WO2018126893A1 - 一种聚氨酯微载体及其制备方法和用途 - Google Patents

一种聚氨酯微载体及其制备方法和用途 Download PDF

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WO2018126893A1
WO2018126893A1 PCT/CN2017/117530 CN2017117530W WO2018126893A1 WO 2018126893 A1 WO2018126893 A1 WO 2018126893A1 CN 2017117530 W CN2017117530 W CN 2017117530W WO 2018126893 A1 WO2018126893 A1 WO 2018126893A1
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polyurethane
reaction
rpm
stirring
isocyanate
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PCT/CN2017/117530
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English (en)
French (fr)
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解慧琪
董丽
龚梅
陈安静
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四川大学华西医院
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Priority to US16/476,211 priority Critical patent/US20200016563A1/en
Publication of WO2018126893A1 publication Critical patent/WO2018126893A1/zh

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    • A—HUMAN NECESSITIES
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/14—Polymerisation; cross-linking
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    • 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
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    • 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
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
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    • C08G18/0819—Manufacture of polymers containing ionic or ionogenic groups containing anionic or anionogenic groups
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    • C08G18/4269—Lactones
    • C08G18/4277—Caprolactone and/or substituted caprolactone
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    • C08G18/40—High-molecular-weight compounds
    • C08G18/48—Polyethers
    • C08G18/4804—Two or more polyethers of different physical or chemical nature
    • C08G18/4808—Mixtures of two or more polyetherdiols
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    • C08G18/40—High-molecular-weight compounds
    • C08G18/48—Polyethers
    • C08G18/4833—Polyethers containing oxyethylene units
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    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
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    • C08G18/48—Polyethers
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    • C08G18/65—Low-molecular-weight compounds having active hydrogen with high-molecular-weight compounds having active hydrogen
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    • C08G18/755—Polyisocyanates or polyisothiocyanates cyclic cycloaliphatic containing only one cycloaliphatic ring containing at least one isocyanate or isothiocyanate group linked to the cycloaliphatic ring by means of an aliphatic group containing one isocyanate or isothiocyanate group linked to the cycloaliphatic ring by means of an aliphatic group having a primary carbon atom next to the isocyanate or isothiocyanate group and at least one isocyanate or isothiocyanate group linked to a secondary carbon atom of the cycloaliphatic ring, e.g. isophorone diisocyanate
    • C—CHEMISTRY; METALLURGY
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Definitions

  • the invention relates to a polyurethane microcarrier, a preparation method thereof and use thereof, and belongs to the field of biological materials.
  • the microcarrier refers to a microbead having a diameter of usually 60 to 300 ⁇ m and suitable for anchorage-dependent cells to adhere to the surface.
  • Microcarrier cultured cells have their unique advantages: they have the surface required for anchorage-dependent cell growth; due to their large surface area/volume ratio, an amplifiable uniform culture system is achieved that allows cells to surface on a small amount of material Adhesion and large-scale amplification are achieved; the expanded cells are easily polymerized to form a complex on the surface of the microcarriers to promote interaction between cells; the secreted extracellular matrix also supports intracellular activities, both of which promote cells Proliferation.
  • microcarriers are commonly used to culture cells in vitro. With the development of repair materials, the repair method of inoculation of cells on the carrier material shows more and more advantages. Since the microcarrier material can carry a large number of cells, it can also promote the proliferation and differentiation of cells, and can overcome the simple cell injection. The problem of massive cell death and cell dispersion has become an object of increasing concern.
  • the cell-carrier complex treats the affected area in a conventional manner by surgical implantation, and thus, the injectable microcarrier can improve the wound caused during the implantation of the cell carrier complex. Injection of cell-carrier complexes is the simplest and straightforward application of vectors, and this method has been extensively studied in tissue engineering.
  • the microcarrier material which is widely used is gelatin, which can effectively culture cells, but since it is a natural polymer material, the mechanical properties are very poor, and it has great limitations for in vivo repair.
  • the mechanical properties of synthetic materials are generally excellent, but it is more difficult to meet the conditions of high-efficiency culture of cells and injectability.
  • polyurethane materials polyurethane foams which are currently reported as microcarriers have no injectability, and the limitations for in vivo repair are very large.
  • the present invention provides a novel polyurethane microsphere microcarrier material, that is, a polyurethane microsphere, a preparation method thereof and use thereof.
  • the polyurethane microsphere of the present invention has a particle diameter of from 150 ⁇ m to 270 ⁇ m.
  • polyurethane microspheres are prepared as follows:
  • the isocyanate is used as a raw material in the step (1), and is added to the reaction vessel and stirred;
  • the polyurethane synthesized in the step (4) is added dropwise to the stirred distilled water and dispersed;
  • the invention also provides the method of the aforementioned polyurethane microspheres, the steps are as follows:
  • the isocyanate is used as a raw material in the step (1), and is added to the reaction vessel and stirred;
  • the polyurethane synthesized in the step (4) is added dropwise to the stirred distilled water and dispersed;
  • the two different oligomer diols used in the step (1) are any two of polyethylene glycol, polycaprolactone diol, and polytetrahydrofuran; preferably, the step (1)
  • the two different oligomer diols used are polyethylene glycol and polycaprolactone diol or polytetrahydrofuran;
  • polycaprolactone diol is polycaprolactone diol 2000
  • polyethylene glycol is polyethylene glycol 200
  • the molar ratio of the polycaprolactone diol to the polyethylene glycol is 1:1 to 2:1.
