CN111068111B

CN111068111B - Injectable self-assembled microsphere gel, application and preparation method thereof

Info

Publication number: CN111068111B
Application number: CN201811219705.7A
Authority: CN
Inventors: 胡尚秀; 许如秀
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-10-19
Filing date: 2018-10-19
Publication date: 2022-03-22
Anticipated expiration: 2038-10-19
Also published as: CN111068111A

Abstract

The invention provides an injectable self-assembled microsphere gel, which comprises a first microgel and a second microgel. The first microgel has a first charge and comprises a plurality of first gel microspheres. The second microgel has a second charge and comprises a plurality of second gel microspheres. The first electric charge and the second electric charge are opposite in electric property, and the average grain diameter of the first gel microspheres is equal to that of the second gel microspheres. Therefore, the injectable self-assembly microsphere gel can be self-assembled due to the characteristic of heteropolar attraction, and can be directly applied by an injection method.

Description

Injectable self-assembled microsphere gel, application and preparation method thereof

Technical Field

The present invention relates to a medical hydrogel and a preparation method thereof, and particularly to an injectable medical hydrogel and a preparation method thereof.

Background

Tissue Engineering (Tissue Engineering) is a medical method in which bioactive substances are cultured in an in vitro construction mode and then implanted into organisms to achieve cell regeneration and Tissue repair. Tissue engineering has three main elements, namely cells, cell growth scaffolds and growth factors, and the cell growth scaffolds are the key points of research in related research fields. The cell growth scaffold is made of biodegradable material, and has sufficient mechanical strength to promote the growth of cells attached therein, in addition to high biocompatibility and porosity.

In order to maintain the mechanical strength or porous structure, the conventional cell growth scaffold is often implanted into the human body by invasive methods such as surgery, which may cause additional damage to the human body and increase the risk of infection, resulting in poor applicability of the conventional cell growth scaffold. In order to solve the above problems, some researchers have proposed a colloid that can be injected into a living body for cell attachment and growth, but the above colloid has insufficient porosity, which may cause poor oxygen and nutrient exchange efficiency of cells around the colloid, and further reduce the growth activity of the cells, and even cause cell aging or death, which may result in cell transplantation failure.

Therefore, how to develop a cell growth scaffold with excellent cell compatibility, porosity and mechanical strength, and convenience for use is a goal of common efforts of those skilled in the art.

Disclosure of Invention

One aspect of the present invention is to provide a method for preparing an injectable self-assembled microsphere gel, comprising the following steps: carrying out a first gel microsphere preparation step, carrying out a second gel microsphere preparation step and carrying out a blending step. The first gel microsphere preparation step comprises the following steps: providing a first solution, providing a first oil phase solution and carrying out a first water-in-oil type emulsification reaction. The first solution is used as a water phase and comprises a first component and a second component, wherein the first component comprises acrylic acid or derivatives thereof, and the second component comprises chitosan or silk. Mixing the first solution and the first oil phase solution in a first water-in-oil type emulsification reaction to form a plurality of first gel microspheres. The second gel microsphere preparation step comprises the following steps: providing a second solution, providing a second oil phase solution and carrying out a second water-in-oil type emulsification reaction. The second solution is used as a water phase and comprises a third component and a fourth component, wherein the third component comprises acrylic acid or derivatives thereof, and the fourth component comprises gelatin, hyaluronic acid or algin. Mixing the second solution and the second oil phase solution in a second water-in-oil type emulsification reaction to form a plurality of second gel microspheres. And a blending step of mixing the first gel microsphere, the second gel microsphere and the aqueous solution to obtain the injectable self-assembled microsphere gel. Wherein the acrylic acid or its derivative comprises methacrylic acid, methacrylic anhydride and glycidyl methacrylate. Wherein, each first gel microsphere has a first charge, each second gel microsphere has a second charge, and the first charge and the second charge are opposite in electrical property. Wherein the average particle size of the first gel microspheres is 30-500 μm, the average particle size of the second gel microspheres is 30-500 μm, and the average pore size of the injectable self-assembled microsphere gel is 30-90 μm.

