Preparation method of polyurethane microspheres capable of actively loading exosome and slowly and controllably releasing exosome

Abstract

The invention provides a preparation method of polyurethane microspheres capable of actively loading and slowly and controllably releasing exosomes, which is characterized in that the polyurethane microspheres comprise polyurethane microspheres and exosomes, the polyurethane microspheres are used as an exosome adsorption carrier, the particle size of the polyurethane microspheres is 3-500 microns, and the surface structure of the polyurethane microspheres is adjustable. Different from traditional hydrogel and microspheres, the polyurethane microspheres provided by the invention can realize high-activity loading and slow-control release of exosomes by regulating and controlling the chemical composition and pore structure of the microspheres, and the molecular weight and hydrophilicity of the polymer are accurately regulated and controlled by a special polymerization process, so that the release of the exosomes by the microspheres is fully adjustable. The prepared exosome-carrying microsphere preparation can be used in the field of tissue repair for treating refractory wounds and the like, and the exosome and the microspheres are used as a novel delivery system and have huge potential in the aspects of treating virus infection, cancers, immune diseases and the like.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and specifically relates to a method for preparing polyurethane microspheres capable of actively loading and slowly and controlled releasing exosomes. Background Art

[0002] Stem cell therapy is an innovative field of scientific research and clinical application. The introduction of stem cells can effectively create a microenvironment for repair and regeneration and improve the treatment effect of disease or injury. However, the source of stem cells is limited, the process of in vitro cell culture is cumbersome, the transportation conditions are harsh, it is difficult to maintain high activity for a long time, and the introduction of a large number of exogenous stem cells has potential tumorigenic risks and ethical approval issues, making it difficult to achieve clinical application. Studies have shown that the role of stem cells in repairing tissue damage depends not only on their migration and differentiation abilities, but also on their powerful paracrine abilities. The main principle of stem cells promoting the repair effect is to regulate the appropriate immune response through the active factors secreted by cells, create a better blood circulation environment, and promote angiogenesis and tissue repair.

[0003] Stem cell exosomes (EXO) are key components in the paracrine secretion of stem cells. They contain a variety of active factors and have biological activities and functions similar to those of stem cells. Despite this, there are still problems such as insufficient stability of active ingredients and inability to maintain their biological activity for a long time when stem cell exosomes are used. Therefore, it is urgent to develop a material that can actively load stem cell exosomes and effectively maintain the long-term activity of stem cell exosomes, so as to provide new active biomaterials and effective treatment technologies for the treatment of major diseases such as refractory wounds. The use of microcarrier technology to achieve active loading of exosomes is an important development direction in the future. As a high-end technology proposed to solve the national bottleneck project, microsphere preparations will be developed as new preparations such as long-acting, sustained-release and controlled-release. The current microsphere production technology is difficult, and the quality parameters such as the morphology, particle size and particle size distribution of the microspheres are difficult to control.

[0004] In order to solve the above technical problems, the present invention forms a series of polyurethane materials with physical and chemical properties through process optimization, and prepares polyurethane microspheres with different microscopic morphologies through microemulsion and phase separation technology. The microspheres have unique advantages in performance, adjustable microsphere particle size, narrow size distribution, and controllable morphology. Exosomes are actively loaded through the surface microstructure of polyurethane microspheres, and then the controlled release of exosomes is achieved through diffusion and gradual disintegration of microspheres, ultimately achieving a better effect of promoting tissue repair. Summary of the invention

[0005] In order to solve the problem of high requirements for the existing preservation conditions of exosomes, as well as the shortcomings of uneven size of polyurethane microspheres and difficulty in precise control of microscopic morphology, the present invention provides a method for preparing polyurethane microspheres that can actively load and slowly release exosomes. Different from traditional hydrogels and microspheres, the polyurethane microspheres of the present invention can achieve high active loading and slow-controlled release of exosomes by regulating the chemical composition and pore structure of the microspheres and precisely regulating the polymer molecular weight and molecular chain structure through a special polymerization process.