  • step (1) the molar ratio of the polytetrahydrofuran to the polyethylene glycol is 1:1 to 2:1;
  • step (2) the molar ratio of isocyanate to total oligomer diol in step (1) is (2 ⁇ 3): 1, preferably 3: 1;
  • the isocyanate is any one or more of isophorone diisocyanate, L-lysine diisocyanate, and diphenylmethane diisocyanate; preferably, the isocyanate Is isophorone diisocyanate;
  • step (2) stirring at a speed of 350-700 rpm, preferably at a stirring speed of 380 rpm; and a reaction time of 2-4 hours, preferably 2.5 hours.
  • the molar ratio of the chain extender to the isocyanate in step (2) is (0.1 ⁇ 1): (1), preferably 0.5: 1;
  • the chain extender is 2,2-dimethylolbutanoic acid or 2,2-dimethylolpropionic acid; preferably, the chain extender is 2,2 - dimethylolbutanoic acid;
  • the cooling is lowered to 45 to 55 ° C, preferably 50 ° C; the stirring is stirred at a speed of 350-700 rpm, preferably the stirring speed is 380 rpm; the reaction time is 1-3 hours. , preferably 1.5h.
  • step (4) the equimolar ratio of the neutralizing agent to the chain extender in step (3);
  • the neutralizing agent is triethylamine or sodium hydroxide
  • the stirring is performed at a speed of 350 to 700 rpm, preferably at a stirring speed of 380 rpm; and the reaction time is 15 minutes.
  • the stirring speed is 350-700 rpm, preferably 500 rpm.
  • the method of the step (6) is: washing the polyurethane particles obtained by the step (5) with distilled water, drying to a constant weight in a vacuum, and sieving the microspheres having a particle diameter of 150 to 270 ⁇ m with a 100 and a 50 mesh sieve.
  • the invention also provides the use of the aforementioned polyurethane microspheres in the preparation of microcarrier materials.
  • the invention also provides an in vivo repairing material, which is a repairing material prepared by using the aforementioned polyurethane microspheres as microcarriers and composite cells.
  • the polyurethane microsphere microcarrier of the invention has good biocompatibility and can provide an excellent base material for adherent cell growth;
  • the invention can optimize the particle size range of the polyurethane microsphere microcarrier to suitable for cell adhesion and amplification on the surface thereof, and the particle size is uniform and controllable, which breaks the application limitation that the polyurethane carrier is only a drug carrier;
  • the polyurethane microsphere microcarrier of the invention does not need to have a high boiling organic solvent as a medium in the preparation process, has no cytotoxicity, and has little environmental pollution;
  • the polyurethane microsphere microcarrier of the invention has good particle dispersibility in the process of suspension culture, does not cause agglomeration, and ensures the effective particle size required for injection repair;
  • the polyurethane microsphere microcarrier system of the invention can achieve high yield of cells in a small culture volume
  • the polyurethane microsphere microcarrier of the invention is low in cost and can be recycled.
  • the polyurethane microspheres prepared by the method of the invention can be used as a microcarrier, can produce cells with high yield, has good biocompatibility, has injectability, can be used for in vivo repair, and has good clinical application prospects.
  • Figure 1 is a general view of the polyurethane microspheres, the polyurethane microspheres are white and uniform spherical, and the particle size distribution ranges from 150 microns to 270 microns;
  • Fig. 2 Surface morphology of polyurethane microspheres.
  • the microspheres of polyurethane were observed by scanning electron microscopy.
  • the microspheres were spherical and smooth.
  • Figure 3 is a nuclear magnetic analysis of polyurethane microspheres, using a CHCl 3 solvent as a solvent to dissolve the polyurethane microspheres, the measured 1 H-NMR spectrum, 4.1 ppm from polycaprolactone, 3.7 ppm from polyethylene glycol;
  • FTIR Polyurethane microsphere infrared analysis
  • Figure 5 shows the cellular activity of cells on the surface of polyurethane microsphere microcarriers and commercially available CultiSpher G vectors.
  • TCP planar culture
  • Cultispher G commercially available microcarriers
  • FIG. 1 Cell surface distribution of polyurethane microsphere microcarriers (7d). After the cells were seeded on the microcarriers, they were subjected to suspension culture for 7 days, and the cells were stained by DAPI, and the nuclei reacted with the dye solution to give a blue color under fluorescence excitation. Under the confocal microscope, the cells were evenly distributed on the surface of the carrier, indicating that the material has good cytocompatibility;
  • Figure 7 is a picture of the injectability of a polyurethane microsphere microcarrier
  • Figure 8 is a picture of the injectability of a polyurethane microsphere microcarrier.
  • Oligomer diol polyethylene glycol, polycaprolactone diol 1000, polytetrahydrofuran
  • Isocyanate isophorone diisocyanate, L-lysine diisocyanate, diphenylmethane diisocyanate
  • Chain extender (2,2-dimethylolbutanoic acid, 2,2-dimethylolpropionic acid);
  • Triethylamine cells (osteoblasts, fibroblasts or stem cells);
  • the preparation method of the carrier-loaded polyurethane microspheres comprises the following steps:
  • the molar ratio of isophorone isocyanate to total oligomer diol is 2:1;
  • step (2) 2,2-dimethylolpropionic acid was added while cooling to 45 ° C, and reacted at a stirring speed of 700 rpm for 2 h;
  • the synthesized polyurethane is added dropwise to the stirred distilled water, and dispersed, wherein the stirring speed is 700 rpm;
  • the polyurethane particles obtained in the step (5) are dried at room temperature, vacuum-dried to constant weight, and sieved to a particle size of 150 by a 100 and 50 mesh sieve. 270 ⁇ m microspheres.
