阅读说明：本技术 一种生物智能的电响应性细胞微载体、其制备方法及应用 (Biological intelligent electric response cell microcarrier, preparation method and application thereof ) 是由 章培标 闫欢欢 王宗良 王宇 于 2020-05-09 设计创作，主要内容包括：本发明提供了一种生物智能的电响应性细胞微载体的制备方法,包括以下步骤：将电活性无规共聚物溶解于醇类溶剂中,加入微载体及氧化剂,进行氧化聚合反应,干燥后得到生物智能的电响应性细胞微载体；所述电活性无规共聚物为含有苯胺四聚体和多巴胺官能团的电活性无规共聚物；所述氧化剂为高碘酸钠和/或高碘酸钾。本发明通过多巴胺邻苯二酚的氧化聚合直接将电活性的苯胺四聚体引入到微载体表面,同时还可将生物活性分子固定到微载体上,且制备得到的电响应性细胞微载体在有机溶剂中依然具有优异的结合力,稳定不脱落。本发明还提供了一种生物智能的电响应性细胞微载体及应用。(The invention provides a preparation method of a biological intelligent electric response cell microcarrier, which comprises the following steps: dissolving the electroactive random copolymer in an alcohol solvent, adding a microcarrier and an oxidant, carrying out oxidative polymerization reaction, and drying to obtain a biological intelligent electric response cell microcarrier; the electroactive random copolymer is an electroactive random copolymer containing aniline tetramers and dopamine functional groups; the oxidant is sodium periodate and/or potassium periodate. According to the invention, the electroactive aniline tetramer is directly introduced to the surface of the microcarrier through oxidative polymerization of dopamine catechol, and meanwhile, bioactive molecules can be fixed on the microcarrier, and the prepared electrically-responsive cell microcarrier still has excellent binding force in an organic solvent and is stable and free of falling off. The invention also provides a biological intelligent electric responsive cell microcarrier and application thereof.)

1. A method for preparing a bio-intelligent electrically responsive cell microcarrier, comprising the steps of:

dissolving the electroactive random copolymer in an alcohol solvent, adding a microcarrier and an oxidant, carrying out oxidative polymerization reaction, and drying to obtain a biological intelligent electric response cell microcarrier;

the electroactive random copolymer is an electroactive random copolymer containing aniline tetramers and dopamine functional groups;

the oxidant is sodium periodate and/or potassium periodate.

2. The production method according to claim 1, wherein the mass ratio of the electroactive random copolymer to the oxidizing agent is 1: (0.1-10).

3. The method according to claim 1, wherein the ratio of the mass of the electroactive random copolymer to the volume of the alcoholic solvent is (0.5 to 2.5) g (1 to 10) m L;

the ratio of the mass of the microcarrier to the volume of the alcohol solvent is (0.05-5) g, (1-10) m L.

4. The method according to claim 1, wherein the temperature of the oxidative polymerization reaction is 30 to 45 ℃; the time of the oxidative polymerization reaction is 12-50 hours;

the oxidative polymerization reaction is carried out under the condition of stirring, and the stirring speed is 130-180 rpm.

5. The method according to claim 4, wherein the alcoholic solvent is methanol and/or ethanol.

6. The preparation method according to claim 1, wherein the microcarrier is one or more of a polylactic acid-glycolic acid copolymer microcarrier, a polylactic acid microcarrier, a polyether ether ketone microcarrier and a hydroxyapatite-compounded polylactic acid-glycolic acid copolymer.

7. The method of claim 1, wherein the electroactive random copolymer is prepared by the steps of:

dissolving polyethylene glycol methyl ether methacrylate, dopamine methacrylamide, aniline tetramer methacrylamide and an initiator in a solvent in an inert gas environment, stirring for 0.4-0.6 hours at the temperature of 20-30 ℃, and then moving to an oil bath for reaction for 20-50 hours at the temperature of 60-85 ℃ to obtain the electroactive random copolymer.

