WO2022111595A1 - 一种核壳微凝胶、氧气缓释材料、药物缓释制剂和一种多功能细胞包裹系统 - Google Patents

一种核壳微凝胶、氧气缓释材料、药物缓释制剂和一种多功能细胞包裹系统 Download PDF

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WO2022111595A1
WO2022111595A1 PCT/CN2021/133226 CN2021133226W WO2022111595A1 WO 2022111595 A1 WO2022111595 A1 WO 2022111595A1 CN 2021133226 W CN2021133226 W CN 2021133226W WO 2022111595 A1 WO2022111595 A1 WO 2022111595A1
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sustained
release
core
preparation
oxygen
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PCT/CN2021/133226
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French (fr)
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王钧平
董华
郑立新
周金生
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华源再生医学(香港)有限公司
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Priority claimed from CN202011343278.0A external-priority patent/CN112472682B/zh
Priority claimed from CN202110648315.7A external-priority patent/CN113476617A/zh
Application filed by 华源再生医学(香港)有限公司 filed Critical 华源再生医学(香港)有限公司
Publication of WO2022111595A1 publication Critical patent/WO2022111595A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • 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

Definitions

  • the invention relates to the technical field of microgels, in particular to a core-shell microgel, an oxygen sustained-release material, a drug sustained-release preparation and a multifunctional cell encapsulation system.
  • Type 1 diabetes mellitus is a disease characterized by immune-mediated destruction and loss of function of beta cells that secrete insulin from the pancreas, also known as insulin-dependent diabetes.
  • the World Health Organization 422 million people are diagnosed with diabetes worldwide; according to the Centers for Disease Control and Prevention, nearly 1.6 million Americans have type 1 diabetes, including about 187,000 children and adolescents.
  • the severe diabetes situation requires efficient and high-quality treatment methods to improve the quality of life.
  • the existing treatment methods include exogenous insulin administration, allogeneic nude islet transplantation, macro-encapsulated islet transplantation, and microencapsulated islet transplantation.
  • Exogenous insulin administration often includes insulin pump therapy (continuous subcutaneous insulin infusion, CSII) and multiple daily insulin injections (MDI).
  • CSII continuous subcutaneous insulin infusion
  • MDI multiple daily insulin injections
  • CSII is an effective and flexible method of insulin administration, equipped with self-monitoring of blood glucose, which can improve blood glucose management and clinical outcomes, but the pump itself may suffer from occlusion caused by twisting of the infusion tube and insulin accumulation, which can cause interruption of insulin infusion, resulting in Hyperglycemia and ketoacidosis; or severe hypoglycemia due to excessive insulin infusion due to faulty instrument calibration; or even pump discontinuation due to fat hypertrophy, infection and inflammation at the infusion site, and instrument wear; Pump incompatibility, sticking problems, and inconvenience in motor activities. MDI is a traditional and effective method to control the level of glycated hemoglobin.
  • Maintaining good glycemic control is susceptible to factors such as inaccurate doses, pain, needle phobia, acceptability and inconvenience, resulting in poor treatment outcomes, especially for I.
  • Children and adolescents with type 2 diabetes often have non-compliance and nocturnal hypoglycemia, which requires close supervision by guardians, which not only causes inconvenience in life but also affects the timeliness of insulin injection therapy.
  • Islet replacement therapy has been proposed for a long time as an alternative to exogenous insulin with the potential to eliminate secondary complications by restoring insulin signaling in patients.
  • intrahepatic transplantation using naked islets isolated from the pancreas of a deceased donor can improve problematic hypoglycemia, stabilize blood sugar levels, maintain target blood sugar control, and thus improve quality of life, and often does not require insulin therapy.
  • the deceased donor islets have the problem of low quantity and poor quality.
  • macro-encapsulated islet transplantation can produce fine geometric structures such as hollow fibers, planes, and cylinders through 3D printing and micromachining techniques for encapsulating a large number of islet cells at the same time.
  • small surface-to-volume ratios often make it difficult to achieve easy implantation and adequate mass transfer, It is prone to cell aggregation and islet death due to insufficient nutrition and oxygen supply due to proximity effect.
  • a large area of host cell infiltration can easily lead to fibrosis, resulting in graft failure.
  • Microencapsulated islet transplantation maximizes the surface-to-volume ratio by encapsulating islet cells separately, reduces the diffusion distance of oxygen and nutrients between cells and the host, and avoids the proximity effect caused by cell aggregation. It can also co-encapsulate anti-inflammatory drugs and pro-angiogenic factors. , oxygen supply particles, etc. to improve the survival rate of transplanted islets, and islet immune isolation in the encapsulation material can avoid or eliminate chronic systemic immunosuppression, so that the application of islet transplantation is more extensive.
  • the traditional electrostatic droplet method is to form droplets under the action of the electric field force and the driving force of the syringe pump and fall into the gel bath to solidify and form.
  • the diameter of microcapsules prepared by electrostatic droplet method is generally 500-1500 ⁇ m, and a large amount of capsule material hinders the transport of key solutes to islets, resulting in core hypoxia and necrotic diffusion barriers, hindering the transport of glucose and insulin, The resulting delay in glucose sensitivity and insulin secretion from the encapsulated islets weakens the advantage of microencapsulation over islet encapsulation and transplantation.
  • the droplet microfluidic method is more controllable in controlling the size of the microspheres, reducing the total transplant volume, and the diversity of the material system.
  • the dispersed phase is sheared by the continuous phase into droplets of uniform size, and the biocompatibility and permeability of the material system can also be optimized by multiple options. Enhance the immune isolation of islets, improve the survival rate of islet grafts, and reduce or completely eliminate immunosuppression. However, for mononuclear microencapsulated islets, it is prone to incomplete encapsulation, the direct exposure of islets to the host causes transplantation failure, and the diverse material system may reduce the quality of the environment around the islets.
  • a major problem with cell transplantation is the need to meet the high nutritional requirements of these cells after transplantation.
  • these cells are unable to function as a network of blood vessels to deliver oxygen to the cells, so the cells are "starved", leading to mass cell death.
  • Hypoxia is considered to be the leading cause of islet transplantation failure.
  • researchers mainly solve the problem of cell hypoxia in islet transplantation in four ways: 1. Reduce the distance that oxygen diffuses to the islet tissue in the islet transplantation device, 2. Increase the oxygen permeability of the cell wrapping material, 3. Before transplantation , to form a vascularized network in advance at the site of the transplant device, so that the blood flow is close to the transplanted tissue, and 4.
  • the last strategy of local oxygen supply is the most used, and the local production of specific oxygen can be realized by electrolysis of water, photosynthetic algae, artificial hemoglobin, etc.
  • the method of electrolyzing water is more complicated and requires the implantation of complex devices, while the second method requires the implantation of live algae into the human body, which is a safety hazard.
  • Artificial hemoglobin can only support oxygen release for up to 48 hours.
  • one of the main problems of cell transplantation therapy is immune rejection, and the most common method for this problem is to suppress the rejection of the transplant by the organ recipient by taking immunosuppressive agents.
  • immunosuppressive agents for example, in the field of diabetes treatment, many artificial pancreas transplants are accompanied by the use of various immunological drugs, such as Viacyte's PEC-direct products and Sernova's artificial pancreas devices are currently in the clinical validation stage, and they are implanted in patients. require immunosuppressants.
  • the use of immunosuppressants began in the 1970s, and after decades of development, its family has grown stronger.
  • the first-generation immunosuppressants are represented by corticosteroid methylprednisolone injection, tripterygium glycosides tablets, azathioprine (Imuran), and anti-lymphocyte immunoglobulin (ALG). differentiation of cells.
  • the first-generation immunosuppressants are characterized by nonspecificity.
  • the second-generation immunosuppressants are represented by cyclosporine (such as cyclosporine A, sanming, cyspin) and tacrolimus (tacrolimus, FK506), whose main role is to block the interleukin 2 of immunocompetent cells
  • the effector link of (IL-2) is dominated by lymphocytes and is relatively specific.
  • the third-generation immunosuppressants are represented by sirolimus (SRL) and mycophenoate mofetil (MMF), which mainly act on antigen presentation and intermolecular interactions, and have synergistic effects with second-generation preparations.
  • the fourth generation of immunosuppressants is represented by anti-IL-2 receptor monoclonal antibodies.
  • the main immunosuppressants that are currently used in clinical practice include tacrolimus, mycophenolate mofetil, rapamycin, and midazoribine.
  • rapamycin is a new potent immunosuppressant with less toxicity and potential discovered so far, which can prolong the survival of patients after transplantation and reduce the occurrence of acute rejection.
  • Rapamycin is an oral drug used systemically, and its main principle of action is to prevent the later stages of T cell activation, inhibiting the entry of cells from the G1 phase into the S phase. It blocks the combination of IL-2 and its receptors, so that Tc and Td cells cannot become sensitized T cells with immune response, and ultimately inhibit the differentiation and reproduction of T cells and exert their immunosuppressive effect.
  • rapamycin The main adverse reactions of rapamycin are bone marrow suppression and hyperlipidemia, mainly manifested as thrombocytopenia, leukopenia, decreased serum creatinine level, increased serum triglyceride and cholesterol levels, long-term use and nephrotoxicity. Another side effect of rapamycin is its toxicity to islets. Several studies have shown that rapamycin is toxic to the islets themselves while prolonging the survival of islet grafts through immunosuppressive effects. The direct damage of rapamycin to pancreatic islets is mainly manifested in three aspects: (1) directly inhibiting the secretion of insulin from islet ⁇ cells; (2) inhibiting islet cell viability and promoting islet cell apoptosis; (3) inhibiting islet cell proliferation.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a core-shell microgel, an oxygen sustained-release material, a drug sustained-release preparation and a multifunctional cell encapsulation system.
  • a first aspect of the present invention provides a core-shell microgel, comprising an inner core and a shell layer, the inner core is made from a core material including a photocurable monomer, an active ingredient and a photoinitiator, and the shell layer is also called a microgel Gel shell layer, the material of the microgel shell layer is anti-nonspecific protein adsorption material.
  • the core material used in the embodiment of the present invention is a photocurable material system and has good biocompatibility.
  • the core material can be used to encapsulate the active ingredients well, and the microgel shell material is used to encapsulate the core.
  • the risk of acute immune rejection from direct exposure of active ingredients such as islets can be avoided, and the shell material used at the same time is a material with anti-non-specific protein adsorption, which can reduce chronic immune rejection and reduce or eliminate long-term immune suppression.
  • the active ingredient is selected from at least one of cells, drugs, and protein-based active factors.
  • the cells can be exemplified by islets, stem cells, etc.
  • the protein-based active factors can be exemplified by various growth factors such as vascular endothelial growth factor, etc.
  • the core material further comprises extracellular matrix material.
  • the extracellular matrix substances can be exemplified by gelatin, collagen, chondroitin sulfate, hyaluronic acid and the like. During preparation, extracellular matrix substances can be mixed into the core material to improve the gel-forming quality and cell compatibility of the microgel.
  • the photocurable monomer includes hyaluronic acid methacrylate (HAMA), gelatin methacrylate, pectin methacrylate, glycidyl methacrylate modified silk protein at least one of the materials.
  • HAMA hyaluronic acid methacrylate
  • gelatin methacrylate gelatin methacrylate
  • pectin methacrylate pectin methacrylate
  • glycidyl methacrylate modified silk protein at least one of the materials.
  • the anti-nonspecific protein adsorption material is a hydrogel.
  • the anti-nonspecific protein adsorption material comprises at least one of polyethylene glycol diacrylate (PEGDA), methacrylated carboxybetaine (CBMA), and methacrylated sulfobetaine A sort of.
  • the anti-nonspecific protein adsorption material is a mixture of polyethylene glycol diacrylate and methacrylated carboxybetaine.
  • the shell material is a blend of CBMA and PEGDA, the adsorption of proteins on the gel surface can be reduced.
  • the mass concentration of CBMA in the blend material does not exceed 12% g/mL, the best anti-adsorption effect can be achieved.
  • the second aspect of the present invention provides the preparation method of the above-mentioned core-shell microgel, comprising the following steps:
  • the inner core is immersed in the solution of the second photoinitiator, then transferred to the material solution of the microgel shell layer, and left to stand in the dark, and then light-cured to obtain a core-shell microgel.
  • an extracellular matrix-like microgel is prepared by using droplet microfluidic technology, which provides an environment suitable for the survival of pancreatic islets and conducive to the secretion of insulin for active ingredients such as pancreatic islets; secondly, the diffusion of photoinitiators is used.
  • a shell structure is constructed on the basis of the single-core microgel core, which realizes the secondary encapsulation of active ingredients such as islets, and at the same time, the use of anti-non-specific protein adsorption materials reduces the immune rejection of the graft and improves transplantation. It provides a feasible technical solution for the realization of blood sugar reversal and insulin independence in patients with type I diabetes.
  • single emulsion droplets are prepared using microfluidic technology.
  • the flow rate ratio of the continuous phase and the dispersed phase is (10-40):1, which may be 40:1, 30:1, 20:1, 10:1.
  • the oil phase includes HFE-7500 fluorinated oil.
  • Surfactants can be added to the oil phase to help improve the stability of the resulting single emulsion droplets.
  • the dispersed phase is formed by dissolving the first photoinitiator in the photocurable monomer solution, and the photocurable monomer solution may be 2, 3, 4, and 5 wt % methacrylated transparent
  • the acid can be 5, 6, 7, 8, 9, 10 wt % of methacrylated gelatin, or a mixed solution of the above two in different proportions.
  • the dispersed phase can also be a blend of the above two with collagen, chondroitin sulfate, gelatin, hyaluronic acid, and the like.
  • the first photoinitiator and the second photoinitiator are LAP blue light initiators.
  • the concentration of the solution of the second photoinitiator is 0.3-3 wt %, and may be 0.3, 0.5, 1, 2, 3 wt %.
  • the soaking time is 10min-12h, which may be 10min, 20min, 30min, 1h, 2h, 3h...12h.
  • the third aspect of the present invention provides the use of the above-mentioned core-shell microgel or the core-shell microgel prepared according to the above-mentioned preparation method of the core-shell microgel in the preparation of a medicine for treating diabetes.
  • the invention provides an oxygen sustained-release material, a preparation method and a medicine thereof.
  • the oxygen sustained-release material can continuously release oxygen for a long time and can be applied to local oxygen supply.
  • the present invention provides an oxygen sustained-release material, the oxygen sustained-release material is microspheres, and the microspheres include a degradable biological material and a peroxide dispersed in the degradable biological material, and the degradable biological
  • the material is polycaprolactone (PCL), polylactic-co-glycolic acid (PLGA), or a mixed material of polycaprolactone and polylactic acid (PLA).