[0006] On the one hand, the present invention provides a method for preparing polyurethane microspheres capable of active loading and controlled release of exosomes, characterized in that the method comprises polyurethane microspheres and exosomes, wherein the polyurethane microspheres are used as exosome adsorption carriers, have a particle size of 3-500 μm, and have an adjustable surface structure;

[0007] The chemical composition of the polyurethane microspheres is: 45%-65% diisocyanate; 20%-45% diol; 5%-10% chain extender; 0.1%-0.6% catalyst;

[0008] The physical structure of the polyurethane microspheres is: particle size 3-500 μm, one or more of solid microspheres, hollow microspheres, and porous microspheres;

[0009] The exosomes are: one or more of human umbilical cord mesenchymal stem cell exosomes, bone marrow mesenchymal stem cell exosomes, and adipose mesenchymal stem cell exosomes;

[0010] Preferably, there are the following steps:

[0011] S1: preparing polyurethane material;

[0012] S2: Preparation of polyurethane microspheres;

[0013] S3: Cell expansion, isolation and purification of stem cell exosomes, and active loading of exosomes by physical adsorption and freeze drying;

[0014] Preferably, the preparation steps of the polyurethane material are:

[0015] S11: mixing diisocyanate, diol, chain extender and catalyst in a certain proportion and dissolving them in a solvent;

[0016] S12: heating the mixed raw materials at 60-110° C. for 4-24 hours;

[0017] The ratio of raw materials used in step S11 is: diisocyanate 45%-65% (w / v); diol 20%-45% (w / v); chain extender 5%-10% (w / v); catalyst 0.1%-0.6% (w / v);

[0018] The solvent used in step S11 is: one or more of dimethylformamide, tetrahydrofuran, xylene, and cyclohexanone;

[0019] Preferably, the preparation steps of polyurethane microspheres are:

[0020] S21: dissolving the polyurethane in an organic solvent to form an organic phase I;

[0021] S22: dissolving the porogen in water to form aqueous phase I;

[0022] S23: dissolving the surfactant in water to form water phase II;

[0023] S24: adding the aqueous phase I dropwise to the organic phase I, and emulsifying for 2-30 min at a stirring speed of 300-3000 rpm to form an emulsion I;

[0024] S25: adding emulsion I dropwise to aqueous phase II, and emulsifying at a stirring speed of 300-3000 rpm for 1-12 h;

[0025] S26: Continue stirring until the organic solvent evaporates, remove the porogen, collect the microspheres by centrifugation or filtration, wash them with pure water for multiple times, and then freeze-dry them for 24-48 hours to obtain polyurethane microspheres;

[0026] The polyurethane concentration used in step S21 is: 0.1-20% (w / v);

[0027] The organic solvent used in step S21 is one or more of dimethylformamide, tetrahydrofuran, xylene, cyclohexanone, and butyl acetate;

[0028] The porogen used in step S22 is: one or more of ammonium bicarbonate and sodium bicarbonate, with a concentration of 0.1-20% (w / v);

[0029] The surfactant in step S23 is one or more of polyvinyl pyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, and sodium lauryl sulfate, with a concentration of 0.1-20% (w / v).

[0030] The volume ratio of the aqueous phase I to the organic phase I in step S24 is: 1:1 to 1:20

[0031] The volume ratio of the emulsion I and the aqueous phase II in step S25 is: 1:1 to 1:20

[0032] Preferably, the steps for preparing exosomes are:

[0033] S31: culturing, passaging and collecting mesenchymal stem cells, and performing cell lysis to obtain cell lysate;

[0034] S32: obtaining mesenchymal stem cell exosomes from the cell lysate by differential centrifugation at a centrifugal force of 300-100000×g;

[0035] S33: performing sterility test, mycoplasma test, endotoxin test and flow cytometry sorting on the stem cell exosomes to obtain qualified exosomes;

[0036] The mesenchymal stem cells used in step S31 are one of: human umbilical cord mesenchymal stem cell exosomes, bone marrow mesenchymal stem cell exosomes, and adipose mesenchymal stem cell exosomes.