According to the preparation method of the injectable self-assembled microsphere gel, the volume percentage of the first gel microspheres and the second gel microspheres in the injectable self-assembled microsphere gel may be 1: 0.5 to 1: 6.

according to the preparation method of the injectable self-assembled microsphere gel, the preparation method can be carried out in a micro-channel system.

According to the preparation method of the injectable self-assembled microsphere gel, the colloid strength of the injectable self-assembled microsphere gel can be 30Pa to 3100 Pa.

According to the preparation method of the injectable self-assembled microsphere gel, the average porosity of the injectable self-assembled microsphere gel can be 35% to 50%.

Therefore, the preparation method of the injectable self-assembled microsphere gel provided by the invention has the advantages that the injectable self-assembled microsphere gel can be directly applied by an injection mode through a mode of blending the first gel microsphere and the second gel microsphere with opposite electric properties, and the injectable self-assembled microsphere gel has excellent self-assembling capability. In addition, the preparation method of the injectable self-assembled microsphere gel uses the acrylic acid or the derivative thereof and the base material with high biocompatibility as the raw materials of the first gel microsphere and the second gel microsphere, so that the toxicity of the raw materials to cells can be avoided, the mass production can be effectively carried out, and the use safety and the application range of the injectable self-assembled microsphere gel prepared by the method of the injectable self-assembled microsphere gel are further improved.

In another aspect, the present invention provides an injectable self-assembled microsphere gel comprising a first microgel and a second microgel. The first microgel has a first charge, wherein the first microgel comprises a plurality of first gel microspheres, and the average particle size of the first gel microspheres is 30 μm to 500 μm. The second microgel has a second charge, wherein the second microgel comprises a plurality of second gel microspheres, and the average particle size of the second gel microspheres is 30 μm to 500 μm. The average pore diameter of the injectable self-assembly microsphere gel is 30-90 μm. The first electric charges and the second electric charges are opposite in electric property, the average particle size of the first gel microspheres of the injectable self-assembly microsphere gel is equal to that of the second gel microspheres, each first gel microsphere comprises an acrylic chitosan polymer, an acrylic silk polymer or a combination thereof, and each second gel microsphere comprises an acrylic gelatin polymer, an acrylic hyaluronic acid polymer, an acrylic alginate polymer or a combination thereof.

The injectable self-assembled microsphere gel according to the above, wherein each of the first gel microspheres may comprise methacrylate chitosan, methacrylate silk, or a combination thereof, and each of the second gel microspheres may comprise methacrylate gelatin, methacrylate hyaluronic acid, methacrylate algin, or a combination thereof.

According to the injectable self-assembled microsphere gel, the volume percentage of the first microgel and the second microgel can be 1: 0.5 to 1: 6.

according to the injectable self-assembled microsphere gel, the colloid strength of the injectable self-assembled microsphere gel can be 30Pa to 3100 Pa.

According to the injectable self-assembled microsphere gel, the average porosity of the injectable self-assembled microsphere gel can be 35% to 50%.

Therefore, the injectable self-assembled microsphere gel of the invention comprises the first microgel and the second microgel with opposite electric properties, so that the microgel can be self-assembled due to the characteristic of heteropolar attraction and can be directly applied by an injection method. Furthermore, the injectable self-assembled microsphere gel of the invention uses acrylic acid or derivatives thereof and a substrate with high biocompatibility as raw materials, can avoid the toxicity of the raw materials to cells, and can adjust the particle sizes of the first gel microsphere and the second gel microsphere according to actual requirements, thereby expanding the use safety and application range of the injectable self-assembled microsphere gel of the invention.

In another aspect, the present invention provides a use of the injectable self-assembled microsphere gel as described above for preparing a biomedical material for promoting tissue repair.

In another aspect, the present invention provides a use of the injectable self-assembled microsphere gel as described above for preparing a pharmaceutical composition for promoting nerve cell growth.