  • the present invention provides an oxygen sustained-release material, which uses polycaprolactone, polylactic acid-glycolic acid copolymer, or a mixed material of polycaprolactone and polylactic acid as a matrix of a degradable material, and has a suitable degradation rate and can better control oxygen
  • the persistence and total amount of release, the formed oxygen sustained-release material can provide oxygen support for a long time when it is subsequently mixed with the cell therapy agent to form a drug, which can effectively prevent the cell therapy agent from dying due to hypoxia and promote cell therapy.
  • the oxygen sustained-release material of the present invention can subsequently provide the required oxygen supplement for the cell therapeutic agent, plays the role of an oxygen transport bridge until the formation of blood vessels, provides natural oxygen for the survival of the cell therapeutic agent, and has a good application prospect.
  • the peroxide is selected from at least one of calcium peroxide, potassium peroxide, sodium peroxide, hydrogen peroxide, magnesium peroxide, and urea peroxide; preferably, the The peroxide is calcium peroxide.
  • the degradable biomaterial is a mixed material of polycaprolactone and polylactic acid, and the mass ratio of the polycaprolactone to the polylactic acid is 0.1-100:1.
  • the degradation rate of polycaprolactone is slow, and the alkaline substances produced by the reaction of peroxides, such as calcium hydroxide produced by the reaction of calcium peroxide, will form an alkaline environment and have an impact on the surrounding cells, and the degradation of polylactic acid is fast and The product is slightly acidic.
  • Adding polylactic acid to the slow-release material can adjust the oxygen release rate and neutralize the alkalinity of alkaline substances such as calcium hydroxide, so as to achieve the purpose of increasing the oxygen release rate and reducing the influence of the reaction product.
  • the mass ratio of the degradable biomaterial to the peroxide is 0.1-100:1 based on the mass of the microspheres.
  • the particle size of the microspheres ranges from 1 ⁇ m to 3 mm. In some further embodiments of the present invention, the particle size of the microspheres is 100-300 ⁇ m.
  • the present invention also proposes a preparation method of the above-mentioned oxygen sustained-release material, comprising the following steps:
  • the oil phase is mixed with the water phase to form microspheres.
  • the water phase is an aqueous solution of polyvinyl alcohol or a surfactant solution; the surfactant solution is an aqueous surfactant solution.
  • the present invention also provides a medicine, comprising an oxygen sustained-release material and a cell therapy agent, wherein the oxygen sustained-release material is the above-mentioned oxygen sustained-release material or the oxygen sustained-release material prepared according to the above-mentioned preparation method.
  • the cell therapeutic agent is one or more combinations of pancreatic islet cells, insulin-producing cells, hydrogel-encapsulated islet cells, and hydrogel-encapsulated islet-producing cells.
  • the used hydrogel is a biocompatible material, which is used to encapsulate and protect islet cells.
  • the above-mentioned pancreatic islet cells can be pancreatic islet cells isolated from human donated organs or pancreatic islet cells isolated from animal organs, and the insulin-producing cells can be gene-edited insulin-producing cells or insulin-producing cells produced by stem cell differentiation. Pancreatic islet cells or insulin-producing cells can be used directly as naked cells or encapsulated in hydrogels.
  • plurality refers to two or more.
  • the hydrogel is selected from one or more combinations of sodium alginate, gelatin, chitin, cellulose, polyethylene glycol, polyvinyl alcohol, and hyaluronic acid.
  • the hydrogel used can be a single material or a mixture of multiple materials.
  • Such hydrogels have inflammatory factors, exudate adsorption or other toxin adsorption, and can improve wound inflammation, prevent wound deterioration, and protect cells when used for wound repair.
  • the invention provides a drug sustained-release preparation which can effectively slow down the release speed of the immunosuppressant and improve its sustained-release effect.
  • the purpose of the present invention is also to propose the preparation method of the above-mentioned drug sustained-release preparation.
  • the purpose of the present invention is also to propose a biological material.
  • Another object of the present invention is to provide a cell transplantation device.
  • the present invention provides a medicine sustained-release preparation, which comprises:
  • Particles including immunosuppressants and acetalized cyclodextrins loaded with immunosuppressants
  • a shell coats the particles, and the composition of the shell includes a surfactant.
  • Cyclodextrin is a hollow near-cylindrical structure with a hydrophobic area inside and a hydrophilic area on both sides of the hydroxyl position, so it can be loaded with immunosuppressants inside, and after the acetal reaction, the acetalization formed.
  • cyclodextrin can play a more stable loading effect with immunosuppressants, so as to play a certain slow release effect; at the same time, the particles formed after loading are re-coated with surfactant to improve the internal
  • the encapsulation effect of the immunosuppressant in the preparation further improves the stability of the immunosuppressant and prolongs the release time of the immunosuppressant.
  • cyclodextrin refers to a polysaccharide composed of several D-type glucopyranose connected head-to-tail with ⁇ -1,4-glycosidic bonds. Since the glycosidic bonds cannot rotate freely, cyclodextrins are slightly conical. Hollow cylindrical structure.
  • acetalized cyclodextrin refers to the acetal reaction (which can be catalyzed by an acid catalyst) between the secondary hydroxyl groups of C2 and C3 of at least one glucose unit in the cyclodextrin and an aldehyde or ketone to form a cyclic acetal. product obtained.
  • the type of aldehyde or ketone and the acetal reaction product can be adaptively adjusted according to specific needs.
  • the acetalized cyclodextrin is obtained by acetalization of ⁇ -cyclodextrin.
  • ⁇ -Cyclodextrin has suitable hydrophilicity and hydrophobicity and can effectively encapsulate immunosuppressants.
  • acetalized cyclodextrin is as follows:
  • x is an integer from 1 to 7, and n is any positive integer;
  • R 1 means R 2 means
  • the surfactant is a nonionic surfactant.
  • the nonionic surfactant is selected from the group consisting of polyethylene glycol, polypropylene glycol, polyvinyl alcohol, fatty alcohol polyoxyethylene ether, Tween, Spann, glycerol monostearate, polyvinyl alcohol At least one of ether, cellulose-based surfactant, starch-based surfactant, and polyvinylpyrrolidone.
  • the use of the above-mentioned nonionic surfactant can form a better encapsulation effect on the particles, so that the immunosuppressant has stronger stability therein and further improves the sustained-release effect.
  • the immunosuppressive agent is selected from at least one of tacrolimus, mycophenolate mofetil, rapamycin, mizoribine, and dexamethasone.
  • the administration of the immunosuppressive agent modulates the immune response of the administered object, reduces the immune rejection of the graft by the organ, so that the graft can function normally in the administered object.
  • the present invention also provides a preparation method of the above-mentioned drug sustained-release preparation, the preparation method comprising the following steps:
  • the drug sustained-release preparation is prepared by the emulsification method.
  • the drug sustained-release preparation prepared by this method is not only loaded with the particles formed by the immunosuppressant, but also forms a good coating of the surfactant, and this coating can also make the drug Sustained release is more durable.
  • step S2 further comprises mixing and stirring the emulsion and the solution of the second surfactant.
  • the encapsulation effect of the shell layer formed by the first surfactant is strengthened by the further mixing of the second surfactant, so that the stability of the drug sustained-release preparation is improved, the release time is prolonged, and the reduction is reduced under the same conditions. It reduces the exposure of immunosuppressants to the body, thereby reducing side effects or adverse reactions when used in large quantities.
  • the manner of mixing and stirring the emulsion and the solution of the second surfactant is magnetic stirring.
  • the temperature at which the emulsion and the solution of the second surfactant are mixed and stirred is 30-60°C, preferably 35-55°C, more preferably 40-50°C.
  • Non-limiting examples thereof include 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C.
  • the first surfactant and the second surfactant are only used to distinguish the surfactants added in different processes, and do not represent any difference between the surfactants used in the two processes.
  • the first surface active agent The active agent and the second surfactant can be the same or different optional surfactants, further each optionally selected from nonionic surfactants.
  • the present invention also provides a biological material, which includes the gel and the above-mentioned drug sustained-release preparation.
  • the biological material can provide the immunosuppressant more stably and lastingly, and prolong the release time of the immunosuppressant.
  • the gel material may be a hydrogel material with optional biocompatibility, such as a gel formed by at least one of betaine, hyaluronic acid, and gelatin.
  • the biological material includes gel particles, and the gel particles include a sustained-release drug formulation and a gel layer coated on the outside of the sustained-release drug formulation.
  • the biomaterial includes a gel and the above-described sustained-release drug formulation dispersed in the gel.
  • Transplanted cells refer to cells that optionally replace damaged cells or tissue in the body of the user for repair, non-limiting examples of which include stem cells (eg, mesenchymal stem cells, bone marrow stem cells, hematopoietic stem cells), dendritic cells, spleen cells , pancreatic islet cells, etc.
  • stem cells eg, mesenchymal stem cells, bone marrow stem cells, hematopoietic stem cells
  • dendritic cells eg., spleen cells, pancreatic islet cells, etc.
  • a fourth aspect of the present invention provides a cell transplantation device, the cell transplantation device comprising the above-mentioned drug sustained-release preparation, or the above-mentioned biological material.
  • the drug sustained-release preparation in the cell transplantation device can provide an immunosuppressant for the user in a lasting and long-term effect, thereby prolonging the immunosuppressive effect while reducing cytotoxicity, improving the survival time and viability of the transplanted cells, and enabling the repair of the cell transplantation device.
  • the effect is more obvious.
  • the cells loaded in the cell transplantation device can be cells that can optionally replace damaged cells or tissues in the body of the user for repair, non-limiting examples of which include stem cells (eg, mesenchymal stem cells, bone marrow stem cells, hematopoietic stem cells) , dendritic cells, spleen cells, islet cells, etc.
  • stem cells eg, mesenchymal stem cells, bone marrow stem cells, hematopoietic stem cells
  • dendritic cells eg., spleen cells, islet cells, etc.
  • the present invention also provides a multifunctional cell encapsulation system, including the core-shell microgel described in the above scheme or the core-shell microgel prepared by the preparation method of the core-shell microgel, and an auxiliary agent;
  • the adjuvant includes one or two of the following formulations:
  • the multifunctional cell encapsulation system of the present invention can deliver therapeutic cells and/or drugs into the body of a patient by injection or minimally invasive implantation, wherein the drug sustained-release preparation is embedded in the core-shell microgel, while oxygen
  • the slow-release material is mixed with the core-shell microgel and implanted in the patient.
  • the biological material of the present invention will be mixed with a medical fibronectin solution after being implanted in a patient, and a fibronectin glue is formed by utilizing the principle of coagulation to fix the core-shell microgel on the transplant site.
  • Example 1 is a schematic diagram of the preparation process of preparing core-shell microgels in Example 1 of the present invention
  • FIG. 2 is a structural diagram of a microfluidic chip used in Embodiment 1 of the present invention.
  • Fig. 3 is the electron microscope picture of the embodiment of the present invention 1 GelMA inner core microgel and GelMA/PEGDA core-shell microgel;
  • Fig. 4 is the electron microscope picture and diameter distribution picture of the core-shell microgel prepared by using HAMA or GelMA as the dispersed phase system in Example 1 of the present invention
  • Fig. 5 is the cell staining experiment diagram under the fluorescence microscope picture of Example 1 of the present invention.
  • Example 6 is a graph of the survival rate of BMSCs under different conditions in Example 1 of the present invention.
  • Fig. 7 is the nuclear magnetic spectrum of the carboxybetaine methacrylate used in the embodiment of the present invention 1;
  • Example 8 is an image of green fluorescent protein on different microgel surfaces in Example 1 of the present invention.
  • Example 9 is a graph showing the adsorption capacity of FITC-BSA on different microgel surfaces in Example 1 of the present invention.
  • Figure 10 is the in vivo staining diagram of four groups of core-shell microgels wrapped for 4 days in Example 1 of the present invention.
  • Figure 11 is a graph of insulin release from core-shell microgels under different glucose concentrations in Example 1 of the present invention.
  • Figure 12 is the concentration ratio of insulin released by the core-shell microgel at high glucose concentration and low glucose concentration in Example 1 of the present invention
  • Fig. 13 is the result diagram of alizarin red dyeing of oxygen sustained-release material in Example 2 of the present invention.
  • Fig. 14 is the result diagram of in vitro oxygen release test performed by the oxygen sustained-release material in Example 2 of the present invention.
  • Fig. 15 is the result diagram of oxygen release test carried out by the oxygen sustained-release material in Example 3 of the present invention.
  • Figure 16 is a graph of pH changes before and after the oxygen sustained-release material releases oxygen in Example 3 of the present invention.
  • 17 is a fluorescent staining diagram of live/dead cells of islet cells in groups 1 to 6 after culture in Example 4 of the present invention.
  • Figure 18 is the survival rate of islet cells in groups 1-6 in Example 4 of the present invention on the 1st day, the 3rd day and the 7th day;
  • Fig. 19 is the hydrogen nuclear magnetic resonance spectrogram of cyclodextrin and acetalized cyclodextrin in Example 5 of the present invention.
  • Figure 20 is a schematic diagram of the preparation and action of a sustained-release drug preparation in Example 6 of the present invention.
  • Fig. 21 is the electron micrograph of the medicine sustained-release preparation of different acetalization time in the embodiment of the present invention 6 and the corresponding diameter statistical result;
  • Figure 22 is the sustained-release effect diagram of different forms of rapamycin in Example 8 of the present invention.
  • Figure 23 is a graph showing the effect of different forms of rapamycin on the inhibition of inflammation in RAW 264.7 cells in Example 9 of the present invention.
  • Figure 24 is a graph of the slow-release effect of different forms of rapamycin on the inflammation inhibition of RAW 264.7 cells in Example 10 of the present invention.
  • Figure 25 is the process of modeling the diabetic mice in Example 11 of the present invention, and the blood sugar changes in the mice after injection of the drug streptozotocin STZ;
  • Figure 26 is the process of modeling the diabetic mice in Example 11 of the present invention, and the weight changes of the mice after injection of the drug streptozotocin STZ;
  • Figure 27 shows the results of glucose control after wrapping rat pancreatic islets with different multifunctional cell encapsulation systems in Example 11 of the present invention and then transplanting them into diabetic mice;
  • Figure 28 is the intraperitoneal glucose tolerance test of rat pancreatic islets wrapped by the multifunctional cell encapsulation system in Example 11 of the present invention in diabetic mice for 90 days;
  • Fig. 29 is a photograph of taking out the graft of the rat pancreatic islet wrapped by the multifunctional cell wrapping system in Example 11 of the present invention.
  • Figure 30 shows the degradation of the oxygen sustained-release material in the multifunctional cell wrapping system in Example 11 of the present invention.
  • Figure 31 shows the situation of the multifunctional cell encapsulation system encapsulating rat pancreatic islets in diabetic mice in Example 11 of the present invention
  • Figure 32 shows the results of hematoxylin-eosin staining of tissue sections taken out 90 days after transplantation of rat pancreatic islets by the multifunctional cell encapsulation system in Example 11 of the present invention.