  • the preparation method of the carrier-loaded polyurethane microspheres comprises the following steps:
  • the molar ratio of isophorone isocyanate to total oligomer diol is 2.5:1;
  • step (2) 2,2-dimethylolpropionic acid was added, and the temperature was lowered to 50 ° C, and the reaction was carried out at a stirring speed of 300 rpm for 3 hours;
  • the synthesized polyurethane is added dropwise to the stirred distilled water, and dispersed, wherein the stirring speed is 300 rpm;
  • the polyurethane particles obtained in the step (5) are dried at room temperature, vacuum-dried to constant weight, and sieved to a particle size of 150 by a 100 and 50 mesh sieve. 270 ⁇ m microspheres.
  • the preparation method of the carrier-loaded polyurethane microspheres comprises the following steps:
  • the molar ratio of isophorone isocyanate to total oligomer diol is 2.5:1;
  • step (2) 2,2-dimethylolpropionic acid was added while cooling to 55 ° C, and reacted at a stirring speed of 380 rpm for 2 h;
  • the synthesized polyurethane is added dropwise to the stirred distilled water, and dispersed, wherein the stirring speed is 500 rpm;
  • the polyurethane particles obtained in the step (5) are dried at room temperature, vacuum-dried to constant weight, and sieved to a particle size of 150 by a 100 and 50 mesh sieve. 270 ⁇ m microspheres.
  • the preparation method of the carrier-loaded polyurethane microspheres comprises the following steps:
  • polycaprolactone diol 1000 and PEG200 Adding polycaprolactone diol 1000 and PEG200 to a three-necked flask, the molar ratio of polycaprolactone diol 1000 to PEG200 is 2:1, and stirring and mixing at 70 ° C;
  • the molar ratio of isophorone isocyanate to total oligomer diol is 3:1;
  • step (2) 2,2-dimethylolbutanoic acid was added, and the temperature was lowered to 50 ° C, and the reaction was carried out at a stirring speed of 380 rpm for 1.5 h;
  • the synthesized polyurethane is added dropwise to the stirred distilled water, and dispersed, wherein the stirring speed is 500 rpm;
  • the polyurethane particles obtained in the step (5) are dried at room temperature, vacuum-dried to constant weight, and sieved to a particle size of 150 by a 100 and 50 mesh sieve. 270 ⁇ m microspheres.
  • the polyurethane microspheres prepared by the method of the present invention have a uniform white spherical shape and a particle size distribution ranging from 150 micrometers to 270 micrometers;
  • the polyurethane microspheres prepared by the method of the present invention are observed by scanning electron microscopy, and the microspheres have a spherical shape and a smooth surface;
  • the polyurethane microspheres were dissolved in CHCl 3 as a solvent, and the measured 1 H-NMR spectrum, 4.1 ppm from polycaprolactone, and 3.7 ppm from polyethylene glycol;
  • cm -1 is -OH stretching vibration
  • amide bond C O stretching vibration absorption peak near 1740 cm -1
  • 1520- 1560 cm -1 is an amide bond NH deformation vibration
  • IPDI has no NCO absorption peak at 2270 cm -1 , indicating that the reaction is complete.
  • the preparation method of the carrier-loaded polyurethane microspheres comprises the following steps:
  • the molar ratio of isophorone isocyanate to total oligomer diol is 3:1;
  • step (2) 2,2-dimethylolpropionic acid was added while cooling to 55 ° C, and reacted at a stirring speed of 400 rpm for 1 h;
  • the synthesized polyurethane is added dropwise to the stirred distilled water, and dispersed, wherein the stirring speed is 600 rpm;
  • the polyurethane particles obtained in the step (5) are dried at room temperature, vacuum-dried to constant weight, and sieved to a particle size of 150 by a 100 and 50 mesh sieve. 270 ⁇ m microspheres.
  • the polyurethane microspheres prepared in Example 4 were tested and tested for the following properties:
  • the prepared polyurethane composite material is taken, and the cells are amplified on the surface of the carrier by the following method:
  • Example 4 50 mg of the dried microcarriers (polyurethane microspheres prepared in Example 4 of the present invention) were irradiated under ultraviolet light for 6 hours, added to a siliconized glass bottle, and then mixed with 10 ml of Ca 2+ -free , Mg 2+ -containing PBS at room temperature. uniform;
  • the microcarrier in the step (1) was centrifuged, mixed with 50 ml of the cell culture medium, and added to a biaxial rotary reactor, and 1 ml of a fibroblast suspension having a total amount of 5 ⁇ 10 6 cells/mL was added.
  • microcarriers Commercially available microcarriers (Cultispher G) and planar culture were used as a control group, and the rest of the conditions were the same as the microcarriers of the present invention.
  • the rotation speed of the rotary bottle shaft was set to 40 rpm, and it was cultured in a 5% CO 2 atmosphere at 37 °C.
