，是通过以壳聚糖为核，透明质酸和HBPL依次为两层外壳，利 用材料的静电吸附作用，层层叠加得到。所述的纳米粒子可以作为促再生药物通过静脉注射、原位注射等手段快速到达病变部位，进行相应的治疗。
在具体实施例中，进一步地，所述的纳米粒子的制备方法，包括以下步骤：
1)称取一定量的壳聚糖溶于去离子水中，再加入冰醋酸，开始加热。不断搅拌促进壳聚糖溶解，然后趁热过滤，得到壳聚糖的醋酸溶液；
2)采用离子交联法制备壳聚糖微粒，将提前配制好的三聚磷酸钠溶液在磁力搅拌的条件下逐滴加入壳聚糖的醋酸溶液，在60℃的温度下反应10min，得到壳聚糖的纳米微粒悬浊液。静置16h后离心，干燥后得到壳聚糖的纳米微粒；
3)将步骤2)得到的壳聚糖微粒分散在透明质酸的溶液中12h以上，混合搅拌，然后离心弃去上清，再用去离子水离心洗涤，最后用去离子水分散离心沉淀物，即可得到具有核壳结构的壳聚糖-透明质酸纳米微粒；
4)将步骤3)得到的纳米微粒分散在HBPL的溶液中12h以上，混合搅拌，然后离心弃去上清，再用去离子水离心洗涤，最后用去离子水分散离心沉淀物，得到具有三层结构的自组装纳米粒子，具有促组织再生功能。
在本发明的具体实施例中，所述促组织再生材料是一种负载超支化聚赖氨酸的金属材料，是通过在铝金属经过等离子体的处理，然后负载可促再生的HBPL实现的。
在具体实施例中，所述的金属材料的制备方法，包括以下步骤：
1)用氧气等离子体处理金属铝表面后，置于甲基丙烯酸甲酯的水溶液中，加入引发剂，在紫外灯下照射进行接枝反应。
2)将接枝好的材料用热水(70℃)冲洗至少48小时以除去均聚物，随后在50℃下真空干燥24小时以上。
3)将接枝后的金属材料泡在HBPL的水溶液中，加入缩合剂二环己基碳二亚胺，室温下反应2h以上，再用大量水冲洗后干燥；或者将接枝后的金属材料和水溶性碳二酰亚胺(如EDAC)进行反应，再和HBPL进行共价偶联；得到负载HBPL的金属材料，具有促组织再生的功能。
在本发明的具体实施例中，所述促组织再生材料是一种负载超支化聚赖氨酸的微凝胶，是通过HBPL的氨基官能团和双键的反应实现的。
在具体实施例中，所述的促组织再生微凝胶的制备方法如下：
1)称取一定量的聚乙二醇双丙烯酸酯(PEGDA)和HBPL混合在二甲亚砜(DMSO)溶液中，40℃搅拌反应6h后，用溶剂甲基叔丁基醚沉淀。旋蒸，充分除去溶剂；
2)向得到的产物中加入引发剂，在微流道装置中用蓝光(405nm)照射1-5min，得到微凝胶，具有促组织再生功能。其结构式如下：
Figure PCTCN2022097157-appb-000002
这类反应可以扩展到其他含有双键的单体。
在本发明的具体实施例中，所述促组织再生材料是一种负载超支化聚赖氨酸的纳米粒子，是通过以聚氨酯为基材而得到的。
在具体实施例中，所述的纳米粒子的制备方法如下：
1)称取一定量的聚富马酸丙二醇酯(PPF)在110℃真空(0.1mbar)中脱气干燥1h。降温至80℃，加入二苯基甲烷-4,4’-二异氰酸酯(MDI)，在氮气气氛中连续搅拌反应2h。
2)继续降温至50℃，将1,6-己二胺的N,N-二甲基乙酰胺溶液缓慢搅拌滴加入聚合物溶液中，直到所有异氰酸酯与扩链剂反应完全，得到透明溶液。
3)将HBPL和三乙胺加入反应物溶液中，40℃搅拌反应4h。最后，将HBPL接枝的聚合物滴加至不断搅拌的水中，得到纳米颗粒。其结构式如下：
Figure PCTCN2022097157-appb-000003
综上，所述促组织再生材料可以是将超支化聚赖氨酸通过物理作用(共混、吸附、渗透、自组装、离子络合等)、化学作用(氨基和双键的点击反应等)中的至少一种方法负载于载体之上，且载体形式包括但不限于金属、聚合物(水凝胶、微凝胶、聚氨酯、纳米粒子等)、无机非金属等材料。
以下结合实例进一步说明本发明的技术方案，但这些实例并不用来限制本发明。
实施例1
本发明的促组织再生的HBPL的制备过程如下：
称取13.73g L-赖氨酸盐酸盐于三口烧瓶中，再加入25mL去离子水。取4.2g KOH溶于15mL水中，加入恒压漏斗中缓慢滴入烧瓶中，反应2h。在氮气保护下，升温至150℃，反应24h后，向体系中加入200mL甲醇，离心，旋蒸，再加适量水，重新溶解产物。用截留分子量为3500的透析袋透析3天以上，冷冻干燥，得到HBPL。