  • references to the terms “one embodiment,” “some embodiments,” “exemplary embodiment,” “example,” “specific example,” or “some examples”, etc., are meant to incorporate the embodiments
  • a particular feature, structure, material, or characteristic described or exemplified is included in at least one embodiment or example of the present application.
  • schematic representations of the above terms do not necessarily refer to the same embodiment or example.
  • the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
  • the existing microfluidic device is used to prepare core-shell microgels.
  • the syringe fixed on the micro-injection pump and the microfluidic chip fixed under the high-speed camera microscope are connected by a needle with a connecting tube , which constitute the sampling system and the collection system.
  • the embodiment of the present invention can improve the preparation process of the core-shell microgel by designing the shape and size of the microfluidic channel on the microfluidic chip, and the channel shape can be a T-shaped channel, a Y-shaped channel, a fluid focusing channel, etc.; Take the T-channel as an example, the size of the T-channel can be 200-100-200, 300-200-300, etc.
  • the preparation method of the microfluidic chip is in the prior art, and the microfluidic chip with different microfluidic channels can be produced by operations such as photolithography, mold turning, sealing, channel modification and the like.
  • the preparation process of preparing core-shell microgels according to the embodiment of the present invention is as follows: the raw materials containing active ingredients and photoinitiators are photocured to form a core, and the core is washed into a photoinitiator solution for pregelation, and then transferred into the monomer solution, post-photocuring and washing to form core-shell microgels.
  • the T-shaped channel has a larger shear force at the meeting point of the continuous phase and the dispersed phase, and the fabrication of the channel is relatively simple.
  • the embodiment of the present invention can use the microfluidic chip as shown in FIG. 2 when wrapping the islet cells, (a) represents the size design diagram of the microfluidic chip, (b) ) represents the physical image of the microfluidic chip, which has a T-shaped channel with a continuous phase channel width of 200 ⁇ m and a channel width narrowing to 150 ⁇ m at the intersection of the two phases.
  • Preparation of core-shell microgels soak the prepared inner core in a deionized aqueous solution of 0.2wt% LAP for 3 min in the dark to allow the initiator to penetrate, wash 2-3 times in deionized water and absorb the excess aqueous solution, followed by It was transferred to 20% polyethylene glycol diacrylate solution (PEGDA), placed in the dark for 30 s, and subjected to blue light irradiation for 1 min to make the microgel interface photo-crosslinking to form a shell structure. After thorough washing with water, microgels with core-shell structure were obtained.
  • PEGDA polyethylene glycol diacrylate solution
  • the core-shell microgel was successfully prepared in the embodiment of the present invention.
  • the shell layer can still maintain a small size after encapsulation, and at the same time, the use of anti-protein adhesion materials can reduce the immune rejection of the graft and improve the survival of the graft.
  • the concentration of the LAP blue light initiator can be 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5 wt%, and the UV irradiation time can be 1, 2, 3, 4, 5, 10min.
  • the link parameters can be used to prepare the inner core microgels with different diameters of 100, 150 and 200 microns.
  • hyaluronic acid, gelatin, collagen, chondroitin sulfate and other ingredients can be mixed to form a hydrogel to encapsulate the cells, simulate the structure of the extracellular matrix, and prevent cell apoptosis.
  • the concentration of the initiator, the soaking time of the initiator, the material of the shell layer, the concentration of the shell layer material, the resting time in the dark, and the illumination time the thickness of the shell layer and the resistance of the shell layer are improved.
  • the blue light initiator LAP concentration can be 0.2, 0.3, 0.5, 1, 2wt%
  • the light time can be 1, 2, 3, 4, 5, 10min
  • the shell material can be poly Ethylene glycol diacrylate (PEGDA), methacrylated carboxybetaine (CBMA), methacrylated sulfobetaine.
  • the electron microscope image (scale bar is 200 microns) and diameter distribution of the core-shell microgels prepared without encapsulating active ingredients with HAMA or GelMA as the dispersed phase system are shown in Figure 4, where (a) represents the core prepared by the GelMA system Electron microscope image of shell hydrogel, (A) shows the diameter distribution statistics of core-shell hydrogel in (a), (b) shows the electron microscope image of core-shell hydrogel prepared by HAMA system, (B) shows (b) Statistical plot of diameter distribution of midcore-shell hydrogels. If the size of the core-shell microgel is too large, it will affect the transport of substances inside and outside the hydrogel and affect the cell activity. If the size is too small, the final volume of the encapsulated islets will be too large.
  • both dispersed phase systems can successfully prepare core-shell microgels. More than 75% of the core-shell microgels of GelMA and HAMA systems have a diameter of about 200 microns, which is conducive to encapsulating active ingredients.
  • the initiator takes blue light LAP (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate) as an example, and the cells take stem cells (BMSC) as an example.
  • LAP lithium phenyl (2,4,6-trimethylbenzoyl) phosphate
  • BMSC stem cells
  • the specific experimental process is: cells wrapped with HAMA/GELMA Soak into 0.5% LAP solution for soaking treatment, then replace the soaking solution, and finally use blue light for 60 s, repeat the operation, and the specific treatment methods are shown in Table 1.
  • Figure 5 shows the cell staining experiments of different groups under the fluorescence microscope, the green on the left represents live cells, and the red on the right represents dead cells.
  • Figure 6 represents the survival rate of BMSCs under different conditions. In the presence of monomer (PEGDA), blue light-induced initiator free radicals had little effect on cell activity.
  • PEGDA monomer
  • CBMA carboxybetaine methacrylate
  • Figure 7 its nuclear magnetic spectrum is shown in Figure 7.
  • the positive and negative charge groups carried by the zwitterionic hydrogel can bind free water molecules through electrostatic interaction and ionized solvent.
  • the formation of a hydration layer on the surface of the material prevents further adsorption of proteins or cells on the surface of the material. Therefore, carboxybetaine methacrylate (CBMA) with non-specific anti-protein adsorption was prepared and can be used to improve the anti-protein adhesion properties of microgels.
  • FIG 8 shows the images of green fluorescent protein with different microgel surfaces
  • green is the fluorescently labeled protein (FITC-BSA)
  • FITC-BSA fluorescently labeled protein
  • Figure 9 shows the different microgels Adsorption amount of FITC-BSA on the gel surface.
  • mouse islet cells Islet
  • mouse bone marrow mesenchymal stem cells BMSCs
  • Islet-GelMA 1GelMA-coated pure islets
  • Islet-HAMA 2HAMA-coated Pure islet
  • 3GelMA co-encapsulates stem cells and islets
  • 4HAMA co-encapsulates stem cells and islets
  • Figure 10 The in vivo staining of the four groups of core-shell microgels after 4 days of encapsulation is shown in Figure 10. It can be seen from the figure that HAMA or GelMA system materials and whether or not encapsulated stem cells (BMSCs) have no negative effect on the activity of pancreatic islets.
  • FIG. 11 shows Insulin release at different glucose concentrations each week. It can be seen from Figure 11 that the insulin release of different groups will have differences in insulin release due to the change of high glucose concentration and low glucose concentration environment, indicating that the corresponding sensitivity of islets to glucose is not obvious after 10 weeks of culture. Variety.
  • Figure 12 shows the insulin concentration and concentration ratio (i.e. stimulation index) at high and low glucose concentrations.
  • This embodiment provides a series of oxygen sustained-release materials, which are prepared according to the following steps:
  • PCL Polycaprolactone
  • CaO 2 , CPO calcium peroxide
  • the reaction raw materials were added to the organic solvent DCM (dichloromethane), and fully stirred and mixed with a homogenizer to form a suspension, which was used as an oil phase.
  • PVA polyvinyl alcohol
  • the oxygen sustained-release material prepared in this example was used for alizarin red dyeing.
  • the dyeing result is shown in Figure 13. It can be seen from the figure that with the increase of the amount of calcium peroxide added, the red brightness increases, indicating that the microspheres are over
  • This embodiment provides a series of oxygen sustained-release materials, which are prepared according to the following steps:
  • PCL with calcium peroxide (CaO 2 , CPO for short) to form the reaction raw materials.
  • the mass fraction of CaO 2 in the reaction raw materials are 0% (PCL group), 25% (PCL+25% CPO group) and 50% (PCL group). +50% CPO group).
  • the mixed material is formed by blending, and the mixed material is mixed with CPO to form the reaction raw material.
  • the mass fraction of CaO2 in the reaction raw material is 50% (marked as PCL/PLA+ 50% CPO group).
  • the reaction raw materials prepared above were added to the organic solvent DCM (dichloromethane), and fully stirred and mixed with a homogenizer to form a suspension, which was used as an oil phase.
  • DCM diichloromethane
  • Dissolve PVA polyvinyl alcohol
  • PVA aqueous solution with a mass fraction of 1.5%, which is used as the water phase.
  • Organic solvent, the oxygen slow-release material is obtained.
  • the oxygen release amount of the PCL group material is 0.0mL/(g ⁇ Day)
  • the oxygen release amount of the PCL+25%CPO group material is 0.238mL/(g ⁇ Day)
  • the PCL+50%CPO group is 0.238mL/(g ⁇ Day).
  • the oxygen release amount of the group material is 0.508mL/(g ⁇ Day)
  • the oxygen release amount of the PCL/PLA+50%CPO group material is 0.625mL/(g ⁇ Day)
  • the results show that the oxygen slow-release material of the embodiment of the present invention Oxygen can be continuously supplied, and the release rate of oxygen is improved after the content of peroxide is increased, and the release rate of oxygen can be further increased after adding PLA.
  • the pH of deionized water itself is about 6.5. Since the calcium peroxide added to the oxygen slow-release material will immediately react with water to form calcium hydroxide, which will increase the pH of the solution, even after adding the oxygen slow-release material When the pH was measured immediately, the pH value also increased, and with the increase of the calcium peroxide content, the increase of the solution pH also increased. Compared with the pH before the sustained release, the pH after the sustained release of oxygen for 40 days was increased, but the pH of the PCL/PLA+50%CPO group did not increase significantly, indicating that the degradation products of PLA had a certain pH-lowering buffering effect.
  • PLA can be considered in the oxygen sustained release material. Slightly acidic, it can adjust the rate of oxygen release and neutralize the alkalinity of calcium hydroxide. From the results, compared with the oxygen sustained-release material without PLA, the oxygen release rate of the oxygen sustained-release material with PLA increased, and the pH value of the solution decreased after 40 days of oxygen release, indicating that in the sustained-release material
  • the addition of polylactic acid can adjust the oxygen retardation rate and neutralize the alkalinity of calcium hydroxide, which can achieve the purpose of increasing the epoxy rate and reducing the influence of reaction products.
  • mice were cultured in a conventional aerobic environment; group 2: islet cells were cultured in a hypoxic environment; group 3: islets and PCL+50% CPO group was cultured in hypoxic environment; group 4: islets and PCL/PLA+50% CPO group were cultured in hypoxic environment; group 5: islets and PCL+50% The oxygen sustained-release material in the CPO group was cultured in a hypoxic environment; group 6: islets and the oxygen sustained-release material in the PCL/PLA+50% CPO group were cultured in a hypoxic environment.
  • the cell therapeutic agent used in the experimental process of this example is islet cells and islet cells wrapped with GelMA (methacrylated hydrogel).
  • the preparation steps of the islet cells wrapped with GelMA are as follows: the islet cells are mixed with GelMA to obtain .
  • Figure 17 shows live/dead cell staining of islet cells in groups 1-6 after culture
  • Figure 18 shows islet cells in groups 1-6 on day 1, day 3 and day 7
  • the survival rate as can be seen from Figures 17 and 18, in the hypoxic environment, the islet cells in group 2 were not packaged, nor were the oxygen sustained-release materials prepared in the embodiment of the present invention added, and the naked islet cells were recovered within one day. Apoptosis was evident, and cell viability had dropped to 26% after three days.
  • oxygen sustained-release material was added. After blending with oxygen sustained-release material, the activity of both naked islet cells and hydrogel-encapsulated islet cells was greatly improved, which was comparable to that of normal environment. There is no significant difference in islet cells, indicating that the oxygen sustained-release material prepared in the example of the present invention can provide the oxygen required for the survival of islet cells, and the hydrogel encapsulation does not affect the diffusion and transport of oxygen.
  • the oxygen sustained-release material provided by the present invention can be mixed with islet cells or hydrogel-encapsulated islet cells to provide local oxygen supply for islet cells for a long time, and can be mixed and used as a medicine.
  • the cell therapy agent used in the above examples is illustrated by taking pancreatic islet cells as an example. Those skilled in the art will know that it is also applicable when using other therapeutic cells to replace pancreatic islet cells.
  • the hydrogel has no effect on the diffusion of oxygen.
  • GelMA those skilled in the art can also choose materials such as gelatin and chitin to wrap the cells, and the corresponding effects can be expected.
  • the present embodiment provides an acetalized cyclodextrin, and the preparation method of the acetalized cyclodextrin is as follows:
  • x is an integer from 1 to 7, and n is any positive integer;
  • R 1 means R 2 means
  • Figure 19 shows the results of proton nuclear magnetic resonance spectroscopy of cyclodextrin and acetalized cyclodextrin, where A is cyclodextrin and B is acetalized cyclodextrin. As can be seen from the figure, the product after the reaction has successfully undergone acetalization reaction.
  • the present embodiment provides a drug sustained-release preparation, and the preparation method of the drug sustained-release preparation is as follows:
  • FIG. 20 The preparation process and action principle of the drug sustained-release preparation are shown in Figure 20.
  • Acetalized cyclodextrin is obtained from cyclodextrin through acetalization reaction, and cyclic acetal and chain acetal are formed on both sides of its openings. aldehyde.
  • a drug sustained-release preparation comprising a surfactant shell layer is prepared by an emulsification method and a polyvinyl alcohol surfactant.
  • the drug sustained-release preparation will gradually and slowly release the rapamycin contained therein, and the sustained-release rapamycin can effectively reduce the inflammatory response caused by immune rejection, and achieve a lasting anti-inflammatory effect.
  • the acetalized cyclodextrin that was added with triethylamine to terminate the reaction after 1h, 3h, 5h, and 12h of magnetic stirring reaction in Example 5 was used for the above preparation reaction, and the finally prepared drug sustained-release preparation was observed by scanning electron microscope, and The particle size of the drug sustained-release preparation was counted, and the results are shown in Figure 21. From left to right, the products prepared from the acetalized cyclodextrin obtained by the acetalization reaction of 1h, 3h, 5h, and 12h are respectively, and the upper side is Electron micrograph, the lower side is the particle size statistics. As can be seen from the figure, the preparation method provided in this example can prepare a sustained-release drug preparation with a size of 100-450 nm.
  • This embodiment provides a microgel.
  • the preparation method of this microgel is as follows:
  • Example 6 The drug sustained-release preparation of Example 6, the microgel of Example 7, and the control group 1 and the control group 2 were respectively prepared into a 150 ⁇ g/mL physiological saline solution, oscillated at a constant temperature in a water bath at 37 ° C, and 1 mL was sampled at different time points, and supplemented. Add 1 mL of normal saline at the same temperature. The absorbance value of the sample was measured by the method in Example 7, and the corresponding release amount of rapamycin was calculated according to the standard curve.