Therefore, the injectable self-assembled microsphere gel provided by the invention has high biocompatibility and the effect of promoting cell growth, so that the injectable self-assembled microsphere gel can be applied to a biomedical material for promoting cell proliferation and tissue repair, has the potential to be further applied to the preparation of a pharmaceutical composition for promoting the growth of nerve cells, and further achieves the aim of providing efficient and safe adjuvant therapy for human bodies.

The above summary is intended to provide a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and is intended to neither identify key/critical elements of the embodiments nor delineate the scope of the embodiments.

Drawings

In order to make the aforementioned and other objects, features, and advantages of the invention, as well as others which will become apparent, reference is made to the following description of the preferred embodiments of the invention in which:

fig. 1 shows a flow chart of steps of a method for preparing an injectable self-assembled microsphere gel according to an embodiment of the present invention.

Fig. 2A shows an image of an injectable self-assembled microsphere gel of the present invention.

Fig. 2B shows another image of the injectable self-assembled microsphere gel of fig. 2A.

Fig. 3 is a graph showing the results of rheological property tests of the injectable self-assembled microsphere gel of the present invention.

Fig. 4A shows a microscope image of injectable self-assembled microsphere gels of the present invention at different average particle sizes.

Fig. 4B is a graph showing the results of an analysis of the average porosity of the injectable self-assembled microsphere gel of fig. 4A at different average particle sizes.

Fig. 5A shows another microscope image of injectable self-assembled microsphere gels of the present invention at different average particle sizes.

FIG. 5B is a graph showing the results of analysis of the average pore size of the injectable self-assembled microsphere gel of FIG. 5A at different average particle sizes.

FIG. 6 is a graph showing the results of cell viability 24 hours after cell culture using the injectable self-assembled microsphere gel of the present invention as a matrix.

FIG. 7 is a graph showing the results of staining cells cultured for various periods of time using the injectable self-assembled microsphere gel of the present invention as a matrix.

FIG. 8 is a graph showing the results of cell proliferation after cell culture for various periods of time using the injectable self-assembled microsphere gel of the present invention as a matrix.

Fig. 9A is a graph showing the results of nerve strength evaluation of regenerated sciatic nerves at different axon sections 7 days after implantation of the nerve conduit of example 4 of the present invention into the hindlimb of a rat.

Fig. 9B is a graph showing analysis results of growth of axon remote sections of regenerated sciatic nerve into nerve conduits of example 4 of the present invention 7 days after implantation of the nerve conduits into hindlimbs of rats.

Fig. 10A shows an image of toes of a rat after a nerve conduit of example 4 of the present invention was implanted for 30 days.

Fig. 10B is a graph showing the analysis results of sciatic nerve function index of the nerve conduit of example 4 of the present invention 30 days and 60 days after the implantation of the hindlimb of the rat.

Fig. 11A is a graph showing the result of nerve conduction velocity test of sciatic nerve of the nerve conduit of example 4 of the present invention 60 days after the implantation of the hindlimb of the rat.

Fig. 11B is a graph showing the result of the compound action potential test of the sciatic nerve of the nerve conduit of example 4 of the present invention 60 days after the implantation of the hindlimb of the rat.

Wherein the description of the symbols of the drawings is as follows:

100: a preparation method of injectable self-assembled microsphere gel;

110. 112, 114, 116, 120, 122, 124, 126, 130: a step of;

200: injectable self-assembled microsphere gels.

Detailed Description

< preparation method of injectable self-assembled microsphere gel >

Referring to fig. 1, a flow chart of steps of a method 100 for preparing an injectable self-assembled microsphere gel according to an embodiment of the present invention is shown. The method 100 for preparing the injectable self-assembled microsphere gel comprises

steps

110, 120 and 130.

Step 110 is to perform a first gel microsphere preparation step, which includes step 112, step 114 and step 116.

Step 112 is to provide a first solution, which is used as an aqueous phase and comprises a first component and a second component, wherein the first component comprises acrylic acid or a derivative thereof, and the second component comprises chitosan or silk. The acrylic acid or its derivative may include methacrylic acid, methacrylic anhydride, glycidyl methacrylate and other monomers of polymer material containing double bond, so as to provide better structural strength for the first gel microsphere.