为了考察HBPL的促再生能力，将HBPL配成25μg/mL的溶液。采用CCK-8法测试细胞在HBPL溶液中的增值情况。取生长状态良好的H9C2细胞，分为两组，一组加入普通的培养基，一组加入混有HBPL的培养基，每孔加入5×10 3个细胞，分别培养1-2天。到各个时间点的时候，将里面的培养基吸出，用无菌的磷酸盐缓冲液(PBS)漂洗三次，每孔加入配好的CCK-8溶液100μL，避光孵育2h后，每孔取出80μL，测试其在450nm波长下的吸光值(附图1)。如图所 示，混有HBPL溶液组有促进细胞增殖的现象，证明了HBPL促心肌细胞增值能力。
实施例2
本发明的负载超支化聚赖氨酸的促组织再生聚氨酯材料，是通过将HBPL与聚氨酯材料通过物理共混的作用得到的，其合成路线如图2。
负载超支化聚赖氨酸的促组织再生聚氨酯材料，以聚ε-己内酯二醇、1,3-双(4-羟基丁基)四甲基二硅氧烷软段，以六亚甲基二异氰酸酯(HDI)、1,4-丁二胺为硬段。聚ε-己内酯二醇的数均分子量为2.0kDa，1,3-双(4-羟基丁基)四甲基二硅氧烷的数均分子量为0.278kDa
其制备包括以下步骤：
1)取3g聚ε-己内酯二醇放入真空烘箱，干燥4h以上。将干燥后的聚ε-己内酯二醇加入到干燥容器中，在110℃真空(0.1mbar)中脱气干燥1h。然后加入435μL六亚甲基二异氰酸酯及催化剂辛酸亚锡45μL，在氮气保护下80～100℃反应至少2h，再加入16mL无水二氧六环溶解，得到固含量为12.5wt％的聚氨酯预聚物溶液。其中饱和脂肪族二异氰酸酯中异氰酸根基团与聚ε-己内酯二醇中羟基的摩尔比为1.8:1；
2)在得到的聚氨酯预聚物溶液中，加入1,3-双(4-羟基丁基)四甲基二硅氧烷(0.167g，0.6mmol)，在氮气氛围的保护下搅拌反应2h。1,3-双(4-羟基丁基)四甲基二硅氧烷为体系的第一组分扩链剂加入，和步骤1)未反应的聚ε-己内酯二醇的摩尔比为0.5:1。反应结束之后，将反应温度降至50℃，降低转速，缓慢滴加第二组分扩链剂1,4-丁二胺(0.0576g，0.6mmol)的1mL无水二氧六环溶液。在完全加入1,4-丁二胺后，恢复搅拌速度直到所有异氰酸酯与扩链剂反应完全，得到澄清透明的聚氨酯溶液。两种扩链剂的摩尔比为1：1；
3)将制得的聚氨酯溶液倒入冰无水乙醇中沉淀，离心、收集。将收集的聚氨酯固体再次溶于二氧六环溶剂中，并再次倒入冰无水乙醇中沉淀，反复数次，最后将所得的聚氨酯固体置于无水乙醇中搅拌4h以上，充分置换出溶剂。再放入去离子水中充分置换无水乙醇，冻干。最后得到具有良好力学性能的聚氨酯材料；
4)取0.5g聚氨酯材料，溶于溶剂六氟异丙醇中。将HBPL溶于一定量的六 氟异丙醇中，然后与聚氨酯的六氟异丙醇溶液混合均匀。倒入聚四氟乙烯的模具中，放入通风橱中充分挥发溶剂，得到负载HBPL的聚氨酯薄膜。
5)为了考察聚氨酯的促再生能力，将得到的聚氨酯薄膜用打孔器制成直径为8mm的圆片。采用CCK-8法测试细胞在材料表面的增值情况。将聚氨酯膜灭菌后放入48孔板中，取生长状态良好的成纤维细胞，每孔加入2×10 4个细胞，分别培养1-3天。到各个时间点的时候，将里面的培养基吸出，用无菌的PBS漂洗三次，每孔加入配好的CCK-8溶液200μL，避光孵育2h后，每孔取出100μL，测试其在450nm波长下的吸光值(附图3)。如图所示，负载了HBPL的聚氨酯材料有促进细胞增殖的现象，证明了HBPL促成纤维细胞增值的能力。
实施例3
本发明的负载超支化聚赖氨酸的促组织再生水凝胶，是通过活性氧响应性的水凝胶的环氧基团和HBPL的氨基进行化学反应所得到的，其制备步骤如下：
在氮气氛围保护下，将5.1g甲氧基聚乙二醇单甲基丙烯酸酯单体(Mn＝300)，1.28g甲基丙烯酸缩水甘油酯单体和0.66g过氧化二苯甲酰腈溶于300mL甲醇溶剂中，磁力搅拌，70℃加热回流10h；反应结束后将反应液在冰乙醚中沉淀，离心收集产物，干燥后得到粗产物；将粗产物溶于甲醇中，再于冰乙醚中沉降除杂，重复三次后收集产物，真空干燥得到产物1；