  • control group 1 adopts the preparation method in Example 7, the difference is only that rapamycin is used instead of drug sustained-release preparation.
  • Control group 2 adopts the preparation method in Example 7, except that the acetalization time of the acetalized cyclodextrin used in the drug sustained-release preparation is 1 h.
  • control group 1 microgel+RAP
  • control group 2 microgel+NPs/1h
  • Example 6 NPs
  • Example 7 microgel+NPs
  • the acetalization treatment for 12h compared with the acetalization treatment for 1h, the time taken for the immunosuppressant to reach the same cumulative release rate was greatly prolonged, indicating that a longer acetalization treatment time can make the drug sustained-release preparation slow down.
  • the release effect is greatly improved. Under the same conditions, there was no significant difference in the sustained-release effect between drug sustained-release preparations and microgels.
  • NPs+LPS group RAP+LPS group
  • the drug sustained-release preparation (RAP-NPs) and the microgel (RAP-NPs-microgel) in Example 7 were stimulated for 1 h, and the expression of TNF- ⁇ was detected by western blot analysis, and the results were shown in Figure 23. It can be seen from the figure that under the stimulation of lipopolysaccharide, macrophages will release the pro-inflammatory factor TNF- ⁇ , and multiple groups using rapamycin have obvious anti-inflammatory and immunosuppressive effects effect.
  • the use of the drug sustained-release preparations or biological materials provided in the examples of the present application can reduce the release amount of immunosuppressants under the same circumstances, and reduce the side effects of systemic use.
  • the above-mentioned drug sustained-release preparation can be applied to the process of cell transplantation, such as direct use or embedding of the drug sustained-release preparation in a corresponding device encapsulating the transplanted cells, for example, water encapsulating the transplanted cells In the gel, with the growth and secretion of the transplanted cells, it is gradually released to the transplant site, achieving long-term immunosuppression and anti-inflammatory effects.
  • Group 1 normal mice
  • Group 3 Naked islet injection, 500IEQ per mouse;
  • Group 4 Ordinary microgel-encapsulated islets
  • Group 5 Multifunctional cell encapsulation system encapsulating pancreatic islets Group: refer to the core-shell microgels of islets encapsulated with drug sustained-release preparations prepared in Examples 7 and 8, and refer to the oxygen sustained-release prepared in Example 3 material, the volume ratio is 1:1;
  • the preparations of groups 3 to 5 were implanted into the epididymal fat pad of diabetic mice respectively.
  • the fat pad has a rich blood vessel network, which can timely reflect the blood glucose level in the body, which is beneficial to the feedback regulation of blood glucose by pancreatic islets.
  • the results are shown in Figure 27. It can be seen from the figure that the diabetic mice have been in a state of hyperglycemia after modeling, and their body weight has been decreasing. In addition, their mental state is also relatively depressed, and their hair color is yellow and dull. After about a month die.
  • the blood sugar dropped significantly on the first day, and reached the blood sugar level of normal mice on the third day, indicating that the implanted nude islets had a certain activity and regulates blood sugar in the early stage of transplantation.
  • the blood glucose level of the mice rebounded and increased, and the blood glucose level was comparable to that of the non-transplanted diabetic mice on the 8th day, indicating that most of the transplanted nude islets died at this time and did not have the ability to regulate blood sugar.
  • the islets encapsulated by ordinary core-shell structure microgels were implanted into diabetic mice, and the blood sugar was controlled for more than 50 days.
  • the functional cell encapsulation system including drug sustained-release preparations, core-shell microgels coated with islets, and oxygen sustained-release materials
  • transplantation group still maintained a stable blood glucose level 90 days after islet transplantation.
  • FIG. 32 The hematoxylin-eosin HE staining of the grafts in the multifunctional cell encapsulation system encapsulated with islets is shown in FIG. 32 . It can be seen from the figure that the islets in the core-shell microgel have obvious cytoplasm and nuclei (the rightmost image is enlarged), while from the leftmost image, the edge of the core-shell microgel has no obvious immune cells. There was no dense fibroblast layer, indicating that after 90 days of transplantation, there was no obvious immune inflammatory response or fibrosis near the multifunctional cell encapsulation system, which was consistent with the results of in vivo blood glucose monitoring. Scale bar length: 200 ⁇ m.

Abstract

提供了一种多功能细胞包裹系统，包括核壳微凝胶以及氧气缓释材料和药物缓释制剂中的一种或两种，该核壳微凝胶包括内核和壳层，所述内核由包括光固化单体、活性成分和光引发剂的内核原料制成，所述壳层的材料为抗非特异性蛋白质吸附材料。用内核材料对活性成分进行包裹，用壳层材料对内核进行封装，能避免活性成分如胰岛细胞直接暴露遭受急性免疫排斥的风险，该壳层材料能降低慢性免疫排斥，减少或消除长期免疫抑制。氧气缓释材料为核壳微凝胶包裹的细胞在体内提供氧气，保证细胞的存活率和活性，药物缓释制剂内嵌在核壳微凝胶中，在移植部位提供抗免疫排斥效果，大大降低了口服抗免疫排斥药物的毒副作用。

Description

一种核壳微凝胶、氧气缓释材料、药物缓释制剂和一种多功能细胞包裹系统
本发明要求申请号为202011343278.0、202110482609.7和202110648315.7，对应发明名称依次为“核壳微凝胶及其制备方法和应用”、“氧气缓释材料及其制备方法和药物”和“药物缓释制剂及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明涉及微凝胶技术领域，尤其是涉及一种核壳微凝胶、氧气缓释材料、药物缓释制剂和一种多功能细胞包裹系统。
背景技术