Step 114 is to provide a first oil phase solution, and specifically, the first oil phase solution may include an oily solution and a surfactant, wherein the surfactant may be, but is not limited to, Polyvinyl alcohol (PVA), tween 20, tween80, or Sodium Dodecyl Sulfate (SDS), so as to further improve the polymerization efficiency of the first component and the second component in the first solution.

Step 116 is to perform a first water-in-oil type emulsification reaction, which mixes the first solution and the first oil phase solution to form a plurality of first gel microspheres, wherein each first gel microsphere is in the shape of a microsphere. Preferably, the first water-in-oil emulsification reaction can be performed in a microfluidic system, and the average particle size of the first gel microspheres can be between 30 μm and 500 μm and maintain a low molecular weight polydispersity by adjusting the flow rates of the first solution as the aqueous phase and the first oil phase solution and the channel size of the microfluidic system.

Step 120 is to perform a second gel microsphere preparation step, which includes step 122, step 124 and step 126.

Step 122 is to provide a second solution, which is used as an aqueous phase and comprises a third component and a fourth component, wherein the third component comprises acrylic acid or a derivative thereof, and the fourth component comprises gelatin, hyaluronic acid or algin.

Step 124 is to provide a second oil phase solution, and specifically, the second oil phase solution may comprise an oily solution and a surfactant, wherein the surfactant may be, but is not limited to, polyvinyl alcohol, tween 20, tween80 or sodium dodecyl sulfate, so as to further improve the polymerization efficiency of the third component and the fourth component in the second solution.

Step 126 is to perform a second water-in-oil type emulsification reaction, which mixes the second solution with the second oil phase solution to form a plurality of second gel microspheres, wherein each second gel microsphere is in the shape of a microsphere. Preferably, the second water-in-oil emulsion reaction can be performed in a microfluidic system, and the average particle size of the second gel microspheres can be adjusted to be between 30 μm and 500 μm by adjusting the ratio of the second solution as an aqueous phase solution to the second oil phase solution, the flow rate and the channel size of the microfluidic system, and the low molecular weight polydispersity can be maintained.

In addition, the first gel microsphere and the second gel microsphere can adjust the degree of substitution of functional groups (such as amino groups and hydroxyl groups) thereof by a modification method to adjust the first charge and the second charge of the first gel microsphere and the second gel microsphere, and can perform a photo-crosslinking reaction by using a photo-activated radical initiator and UV light to connect biomolecules such as cell growth factors and cell attachment peptides thereon, so as to increase the application range of the first gel microsphere and the second gel microsphere.

Step 130 is to perform a blending step, which mixes the first gel microsphere, the second gel microsphere and the aqueous solution to obtain the injectable self-assembled microsphere gel of the present invention. In detail, since the second component of the first gel microsphere and the fourth component of the second gel microsphere have different functional groups, the first gel microsphere and the second gel microsphere prepared through the

above steps

110 and 120 have a first charge and a second charge, respectively, the first charge and the second charge have opposite electrical properties, and the average particle size of the first gel microsphere and the average particle size of the second gel microsphere of the injectable self-assembled microsphere gel of the present invention are equal. In detail, the injectable self-assembled microsphere gel prepared by the method can be mutually attracted and regularly arranged by the electrostatic attraction between the first gel microsphere and the second gel microsphere to form a plastic colloid, and can be readjusted and assembled again by the electrostatic attraction between the first gel microsphere and the second gel microsphere and the shear thinning force of the colloid after the colloid is changed in shape, so that the injectable self-assembled microsphere gel has injectable property.

Preferably, the volume percentage of the first gel microspheres and the second gel microspheres in the injectable self-assembled microsphere gel of the invention may be 1: 0.5 to 1: 6, the volume percentage of the first microgel to the second microgel may be 1: 0.5 to 1: 6, so as to have different colloid strengths, and the injectable self-assembled microsphere gel of the present invention may have a colloid strength of 30 to 3100Pa, an average porosity of 35 to 50%, and an average pore diameter of 30 to 90 μm.