  • the absorbance of the cells was measured at 3h, 1d, 3d and 7d of the culture.
  • the cell compatibility test was performed, that is, the cells were stained by DAPI, and the nuclei reacted with the dye solution to give a blue color under fluorescence excitation.
  • the dried microcarriers (polyurethane microspheres prepared in Example 4 of the present invention) were irradiated under ultraviolet light for 6 hours, added to a siliconized glass bottle, and then hydrated with 10 ml of Ca 2+ -free , Mg 2+ -containing PBS at room temperature. , suction with a syringe to check whether it has injectability.
  • the assay for the performance of the expanded cells is shown in Figure 5: Cells cultured in planar culture (TCP) and commercially available microcarriers (Cultispher G) were used as controls, and cells in the same culture volume were tested with CCK-8 at different time points.
  • TCP planar culture
  • Cultispher G commercially available microcarriers
  • the absorbance of the polyurethane microsphere microcarrier of the invention can effectively promote the expansion of cells in a short time, and the effect is obviously superior to the commercially available gelatin microcarrier.
  • the cells were seeded on the polyurethane microsphere microcarriers of the present invention, and after suspension culture for 7 days, the cells were stained by DAPI, and the nuclei reacted with the dye solution to give a blue color under fluorescence excitation. Under the laser confocal microscope, the cells were uniformly distributed on the surface of the carrier, indicating that the material of the present invention is non-toxic and has good cell compatibility.
  • the polyurethane microsphere microcarrier of the present invention can pass through a syringe and a needle of a syringe, and has an injectability and is convenient for in vivo repair.
  • the polyurethane microspheres are prepared by the method of the invention, can be used as microcarriers, can produce cells with high yield, have good biocompatibility, and have injectability, can be used for convenient and safe repair in vivo, and has good repair effect and better. Application prospects.

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Abstract