8. The preparation method according to claim 7, wherein the mass ratio of the dopamine methacrylamide, the aniline tetramer methacrylamide, the polyethylene glycol methyl ether methacrylate and the initiator is (15-40): (0.1-18): (50-80): (0.1-2).

9. A biologically-intelligent electrically-responsive cell microcarrier prepared by the method of any one of claims 1 to 8.

10. Use of the biosensing electrically responsive cell microcarrier of claim 9 for bone tissue damage repair.

Technical Field

The invention belongs to the technical field of preparation of cell microcarriers, and particularly relates to a biological intelligent electric responsiveness cell microcarrier, and a preparation method and application thereof.

Background

The microcarrier is a spherical particle which can support the adherence type cells to adhere to, grow and metabolize on the surface of the microcarrier in a three-dimensional space. In the large-scale culture technology of animal cells, the microcarrier culture method has various advantages: the surface area/volume ratio is large, the area of cell adhesion growth is increased, the space occupied by cell culture is reduced, the large-scale culture and collection of cells are facilitated, and the cell yield of a culture solution in unit volume is high; the cell harvesting process is relatively simple, and the labor intensity is low; the damage of pancreatin to cells is avoided during cell passage, and only a new microcarrier needs to be added; the culture medium has high utilization rate, easy amplification and the like, and is a culture mode which is recognized as the most promising development mode at present. The microcarrier cell culture technology can realize rapid and large-scale high-quality amplification of cells in a dynamic three-dimensional culture environment so as to meet the requirements of tissue engineering and stem cell treatment.

In recent years, with the progress of technology, microcarriers prepared from non-toxic and biocompatible degradable polymer materials such as polylactic-co-glycolic acid (P L GA) and polylactic acid (P L a) can be used to prepare non-degradable microcarriers from special polymer materials such as Polyetheretherketone (PEEK).

At present, functional materials for modifying the surface of the microcarrier with electric activity, biological activity and the like have two methods of physical adsorption and chemical bond combination. Among them, as a commonly used method for immobilizing functional groups, physical adsorption is generally nonspecific and inefficient. Conventional chemical covalent bonding often requires multiple steps and complicated procedures, such as activation of the material surface by pretreatment, or irradiation with oxygen plasma, ultraviolet light, or other chemicals. Such modifications can significantly alter the bulk properties of the material and even result in surface denaturation of the material. Therefore, the method for fixing the functional material to modify the surface of the microcarrier has good application prospect, and is simple, reliable and efficient.

Disclosure of Invention

The invention aims to provide a biological intelligent electric responsive cell microcarrier, a preparation method and application thereof.

The invention provides a preparation method of a biological intelligent electric response cell microcarrier, which comprises the following steps:

dissolving the electroactive random copolymer in an alcohol solvent, adding a microcarrier and an oxidant, carrying out oxidative polymerization reaction, and drying to obtain a biological intelligent electric response cell microcarrier;

the electroactive random copolymer is an electroactive random copolymer containing aniline tetramers and dopamine functional groups;

the oxidant is sodium periodate and/or potassium periodate.

Preferably, the mass ratio of the electroactive random copolymer to the oxidizing agent is 1: (0.1-10).

Preferably, the ratio of the mass of the electroactive random copolymer to the volume of the alcoholic solvent is (0.5-2.5) g, (1-10) m L;

the ratio of the mass of the microcarrier to the volume of the alcohol solvent is (0.05-5) g, (1-10) m L.

Preferably, the temperature of the oxidative polymerization reaction is 30-45 ℃; the time of the oxidative polymerization reaction is 12-50 hours;

the oxidative polymerization reaction is carried out under the condition of stirring, and the stirring speed is 130-180 rpm.

Preferably, the alcohol solvent is methanol and/or ethanol.

Preferably, the microcarrier is one or more of a polylactic acid-glycolic acid copolymer microcarrier, a polylactic acid microcarrier, a polyether-ether-ketone microcarrier and a polylactic acid-glycolic acid copolymer compounded with hydroxyapatite.