The following will further illustrate the preparation method 100 of the injectable self-assembled microsphere gel by examples, and evaluate the physical properties and effects of the injectable self-assembled microsphere gel of the present invention by using a plurality of examples and a plurality of comparative examples, but the conditions listed in the examples are not intended to limit the scope of the present invention, and are described in the following.

< test example >

Firstly, preparing injectable self-assembly microsphere gel

The injectable self-assembled microsphere gel according to an embodiment of the present invention comprises a first microgel and a second microgel, wherein the first microgel has a first charge and comprises a plurality of first gel microspheres, the second microgel has a second charge and comprises a plurality of second gel microspheres, and the first charge and the second charge are opposite in electrical property. The details of the preparation of the first gel microsphere, the second gel microsphere and the blending of the first gel microsphere and the second gel microsphere will be described below.

1. Preparation of first gel microspheres

The first gel microspheres may be classified into acrylic chitosan polymer and acrylic silk polymer according to the difference between the first component and the second component, and in this experimental example, the acrylic chitosan polymer may be methacrylate-modified chitosan (Chito-MA), and the acrylic silk polymer may be methacrylate-modified silk (Sil-MA), and the preparation methods of the first gel microspheres with different materials will be described below.

(1) Preparing the first gel microspheres made of methacrylate chitosan

Firstly, 1.5g of water-soluble chitin is added into 50mL of PBS buffer solution and stirred at 60 ℃ until the water-soluble chitin is completely dissolved, then a proper amount of Methacrylic Anhydride (MA) is measured and added into the water-soluble chitin solution dropwise at the speed of 0.5mL/min, and the mixture is continuously stirred in a thermostatic bath at 50 ℃ for 3 hours to completely react. After 3 hours of reaction, 200mL of PBS buffer solution at 50 ℃ was further diluted and stirred continuously until the reaction was terminated, and then the solution was dialyzed with pure water at 50 ℃ in a dialysis bag having a cut-off (MCO) of 12 to 14kDa, and the dialyzed solution was put into a centrifuge tube and centrifuged at 2500rpm for 15 minutes to remove the precipitate, to obtain a first solution containing a methacrylate chitosan prepolymer. The first solution is further added into a first oil phase solution containing an oily solution and a surfactant to prepare the first gel microsphere made of methacrylate chitosan.

(2) Preparing first gel microspheres made of methacrylate silk

First, 20g of degummed silk fiber was added to 100mL of a 60 ℃ 9.3M lithium bromide solution and reacted for 1 hour, and then 2mL, 4mL, 6mL and 10mL (141 mM, 282mM, 424mM and 705mM, respectively) of Glycidyl methacrylate solution (GMA) were added to the degummed silk fiber solution and reacted for 3 hours at 60 ℃ and 300 rpm. After completion of the reaction, filtration was performed with a Miracloth filter (Calbiochem, San Diego, USA) and dialysis was performed with distilled water using a cut-off dialysis tube having a cut-off amount of 12 to 14kDad to obtain a first solution containing methacrylate silk high molecular prepolymer. The first solution is further added into a first oil phase solution containing an oily solution and a surfactant to prepare the first gel microspheres made of methacrylate silk.

In this experiment, the first water-in-oil emulsion reaction was performed in the microfluidic system, and the average particle size of the first gel microspheres was between 30 μm and 500 μm and the low molecular weight polydispersity was maintained by adjusting the ratio of the first solution to the first oil phase solution, the flow rate, and the channel size of the microfluidic system.

2. Preparation of second gel microspheres

The second Gel microspheres may be classified into an acrylic gelatin polymer, an acrylic hyaluronic acid polymer and an acrylic alginate polymer according to the difference between the third component and the fourth component, in this experimental example, the acrylic gelatin polymer may be methacrylate-modified gelatin (Gel-MA), the acrylic hyaluronic acid polymer may be methacrylate-modified hyaluronic acid (HA-MA), and the acrylic alginate polymer may be methacrylate-modified alginate (Algi-MA), and the preparation methods of the second Gel microspheres of different materials will be described below.