本发明公开了一种聚氨酯微球，它的粒径为150μm～270μm。本发明还公开了该聚氨酯微球的制备方法和用途。本发明方法制备得到的聚氨酯微球，可以作为微载体使用，能够得到高产量的细胞，同时生物相容性良好，可注射，能用于体内修复，具有较好的应用前景。

Description

一种聚氨酯微载体及其制备方法和用途 技术领域
本发明涉及一种聚氨酯微载体及其制备方法及其用途，属于生物材料领域。
背景技术
微载体是指直径通常在60～300μm，适用于贴壁依赖型细胞在其表面贴壁生长的微珠。微载体培养细胞有其独特的优势：它们有贴壁依赖型细胞生长所需的表面；由于它们具有大的表面积/体积比率，实现了一个可放大的均一培养系统，使细胞能在少量材料表面实现粘附及大规模扩增；扩增后的细胞容易聚合在微载体表面形成复合体，促进细胞间的相互作用；分泌的胞外基质也支持胞内活动，此两种作用都能够促进细胞的增殖。
目前的微载体通常用于体外培养细胞。随着修复材料的发展，在载体材料上接种细胞的修复方式表现出越来越多的优势，由于微载体材料可以携带大量细胞，还能促进细胞的增殖分化，能够克服单纯细胞注入带来的细胞大量死亡及细胞分散的问题，成为越来越多人关注的对象。细胞-载体复合物对患处进行治疗，常规的方式是通过外科手术植入，因此，可注射性的微载体能够改善细胞载体复合物植入过程中引发的创伤。注射细胞-载体复合物是载体最简单直接的应用，这种方法已在组织工程中被广泛研究。
目前应用较多的微载体材料是明胶，其可以有效培养细胞，但是由于其是天然聚合物材料，力学性能非常差，用于体内修复具有很大的局限性。
人工合成材料的力学性能通常比较优良，但是要满足高效培养细胞和可注射性能两个条件就比较困难。比如，聚氨酯材料中，目前报道的能够作为微载体的是聚氨酯泡沫材料，其就不具备可注射性能，用于体内修复的局限性非常大。
发明内容
为了解决上述问题，本发明提供了一种新的聚氨酯微球微载体材料，即聚氨酯微球及其制备方法和用途。
本发明聚氨酯微球，它的粒径为150μm～270μm。
其中，所述聚氨酯微球按照如下方法制备：
(1)两种低聚物二元醇预混
(2)预聚反应：
异氰酸酯与步骤(1)中二元醇为原料，加入反应容器中，搅拌；
(3)扩链反应
在上述步骤(2)后加入亲水扩链剂，同时降温，搅拌反应；
(4)中和：
加入中和剂，继续搅拌反应；
(5)乳化：
将步骤(4)合成好的聚氨酯逐滴加入到搅拌中的蒸馏水中，分散；
(6)纯化、过筛收集粒径为150μm～270μm的聚氨酯微球。
本发明还提供了前述聚氨酯微球的方法，步骤如下：
(1)两种低聚物二元醇预混
(2)预聚反应：
异氰酸酯与步骤(1)中二元醇为原料，加入反应容器中，搅拌；
(3)扩链反应
在上述步骤(2)后加入亲水扩链剂，同时降温，搅拌反应；
(4)中和：
加入中和剂，继续搅拌反应；
(5)乳化：
将步骤(4)合成好的聚氨酯逐滴加入到搅拌中的蒸馏水中，分散；
(6)纯化、过筛收集粒径为150μm～270μm的聚氨酯微球。
优选地，步骤(1)中，步骤(1)采用的两种不同低聚物二元醇为聚乙二醇、聚己内酯二元醇、聚四氢呋喃中的任意两种；优选地，步骤(1)采用的两种不同低聚物二元醇为聚乙二醇与聚己内酯二元醇或聚四氢呋喃；
进一步地，所述聚己内酯二元醇是聚己内酯二元醇2000，和/或所述聚乙二醇是聚乙二醇200；
进一步地，步骤(1)中，所述聚己内酯二元醇和聚乙二醇的摩尔比为1:1～2:1
进一步地，步骤(1)中，所述聚四氢呋喃和聚乙二醇的摩尔比为1:1～2:1；
优选地，步骤(2)中，异氰酸酯与步骤(1)中总的低聚物二元醇的摩尔比为(2～3)：1，优选为3：1；
和/或，步骤(2)中，所述异氰酸酯为异佛尔酮二异氰酸酯、L-赖氨酸二异氰酸酯、二苯基甲烷二异氰酸酯中的任意一种或多种；优选地，所述异氰酸酯为异佛尔酮二异氰酸酯；
和/或，步骤(2)中，以350-700rmp的速度搅拌，优选搅拌速度为380rmp；反应时间是2-4小时，优选2.5h。
优选地，步骤(3)中，所述扩链剂与步骤(2)中异氰酸酯的摩尔比为(0.1～1):(1)，优选0.5：1；
和/或，步骤(3)中，所述扩链剂为2,2-二羟甲基丁酸或2,2-二羟甲基丙 酸；优选地，所述扩链剂为2,2-二羟甲基丁酸；
和/或，步骤(3)中，所述降温是降至45～55℃，优选50℃；所述搅拌是以350-700rmp的速度搅拌，优选搅拌速度为380rmp；反应时间是1-3小时，优选1.5h。
优选地，步骤(4)中，中和剂与步骤(3)中扩链剂等摩尔比；
和/或，步骤(4)中，所述中和剂为三乙胺或氢氧化钠；
和/或，步骤(4)中，所述搅拌是以350-700rmp的速度搅拌，优选搅拌速度为380rmp；反应时间是15min。
优选地，步骤(5)中，所述搅拌的速度为350-700rmp，优选为500rmp。
优选地，步骤(6)的方法是：用蒸馏水清洗步骤(5)反应所得聚氨酯颗粒，真空干燥至恒重，并用100及50目网筛筛出粒径为150-270μm的微球。
本发明还提供了前述的聚氨酯微球在制备微载体材料中的用途。
本发明还提供了一种体内修复材料，它是以前述聚氨酯微球为微载体，复合细胞制备而成的修复材料。
本发明聚氨酯微球微载体，具有以下有益效果：
本发明聚氨酯微球微载体具有良好的生物相容性，能够为贴壁细胞生长提供优良基底材料；