Preferably, the electroactive random copolymer is prepared by the following steps:

dissolving polyethylene glycol methyl ether methacrylate, dopamine methacrylamide, aniline tetramer methacrylamide and an initiator in a solvent in an inert gas environment, stirring for 0.4-0.6 hours at the temperature of 20-30 ℃, and then moving to an oil bath for reaction for 20-50 hours at the temperature of 60-85 ℃ to obtain the electroactive random copolymer.

Preferably, the mass ratio of the dopamine methacrylamide, the aniline tetramer methacrylamide, the polyethylene glycol methyl ether methacrylate to the initiator is (15-40): (0.1-18): (50-80): (0.1-2).

A biologically-active electrically-responsive cell microcarrier is prepared by the above-mentioned preparation method.

Use of a biosensing electrically responsive cell microcarrier as described above for bone tissue damage repair.

The invention provides a preparation method of a biological intelligent electric response cell microcarrier, which comprises the following steps: dissolving the electroactive random copolymer in an alcohol solvent, adding a microcarrier and an oxidant, carrying out oxidative polymerization reaction, and drying to obtain a biological intelligent electric response cell microcarrier; the electroactive random copolymer is an electroactive random copolymer containing aniline tetramers and dopamine functional groups; the oxidant is sodium periodate and/or potassium periodate. According to the invention, an electroactive random copolymer containing aniline tetramer and dopamine functional group is directly introduced to the surface of a microcarrier in a sodium periodate oxidant environment through oxidative polymerization of dopamine catechol, and a bioactive molecule can be fixed on the microcarrier to prepare the bioactive electroactive microcarrier, and the prepared electrically-responsive cell microcarrier still has excellent binding force in an organic solvent and is stable and does not fall off. The method is simple and convenient, can be suitable for the surfaces of various microcarriers, and has a wide application range.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a cyclic voltammogram of the PAT electroactive random copolymer of example 1 of the present invention;

FIG. 2 is a stereomicroscope photograph of P L GA microcarriers according to example 1 of the present invention;

FIG. 3 is a stereomicroscope picture of PAT @ P L GA microcarrier in example 1 of the present invention;

FIG. 4 is an infrared spectrum of PAT @ P L GA microcarrier according to example 1 of the present invention;

FIG. 5 is a stereomicroscope photograph of PEEK microcarriers according to example 2 of the invention;

FIG. 6 is a stereomicroscope photograph of PAT @ PEEK microcarriers according to example 2 of the present invention;

FIG. 7 is an infrared spectrum of the PAT @ PEEK microcarrier of example 2 according to the present invention;

FIG. 8 is a stereomicroscope photograph of P L GA/HA microcarriers according to example 3 of the present invention;

FIG. 9 is a stereomicroscope photograph of PAT @ P L GA/HA microcarriers in example 3 of the invention;

FIG. 10 is an X-ray photoelectron Spectroscopy (XPS) of the PAT @ P L GA/HA microcarrier of example 3 of the invention;

FIG. 11 is an infrared spectrum (FT-IR) of PAT @ P L GA/HA microcarrier in example 3 of the invention;

FIG. 12 is an SEM photograph of the surface of an electroactive PAT @ P L GA/HA microcarrier in example 3 of the invention;

FIG. 13 is an SEM photograph of a section of the electroactive PAT @ P L GA/HA microcarrier of example 3 of the invention;

FIG. 14 is a chart of the infrared spectrum of the microcarrier product of comparative example 1 of the invention;

FIG. 15 is an XPS spectrum of a microcarrier product according to comparative example 1 of the invention;

FIG. 16 is a chart of the infrared spectrum of a microcarrier product according to comparative example 2 of the invention;

FIG. 17 is a chart of the infrared spectrum of a microcarrier product according to comparative example 3 of the invention;

FIG. 18 shows the proliferation of MC3T3-E1 preosteoblasts of PAT @ P L GA/HA in example 3 of the present invention at 1,3 and 7 days;

FIG. 19 shows the alkaline phosphatase (A L P) activity of PAT @ P L GA/HA in example 3 of the present invention at 7 and 14 days;

FIG. 20 shows the quantitation result of alizarin red calcium at 14 and 21 days for PAT @ P L GA/HA in example 3 of the present invention.