(1) Preparing the second gel microspheres made of methacrylate gelatin

Firstly, 5g of gelatin is added into 50mL of PBS buffer solution and stirred at 60 ℃ until the gelatin is completely dissolved, then a proper amount of methacrylic anhydride is weighed and dripped into the gelatin solution at the speed of 0.5mL/min, and the stirring is continued for 3 hours in a thermostatic bath at 50 ℃. After 3 hours of reaction, 200mL of PBS buffer solution at 50 ℃ was further diluted and stirred until the reaction was terminated, and then the solution was dialyzed against pure water at 50 ℃ in a dialysis bag having a cut-off of 12 to 14kDa, and the dialyzed solution was centrifuged at 2500rpm for 15 minutes in a centrifugal tube to remove the precipitate, to obtain a second solution containing a methacrylate gelatin high molecular prepolymer. The second solution is further added into a second oil phase solution containing an oily solution and a surfactant to prepare second gel microspheres made of methacrylate gelatin.

(2) Preparing second gel microspheres made of methacrylate hyaluronic acid

0.5g of hyaluronic acid is initially taken and added to 50mL of distilled water and stirring is continued at room temperature until the next day. Then, 1mL of Triethylamine (TEA) solution, 1mL of glycidyl methacrylate solution with purity of more than 92% and 1g of Tetrabutylammonium bromide (TBAB) are added into the hyaluronic acid aqueous solution, and the mixture is continuously stirred and reacted for 1 hour at 55 ℃, after the mixture is cooled, two extraction and precipitation steps are carried out in acetone, the obtained precipitate is dried, the dried precipitate is dissolved back into distilled water, and dialysis is carried out by using distilled water through a cut-off dialysis tube with cut-off amount of 12-14kDa, so as to obtain a second solution containing the methacrylate hyaluronic acid high polymer prepolymer. The second solution is further added into a second oil phase solution containing an oily solution and a surfactant to prepare second gel microspheres made of methacrylate hyaluronic acid.

(3) Preparing second gel microspheres made of methacrylate algin

First, 4.0g of alginate was added to 200mL of distilled water and stirred until the alginate was completely dissolved, and then 15mL of a 2.0% by weight aqueous methacrylic anhydride solution was added in one portion and the pH of the solution was adjusted to 8, followed by incubation at 5 ℃ for 24 hours. After 24 hours of reaction, the solution is purified by ethanol and dried in vacuum for 3 days at room temperature, and the dried product is dissolved back in distilled water and dialyzed by distilled water by using a cut-off dialysis tube with the cut-off amount of 12-14kDa, so as to obtain a second solution containing the methacrylate alginate high molecular prepolymer. The second solution is further added into a second oil phase solution containing an oily solution and a surfactant to prepare second gel microspheres made of methacrylate algin.

In this experiment, the second water-in-oil emulsion reaction was performed in the microfluidic system, and the average particle size of the second gel microspheres was between 30 μm and 500 μm while maintaining the low molecular weight polydispersity by adjusting the ratio of the second solution to the second oil phase solution, the flow rate, and the channel size of the microfluidic system.

3. Blending the first gel microspheres with the second gel microspheres

After the preparation of the first gel microspheres and the second gel microspheres is completed, the volume percentage of the first gel microspheres to the second gel microspheres is 1: 0.5 to 1: 6, and adding the aqueous solution, wherein the average grain diameter of the first gel microspheres of the injectable self-assembled microsphere gel is equal to the average grain diameter of the second gel microspheres, so as to obtain the injectable self-assembled microsphere gel.

Referring to fig. 2A and 2B, fig. 2A shows an image of an injectable self-assembled microsphere gel 200 of the present invention, and fig. 2B shows another image of the injectable self-assembled microsphere gel 200 of fig. 2A. As shown in fig. 2A and 2B, the injectable self-assembled microsphere gel 200 is in the form of colloid that can be injected through a needle, and can be self-assembled without applying an external force after the needle is discharged to form a colloid form, and can be shaped to obtain a specific form (such as the shape shown in fig. 2B), so that the injectable self-assembled microsphere gel 200 can be self-assembled in a cavity with a complex shape, such as a bone gap and a tissue gap, and the injectable self-assembled microsphere gel 200 can be adjusted according to the requirement, thereby having both excellent convenience in use and wide applicability.