本发明可优化聚氨酯微球微载体的粒径范围到适合细胞在其表面的粘附扩增，且粒径均匀可控，打破了聚氨酯载体仅为药物载体的应用局限；
本发明聚氨酯微球微载体制备过程中不需要有高沸点的有机溶剂作为介质，无细胞毒性，对环境污染小；
本发明聚氨酯微球微载体在悬浮培养的过程中颗粒分散性好，不会产生团聚的现象，保证了注射修复所需的有效粒径；
本发明聚氨酯微球微载体系统可实现在小的培养体积中得到高产量的细胞；
本发明聚氨酯微球微载体成本低廉，可回收。
综上，本发明方法制备得到的聚氨酯微球可以作为微载体使用，能够高产细胞，同时生物相容性良好，具备可注射性能，可用于体内修复，临床应用前景良好。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。 凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1聚氨酯微球大体图，聚氨酯微球呈白色均匀球形，粒径分布范围在150微米至270微米之间；
图2聚氨酯微球表面形貌，用扫描电镜观察聚氨酯微球形貌，微球呈圆球形，且表面光滑；
图3聚氨酯微球核磁分析，用CHCl 3作溶剂溶解聚氨酯微球，测得的 1H-NMR图谱，4.1ppm来自聚己内酯，3.7ppm来自聚乙二醇；
图4聚氨酯微球红外分析(FTIR)。3250-3500cm -1为-OH伸缩振动、IPDI中NHCO的NH伸缩振动峰，在1740cm -1附近出现酯基C＝O、酰胺键C＝O伸缩振动吸收峰，1520-1560cm -1为酰胺键N-H变形振动，IPDI在2270cm -1处无NCO的吸收峰，说明反应完全；
图5细胞在聚氨酯微球微载体及市售CultiSpher G载体表面的细胞活性。以平面培养(TCP)及市售微载体(Cultispher G)上培养的细胞作为对照，在不同时间点用CCK-8测试相同培养体积中细胞的吸光度，结果证明聚氨酯微球微载体无毒性，并能够有效促进细胞短时间内的扩增；
图6聚氨酯微球微载体表面细胞分布(7d)。细胞接种在微载体上后经过悬浮培养7天，将细胞通过DAPI染色，细胞核与染液反应，在荧光激发下呈现蓝色。激光共聚焦显微镜下观察细胞均匀分布于载体表面，说明材料具有良好的细胞相容性；
图7聚氨酯微球微载体可注射性能的图片；
图8聚氨酯微球微载体可注射性能的图片。
具体实施方式
主要材料、试剂与仪器：
Figure PCTCN2017117530-appb-000001
低聚物二元醇(聚乙二醇、聚己内酯二元醇1000、聚四氢呋喃)；
异氰酸酯(异佛尔酮二异氰酸酯、L-赖氨酸二异氰酸酯、二苯基甲烷二异氰酸酯)；
扩链剂(2,2-二羟甲基丁酸、2,2-二羟甲基丙酸)；
三乙胺、细胞(成骨细胞、成纤维细胞或干细胞)；
无Ca 2+，Mg 2+的PBS。
仪器:CELLSPIN旋转瓶及双轴旋转反应器(INTEGRABiosciencesAG)、增力电动搅拌器(江苏金坛佳美仪器)。
实施例1 本发明聚氨酯微球的制备
1、制备方法
载细胞聚氨酯微球的制备方法包括以下步骤：
(1)低聚物二元醇预混
在三口烧瓶中加入聚己内酯二元醇1000和PEG200，二者的摩尔比为1:1，70℃下搅拌混匀；
(2)预聚反应：
异佛尔酮二异氰酸酯与步骤(1)中二元醇为原料，加入反应容器中，以300rmp的速度搅拌，反应2h；
异佛尔酮异氰酸酯与总的低聚物二元醇的摩尔比为2:1；
(3)扩链反应
在上述步骤(2)后加入2,2-二羟甲基丙酸，同时降温至45℃，在700rmp的搅拌速度下反应2h；
其中扩链剂与步骤(2)中异氰酸酯的摩尔比为0.1:1；
(4)中和：
加入三乙胺中和剂，继续在300rmp的搅拌速度下反应15min；
其中，中和剂与步骤(3)中扩链剂等摩尔比；
(5)乳化：
将合成好的聚氨酯逐滴加入到搅拌中的蒸馏水中，分散，其中搅拌速度为700rmp；
(6)纯化和过筛收集
用蒸馏水反复清洗(室温超声清洗，3次以上，每次10分钟)步骤(5)反应所得聚氨酯颗粒，室温下，真空干燥至恒重，并用100及50目网筛筛出粒径为150-270μm的微球。
实施例2 本发明聚氨酯微球的制备
1、制备方法
载细胞聚氨酯微球的制备方法包括以下步骤：
(1)低聚物二元醇预混
在三口烧瓶中加入聚四氢呋喃和PEG200，二者的摩尔比为1.5:1，70℃下搅拌混匀；
(2)预聚反应：
异佛尔酮二异氰酸酯与步骤(1)中二元醇为原料，加入反应容器中，以700rmp的速度搅拌，反应3h；
异佛尔酮异氰酸酯与总的低聚物二元醇中的摩尔比为2.5:1；
(3)扩链反应
在上述步骤(2)后加入2,2-二羟甲基丙酸，同时降温至50℃，在300rmp的搅拌速度下反应3h；
其中扩链剂与步骤(2)中异氰酸酯的摩尔比为1：1；
(4)中和：
加入三乙胺中和剂，继续在700rmp的搅拌速度下反应15min；
其中，中和剂与步骤(3)中扩链剂等摩尔比；
(5)乳化：
将合成好的聚氨酯逐滴加入到搅拌中的蒸馏水中，分散，其中搅拌速度为300rmp；
(6)纯化和过筛收集
用蒸馏水反复清洗(室温超声清洗，3次以上，每次10分钟)步骤(5)反应所得聚氨酯颗粒，室温下，真空干燥至恒重，并用100及50目网筛筛出粒径为150-270μm的微球。
实施例3 本发明聚氨酯微球的制备
1、制备方法