Detailed Description

The invention provides a preparation method of a biological intelligent electric response cell microcarrier, which comprises the following steps:

dissolving the electroactive random copolymer in an alcohol solvent, adding a microcarrier and an oxidant, carrying out oxidative polymerization reaction, and drying to obtain a biological intelligent electric response cell microcarrier;

the electroactive random copolymer is an electroactive random copolymer containing aniline tetramers and dopamine functional groups;

the oxidant is sodium periodate and/or potassium periodate.

In the present invention, the electroactive random copolymer is an electroactive random copolymer (PAT) containing aniline tetramers and dopamine functional groups, preferably prepared according to the following steps:

mixing polyethylene glycol methyl ether methacrylate, dopamine methacrylamide, aniline tetramer methacrylamide and an initiator with a solvent in an inert gas environment, stirring for 0.4-0.6 hours at the temperature of 20-30 ℃, and then moving to an oil bath for reaction for 20-50 hours at the temperature of 60-85 ℃ to obtain the electroactive random copolymer.

Preferably, polyethylene glycol methyl ether methacrylate, dopamine methacrylamide, aniline tetramer methacrylamide and Azobisisobutyronitrile (AIBN) are added into a Schlenk bottle in a nitrogen environment, then dried and deoxidized N, N-Dimethylformamide (DMF) solvent is injected, stirred for half an hour at room temperature in the nitrogen environment, and moved into an oil bath for reaction at 60-85 ℃ for 20-50 hours. After the reaction is finished, cooling to room temperature, adding a little methanol to stop the reaction, then adding dichloromethane to dilute the solution, settling in ether for 2-5 times, and drying to obtain the electroactive random copolymer.

The specific reaction process is as follows:

specifically, the electroactive random copolymer used in the present invention may be prepared by radical polymerization with reference to the preparation method disclosed in chinese patent application No. 201811509073.8, the disclosure of which is incorporated herein by reference.

In the present invention, the microcarrier is preferably one or more of a polylactic acid-glycolic acid copolymer microcarrier (P L GA), a polylactic acid microcarrier, a polyetheretherketone microcarrier (PEEK) and a polylactic acid-glycolic acid copolymer compounded with hydroxyapatite (P L GA/HA).

P L GA microcarriers

The P L GA microcarrier is preferably prepared by a high-voltage electrostatic method, P L GA is dissolved in an organic solvent N-methyl pyrrolidone, the solution is placed in a push injection device provided with a needle, the P L GA solution is charged by a high-voltage electrostatic field, a liquid drop is formed at the tail end of the needle, the formed liquid drop is dropped into an extraction solvent (60% ethanol), the distance between the needle and the liquid surface is 7.5 cm., and NMP solvent in the liquid drop is rapidly removed by a solvent extraction principle, so that the P L GA microcarrier is prepared, and the P L GA microsphere is obtained by filtering with a screen mesh.

PEEK microcarrier

The PEEK microsphere is prepared by preferably preparing a PEEK microcarrier through an air flow method, dissolving PEEK powder in concentrated sulfuric acid, mechanically stirring for 2 hours at room temperature, standing for ten minutes, adding mixed liquid into a cylinder of 50m L, wherein the settling liquid is 30% ethanol (1000m L), performing ice bath, the height of a needle from the liquid level of the settling liquid is 14cm, preparing the PEEK microsphere through the air flow method, the air flow rate is 6-7L/min, the pump pressure is 0.26-0.28 MPa, extracting and replacing the obtained PEEK microsphere through a solvent, and filtering the obtained product through a screen mesh to obtain the PEEK microsphere.