[0037] Preferably, the step of loading exosomes with activity of exosomes is: dispersing the exosomes in a buffer, adding polyurethane microspheres, and incubating in a light-proof, 4°C constant temperature shaker for 1-24 hours to obtain polyurethane microspheres carrying exosomes.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The present invention synthesizes polyurethane materials with different chemical compositions through a precise polymerization process to achieve materials with different hydrophilicity, different molecular weight and different molecular chain structures, thereby achieving different binding strengths to exosomes.

[0040] 2. The present invention uses microemulsion technology to prepare polyurethane microspheres with adjustable particle size and uniform size, and uses phase transition technology to achieve precise control of the pore structure of the polyurethane microspheres, and combines the different microstructures of the microspheres to achieve active loading and controlled release of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is the particle size distribution diagram of polyurethane porous microspheres. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further specifically described below through examples, but the protection scope of the present invention is not limited thereto.

[0043] Example 1

[0044] The present invention provides a method for preparing polyurethane microspheres capable of active loading and controlled release of exosomes, characterized in that the method comprises polyurethane microspheres and exosomes, wherein the polyurethane microspheres are used as exosome adsorption carriers, have a particle size of 3-500 μm, and have an adjustable surface structure;

[0045] The chemical composition of the polyurethane microspheres is: 45%-65% diisocyanate; 20%-45% diol; 5%-10% chain extender; 0.1%-0.6% catalyst;

[0046] The physical structure of the polyurethane microspheres is: particle size 3-500 μm, one or more of solid microspheres, hollow microspheres, and porous microspheres;

[0047] The exosomes are: one or more of human umbilical cord mesenchymal stem cell exosomes, bone marrow mesenchymal stem cell exosomes, and adipose mesenchymal stem cell exosomes;

[0048] Specifically, there are the following steps:

[0049] S1: preparing polyurethane material;

[0050] S11: mixing diisocyanate, diol, chain extender and catalyst in a certain proportion and dissolving them in a solvent;

[0051] S12: heating the mixed raw materials at 60-110° C. for 4-24 hours;

[0052] The ratio of raw materials used in step S11 is: diisocyanate 45%-65% (w / v); diol 20%-45% (w / v); chain extender 5%-10% (w / v); catalyst 0.1%-0.6% (w / v);

[0053] The solvent used in step S11 is: one or more of dimethylformamide, tetrahydrofuran, xylene, and cyclohexanone;

[0054] S2: Preparation of polyurethane microspheres;

[0055] S21: dissolving polyurethane in an organic solvent to form an organic phase I, wherein the polyurethane concentration is 0.1-20% (w / v);

[0056] S22: dissolving the porogen in water to form aqueous phase I;

[0057] S23: dissolving the surfactant in water to form water phase II;

[0058] S24: adding the aqueous phase I dropwise to the organic phase I, and emulsifying for 2-30 min at a stirring speed of 300-3000 rpm to form an emulsion I;

[0059] S25: adding emulsion I dropwise to aqueous phase II, and emulsifying at a stirring speed of 300-3000 rpm for 1-12 h;

[0060] S26: Continue stirring until the organic solvent evaporates, remove the porogen, collect the microspheres by centrifugation or filtration, wash them with pure water for multiple times, and then freeze-dry them for 24-48 hours to obtain polyurethane microspheres;

[0061] The organic solvent used in step S21 is one or more of dimethylformamide, tetrahydrofuran, xylene, cyclohexanone, and butyl acetate;

[0062] The porogen used in step S22 is: one or more of ammonium bicarbonate and sodium bicarbonate, with a concentration of 0.1-20% (w / v);

[0063] The surfactant in step S23 is one or more of polyvinyl pyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, and sodium lauryl sulfate, with a concentration of 0.1-20% (w / v).