载细胞聚氨酯微球的制备方法包括以下步骤：
(1)低聚物二元醇预混
在三口烧瓶中加入聚己内酯二元醇1000和PEG200，二者的摩尔比为2:1，70℃下搅拌混匀；
(2)预聚反应：
异佛尔酮二异氰酸酯与步骤(1)中二元醇为原料，加入反应容器中，以380rmp的速度搅拌，反应4h；
异佛尔酮异氰酸酯与总的低聚物二元醇中的摩尔比为2.5:1；
(3)扩链反应
在上述步骤(2)后加入2,2-二羟甲基丙酸，同时降温至55℃，，在380rmp的搅拌速度下反应2h；
其中扩链剂与步骤(2)中异氰酸酯的摩尔比为1：1；
(4)中和：
加入三乙胺中和剂，继续在380rmp的搅拌速度下反应15min；
其中，中和剂与步骤(3)中扩链剂等摩尔比；
(5)乳化：
将合成好的聚氨酯逐滴加入到搅拌中的蒸馏水中，分散，其中搅拌速度为500rmp；
(6)纯化和过筛收集
用蒸馏水反复清洗(室温超声清洗，3次以上，每次10分钟)步骤(5)反应所得聚氨酯颗粒，室温下，真空干燥至恒重，并用100及50目网筛筛出粒径为150-270μm的微球。
实施例4 本发明聚氨酯微球的制备
1、制备方法
载细胞聚氨酯微球的制备方法包括以下步骤：
(1)低聚物二元醇预混
在三口烧瓶中加入聚己内酯二元醇1000和PEG200，聚己内酯二元醇1000与PEG200的摩尔比是2:1，70℃下搅拌混匀；
(2)预聚反应：
异佛尔酮异氰酸酯与步骤(1)中二元醇为原料，加入反应容器中，以380rmp的速度搅拌，反应2.5h；
异佛尔酮异氰酸酯与总的低聚物二元醇的摩尔比为3:1；
(3)扩链反应
在上述步骤(2)后加入2,2-二羟甲基丁酸，同时降温至50℃，在380rmp的搅拌速度下反应1.5h；
其中扩链剂与步骤(2)中异氰酸酯的摩尔比为0.5：1；
(4)中和：
加入三乙胺中和剂，继续在380rmp的搅拌速度下反应15min；
其中，中和剂与步骤(3)中扩链剂等摩尔比；
(5)乳化：
将合成好的聚氨酯逐滴加入到搅拌中的蒸馏水中，分散，其中搅拌速度为500rmp；
(6)纯化和过筛收集
用蒸馏水反复清洗(室温超声清洗，3次以上，每次10分钟)步骤(5)反应所得聚氨酯颗粒，室温下，真空干燥至恒重，并用100及50目网筛筛出粒径为150-270μm的微球。
2、性质
如图1所示，本发明方法制备的聚氨酯微球呈白色均匀球形，粒径分布范围在150微米至270微米之间；
如图2所示，本发明方法制备的聚氨酯微球用扫描电镜观察聚氨酯微球形貌，微球呈圆球形，且表面光滑；
如图3所示，用CHCl 3作溶剂溶解聚氨酯微球，测得的 1H-NMR图谱，4.1ppm来自聚己内酯，3.7ppm来自聚乙二醇；
如图4所示，3250-3500cm -1为-OH伸缩振动、IPDI中NHCO的NH伸缩振动峰，在1740cm -1附近出现酯基C＝O、酰胺键C＝O伸缩振动吸收峰，1520-1560cm -1为酰胺键N-H变形振动，IPDI在2270cm -1处无NCO的吸收峰，说明反应完全。
实施例5 本发明聚氨酯微球的制备
1、制备方法
载细胞聚氨酯微球的制备方法包括以下步骤：
(1)低聚物二元醇预混
在三口烧瓶中加入聚己内酯二元醇1000和PEG200，二者的摩尔比为1:1，70℃下搅拌混匀；
(2)预聚反应：
异佛尔酮异氰酸酯与步骤(1)中二元醇为原料，加入反应容器中，以400rmp的速度搅拌，反应3.5h；
异佛尔酮异氰酸酯与总的低聚物二元醇的摩尔比为3:1；
(3)扩链反应
在上述步骤(2)后加入2,2-二羟甲基丙酸，同时降温至55℃，在400rmp的搅拌速度下反应1h；
其中扩链剂与步骤(2)中异氰酸酯的摩尔比为1：1；
(4)中和：
加入三乙胺中和剂，继续在400rmp的搅拌速度下反应15min；
其中，中和剂与步骤(3)中扩链剂等摩尔比；
(5)乳化：
将合成好的聚氨酯逐滴加入到搅拌中的蒸馏水中，分散，其中搅拌速度为600rmp；
(6)纯化和过筛收集
用蒸馏水反复清洗(室温超声清洗，3次以上，每次10分钟)步骤(5)反应所得聚氨酯颗粒，室温下，真空干燥至恒重，并用100及50目网筛筛出粒径为150-270μm的微球。
以下用实验例的方式来证明本发明的有益效果：
实验例1 本发明聚氨酯微球的性能检测
取实施例4制备得到的聚氨酯微球，检测其如下性能：
一、实验方法
1、扩增细胞的性能和细胞相容性
取制备得到的聚氨酯复合材料，取细胞在载体表面扩增，通过如下方法实现：
(1)本发明微载体材料灭菌及水合
将干燥的微载体(本发明实施例4制备得到的聚氨酯微球)50mg于紫外下照射6h，加入到硅化过的玻璃瓶，然后用10ml无Ca 2+，Mg 2+的PBS在室温下混匀；
(2)接种细胞
离心出步骤(1)中的微载体，与50ml细胞培养基混合，加入到双轴旋转反应器中，加入总量为5×10 6cells/mL的成纤维细胞悬液1ml。
以市售微载体(Cultispher G)和平面培养为对照组，其余条件同本发明微载体。
(3)细胞扩增
设置转瓶轴转速为40rpm，置于5％CO 2，37℃环境中培养。
(4)检测
于培养的第3h、1d、3d和7d检测细胞的吸光度。
培养7天后，进行细胞相容性检测，即，将细胞通过DAPI染色，细胞核与染液反应，在荧光激发下呈现蓝色。
2、可注射性能