Specifically, in each example of the present experiment, the first gel microspheres were positively charged methacrylate chitosan gel microspheres, and the second gel microspheres were negatively charged methacrylate gelatin gel microspheres, wherein the volume percentage between the first gel microspheres and the second gel microspheres of example 1 was 1: 1, the volume percentage of the first gel microspheres and the second gel microspheres of example 2 is 1: 2, the volume percentage of the first gel microspheres and the second gel microspheres of example 3 is 1: and the injectable self-assembled microsphere gels of examples 1 to 3 were evaluated for physical properties, biocompatibility, and the ability to promote nerve cell growth, as follows.

Second, evaluation of physical Properties of injectable self-assembled microsphere gels of the invention

1. Evaluation of colloidal Strength of injectable self-assembled microsphere gels of the invention

The colloidal strength of the injectable self-assembled microsphere gels of the present invention was evaluated by performing rheological property tests on the injectable self-assembled microsphere gels of examples 1 to 3 described above. A rheometer (AR 2000ex, TA Instruments) was used for the test at 25 ℃ with a 20mm diameter geometrically flat steel plate as the upper plate, after the gap distance is adjusted to be 0.5mm, the constant stress is 1Pa and the constant frequency is 1Hz, to measure the storage modulus (G ') and loss modulus (G'), the storage modulus is also called elastic modulus, which refers to the amount of energy stored by elastic deformation when a material is deformed, and is used to represent the elasticity of the material, and the loss modulus is also called viscous modulus, the energy stored by viscous deformation when the material deforms is used for representing the viscosity of the material, and the colloid strength of the injectable self-assembled microsphere gel is described by the measured values of the storage modulus and the loss modulus in the test.

In addition, the test also includes comparative examples 1 and 2, wherein comparative example 1 is a conventional positively charged hydrogel, and comparative example 2 is a conventional negatively charged hydrogel, so as to further compare and demonstrate the colloidal strength of the injectable self-assembled microsphere gel of the present invention compared with comparative examples 1 and 2.

Referring to fig. 3, a graph showing the results of rheological property tests of the injectable self-assembled microsphere gel of the present invention is shown. As shown in fig. 3, the injectable self-assembled microsphere gels of examples 1 to 3 have high storage modulus and loss modulus, while the storage modulus of comparative examples 1 and 2 is not 100Pa, wherein the storage modulus of example 1 is 3100Pa, the storage modulus of example 3 is 350Pa, which is 17.5 times and 155 times of the storage modulus (Pa ═ 20) of comparative example 2, respectively, showing that the injectable self-assembled microsphere gels of the present invention have good elasticity and proper viscosity, and the colloid strength can be 30Pa to 3100Pa according to different mixing ratios of the first gel microspheres and the second gel microspheres.

In addition, it is noted that the injectable self-assembled microsphere gel of the present invention has adjustable cohesiveness through the charge strength of the positively charged first gel microspheres and the negatively charged second gel microspheres, which can further expand the stiffness adjustment range of the cell growth scaffold, thereby further improving the application scope of the injectable self-assembled microsphere gel of the present invention.

2. Evaluation of average porosity and average pore diameter of injectable self-assembled microsphere gel of the invention

The evaluation of average porosity and average pore size of injectable self-assembled microsphere gels of the present invention was conducted by testing the injectable self-assembled microsphere gels of example 1 and adjusting the average particle size of the gel microspheres of example 1 (including the first gel microspheres and the second gel microspheres) to test the average porosity and average pore size of the injectable self-assembled microsphere gels of the present invention when the gel microspheres have different average particle sizes, wherein the average particle sizes of example 1 are 115 μm, 125 μm, 156 μm, 175 μm and 210 μm from small to large, respectively. In the experiment, after the injectable self-assembled microsphere gel is dyed in a fluorescent dyeing mode, a conjugated focus microscope is used for shooting a microscopic fluorescence image of a colloid, and image analysis is carried out according to the microscopic fluorescence image so as to calculate the average porosity and the average pore diameter of the injectable self-assembled microsphere gel when the average particle diameters are different.