I型糖尿病是一种以免疫介导破坏和导致胰腺内分泌胰岛素的β细胞功能丧失为特征的疾病，又称胰岛素依赖糖尿病。根据世界卫生组织的报告，全球有4.22亿人被诊断患有糖尿病；根据美国疾病控制与预控中心估计，近160万美国人患有I型糖尿病，其中包括约18.7万名儿童和青少年。严峻的糖尿病形势需要高效高质量的治疗手段来提高生命质量，现有的治疗手段中，包括外源胰岛素给药、异体裸胰岛移植、宏封装胰岛移植、微囊化胰岛移植。外源胰岛素给药常包括胰岛素泵治疗(持续皮下胰岛素输注，CSII)与每日多次胰岛素注射(MDI)。CSII是一种有效而灵活的胰岛素给药方法，配备血糖自我监测，可以改善血糖管理和临床疗效，但泵本身可能出现输注管扭曲和胰岛素聚集导致的闭塞，从而引起胰岛素输注中断，导致高血糖和酮酸症中毒；或是仪器故障校准失误引起的过量胰岛素注入导致严重低血糖；甚至是输注部位的脂肪肥大、感染和炎症以及仪器损耗导致泵的停用；还包括接受者对泵的不适应性、粘连问题以及运动活动上的不便之处。MDI是控制糖化血红蛋白水平的一种传统有效方法，维持良好血糖控制的同时容易受到剂量不准确、疼痛、针头恐惧症、可接受性和不便等因素的影响，导致治疗结果不佳，特别对于I型糖尿病儿童和青少年，常有不依从性和夜间低血糖的发生，非常需要监护人的密切监督，既造成生活上的不便又影响胰岛素注射治疗的时效性。胰岛替代疗法作为外源性胰岛素的替代疗法已经被提出很长时间了，它有可能通过恢复患者体内的胰 岛素信号来消除继发性并发症。对于I型糖尿病患者，利用死者供体胰腺中分离出来的裸胰岛进行肝内移植，可以改善问题性低血糖，稳定血糖水平，维持靶血糖控制，从而提高生活质量，且往往不需要胰岛素治疗，但死者供体胰岛存在着数量少、质量差的问题，即使受者可以接受胰岛移植，要实现胰岛素独立的目的还要求移植来自多个供体胰腺的胰岛以满足数量与功能的匹配，甚至胰岛移植后可能面临急性和慢性免疫排斥反应，须要采用T淋巴细胞消耗剂诱导免疫抑制、长期使用免疫抑制药物维持免疫抑制，减少炎症、最小化血栓和出血风但增加了肾功能损伤、相关感染和恶性肿瘤产生的风险，同时要促进移植胰岛的血运重建并保证一定数量的胰岛β细胞存活，以真正达到糖尿病逆转的效果。胰岛封装有望克服裸胰岛移植的限制，商业化的宏封装胰岛移植，能通过3D打印、微加工技术制备出中空纤维、平面和圆柱形等精细的几何结构用于同时封装大量胰岛细胞，使移植物监测更直接，且能在发生不良事件时通过移除设备确保细胞的全部回收；相反地，对于宏观封装构造来说，较小的表面体积比，常难以实现简易植入和充分传质，易发生细胞聚集和由于邻近效应造成的营养、供氧不足导致胰岛死亡。此外，大面积的宿主细胞浸润极易引起纤维化，造成移植失败。微囊化胰岛移植通过对胰岛细胞单独封装，最大化表面体积比，减少细胞与宿主间氧气、营养扩散距离，避免了细胞聚集产生邻近效应，还能通过共封装抗炎药物、促血管生成因子、供氧粒子等提高移植胰岛的存活率，并在包封材料内进行胰岛免疫隔离可避免或消除慢性全身免疫抑制，从而使胰岛移植的应用更加广泛。传统静电液滴方法是在电场力和注射器泵的推动力作用下形成液滴并落在凝胶浴中固化成型，是基于藻酸盐在钙离子浴中快速交联微囊化胰岛最常用的方法，该方法存在一定局限性，一方面材料体系上依赖于藻酸盐的快速交联，而藻酸盐微胶囊移植后会因渗透应激肿胀破裂，从而导致免疫隔离丧失和移植物排斥反应；另一方面，静电液滴法制备的微胶囊直径一般在500～1500μm，大量的胶囊物质阻碍了关键溶质向胰岛的转运，导致核心缺氧和坏死扩散障碍，阻碍了葡萄糖和胰岛素的运输，导致糖敏感的延迟和被包裹胰岛的胰岛素分泌，弱化了微囊化在胰岛封装移植上了优势。相对于静电液滴法，液滴微流控法在把控微球尺寸、减小总移植体积、材料体系多样性上更加可控，液滴微流控制备微囊化胰岛是通过两个或多个不互溶液相，在剪切力和界面张力共同作用下，分散相被连续相剪切成大小均匀的液滴形成 的，还可以多选择优化材料体系的生物相容性和通透性增强对胰岛的免疫隔离，提高胰岛移植物的存活率，减少或完全消除免疫抑制。但对于单核微囊化胰岛而言，容易发生封装不完全，胰岛直接暴露于宿主引起移植失败，且多样性材料体系可能会降低胰岛周围的环境质量。
其次，细胞移植所面临的一个主要问题就是，需要满足这些细胞在移植后较高的营养要求。在移植完成的最初时期，这些细胞无法发挥血管网络的功能，将氧气输送至细胞，因此细胞就会处于“饥饿”状态，导致细胞的大批量死亡。缺氧被认为是造成胰岛移植失败的最主要原因。目前研究者们主要通过四种方式解决胰岛移植中细胞缺氧的问题：1.减少胰岛移植装置中氧气扩散到胰岛组织的距离，2.增加细胞包裹材料的氧气通透性，3在移植之前，在移植装置部位预先形成一个血管化网络，使血流靠近移植组织，4.提供外部氧气供应给移植组织。其中，最后一种局部供氧的策略被使用最多，具体氧气的局部产生可以通过电解水、光合作用的藻类、人造血红蛋白等方式实现。但是电解水的方式比较复杂，需要植入复杂装置，而第二类方式则需要植入活的藻类到人体，具有安全隐患，人造血红蛋白只能最多支持48小时的氧气释放。
另外，细胞移植治疗的一个主要问题就是免疫排斥，而针对这一问题目前最通用的方法是通过服用免疫抑制剂去抑制器官接受者对移植物的排斥。例如在糖尿病治疗领域，很多人工胰腺的移植都伴随着各种免疫药物的使用，比如Viacyte公司的PEC-direct产品、以及Sernova的人工胰腺装置目前都在临床验证阶段，他们在植入病人体内过程中都需要免疫抑制剂。
免疫抑制剂的使用开始于本世界70年代，经过几十年的发展，其家族越来越壮大。第一代免疫抑制剂以皮质激素甲强龙针剂、雷公藤多苷片、硫唑嘌呤(依木兰)、抗淋巴细胞免疫球蛋白(ALG)为代表，主要作用为溶解免疫活性细胞，阻断细胞的分化。作为广泛的免疫抑制剂，第一代免疫抑制剂的特点是非特异性。第二代免疫抑制剂以环孢素(如环孢素A、山地明、赛斯平)和他克莫司(tacrolimus，FK506)为代表，主要作用是阻断免疫活性细胞的白细胞介素2(IL-2)的效应环节，以淋巴细胞为主而具有相对特异性。第三代免疫抑制剂以西罗莫司(sirolimus，SRL)、霉酚酸酯(mycophenoate mofetil，MMF)为代表，主要作用于抗原呈递和分子间的相互作用，与第二代制剂有协同作用。 第四代免疫抑制剂以抗IL-2受体单克隆抗体为代表。
目前临床上应用较多的主要的免疫抑制剂包括他克莫司、麦考酚酸酯、雷帕霉素、咪唑立宾等等。其中，雷帕霉素是迄今为止发现的毒性较小且有潜力的新型强效免疫抑制剂，可延长移植术后患者的生存期，减少急性排斥反应的发生。雷帕霉素是一种全身使用的口服药，它的主要作用原理是是阻止T细胞活化的后期阶段，抑制细胞从G1期进入S期。它阻断IL-2与其受体的结合，使Tc、Td细胞不能成为具有免疫应答作用的致敏性T细胞，最终抑制T细胞的分化繁殖，发挥其免疫抑制作用。
雷帕霉素的主要不良反应是骨髓抑制和高血脂，主要表现为血小板、白细胞减少，血肌酐水平降低，血甘油三酯、胆固醇水平升高，长期使用还有肾毒性等不良反应。雷帕霉素的副作用另外还表现为对于胰岛的毒性，多项研究显示，雷帕霉素在通过免疫抑制作用延长胰岛移植物存活的同时也对胰岛本身产生毒性。雷帕霉素对胰岛直接的损伤主要表现在三方面：(1)直接抑制胰岛β细胞分泌胰岛素；(2)抑制胰岛细胞活力，促进胰岛细胞凋亡；(3)抑制胰岛细胞的增殖。为了保证胰岛移植在I型糖尿病中的效能，减少雷帕霉素在单位时间内的用量是避免引起上述不良反应的一种解决方案。因此，有必要提供一种能够有效减慢作为免疫抑制剂的雷帕霉素的释放速度、提高其缓释效果的制剂。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明提出一种核壳微凝胶、氧气缓释材料、药物缓释制剂和一种多功能细胞包裹系统。
本发明的第一方面，提供一种核壳微凝胶，包括内核和壳层，所述内核由包括光固化单体、活性成分和光引发剂的内核原料制成，所述壳层又称微凝胶壳层，所述微凝胶壳层的材料为抗非特异性蛋白质吸附材料。
根据本发明实施例的核壳微凝胶，至少具有如下有益效果：
本发明实施例使用的内核材料为光固化材料体系且具有很好的生物相容性材料，利用该内核材料能够对活性成分进行很好的包裹，使用微凝胶壳层材料对内核进行封装，能够避免活性成分如胰岛直接暴露遭受急性免疫排斥的风险， 同时使用的壳层材料为具有抗非特性蛋白吸附材料，能够降低慢性免疫排斥，减少或消除长期免疫抑制。
根据本发明的一些实施例，所述活性成分选自细胞、药物、蛋白质类活性因子中的至少一种。其中，细胞可以例举的有胰岛、干细胞等，蛋白质类活性因子可以例举的有各种生长因子比如血管内皮生长因子等，胰岛发育转录因子调控类物质，防止胰岛细胞凋亡类因子等。
根据本发明的一些实施例，所述内核原料还包括细胞外基质物质。所述细胞外基质物质可以例举的有明胶、胶原、硫酸软骨素、透明质酸等。制备时可以在内核原料中混入细胞外基质物质，以改善微凝胶的成胶质量和细胞相容性。
根据本发明的一些实施例，所述光固化单体包括甲基丙烯酰化透明质酸(HAMA)、甲基丙烯酸化明胶、甲基丙烯酸果胶、甲基丙烯酸缩水甘油酯改性的丝蛋白材料中的至少一种。
根据本发明的一些实施例，所述抗非特异性蛋白质吸附材料为水凝胶。
根据本发明的一些实施例，所述抗非特异性蛋白质吸附材料包括聚乙二醇二丙烯酸酯(PEGDA)、甲基丙烯酸化羧基甜菜碱(CBMA)、甲基丙烯酸化磺基甜菜碱中的至少一种。
根据本发明的一些实施例，所述抗非特异性蛋白质吸附材料为聚乙二醇二丙烯酸酯和甲基丙烯酸化羧基甜菜碱的混合物。壳层材料为CBMA和PEGDA共混材料时，能够减少凝胶表面对蛋白的吸附。CBMA占共混材料的质量浓度不超过12％g/mL时，能够达到最佳抗吸附效果。
本发明的第二方面，提供上述的核壳微凝胶的制备方法，包括以下步骤：
取包括油相和活性成分的原料混合，形成连续相；
取包括光固化单体和第一光引发剂的原料混合，形成分散相；
取所述连续相和所述分散相，反应制备得到单乳液滴，对所述单乳液滴进行光固化，制得内核；
将所述内核浸泡至第二光引发剂的溶液中，后转移至所述微凝胶壳层的材 料溶液中避光静置，然后对其进行光固化得到核壳微凝胶。
根据本发明实施例的核壳微凝胶的制备方法，至少具有如下有益效果：
本发明实施例首先利用液滴微流控技术制备出类细胞外基质微凝胶，为活性成分如胰岛提供了一个适合胰岛生存、利于发挥胰岛素分泌功能的环境；其次，利用光引发剂的扩散特性通过界面聚合反应，在单核微凝胶内核的基础上构建壳层结构，实现对活性成分如胰岛进行二次封装，同时利用抗非特性蛋白吸附材料降低移植物的免疫排斥反应，提高移植物的存活，为实现I型糖尿病患者的血糖逆转、胰岛素独立提供可行的技术方案。
根据本发明的一些实施例，利用微流控技术制备得到单乳液滴。
根据本发明的一些实施例，连续相和分散相流速比为(10～40)：1，可以是40:1，30:1，20:1，10:1。
根据本发明的一些实施例，油相包括HFE-7500氟化油。可以在油相中加入表面活性剂，有助于提高产生的单乳液滴的稳定性。
根据本发明的一些实施例，采用将第一光引发剂溶解于光固化单体溶液中的方式形成分散相，光固化单体溶液可以是2，3，4，5wt％的甲基丙烯酸化透明质酸，可以是5，6，7，8，9，10wt％的甲基丙烯酸化明胶，也可以是上述两者的不同比例混合溶液。分散相也可以是上述两者与胶原、硫酸软骨素、明胶、透明质酸等的共混物。
根据本发明的一些实施例，所述第一光引发剂和所述第二光引发剂为LAP蓝光引发剂。
根据本发明的一些实施例，所述第二光引发剂的溶液的浓度为0.3～3wt％，可以是0.3，0.5，1，2，3wt％。
根据本发明的一些实施例，浸泡的时间为10min～12h，可以是10min，20min，30min，1h，2h，3h........12h。
本发明的第三方面，提供上述的核壳微凝胶或者根据上述的核壳微凝胶的制备方法制得的核壳微凝胶在制备治疗糖尿病药物中的应用。
本发明提出一种氧气缓释材料及其制备方法和药物，该氧气缓释材料能够长时间持续释放氧气，可应用于局部供氧。
本发明提出了一种氧气缓释材料，所述氧气缓释材料为微球，所述微球包括可降解生物材料和分散在所述可降解生物材料中的过氧化物，所述可降解生物材料为聚己内酯(PCL)，聚乳酸-羟基乙酸共聚物(PLGA)，或者聚己内酯与聚乳酸(PLA)的混合材料。
根据本发明实施例的氧气缓释材料，至少具有如下有益效果：
可降解生物材料若降解速率过快，则分解产生的降解物容易来不及排出而积累较高浓度，进而容易引发炎症反应发生，也不利于氧气的缓慢释放。本发明提供一种氧气缓释材料，使用聚己内酯，聚乳酸-羟基乙酸共聚物，或者聚己内酯与聚乳酸的混合材料作为降解材料基体，降解速率合适，能够较好地控制氧气释放的持续性和总量，形成的氧气缓释材料后续与细胞治疗剂混合形成药物使用时，能够较长时间地提供氧气支持，能够有效地防止细胞治疗剂因缺氧导致死亡，促进细胞治疗剂的效果。本发明的氧气缓释材料后续能够为细胞治疗剂提供所需要的氧气补充，起到了输氧桥梁的作用，直至血管形成，为细胞治疗剂的存活提供自然氧气，具有较好的应用前景。
在本发明的一些实施方式中，所述过氧化物选自过氧化钙、过氧化钾、过氧化钠、过氧化氢、过氧化镁、过氧化脲中的至少一种；优选地，所述过氧化物为过氧化钙。
在本发明的一些实施方式中，所述可降解生物材料为聚己内酯与聚乳酸的混合材料，所述聚己内酯与所述聚乳酸的质量比为0.1～100：1。考虑到聚己内酯的降解速率较慢，并且过氧化物反应产生的碱性物质如过氧化钙反应产生的氢氧化钙会形成碱性环境，对周围细胞存在影响，聚乳酸降解较快且产物偏酸性，在缓释材料中加入聚乳酸可以调节氧气释放速率和中和碱性物质如氢氧化钙的碱性，从而达到提升氧气释放速率和降低反应产物影响的目的。
在本发明的一些实施方式中，基于所述微球的质量，所述可降解生物材料与所述过氧化物的质量比为0.1～100：1。
在本发明的一些实施方式中，所述微球的粒径尺寸为1μm～3mm。在本发 明进一步的一些实施例方式中，所述微球的粒径尺寸为100～300μm。
本发明还提出了上述的氧气缓释材料的制备方法，包括以下步骤：
取所述可降解生物材料和所述过氧化物，与有机溶剂混合形成油相；
将所述油相与水相混合，形成微球。
在本发明的一些实施方式中，所述水相为聚乙烯醇的水溶液或者表面活性剂溶液；所述表面活性剂溶液为表面活性剂水溶液。
本发明还提出了一种药物，包括氧气缓释材料和细胞治疗剂，所述氧气缓释材料为上述的氧气缓释材料或根据上述的制备方法制得的氧气缓释材料。
在本发明的一些实施方式中，所述细胞治疗剂为胰岛细胞、胰岛素生成细胞、水凝胶包裹的胰岛细胞、水凝胶包裹的胰岛生成细胞中的一种或多种组合。使用的水凝胶为生物相容性材料，用以对胰岛细胞形成包裹保护。上述的胰岛细胞可以是人体捐献器官分离后的胰岛细胞或者动物器官分离得到的胰岛细胞，胰岛素生成细胞可以是基因编辑的胰岛素生成细胞或者干细胞分化产生的胰岛素生成细胞。胰岛细胞或胰岛素生成细胞可以作为裸细胞直接使用，也可以对其进行水凝胶包裹后使用。
本发明中“多种”是指两种及以上。
在本发明的一些实施方式中，所述水凝胶选自海藻酸钠、明胶、甲壳素、纤维素、聚乙二醇、聚乙烯醇、透明质酸中的一种或多种组合。使用的水凝胶可以是单独一种材料，也可以是多种材料混合使用。此类水凝胶具有炎症因子，渗出液吸附作用或者其他毒素吸附作用，用于创面修复时，可以改善创面炎症情况，防止创面恶化，保护细胞。
本发明提出一种能够有效减慢免疫抑制剂释放速度、提高其缓释效果的药物缓释制剂。
本发明的目的还在于提出上述药物缓释制剂的制备方法。
本发明的目的还在于提出一种生物材料。
本发明的目的还在于提出一种细胞移植装置。
本发明提供一种药物缓释制剂，该药物缓释制剂包括：
粒子，粒子包括免疫抑制剂和负载免疫抑制剂的缩醛化环糊精；
壳层，壳层包覆粒子，壳层的组成包括表面活性剂。
根据本发明实施例的药物缓释制剂，至少具有如下有益效果：
环糊精为中空的近圆筒状结构，内部为疏水区而两侧开口的羟基位置为亲水区，因而可以在内部装载免疫抑制剂，而在经过缩醛反应后，形成的缩醛化环糊精相比于环糊精能够与免疫抑制剂起到更稳定的装载效果，从而起到一定的缓释作用；同时，对于装载后形成的粒子采用表面活性剂进行再次包覆，提高内部免疫抑制剂在制剂中的包裹效果，进一步提升免疫抑制剂在其中的稳定性，延长免疫抑制剂的释放时间。