取干燥的微载体(本发明实施例4制备得到的聚氨酯微球)50mg于紫外下照射6h，加入到硅化过的玻璃瓶，然后用10ml无Ca 2+，Mg 2+的PBS在室温下水合，用注射器吸取，检测其是否具备可注射性能。
二、实验结果
1、扩增细胞的性能
扩增细胞的性能的检测图如图5所示：以平面培养(TCP)及市售微载体(Cultispher G)上培养的细胞作为对照，在不同时间点用CCK-8测试相同培养体积中细胞的吸光度，本发明聚氨酯微球微载体能够有效促进细胞短时间内的扩增，且效果明显优于市售的明胶微载体。
2、细胞相容性
如图6所示，细胞接种在本发明聚氨酯微球微载体上后经过悬浮培养7天，将细胞通过DAPI染色，细胞核与染液反应，在荧光激发下呈现蓝色。激光共聚焦显微镜下观察细胞均匀分布于载体表面，说明本发明材料无毒，具有良好的细胞相容性。
3、可注射性能
如图7和图8所示，本发明聚氨酯微球微载体可以通过注射器以及注射器的针头，且具备可注射性能，用于体内修复方便。
综上，本发明方法制备得到了聚氨酯微球，可以作为微载体使用，能够高产细胞，同时生物相容性良好，具备可注射性能，可用于体内修复方便、安全，修复效果好，具有较好的应用前景。

Claims (11)

  1. 一种聚氨酯微球，其特征在于：它的粒径为150μm～270μm。
  2. 根据权利要求1所述的聚氨酯微球，其特征在于：它是按照如下方法制备：
    (1)两种不同低聚物二元醇预混；
    (2)预聚反应：
    异氰酸酯与步骤(1)中低聚物二元醇为原料，加入反应容器中，搅拌；
    (3)扩链反应
    在上述步骤(2)后加入亲水扩链剂，同时降温，搅拌反应；
    (4)中和：
    加入中和剂，继续搅拌反应；
    (5)乳化：
    将步骤(4)合成好的聚氨酯逐滴加入到搅拌中的蒸馏水中，分散；
    (6)纯化、过筛收集粒径为150μm～270μm的聚氨酯微球。
  3. 一种制备权利要求1或2所述聚氨酯微球的方法，其特征在于：步骤如下：
    (1)两种不同低聚物二元醇预混；
    (2)预聚反应：
    异氰酸酯与步骤(1)中低聚物二元醇为原料，加入反应容器中，搅拌；
    (3)扩链反应
    在上述步骤(2)后加入亲水扩链剂，同时降温，搅拌反应；
    (4)中和：
    加入中和剂，继续搅拌反应；
    (5)乳化：
    将步骤(4)合成好的聚氨酯逐滴加入到搅拌中的蒸馏水中，分散；
    (6)纯化、过筛收集粒径为150μm～270μm的聚氨酯微球。
  4. 根据权利要求3所述的方法，其特征在于：
    步骤(1)中，步骤(1)采用的两种不同低聚物二元醇为聚乙二醇、聚己内酯二元醇、聚四氢呋喃中的任意两种；优选地，步骤(1)采用的两种不同低聚物二元醇为聚乙二醇与聚己内酯二元醇或聚四氢呋喃；
    进一步地，所述聚己内酯二元醇是聚己内酯二元醇2000，和/或，所述聚乙二醇是聚乙二醇200；
    进一步地，步骤(1)中，所述聚己内酯二元醇和聚乙二醇的摩尔比为1:1～2:1；
    进一步地，步骤(1)中，所述聚四氢呋喃和聚乙二醇的摩尔比为1:1～2:1；
    和/或，步骤(1)中，所述搅拌是70℃下搅拌混匀。
  5. 根据权利要求3所述的方法，其特征在于：
    步骤(2)中，异氰酸酯与步骤(1)中总的低聚物二元醇的摩尔比为(2～3)：1，优选为3：1；
    和/或，步骤(2)中，所述异氰酸酯为异佛尔酮二异氰酸酯、L-赖氨酸二异氰酸酯、二苯基甲烷二异氰酸酯中的任意一种或多种；优选地，所述异氰酸酯为异佛尔酮二异氰酸酯；
    和/或，步骤(2)中，以350-700rmp的速度搅拌，优选搅拌速度为380rmp；反应时间是2-4小时，优选2.5h。
  6. 根据权利要求3所述的方法，其特征在于：步骤(3)中，所述扩链剂与步骤(2)中异氰酸酯的摩尔比为(0.1～1):(1)，优选0.5：1；
    和/或，步骤(3)中，所述扩链剂为2,2-二羟甲基丁酸或2,2-二羟甲基丙酸；优选地，所述扩链剂为2,2-二羟甲基丁酸；
    和/或，步骤(3)中，所述降温是降至45～55℃，优选50℃；所述搅拌是以350-700rmp的速度搅拌，优选搅拌速度为380rmp；反应时间是1-3小时，优选1.5h。
  7. 根据权利要求3所述的方法，其特征在于：步骤(4)中，中和剂与步骤(3)中扩链剂等摩尔比；
    和/或，步骤(4)中，所述中和剂为三乙胺或氢氧化钠；
    和/或，步骤(4)中，所述搅拌是以350-700rmp的速度搅拌，优选搅拌速度为380rmp；反应时间是15min。
  8. 根据权利要求3所述的方法，其特征在于：步骤(5)中，所述搅拌的速度为350-700rmp，优选为500rmp。
  9. 根据权利要求3所述的方法，其特征在于：步骤(6)的方法是：用蒸馏水清洗步骤(5)反应所得聚氨酯颗粒，真空干燥至恒重，并用100及50目网筛筛出粒径为150-270μm的微球。
  10. 权利要求1或2所述的聚氨酯微球在制备微载体材料中的用途。
  11. 一种体内修复材料，其特征在于：它是以权利要求1或2所述聚氨酯微球为微载体，复合细胞制备而成的修复材料。
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