[0064] The volume ratio of the aqueous phase I to the organic phase I in step S24 is: 1:1 to 1:20

[0065] The volume ratio of the emulsion I and the aqueous phase II in step S25 is: 1:1 to 1:20

[0066] S3: Cell expansion, isolation and purification of stem cell exosomes, and active loading of exosomes by physical adsorption and freeze drying;

[0067] The steps for preparing exosomes are:

[0068] S31: culturing, passaging and collecting mesenchymal stem cells, and performing cell lysis to obtain cell lysate;

[0069] S32: obtaining mesenchymal stem cell exosomes from the cell lysate by differential centrifugation at a centrifugal force of 300-100000×g; the mesenchymal stem cells are: human umbilical cord mesenchymal stem cell exosomes, bone marrow mesenchymal stem cell exosomes, and adipose mesenchymal stem cell exosomes;

[0070] S33: performing sterility test, mycoplasma test, endotoxin test and flow cytometry sorting on the stem cell exosomes to obtain qualified exosomes;

[0071] Specifically, the step of loading exosomes with active exosomes is as follows: dispersing the exosomes in a buffer solution, adding polyurethane microspheres, and incubating in a light-proof, 4°C constant temperature shaker for 1-24 hours to obtain polyurethane microspheres carrying exosomes.

[0072] Example 2

[0073] This embodiment provides a method for preparing polyurethane microspheres capable of active loading and controlled release of exosomes. The difference between this embodiment and embodiment 1 is that:

[0074] 2g of polyurethane was dissolved in 20mL of dimethylformamide to prepare organic phase I, 0.5g of sodium bicarbonate was dissolved in 10mL of pure water to prepare aqueous phase I, and 1g of polyvinyl alcohol was dissolved in 50mL of pure water to prepare aqueous phase II. Then the sodium bicarbonate solution was dripped into the dimethylformamide solution of polyurethane, stirred at 1500rpm for 15min to form emulsion I, and then emulsion I was dripped into aqueous phase II, stirred at 500rpm for 12h to emulsify and evaporate the solvent. The microspheres were collected by centrifugation and washed with pure water 3 times and freeze-dried for 48h to obtain polyurethane microspheres.

[0075] Example 3

[0076] This embodiment provides a method for preparing polyurethane microspheres capable of active loading and controlled release of exosomes. The difference between this embodiment and embodiment 1 is that:

[0077] 3g of polyurethane was dissolved in 20mL of dimethylformamide to prepare organic phase I, 0.5g of sodium bicarbonate was dissolved in 8mL of pure water to prepare aqueous phase I, and 2g of polyvinyl alcohol was dissolved in 100mL of pure water to prepare aqueous phase II. Then the sodium bicarbonate solution was dripped into the dimethylformamide solution of polyurethane, stirred at 1000rpm for 15min to form emulsion I, and then emulsion I was dripped into aqueous phase II, stirred at 300rpm for 12h to emulsify and evaporate the solvent. The microspheres were collected by centrifugation and washed with pure water 3 times and freeze-dried for 48h to obtain polyurethane microspheres.

[0078] Example 4

[0079] This embodiment provides a method for preparing polyurethane microspheres capable of active loading and controlled release of exosomes. The difference between this embodiment and embodiment 1 is that:

[0080] The exosomes of umbilical cord mesenchymal stem cells were separated, dispersed in a buffer solution, and 10 times the mass of polyurethane microspheres were added. The microspheres were incubated in a light-proof, 4°C constant temperature shaker for 4 h to obtain polyurethane microspheres carrying exosomes.

[0081] The above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. Any variation directly derived from the contents disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for preparing polyurethane microspheres capable of active loading and controlled release of exosomes, characterized in that: It includes polyurethane microspheres and exosomes, wherein the polyurethane microspheres are used as exosome adsorption carriers, have a particle size of 3-500 μm, and an adjustable surface structure; The chemical composition of the polyurethane microspheres is: 45%-65% diisocyanate; 20%-45% diol; 5%-10% chain extender; 0.1%-0.6% catalyst; The physical structure of the polyurethane microspheres is: particle size 3-500 μm, one or more of solid microspheres, hollow microspheres, and porous microspheres; The exosomes are: one or more of human umbilical cord mesenchymal stem cell exosomes, bone marrow mesenchymal stem cell exosomes, and adipose mesenchymal stem cell exosomes.