其中，环糊精是指由若干个D型吡喃葡萄糖以α-1,4-糖苷键首尾连接而成的聚糖，由于糖苷键不能自由旋转，因此，环糊精为略呈圆锥形的中空圆筒状结构。而本发明实施例中缩醛化环糊精则是指环糊精中至少一个葡萄糖单元的C2和C3的仲羟基与醛或酮发生缩醛反应(可以经酸催化剂催化)形成环状缩醛而得到的产物。其中，醛或酮的种类以及缩醛反应产物可以根据具体的需要进行适应性调整。
在本发明的一些实施方式中，缩醛化环糊精由β-环糊精经缩醛化处理得到。β-环糊精具有合适的亲疏水性能够对免疫抑制剂进行有效包载。
在本发明的一些实施方式中，缩醛化环糊精的通式如下所示：
Figure PCTCN2021133226-appb-000001
其中，x为1～7的整数，n为任意正整数；
R 1表示
Figure PCTCN2021133226-appb-000002
R 2表示
Figure PCTCN2021133226-appb-000003
在本发明的一些实施方式中，表面活性剂为非离子型表面活性剂。
在本发明的一些实施方式中，非离子型表面活性剂选自聚乙二醇、聚丙二醇、聚乙烯醇、脂肪醇聚氧乙烯醚、吐温、司盘、单硬脂酸甘油酯、聚醚、纤维素类表面活性剂、淀粉基表面活性剂、聚乙烯吡咯烷酮中的至少一种。采用上述的非离子表面活性剂可以对粒子形成更好的包裹效果，从而使得免疫抑制剂在其中具有更强的稳定性，进一步提高缓释效果。
在本发明的一些实施方式中，免疫抑制剂选自他克莫司、麦考酚酸酯、雷帕霉素、咪唑立宾、地塞米松中的至少一种。通过免疫抑制剂的施用，调节施用对象的免疫应答，减少器官对移植物的免疫排斥，从而使移植物能够在施用对象体内正常发挥作用。
本发明还提供上述药物缓释制剂的制备方法，该制备方法包括以下步骤：
S1：取免疫抑制剂和缩醛化环糊精与有机溶剂混合，形成油相混合液；
S2：将油相混合液与第一表面活性剂的溶液混合，超声乳化得到乳液；
S3：干燥处理所述乳液，得到药物缓释制剂。
根据本发明实施例的药物缓释制剂的制备方法，至少具有如下有益效果：
采用乳化法制备药物缓释制剂，通过该方法制备得到的药物缓释制剂在负载免疫抑制剂形成的粒子外，还形成了表面活性剂的良好包覆，而这种包覆也可以使药物的缓释更为持久。
在本发明的一些实施方式中，步骤S2还包括将乳液与第二表面活性剂的溶液混合搅拌。在形成乳液后，通过第二表面活性剂的进一步混合，强化了第一表面活性剂形成的壳层的包裹效果，使得药物缓释制剂的稳定性提高，释放时间得以延长，在相同条件下减少了免疫抑制剂与机体的接触量，从而减轻了大量使用时的副作用或不良反应。
在本发明的一些实施方式中，乳液与第二表面活性剂的溶液混合搅拌的方式为磁力搅拌。
在本发明的一些实施方式中，乳液与第二表面活性剂的溶液混合搅拌的温度为30～60℃，优选为35～55℃，进一步优选为40～50℃。其非限制性实例包括30℃、35℃、40℃、45℃、50℃、55℃、60℃。通过在上述温度范围内混合搅拌，使第二表面活性剂进一步参与形成壳层的同时，加速反应体系中溶剂的挥发。
其中，第一表面活性剂和第二表面活性剂仅用于区分在不同过程中加入的表面活性剂，而不表示这两个过程中使用的表面活性剂的任何区别，实际上，第一表面活性剂和第二表面活性剂可以是相同或不同的任选的表面活性剂，进一步分别任选自非离子型表面活性剂。
本发明还提供一种生物材料，该生物材料包括凝胶和上述的药物缓释制剂。
根据本发明实施例的生物材料，至少具有如下有益效果：
当采用上述药物缓释制剂时，生物材料能够更稳定持久地提供免疫抑制剂，延长了免疫抑制剂的释放时间。
其中，凝胶材料可以是任选具有生物相容性的水凝胶材料，例如甜菜碱类、透明质酸类、明胶类中的至少一种形成的凝胶。
在本发明的一些实施方式中，生物材料包括凝胶颗粒，凝胶颗粒包括药物缓释制剂和包覆在药物缓释制剂外侧的凝胶层。
在本发明的一些实施方式中，生物材料包括凝胶和分散在凝胶中的上述药物缓释制剂。
在本发明的一些实施方式中，还包括移植细胞。移植细胞是指任选向施用者体内替换损伤的细胞或组织、以进行修复的细胞，其非限制性实例包括干细胞(如间充质干细胞、骨髓干细胞、造血干细胞)、树突细胞、脾细胞、胰岛细胞等。
本发明的第四方面，提供细胞移植装置，该细胞移植装置包括上述的药物缓释制剂，或包括上述的生物材料。
根据本发明实施例的细胞移植装置，至少具有如下有益效果：
该细胞移植装置中的药物缓释制剂可以持久长效地为施用者提供免疫抑制剂，从而在延长免疫抑制效果的同时减少细胞毒性，提高移植细胞的存活时间和活力，使细胞移植装置的修复效果有更为明显的提升。
其中，细胞移植装置中装载的细胞可以是任选向施用者体内替换损伤的细胞或组织、以进行修复的细胞，其非限制性实例包括干细胞(如间充质干细胞、骨髓干细胞、造血干细胞)、树突细胞、脾细胞、胰岛细胞等。
本发明还提供了一种多功能细胞包裹系统，包括上述方案所述的核壳微凝胶或者所述的核壳微凝胶的制备方法制得的核壳微凝胶，还包括助剂；
所述助剂包括如下制剂的一种或两种：
1)上述方案所述的氧气缓释材料或根据所述的制备方法制得的氧气缓释材料；
2)上述方案所述的药物缓释制剂或根据所述的制备方法制得的药物缓释制剂。
本发明的多功能细胞包裹系统可以通过注射或者微创植入的方式，将治疗用的细胞和/或药物递送到病人体内，其中药物缓释制剂会镶嵌在核壳微凝胶内，而氧气缓释材料会和核壳微凝胶混合，一起植入病人体内。本发明的生物材料在植入病人体内后会和医用纤维粘连蛋白溶液混合，利用凝血原理形成一种粘纤蛋白胶水将核壳微凝胶固定在移植部位。
附图说明
下面结合附图和实施例对本发明做进一步的说明，其中：
图1为本发明实施例1制备核壳微凝胶的制备流程示意图；
图2为本发明实施例1使用的一种微流控芯片的结构图；
图3为本发明实施例1GelMA内核微凝胶和GelMA/PEGDA核壳微凝胶的电镜图；
图4为本发明实施例1以HAMA或GelMA作为分散相体系，制备出的核壳微凝胶的电镜图和直径分布图；
图5为本发明实施例1荧光显微镜图下的细胞染色实验图；
图6为本发明实施例1中不同条件处理下BMSC的存活率图；
图7为本发明实施例1使用的甲基丙烯酸羧基甜菜碱的核磁图谱；
图8为本发明实施例1中不同微凝胶表面的绿色荧光蛋白成像图；
图9为本发明实施例1中不同微凝胶表面对FITC-BSA的吸附量图；
图10为本发明实施例1中四组核壳微凝胶包裹4天后的活体染色图；
图11为本发明实施例1中核壳微凝胶在不同葡萄糖浓度下胰岛素释放情况图；
图12为本发明实施例1中核壳微凝胶在高葡萄糖浓度与低葡萄糖浓度时释放的胰岛素的浓度比；
图13为本发明实施例2中氧气缓释材料进行茜素红染色的结果图；
图14为本发明实施例2中氧气缓释材料进行体外氧气释放试验的结果图；
图15为本发明实施例3中氧气缓释材料进行氧气释放试验的结果图；
图16为本发明实施例3中氧气缓释材料释放氧气前后的pH变化图；
图17为本发明实施例4中组别1～6培养后胰岛细胞的活/死细胞荧光染色图；
图18为本发明实施例4中组别1～6的胰岛细胞在第1天、第3天和第7天的存活率；
图19是本发明的实施例5中环糊精和缩醛化环糊精的核磁共振氢谱图；
图20是本发明的实施例6中药物缓释制剂的制备和作用的示意图；
图21是本发明的实施例6中不同缩醛化时间的药物缓释制剂的电镜图和对应的直径统计结果；
图22是本发明的实施例8中不同形式雷帕霉素的缓释效果图；
图23是本发明的实施例9中不同形式雷帕霉素对RAW 264.7细胞的炎症抑制效果图；
图24是本发明的实施例10中不同形式雷帕霉素对RAW 264.7细胞的炎症抑制的缓释效果图；
图25是本发明的实施例11中糖尿病小鼠建模的过程，注射了药物链脲佐菌素STZ之后小鼠体内血糖变化；
图26是本发明的实施例11中糖尿病小鼠建模的过程，注射了药物链脲佐菌素STZ之后小鼠体重变化；
图27是本发明的实施例11中用不同多功能细胞包裹系统包裹大鼠胰岛然后移植到糖尿病小鼠体内的控糖结果；
图28是本发明的实施例11中多功能细胞包裹系统包裹大鼠胰岛在糖尿病小鼠体内90天时的腹腔葡萄糖耐受测试；
图29是本发明的实施例11中多功能细胞包裹系统包裹大鼠胰岛的移植物取出的照片；
图30是本发明的实施例11中多功能细胞包裹系统中的氧气缓释材料在体内降解情况；
图31是本发明的实施例11中多功能细胞包裹系统包裹大鼠胰岛在糖尿病小鼠体内的情况；
图32是本发明的实施例11中多功能细胞包裹系统包裹大鼠胰岛在糖尿病小鼠体内移植90天后取出的组织切片做苏木精-伊红染色的结果。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护 的范围。
下面详细描述本申请的实施例，描述的实施例是示例性的，仅用于解释本申请，而不能理解为对本申请的限制。
在本申请的描述中，若干的含义是一个以上，多个的含义是两个以上，大于、小于、超过等理解为不包括本数，以上、以下、以内等理解为包括本数。如果有描述到第一、第二只是用于区分技术特征为目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。
本申请的描述中，除非另有明确的限定，设置、安装、连接等词语应做广义理解，所属技术领域技术人员可以结合技术方案的具体内容合理确定上述词语在本申请中的具体含义。
本申请的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本申请的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
以下实施例中采用现有的微流控装置进行制备核壳微凝胶，制备时将固定在微量注射泵的注射器和固定在高速摄像显微镜下的微流控芯片通过带有连接管的针头连接，构成进样系统和收集系统。本发明实施例可以通过设计微流控芯片上的微流控通道形状和尺寸来改进核壳微凝胶的制备过程，通道形状可以是T型通道、Y型通道、流体聚焦型通道等；以T型通道为例，T型通道尺寸可以是200-100-200、300-200-300等。微流控芯片的制备方法为现有技术，可通过光刻、翻模、封接、通道改性等操作，制作出具有不同微流控通道的微流控芯片。参见图1，本发明实施例制备核壳微凝胶的制备流程为：将含有活性成分和光引发剂的原料光固化形成内核，内核洗涤后进入到光引发剂溶液中进行预凝胶，然后转移至单体溶液中，后光固化并洗涤制备形成核壳微凝胶。
可以理解的是，本发明实施例提供的核壳微凝胶，可按照以下步骤制备：
(1)多种微流控通道类型中，T型通道在连续相与分散相的相遇处具有较 大的剪切力，且通道的制作较为简单。考虑到一般胰岛细胞团的大小在50～500μm左右，包裹胰岛细胞时本发明实施例可使用如图2所示的微流控芯片，(a)表示微流控芯片的尺寸设计图，(b)表示微流控芯片的实物图，其具有连续相通道宽度为200微米，并在两相相交处的通道宽度收窄为150微米的T型通道。
(2)制备连续相：将含8wt％表面活性剂的HFE-7500氟化油溶液和活性成分混合，然后分装在5mL的注射器待用。活性成分可选自细胞如胰岛细胞、干细胞，药物和蛋白质类活性因子等，并根据需求进行选择。
(3)制备分散相：称取50mg甲基丙烯酸酐化明胶(GelMA)溶于1mL PBS中，在50℃下搅拌过夜，得到充分溶解的5wt％GelMA溶液；随之称取1.5mg LAP蓝光引发剂溶于1mL GelMA溶液中，充分溶解后分装到1mL的注射器中待用。
(4)将连续相和分散相通过聚四氟乙烯管接入微流控芯片中，通过调节两相流速比控制液滴尺寸，即以连续相流速8mL/h、分散相流速0.25mL/h时剪切成油包水的单乳液滴。
(5)制备内核：将收集的单乳液滴用紫外光照5min交联得到单核微凝胶，利用高挥发性的HFE-7300氟化油洗涤后转移到去离子水溶液中待用。
(6)制备核壳微凝胶：将制备的内核避光浸泡在0.2wt％LAP的去离子水溶液中3min使引发剂渗透，在去离子水中荡洗2-3次并吸去多余水溶液，随之转移到20％的聚乙二醇二丙烯酸酯溶液(PEGDA)中，避光静置等待30s，对其进行蓝光光照1min，使微凝胶界面发生光交联形成壳层结构，用去离子水充分清洗后得到具有核壳结构的微凝胶。
参见图3，其中(a)表示GelMA内核微凝胶，(b)表示GelMA/PEGDA核壳微凝胶，从图中可以看出，本发明实施例成功制备出核壳微凝胶，在进行壳层封装后仍可以保持较小的尺寸，同时利用抗蛋白粘附材料降低移植物的免疫排斥反应，提高移植物的存活。制备内核的过程中，LAP蓝光引发剂的浓度可以是0.05，0.1，0.15，0.2，0.3，0.4，0.5wt％，紫外光照时间可以是1，2，3，4，5，10min，通过优化不同环节参数可以制备出结构稳定直径分布在100， 150，200微米不同尺寸的内核微凝胶。此外，在制备内核时，可以混入透明质酸、明胶、胶原、硫酸软骨素等成分，以形成水凝胶包裹细胞，模拟细胞外基质结构，防止细胞凋亡。步骤(6)制备核壳微凝胶时，通过改变引发剂浓度、引发剂浸泡时间、壳层材料、壳层材料浓度、避光静置时间、光照时间，进而改进壳层厚度和壳层抗蛋白粘附能力，在具体制备时，蓝光引发剂LAP浓度可以是0.2，0.3，0.5，1，2wt％，光照时间可以是1，2，3，4，5，10min，壳层材料可以是聚乙二醇二丙烯酸酯(PEGDA)，甲基丙烯酸化羧基甜菜碱(CBMA)，甲基丙烯酸化磺基甜菜碱。
以HAMA或GelMA作为分散相体系，未包裹活性成分时制备出的核壳微凝胶的电镜图(比例尺为200微米)和直径分布如图4所示，其中(a)表示GelMA体系制备的核壳水凝胶的电镜图，(A)表示(a)中核壳水凝胶的直径分布统计图，(b)表示HAMA体系制备的核壳水凝胶的电镜图，(B)表示(b)中核壳水凝胶的直径分布统计图。核壳微凝胶如果尺寸过大，会影响水凝胶内外物质传输，影响细胞活性，如果尺寸过小，则导致最终包裹胰岛体积过大。从图中可以看出，两种分散相体系均能成功制备出核壳微凝胶，GelMA和HAMA体系的核壳微凝胶有75％以上直径在200微米左右，利于包裹活性成分。
考察引发剂对细胞活性的影响：
引发剂以蓝光LAP(苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐)为例，细胞以干细胞(BMSC)为例，具体实验过程为：将HAMA/GELMA包裹的细胞浸泡至0.5％LAP溶液中浸泡处理，然后更换浸泡溶液，最后使用蓝光光照60s，重复操作，具体处理方式如表1所示。图5表示不同组别在荧光显微镜图下的细胞染色实验图，左侧绿色表示活的细胞，右侧红色表示死亡细胞，图6表示不同条件处理下BMSC的存活率，结果表明在水凝胶单体(PEGDA)存在时，蓝光引发的引发剂自由基对细胞的活性影响不大。