将0.5g第一步制备的产物、82.8mg丙酮[双-(2-氨基-乙基)-二硫缩醇]及51.4mg 3,3’-二硫代双(丙酰肼)溶解于4mL 0.5wt％聚乙烯醇溶液，得到水凝胶预溶液；
将水凝胶预溶液注入直径1.1cm的模具中，密封后置于37℃恒温水浴箱中，反应24h得到水凝胶。然后放入-20℃冰箱中冷冻24h，再取出放入4℃冰箱解冻12h，重复3次。将制得的水凝胶切成1mm厚的薄片，冷冻干燥得到干燥水凝胶片。然后置0.5mg/mL的超支化聚赖氨酸溶液中溶胀平衡，得到具有活性氧响应性的促组织再生水凝胶。
为了考察活性氧响应性的水凝胶的生物相容性，将得到的水凝胶放入无血清培养基浸泡24h，灭菌后加入血清备用。采用CCK-8法测试细胞在水凝胶浸提液的细胞毒性情况。取生长状态良好的成纤维细胞，分为三组，分别加入普通培养基、活性氧响应的水凝胶的浸提液、负载了HBPL的水凝胶的浸提液200μL。 每孔加入5×10 3个细胞，共培养1天后，将里面的培养基吸出，用无菌的PBS漂洗三次，每孔加入配好的CCK-8溶液100μL，避光孵育2h后，每孔取出80μL，测试其在450nm波长下的吸光值(附图4)。如图所示，负载了HBPL的水凝胶没有明显的细胞毒性，且负载了HBPL的水凝胶有促进细胞增值的趋势。

Claims (10)

  1. 一种具有自适应性的促组织再生材料，其特征在于，所述材料兼具有可提供氢键作用的酰胺键和可反应的氨基官能团。
  2. 根据权利要求1所述的一种具有自适应性的促组织再生材料，其特征在于，所述材料中含有超支化聚赖氨酸，且每个超支化聚赖氨酸含有的氨基数量为22～36个，所述的组织为心肌组织、神经组织、血管组织中的至少一种。
  3. 根据权利要求2所述的一种具有自适应性的促组织再生材料，其特征在于，所述材料在pH为5-10的环境下均可发挥作用。
  4. 根据权利要求1所述的一种具有自适应性的促组织再生材料，其特征在于，所述材料溶于溶液中，直接作为促组织再生药物。
  5. 根据权利要求1所述的一种具有自适应性的促组织再生材料，其特征在于，所述材料通过物理作用和/或化学作用负载于载体材料上形成促组织再生药物。
  6. 根据权利要求5所述的一种具有自适应性的促组织再生材料，其特征在于，所述的物理作用为共混、吸附、渗透、自组装、离子络合、链缠结中的一种或多种；所述的化学作用为本体接枝、表面接枝、共聚中的一种或多种。
  7. 根据权利要求5所述的一种具有自适应性的促组织再生材料，其特征在于，所述的载体材料为聚合物，存在形式为水凝胶、微凝胶、聚氨酯、纳米粒子中的一种或多种。
  8. 根据权利要求5所述的一种具有自适应性的促组织再生材料，其特征在于，所述的载体材料为金属或非金属材料。
  9. 根据权利要求1所述的一种具有自适应性的促组织再生材料，其特征在于，所述促组织再生材料是一种负载超支化聚赖氨酸的聚氨酯材料，以二醇和二胺为软段，以二异氰酸酯和扩链剂为硬段、超支化聚赖氨酸以物理共混的手段负载其中；
    或者是一种负载超支化聚赖氨酸的可抗粘连的聚氨酯纤维膜，是在聚氨酯弹性体材料层的外表面接枝亲水性聚乙二醇得到，所述聚氨酯弹性体包括由聚富马酸丙二醇酯和聚ε-己内酯二醇组成的软段和由饱和脂肪族二异氰酸酯及L-赖氨 酸二甲酯盐酸盐组成的硬段，具有多孔结构，超支化聚赖氨酸吸附于其中；
    或者是一种负载超支化聚赖氨酸的可注射水凝胶，超支化聚赖氨酸在浓度梯度的驱动下，渗透进入水凝胶内部；
    或者是一种负载超支化聚赖氨酸的自组装纳米粒子，是通过以壳聚糖为核，透明质酸和超支化聚赖氨酸依次为两层外壳，利用静电吸附作用层层叠加得到。
  10. 根据权利要求1所述的一种具有自适应性的促组织再生材料，其特征在于，所述的促再生材料是通过在微流道中聚乙二醇双丙烯酸酯的双键和超支化聚赖氨酸的氨基官能团反应得到的；
    或者是在以聚富马酸丙二醇酯为基础的聚氨酯材料上，通过聚富马酸丙二醇酯的双键和超支化聚赖氨酸的氨基官能团反应得到的。
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