P L GA/HA microcarriers

The method comprises the steps of dispersing HA in NMP, carrying out ultrasonic treatment for 30min, dissolving P L GA in the solution, arranging a mixed solution in a syringe device with a needle, wherein the settling solution is a 60% ethanol solution, the height of the needle from the liquid level of the settling solution is 6cm, enabling the P L GA/HA solution to form a liquid drop at the tail end of the needle by using a high-voltage electrostatic field, dropping the formed liquid drop into the settling solution, and rapidly removing the NMP solvent in the liquid drop by using a solvent extraction principle to rapidly prepare the P L GA/HA microcarrier, and filtering by using a screen to obtain the P L GA/HA microspheres with good sphericity and uniform size.

The present invention uses alcohols as oxidative polymerization solvents for the microcarriers and electroactive random copolymers, said alcohols being capable of dissolving the random copolymer and being miscible with the oxidant solution. In the present invention, the alcohol solvent is preferably methanol and/or ethanol.

In the present invention, the oxidizing agent is preferably sodium periodate and/or potassium periodate.

In the present invention, the mass ratio of the electroactive random copolymer to the oxidizing agent is preferably 1 (0.1 to 10), more preferably 1 (0.5 to 8), and most preferably 1 (1 to 5), in an embodiment of the present invention, 1: 1, the mass ratio of the electroactive random copolymer to the volume of the alcohol solvent is preferably (0.5 to 2.5) g of (1 to 10) m L, more preferably (1 to 2) g of (2 to 8) m L, most preferably (1 to 2) g of (3 to 5) m L, and particularly preferably 1g of (5) m L, and the mass ratio of the microcarrier to the volume of the alcohol solvent is preferably (0.05 to 5) g of (1 to 10) m L, more preferably (0.1 to 4) g of (2 to 8) m L, most preferably (1 to 3) g of (3 to 5) m L, and particularly preferably (1 to 3) m L).

In the invention, the temperature of the oxidative polymerization reaction is preferably 30-45 ℃, more preferably 35-40 ℃, and most preferably 37 ℃; the time of the oxidative polymerization reaction is preferably 12 to 50 hours, more preferably 15 to 45 hours, and most preferably 20 to 40 hours. In the present invention, the oxidative polymerization reaction is preferably carried out under a stirring condition, and the stirring speed is preferably 130 to 180rpm, more preferably 140 to 170rpm, and most preferably 150 to 160 rpm.

After the reaction is finished, deionized water and ethanol are preferably used for washing for 2-5 times respectively, and then the electric response cell microcarrier with good sphericity and uniform size is obtained by freeze drying.

The invention also provides a biological intelligent electric response cell microcarrier prepared according to the preparation method. The electrically responsive cell microcarrier of the present invention is spherical, and the diameter thereof is preferably 200 to 600 μm, more preferably 300 to 400 μm.

The invention also provides an application of the biological intelligent electric response cell microcarrier in repairing the damaged bone tissue, the prepared electric response microcarrier fixes cell factors or other active proteins on the surface of the microcarrier through the catechol group of dopamine.

The invention introduces the conductive high molecular material to the surface of the micro-carrier through the oxidation reaction of dopamine, so that the micro-carrier has electric activity, and simultaneously can fix growth factors, and the method is simple and efficient. The complex process of chemical covalent bonding and the possible denaturation of the surface of the microcarrier caused by the chemical covalent bonding are avoided. The electric active micro-carrier is beneficial to the proliferation and differentiation of histiocytes such as MC3T3-E1 osteoblasts and the like, the fixation of the growth factors can further improve the proliferation and differentiation capability of the cells, and the electric active micro-carrier has wide application prospect in the aspects of treating bone tissue injuries and the like.

To further illustrate the present invention, the following examples are provided to describe the bio-intelligent electrically responsive cell microcarrier, its preparation method and application in detail, but should not be construed as limiting the scope of the present invention.