表1 对BMSC进行处理的条件
实验组别 LAP浸泡3min 更换浸泡溶液 蓝光光照60s 重复操作次数
a ------ ------ ------ 0
b ------ ------ √ 0
c √ ------ √ 2
d √ PBS √ 0
e √ PEGDA √ 0
考察材料对蛋白吸附的影响
以甲基丙烯酸羧基甜菜碱(CBMA)为例，其核磁图谱如图7所示，两性离子水凝胶所携带的正负电荷基团可以通过静电相互作用和离子化溶剂作用结合自由水分子，在材料表面形成水化层阻止蛋白或细胞在材料表面的进一步吸附。因此，制备了具有非特异性抗蛋白吸附的甲基丙烯酸羧基甜菜碱(CBMA)能够用于改善微凝胶的抗蛋白粘附性能。
图8示出了具有不同微凝胶表面的绿色荧光蛋白成像图，绿色是荧光标记的蛋白(FITC-BSA)，绿色越多表明微凝胶表面吸附蛋白越多，图9示出了不同微凝胶表面对FITC-BSA的吸附量。通过对材料的蛋白吸附特性进行比较发现，GelMA、HAMA、PEGDA三种材料中PEGDA的抗蛋白吸附性能最好，且与CBMA的混合共聚能减少凝胶表面对蛋白的吸附。增大CBMA含量后发现BSA的吸附反而增多了；溶胀实验结果发现，随着CBMA含量的增加，凝胶的溶胀程度增加。结果表明CBMA和PEGDA的共混时，CBMA的质量浓度不超过12％g/mL时，能够达到最佳抗吸附目的。
胰岛与干细胞的共包裹活性以及功能维持探究：
本发明实施例以老鼠胰岛细胞(Islet)和老鼠骨髓间充质干细胞(BMSCs)为活性成分，设置了四个不同组别的核壳微凝胶①GelMA包裹纯胰岛(Islet-GelMA)；②HAMA包裹纯胰岛(Islet-HAMA)；③GelMA共包裹干细胞与胰岛(Co-culture-GelMA)；④HAMA共包裹干细胞与胰岛(Co-culture-HAMA)。四组核壳微凝胶包裹4天后的活体染色如图10所示，从图中可以看出，HAMA或GelMA体系材料以及是否包裹干细胞(BMSCs)对于胰岛的活性都没有负面影响。
将上述四组核壳微凝胶分别置于高葡萄糖浓度(16.7mmol/L)和低葡萄糖 浓度(2.8mmol/L)环境中培养1至10周，测定胰岛素的释放浓度，图11示出了每周在不同葡萄糖浓度下胰岛素释放情况。从图11可以看出，不同组别的胰岛素释放都会由于高葡萄糖浓度与低葡萄糖浓度环境的变化而出现胰岛素释放的差异，表明胰岛对于葡糖糖的相应敏感度在经过10周培养后没有明显变化。图12示出了高葡萄糖浓度与低葡萄糖浓度时胰岛素的浓度和浓度比(即刺激指数)，从图中可以看出，不同的实验组别中的胰岛并没有产生功能上的不同，表明包裹初期胰岛的活性不受水凝胶材料和干细胞的影响，表明水凝胶材料可以有效保持细胞胰岛活性。同时也表明了该核壳水凝胶可以同时包裹胰岛和干细胞，葡糖糖和胰岛素的传输不受微凝胶的影响，从而使得包裹的胰岛对于葡萄糖响应没有延迟。
实施例2
本实施例提供了系列氧气缓释材料，按照以下步骤制备：
取聚己内酯(PCL)和过氧化钙(CaO 2，简称CPO)混合形成反应原料，反应原料中CaO 2的质量分数分别为0％(PCL组)、10％(PCL+10％CPO组)、30％(PCL+30％CPO组)和50％(PCL+50％CPO组)，其中CaO 2的质量分数为0％的一组作为对比。将反应原料加入到有机溶剂DCM(二氯甲烷)中，并用均质仪充分搅拌混合均匀形成悬浊液，作为油相。取PVA(聚乙烯醇)溶解于水中形成质量分数为1.5％的PVA水溶液，作为水相。然后将油相滴入水相，并用均质仪充分搅拌，使有机溶剂挥发，形成100～300微米大小的微球颗粒，之后将微球离心收集，100度烘干，去除水分和未挥发的有机溶剂，即得到氧气缓释材料。
取本实施例制备得到的氧气缓释材料进行茜素红染色，染色结果如图13所示，从图中可以看出随着过氧化钙加入量的增多，红色亮度增加，表明微球中过氧化钙负载量增多，释放氧气量增多，其中过氧化钙进行氧气释放的反应为：2CaO 2+2H 2O＝2Ca(OH) 2+O 2。
取PCL+50％CPO组的氧气缓释材料进行体外氧气释放实验，具体过程为：将1g干燥的氧气缓释材料放入容器中，加入20mL去离子水，释放出的氧气用溶解氧浓度测试仪观测，连续20天每天进行释氧浓度测试，测定结果如图14 所示。从图14中可以看出，本发明实施例制备得到的氧气缓释材料能够持续20天进行供氧，第20天单位质量氧气缓释材料的氧气释放量达到理论释放量的15％，具有较好的氧气持续供应效果。
实施例3
本实施例提供了系列氧气缓释材料，按照以下步骤制备：
取PCL与过氧化钙(CaO 2，简称CPO)混合形成反应原料，反应原料中CaO 2的质量分数分别为0％(PCL组)、25％(PCL+25％CPO组)和50％(PCL+50％CPO组)。按照PCL与PLA(聚乳酸)的质量比为3：1共混形成混合材料，将混合材料与CPO混合形成反应原料，反应原料中CaO 2的质量分数分别为50％(标记为PCL/PLA+50％CPO组)。
将上述制得的反应原料加入到有机溶剂DCM(二氯甲烷)中，并用均质仪充分搅拌混合均匀形成悬浊液，作为油相。取PVA(聚乙烯醇)溶解于水中形成质量分数为1.5％的PVA水溶液，作为水相。然后将油相滴入水相，并用均质仪充分搅拌，使有机溶剂挥发，形成100～300微米大小的微球颗粒，之后将微球离心收集，100度烘干，去除水分和未挥发的有机溶剂，即得到氧气缓释材料。
取1g本实施例制得的氧气缓释材料放入容器中，加入20mL去离子水，释放出的氧气用溶解氧浓度测试仪观测，连续40天每天进行释氧浓度测试，结果如图15所示。取1g本实施例制得的氧气缓释材料放入容器中，加入20mL去离子水，测定氧气缓释材料放入去离子水中后的pH(标记为缓释前)以及释放氧气40天后的pH(标记为缓释后)，结果如图16所示。从图15可以看出，PCL组材料的释氧量为0.0mL/(g·Day)，PCL+25％CPO组材料的释氧量为0.238mL/(g·Day)，PCL+50％CPO组材料的释氧量为0.508mL/(g·Day)，PCL/PLA+50％CPO组材料的释氧量为0.625mL/(g·Day)，结果显示本发明实施例的氧气缓释材料能够持续提供氧气，提高过氧化物的含量后氧气的释放速率得到了提升，加入PLA后能够进一步提升氧气的释放速率。参见图16，去离子水本身的pH大概在6.5左右，由于加入氧气缓释材料中的过氧化钙会立即和水反应生成氢氧化钙，从而会提高溶液pH，因此加入氧气缓释材料后即使立即 进行测定pH，pH值也会提升，并且随着过氧化钙的含量的提高，溶液pH提高的幅度也增大。相较于缓释前的pH，缓释氧气40天后的pH得到了提高，但PCL/PLA+50％CPO组别的pH没有明显升高，表明PLA的降解产物具有一定的pH降低缓冲作用，考虑到纯PCL的降解速度较慢，而且CPO反应产生的氢氧化钙会形成碱性环境，对周围细胞存在一定的影响，因此在氧气缓释材料中可以考虑加入PLA，PLA降解较快且产物偏酸性，可以调节释氧速率和中和氢氧化钙碱性的作用。从结果来看，相较于未加入PLA的氧气缓释材料，加入了PLA的氧气缓释材料的释氧速率有所提升，并且释氧40天后溶液pH值有所下降，表明在缓释材料中加入聚乳酸可以调节缓氧速率和中和氢氧化钙的碱性，能够达到提升环氧速率和降低反应产物影响的目的。
实施例4
本实施例通过营造体外缺氧环境，评估胰岛细胞在缺氧环境下的活性及氧气缓释材料的作用，具体实验设计如下：
将100IEQ胰岛(或者GelMA包裹的胰岛)放于48孔板培养后，分别加入实施例2中PCL+50％CPO组或者PCL/PLA+50％CPO组的氧气缓释材料，将其放进细胞培养箱中进行常规有氧培养，或者将其转移至厌氧产气袋中密封，后放入细胞培养箱中进行培养，然后分别在培养的第1天、第3天以及第7天对胰岛活性进行表征。本实施例按照表2的实验组别设计实验，其中组别1：胰岛细胞进行常规有氧培养；组别2：胰岛细胞在缺氧环境中进行培养；组别3：胰岛和PCL+50％CPO组氧气缓释材料在缺氧环境中进行培养；组别4：胰岛和PCL/PLA+50％CPO组氧气缓释材料在缺氧环境中进行培养；组别5：胰岛和PCL+50％CPO组氧气缓释材料在缺氧环境中进行培养；组别6：胰岛和PCL/PLA+50％CPO组氧气缓释材料在缺氧环境中进行培养。本实施例实验过程中使用的细胞治疗剂是胰岛细胞和GelMA(甲基丙烯酸化水凝胶)包裹的胰岛细胞，其中GelMA包裹的胰岛细胞的制备步骤为：将胰岛细胞与GelMA混合后制得。图17示出了组别1-6培养后的胰岛细胞的活/死细胞染色图，图18示出了组别1-6中的胰岛细胞在第1天、第3天和第7天的存活率，从图17和18中可以看出，在缺氧环境下，组别2中胰岛细胞未进行包裹，也未加入本发明实施例制备的氧气缓释材料，裸胰岛细胞在一天内就出现明显的凋亡，三天 后细胞活性已经下降到26％。而组别3-6中均加入了氧气缓释材料，与氧气缓释材料共混后，无论是裸胰岛细胞还是水凝胶包裹的胰岛细胞，其活性均有大幅提高，与正常环境下的胰岛细胞无显著差异，表明本发明实施例制备得到的氧气缓释材料可以提供胰岛细胞生存所需的氧气，并且水凝胶包裹并不影响氧气的扩散运输。
表2 细胞活性观察实验的实验条件
Figure PCTCN2021133226-appb-000004
上述实验结果显示，本发明提供的氧气缓释材料可以与胰岛细胞或水凝胶包裹的胰岛细胞混合使用，用以为胰岛细胞长期持续地进行局部供氧，可以混合作为药物使用。上述实施例中使用的细胞治疗剂以胰岛细胞为例进行说明，本领域技术人员可以知晓当使用其他具有治疗作用的细胞替换胰岛细胞时同样可以适用，此外水凝胶对氧气的扩散并没有影响，除了GelMA外，本领域技术人员也可以选择明胶、甲壳素等材料对细胞进行包裹，其相应效果可以预期。
实施例5
本实施例提供一种缩醛化环糊精，该缩醛化环糊精的制备方法如下：
(1)在氮气保护下，将0.01mmol吡啶对甲苯磺酸盐加入到含有1mmol β-环糊精的10ml N,N-二甲基甲酰胺溶液中，同时加入3mmol 2-甲氧基丙烯后磁力搅拌下反应。
(2)12小时后向该反应体系中加入0.02mmol三乙胺停止反应，离心收集沉淀，真空干燥得到缩醛化环糊精。
上述反应过程中具体的反应方程式如下所示：
Figure PCTCN2021133226-appb-000005
其中，x为1～7的整数，n为任意正整数；
R 1表示
Figure PCTCN2021133226-appb-000006
R 2表示
Figure PCTCN2021133226-appb-000007
环糊精和缩醛化环糊精的核磁共振氢谱检验结果如图19所示，其中A为环糊精，B为缩醛化环糊精。从图中可以看出，反应后的产物成功地发生了缩醛化反应。
实施例6
本实施例提供一种药物缓释制剂，该药物缓释制剂的制备方法如下：
(1)取雷帕霉素50mg溶解在0.5mL的二甲基亚砜(DMSO)中，取实施例1制备的缩醛化环糊精200mg溶解在0.5mL的四氯化碳中，将两者混合形成均一的油相混合液。
(2)将油相混合液加入到6mL的1wt％的聚乙烯醇水溶液(聚乙烯醇分子量25kDa)中，超声乳化，形成乳化液滴，再加入到20mL的0.3wt％的聚乙烯醇水溶液(聚乙烯醇分子量25kDa)中，在45℃下磁力搅拌5h后，高速离心收集颗粒。
(3)用去离子水洗涤3次，冷冻干燥，有机溶剂挥发后形成含雷帕霉素的药物缓释制剂。
该药物缓释制剂的制备过程和作用原理如图20所示，由环糊精经缩醛化反应得到缩醛化环糊精，在其大小两侧开口分别形成环状缩醛和链状缩醛。缩醛化环糊精对雷帕霉素进行负载形成粒子后，通过乳化法与聚乙烯醇表面活性剂制备得到包含表面活性剂壳层的药物缓释制剂。该药物缓释制剂在水解作用下，会逐渐缓慢释放出其中的雷帕霉素，而缓释出的雷帕霉素能够有效降低免疫排斥产生的炎症反应，达到持久抗炎的效果。
分别取实施例5中磁力搅拌反应1h、3h、5h、12h后加入三乙胺终止反应的缩醛化环糊精进行上述制备反应，最终制备得到的药物缓释制剂采用扫描电子显微镜观察，并统计药物缓释制剂的粒径大小，结果如图21所示，从左到右分别为1h、3h、5h、12h缩醛化反应得到的缩醛化环糊精制备得到的产物，上侧为电镜图，下侧为粒径统计结果。从图中可以看出，本实施例所提供的制备方法能够制备得到大小在100～450nm的药物缓释制剂。
实施例7
本实施例提供一种微凝胶。该微凝胶的制备方法如下：
(1)利用液滴微流体装置，以分散有1mg/mL的实施例6制备得到的药物缓释制剂的30mg/mL的甲基丙烯酸酯化透明质酸(HAMA)溶液和1.5mg/mL的光引发剂苯基-2,4,6-三甲基苯甲酰基亚磷酸锂(LAP)以及胰岛的混合前体溶液作为分散相，以氟化油HFE7500(美国3M)作为连续相。将分散相和连续相的流速分别设定为0.3mL/h和2.7mL/h，在微流体通道中产生液滴后暴露在蓝光照射下2min使其交联，收集交联产物，得到核微凝胶，并重新分散到水性介质中。
(2)将核微凝胶浸泡到2mg/mL的LAP溶液中2min，用磷酸盐缓冲液(PBS)清洗2次，随后将核微凝胶置于150mg/mL的聚乙二醇二丙烯酸酯(PEGDA)和100mg/mL的羧酸甜菜碱(CBMA)的混合溶液中，静置15s后蓝光照射2min，从而在核微凝胶的表面形成PEGDA和CBMA的壳层包覆，得到核壳结构微凝胶。
实施例8
分别取实施例6的药物缓释制剂、实施例7的微凝胶以及对照组1和对照组2制备成150μg/mL的生理盐水溶液，37℃水浴恒温振荡，不同时间点取样1mL，并补加同温度的生理盐水1mL。所取样本采用实施例7中的方法测定其中的吸光值，根据标准曲线计算得出对应的雷帕霉素的释放量。
其中，对照组1采用实施例7中的制备方法，区别仅在于使用雷帕霉素代替药物缓释制剂。对照组2采用实施例7中的制备方法，区别仅在于，药物缓释制剂中所用的缩醛化环糊精的缩醛化时间为1h。
实验结果如图22所示，从上到下分别为对照组1(微胶+RAP)、对照组2(微胶+NPs/1h)实施例6(NPs)、实施例7(微胶+NPs/12h)。从图中可以看出，采用微凝胶直接包裹雷帕霉素，其累积释放率在第14天已经达到约90％，而采用环糊精负载、表面活性剂包裹的微凝胶的缓释效果则明显优于对照组1。其中，缩醛化处理12h相比于缩醛化处理1h，免疫抑制剂在达到相同的累积释放率时所用的时间大大延长，表明更长的缩醛化处理时间可以使药物缓释制剂的缓释效果大大提高。而相同条件下，药物缓释制剂和微凝胶在缓释效果方面并无明显区别。