2. A method for preparing polyurethane microspheres capable of active loading and controlled release of exosomes as claimed in claim 1, characterized in that: There are the following steps: S1: preparing polyurethane material; S2: Preparation of polyurethane microspheres; S3: Cell expansion, separation and purification of stem cell exosomes, and active loading of exosomes through physical adsorption and freeze-drying.

3. A method for preparing polyurethane microspheres capable of active loading and controlled release of exosomes as claimed in claim 2, characterized in that: The preparation steps of polyurethane material are: S11: mixing diisocyanate, diol, chain extender and catalyst in a certain proportion and dissolving them in a solvent; S12: heating the mixed raw materials at 60-110° C. for 4-24 hours; The ratio of raw materials used in step S11 is: diisocyanate 45%-65% (w / v); diol 20%-45% (w / v); chain extender 5%-10% (w / v); catalyst 0.1%-0.6% (w / v); The solvent used in step S11 is one or more of dimethylformamide, tetrahydrofuran, xylene, and cyclohexanone.

4. A method for preparing polyurethane microspheres capable of active loading and controlled release of exosomes as claimed in claim 2, characterized in that: The preparation steps of polyurethane microspheres are as follows: S21: dissolving the polyurethane in an organic solvent to form an organic phase I; S22: dissolving the porogen in water to form aqueous phase I; S23: dissolving the surfactant in water to form water phase II; S24: adding the aqueous phase I dropwise to the organic phase I, and emulsifying for 2-30 min at a stirring speed of 300-3000 rpm to form an emulsion I; S25: adding emulsion I dropwise to aqueous phase II, and emulsifying at a stirring speed of 300-3000 rpm for 1-12 h; S26: Continue stirring until the organic solvent evaporates, remove the porogen, collect the microspheres by centrifugation or filtration, wash them with pure water for multiple times, and then freeze-dry them for 24-48 hours to obtain polyurethane microspheres; The polyurethane concentration used in step S21 is: 0.1-20% (w / v); The organic solvent used in step S21 is one or more of dimethylformamide, tetrahydrofuran, xylene, cyclohexanone, and butyl acetate; The porogen used in step S22 is: one or more of ammonium bicarbonate and sodium bicarbonate, with a concentration of 0.1-20% (w / v); The surfactant in step S23 is one or more of polyvinyl pyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, and sodium lauryl sulfate, with a concentration of 0.1-20% (w / v). The volume ratio of the aqueous phase I to the organic phase I in step S24 is: 1:1 to 1:20 The volume ratio of the emulsion I and the aqueous phase II in the step S25 is 1:1 to 1:

20.

5. A method for preparing polyurethane microspheres capable of active loading and controlled release of exosomes as claimed in claim 2, characterized in that: The steps for preparing exosomes are: S31: culturing, passaging and collecting mesenchymal stem cells, and performing cell lysis to obtain cell lysate; S32: obtaining mesenchymal stem cell exosomes from the cell lysate by differential centrifugation at a centrifugal force of 300-100000×g; S33: performing sterility test, mycoplasma test, endotoxin test and flow cytometry sorting on the stem cell exosomes to obtain qualified exosomes; The mesenchymal stem cells used in step S31 are one of: human umbilical cord mesenchymal stem cell exosomes, bone marrow mesenchymal stem cell exosomes, and adipose mesenchymal stem cell exosomes.

6. A method for preparing polyurethane microspheres capable of active loading and controlled release of exosomes as claimed in claim 2, characterized in that: The steps of loading exosomes with activity of exosomes are as follows: dispersing the exosomes in a buffer solution, adding polyurethane microspheres, and incubating for 1-24 hours in a light-proof, 4°C constant temperature shaker to obtain polyurethane microspheres carrying exosomes.

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