实施例9
抗炎实验
RAW 264.7巨噬细胞用DMEM完全培养基置于37℃/5％CO 2培养箱中培养，取对数生长期细胞，按5×10 5/孔接种，培养16h使其贴壁。巨噬细胞分为对照组、LPS组(脂多糖LPS＝0.1μg/mL)、NPs+LPS组、RAP+LPS组(RAP＝30nM)、RAP-NPs+LPS组(药物缓释制剂中RAP＝30nM)、RAP-NPs-微胶+LPS组(微凝胶中RAP＝30nM)，视分组情况分别加入RAP、LPS、不包载雷帕霉素的药物缓释制剂(NPs)、实施例6中的药物缓释制剂(RAP-NPs)、实施例7的微凝胶(RAP-NPs-微胶)刺激1h，免疫印迹分析检测TNF-α的表达情况，结果见图23。从图中可以看出，在脂多糖的刺激下，巨噬细胞会释放出促炎因子TNF-α，而使用了雷帕霉素的多个组，均出现了比较明显的抗炎和免疫抑制作用。
实施例10
抗炎缓释实验
RAW 264.7巨噬细胞用DMEM完全培养基置于37℃/5％CO 2培养箱中培养，取对数生长期细胞，按5×10 5/孔接种，培养16h使其贴壁。巨噬细胞分为对照组、LPS组(LPS＝0.1μg/mL)、RAP+LPS组(RAP＝30nM)、RAP-NPs-微胶+LPS组(微凝胶中RAP＝30nM)，视分组情况分别加入RAP、LPS、实施例3的微凝胶(RAP-NPs-微胶)共培养10天，免疫印迹分析检测第1天、第3天、第7天和第10天TNF-α的表达情况，结果见图24。从图中可以看出，一次性RAP投药(RAP+LPS组)情况下，到了第7天促炎性因子TNF-α的表达量有明显上 升，表明这种情况下RAP会在3-7天内失效，不再具有炎症抑制效果。而载RAP纳米颗粒的微凝胶(RAP-NPs-微胶+LPS组)缓释作用第1天与第10天促炎性因子TNF-α的表达情况并没有显著的差异，表明RAP的释放能维持一段较长的时间，在第10天依然有较好的炎症抑制效果。同时，对比第10天的数据，RAP-NPs-微胶+LPS组与RAP+LPS组之间同样存在明显差异，表明制成药物缓释制剂的形式后，RAP在第10天仍然维持有较为明显的释放，从而抑制了TNF-α的表达，其缓释效果大大提高。
综合上述实施例可以看到，采用本申请实施例所提供的药物缓释制剂或生物材料，能够在相同情况下减少免疫抑制剂的释放量，降低了全身使用时的副作用，通过其缓释作用达到更明显的持久抗炎效果。因此，可以将上述的药物缓释制剂应用到细胞移植过程中去，可以采用诸如直接使用或将药物缓释制剂包埋在对应的封装有移植细胞的装置中，例如，封装有移植细胞的水凝胶中，随移植细胞的生长、分泌而逐渐缓释到移植部位，实现长效免疫抑制和抗炎等效果。
实施例11
糖尿病小鼠建模
实验步骤：配置了0.1mM的链脲佐菌素(Streptozotocin，简称STZ)的柠檬酸/柠檬酸钠溶液，提前对大鼠进行过夜禁食不禁水处理，按照65mg/Kg的注射量对大鼠进行药物注射，并在注射后对其血糖水平和体重情况进行了观察测定。Trested 1、2、3分别是不同的大鼠，从图25和图26可见，未经注射药物的对照组大鼠的血糖一直维持在较低的浓度(文献参考范围：6.0±2.0mM)，而注射了药物的大鼠则在连续三次测定中均高于16.7mM，此外通过体重测定可见，虽然前期体重并没有明显变化，但是在药物注射两周后，体重明显减轻，而对照组大鼠的体：1重一直逐步提升，并且通过对大鼠活动状态的观察，注射药物后每两天需要进行饲料和水的补充，且垫料也出现了明显的排泄物，符合了糖尿病患的“三多一少”特点，说明糖尿病大鼠模型的成功建立。
组别1：正常老鼠；
组别2：糖尿病鼠
组别3：裸胰岛注射，500IEQ每只老鼠；
组别4：普通微凝胶包裹胰岛组：参照实施例7中的制备方法制得的核壳微凝胶包裹大鼠胰岛，500IEQ每只老鼠
组别5：多功能细胞包裹系统包裹胰岛组：参照实施例7和实施例8中制得的装载药物缓释制剂的包裹胰岛的核壳微凝胶、参照实施例3制得的氧气缓释材料，体积比为1：1；
将组别3～5的制剂分别植入糖尿病鼠的附睾脂肪垫，其脂肪垫有丰富的血管网络，可及时反应体内的血糖水平，有利于胰岛对血糖的反馈调节。结果参见图27，从图中可以看出，造模后的糖尿病小鼠一直处于高血糖状态，且体重一直在下降，另外精神状态也较为颓靡，毛色偏黄而无光泽，在约一个月后死亡。将大鼠裸胰岛(500IEQ)植入糖尿病小鼠后，第一天血糖出现了明显下降，第三天达到正常小鼠血糖水平，说明植入的裸胰岛在移植初期具有一定的活性和调节血糖的能力，但移植4天后，小鼠血糖水平反弹上升，第8天与未移植的糖尿病小鼠血糖水平相当，说明此时移植的裸胰岛大部分死亡，不具有调节血糖的能力。与此同时，将普通核壳结构微凝胶包裹胰岛植入到糖尿病鼠体内，血糖控制超过50天，57天之后血糖上升，糖尿病小鼠出现高血糖，而组别5的包裹有胰岛的多功能细胞包裹系统(包括药物缓释制剂以及包裹有胰岛的核壳微凝胶和氧气缓释材料)移植组在胰岛移植后90天依然保持稳定的血糖水平。
实施例12 动态葡萄糖耐受测试(IPGTT)
1)对于达到动物实验监测终点(实施例11中的包裹有胰岛的多功能细胞包裹系统移植90天)的小鼠，在移除移植物之前对其进行葡萄糖耐受测试，绘制了曲线并计算曲线下面积(Area Under Curve，AUC)来衡量代谢能力的强弱，将其与正常小鼠以及糖尿病小鼠的曲线进行了对比。结果参见图28，由曲线a显示，可见移植后90天的小鼠的血糖恢复速度与正常小鼠的相接近，而糖尿病大鼠的曲线则在注射后的15分钟达到顶峰后均维持在较高水平。而通过计算AUC也可见，糖尿病小鼠的AUC显著大于另外两组，而包裹有胰岛的多功能细胞包裹系统移植组的小鼠与正常小鼠的面积相接近。说明移植90天的移植物中封装的胰岛依旧保持有良好的调节葡萄糖代谢的能力。
2)包裹有胰岛的多功能细胞包裹系统移植组的移植物外观形貌参见图29，外形没有肿胀，和植入之前尺寸相差不大。
3)包裹有胰岛的多功能细胞包裹系统移植组的移植物茜素红染色结果参见图30。茜素红对于含有钙盐的释氧微球会生成红色沉淀。从图上可见，移植90天的移植物中，大部分释氧微球已经降解了，剩余部分呈现不规则形状，说明释氧微球在移植后不断降解并产生氧气。(比例尺长度：50μm)
4)包裹有胰岛的多功能细胞包裹系统移植组的移植物胰岛素荧光染色结果参见图31。从图中染色结果可见，移植90天后移植物中胰岛细胞依然保持明显的胰岛素阳性，且依然保持当初类似的植入尺寸(图中胰岛直径约为150μm，比例尺长度：为100μm)，说明植入后的移植物在长达三个月的植入后仍然维持较好的细胞活性，并发挥胰岛素释放功能。
5)包裹有胰岛的多功能细胞包裹系统移植组的移植物苏木精-伊红HE染色参见图32。从图上可以看到核壳微凝胶中的胰岛具有明显的细胞质和细胞核(最右图放大)，而从最左边的图来看，核壳微凝胶的边缘并没有明显的免疫细胞的堆积，也没有致密的成纤维细胞层，说明90天的移植后，多功能细胞包裹系统附近并没有明显的免疫炎症反应或者纤维化，这与体内血糖监测结果相吻合。比例尺长度：200μm。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (34)

  1. 一种核壳微凝胶，其特征在于，包括内核和壳层，所述内核由包括光固化单体、活性成分和光引发剂的内核原料制成，所述壳层的材料为抗非特异性蛋白质吸附材料。
  2. 根据权利要求1所述的核壳微凝胶，其特征在于，所述活性成分选自细胞、药物、蛋白质类活性因子中的至少一种。
  3. 根据权利要求1所述的核壳微凝胶，其特征在于，所述细胞为胰岛细胞。
  4. 根据权利要求1所述的核壳微凝胶，其特征在于，所述内核原料还包括细胞外基质物质。
  5. 根据权利要求1至4任一项所述的核壳微凝胶，其特征在于，所述光固化单体包括甲基丙烯酰化透明质酸、甲基丙烯酸化明胶、甲基丙烯酸果胶和甲基丙烯酸缩水甘油酯改性的丝蛋白材料中的至少一种。
  6. 根据权利要求1至4任一项所述的核壳微凝胶，其特征在于，所述抗非特异性蛋白质吸附材料包括聚乙二醇二丙烯酸酯、甲基丙烯酸化羧基甜菜碱和甲基丙烯酸化磺基甜菜碱中的至少一种。
  7. 根据权利要求6所述的核壳微凝胶，其特征在于，所述抗非特异性蛋白质吸附材料为聚乙二醇二丙烯酸酯和甲基丙烯酸化羧基甜菜碱的混合物。
  8. 权利要求1至7任一项所述的核壳微凝胶的制备方法，其特征在于，包括以下步骤：
    取包括油相和活性成分的原料混合，形成连续相；
    取包括光固化单体和第一光引发剂的原料混合，形成分散相；
    取所述连续相和所述分散相，反应制备得到单乳液滴，对所述单乳液滴进行光固化，制得内核；
    将所述内核浸泡至第二光引发剂的溶液中，后转移至所述壳层的材料溶液中避光静置，然后对其进行光固化得到核壳微凝胶。
  9. 根据权利要求8所述的核壳微凝胶的制备方法，其特征在于，所述第一光引发剂和所述第二光引发剂为LAP蓝光引发剂。
  10. 根据权利要求8所述的核壳微凝胶的制备方法，其特征在于，所述第二光引发剂的溶液的浓度为0.3～3wt％。
  11. 根据权利要求8所述的核壳微凝胶的制备方法，其特征在于，所述浸泡的时间为10min～12h。
  12. 权利要求1至7任一项所述的核壳微凝胶或者根据权利要求8至11任一项所述的核壳微凝胶的制备方法制得的核壳微凝胶在制备治疗糖尿病药物中的应用。
  13. 一种氧气缓释材料，其特征在于，所述氧气缓释材料为微球，所述微球包括可降解生物材料和分散在所述可降解生物材料中的过氧化物，所述可降解生物材料为聚己内酯，聚乳酸-羟基乙酸共聚物，或者聚己内酯与聚乳酸的混合材料。
  14. 根据权利要求13所述的氧气缓释材料，其特征在于，所述过氧化物选自过氧化钙、过氧化钾、过氧化钠、过氧化氢、过氧化镁和过氧化脲中的至少一种。
  15. 根据权利要求13所述的氧气缓释材料，其特征在于，所述可降解生物材料为聚己内酯与聚乳酸的混合材料，所述聚己内酯与所述聚乳酸的质量比为0.1～100：1。
  16. 根据权利要求13至15任一项所述的氧气缓释材料，其特征在于，基于所述微球的质量，所述可降解生物材料与所述过氧化物的质量比为0.1～100：1。
  17. 根据权利要求13至15任一项所述的氧气缓释材料，其特征在于，所述微球的粒径尺寸为1μm～3mm。
  18. 权利要求13至17任一项所述的氧气缓释材料的制备方法，其特征在于，包括以下步骤：
    取所述可降解生物材料和所述过氧化物，与有机溶剂混合形成油相；
    将所述油相与水相混合，形成微球。
  19. 根据权利要求18所述的氧气缓释材料的制备方法，其特征在于，所述 水相为聚乙烯醇的水溶液或者表面活性剂溶液。
  20. 一种药物，其特征在于，包括氧气缓释材料和细胞治疗剂，所述氧气缓释材料为权利要求13至17任一项所述的氧气缓释材料或根据权利要求18或19所述的制备方法制得的氧气缓释材料。
  21. 根据权利要求20所述的药物，其特征在于，所述细胞治疗剂为胰岛细胞、胰岛素生成细胞、水凝胶包裹的胰岛细胞和水凝胶包裹的胰岛生成细胞中的一种或多种组合。
  22. 根据权利要求21所述的药物，其特征在于，所述水凝胶选自海藻酸钠、明胶、甲壳素、纤维素、聚乙二醇、聚乙烯醇和透明质酸中的一种或多种组合。
  23. 药物缓释制剂，其特征在于，包括：
    粒子，所述粒子包括免疫抑制剂和负载所述免疫抑制剂的缩醛化环糊精；
    壳层，所述壳层包覆所述粒子，所述壳层的组成包括表面活性剂。
  24. 根据权利要求23所述的药物缓释制剂，其特征在于，所述缩醛化环糊精由β-环糊精经缩醛化处理得到。
  25. 根据权利要求24所述的药物缓释制剂，其特征在于，所述缩醛化环糊精的通式如下所示：
    Figure PCTCN2021133226-appb-100001
    其中，x为1～7的整数，n为任意正整数；
    R 1表示
    Figure PCTCN2021133226-appb-100002
    R 2表示
    Figure PCTCN2021133226-appb-100003
  26. 根据权利要求23至25任一项所述的药物缓释制剂，其特征在于，所述 表面活性剂为非离子型表面活性剂；
    所述非离子型表面活性剂选自聚乙二醇、聚丙二醇、聚乙烯醇、脂肪醇聚氧乙烯醚、吐温、司盘、单硬脂酸甘油酯、聚醚、纤维素类表面活性剂、淀粉基表面活性剂和聚乙烯吡咯烷酮中的至少一种。
  27. 根据权利要求23至25任一项所述的药物缓释制剂，其特征在于，所述免疫抑制剂选自他克莫司、麦考酚酸酯、雷帕霉素、咪唑立宾、地塞米松中的至少一种。
  28. 权利要求23至27任一项所述的药物缓释制剂的制备方法，其特征在于，包括以下步骤：
    S1：取免疫抑制剂和缩醛化环糊精与有机溶剂混合，形成油相混合液；
    S2：将所述油相混合液与第一表面活性剂的溶液混合，超声乳化得到乳液；
    S3：干燥处理所述乳液，得到所述药物缓释制剂。
  29. 根据权利要求28所述的药物缓释制剂的制备方法，其特征在于，步骤S2中还包括将所述乳液与第二表面活性剂的溶液混合搅拌。
  30. 生物材料，其特征在于，包括凝胶和权利要求23至27中任一项所述的药物缓释制剂或权利要求28～29任意一项所述制备方法得到的药物缓释制剂。
  31. 根据权利要求30所述的生物材料，其特征在于，所述生物材料包括凝胶颗粒，所述凝胶颗粒包括所述药物缓释制剂和包覆在所述药物缓释制剂外侧的凝胶层。
  32. 根据权利要求30所述的生物材料，其特征在于，还包括移植细胞。
  33. 细胞移植装置，其特征在于，包括权利要求23至27任一项所述的药物缓释制剂，或包括权利要求28～29任意一项所述制备方法得到的药物缓释制剂，或包括权利要求30至31任一项所述的生物材料。
  34. 一种多功能细胞包裹系统，包括权利要求1至7任一项所述的核壳微凝胶或者权利要求8至11任一项所述的核壳微凝胶的制备方法制得的核壳微凝胶，还包括助剂；
    所述助剂包括氧气缓释材料和/或药物缓释制剂，所述氧气缓释材料为权利要求12至16任一项所述的氧气缓释材料或根据权利要求17或18所述的制备方法制得的氧气缓释材料；
    所述药物缓释剂为权利要求22至26任一项所述的药物缓释制剂或根据权利要求27或28所述的制备方法制得的药物缓释制剂。
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