Referring to fig. 4A and 4B, fig. 4A shows a microscope image of the injectable self-assembled microsphere gel of the present invention at different average particle sizes, and fig. 4B shows a graph of the analysis result of the average porosity of the injectable self-assembled microsphere gel of fig. 4A at different average particle sizes. In detail, the microscope image of fig. 4A represents the microscope images of the gel microspheres with an average particle size of 125 μm and the gel microspheres with an average particle size of 175 μm, respectively, from left to right. As shown in fig. 4A and 4B, the injectable self-assembled microsphere gel of the present invention has interconnected and aligned pores in the structure, and has an average porosity of 50% or more when the average particle size of the gel microspheres is 125 μm and 175 μm, indicating that the injectable self-assembled microsphere gel of the present invention has excellent pore-forming ability.

Referring to fig. 5A and 5B, fig. 5A shows another microscope image of the injectable self-assembled microsphere gel of the present invention at different average particle sizes, and fig. 5B shows a graph of the analysis result of the average pore diameter of the injectable self-assembled microsphere gel of fig. 5A at different average particle sizes. Specifically, the microscope image of fig. 5A represents the microscope images of the gel microspheres with an average particle size of 115 μm, the gel microspheres with an average particle size of 156 μm, and the gel microspheres with an average particle size of 210 μm, respectively, from left to right. As shown in fig. 5A and 5B, the first gel microspheres and the second gel microspheres of the injectable self-assembled microsphere gel of the present invention are tightly stacked on each other on the premise of the same average particle size, and when the average particle size of the gel microspheres is 115 μm, 156 μm and 210 μm, the average pore size is 40.8 μm, 57.1 μm and 85.5 μm, respectively, which shows that the injectable self-assembled microsphere gel of the present invention not only has excellent pore forming ability, but also can adjust the particle size of the gel microspheres to change the pore size thereof according to the requirement, so that it has potential as a biomedical material for promoting tissue repair.

Furthermore, the injectable self-assembled microsphere gel of the invention is stacked with the first gel microsphere and the second gel microsphere with the same particle size, and mutually attracted through the electrostatic attraction between the first gel microsphere and the second gel microsphere, so as to form a pore network which is nearly 100% interconnected, thereby effectively improving the defect of insufficient exchange of oxygen and nutrients of the conventional hydrogel.

Third, the evaluation of biocompatibility of the injectable self-assembled microsphere gel of the invention

The evaluation of biocompatibility of the injectable self-assembled microsphere gel of the present invention was performed based on the cell viability of human adipose stem cells (hADSC), Schwann cells (Schwann cell) and fibroblasts (Fibroblast) and their ability to promote cell proliferation.

1. Cell viability assay for injectable self-assembled microsphere gels of the invention

The cell viability test of the injectable self-assembled microsphere gels of the present invention was performed with the injectable self-assembled microsphere gel of example 2 described above. The injectable self-assembled microsphere gel of example 2 was experimentally cut into gel sheets with a thickness of 2mm, and placed into different wells of a cell culture plate to contain 3X 10⁶Different cell culture solutions of individual human adipose-derived stem cells, Schwann cells or fibroblasts are injected into the gel sheets with different holes at different angles, and the gel sheets are immersed in 5% CO at 37 DEG C₂Cultured under the conditions of (1) for 24 hours. After 24 hours of incubation, 100. mu.L of MTT reagent was further added to each well and incubated at 37 ℃ for 4 hours, and then absorbance at a wavelength of 570nm was measured by a spectrophotometer and converted into cell viability data.

In addition, the experiment also includes the comparative examples 1 and 2, so as to further illustrate the biocompatibility of the injectable self-assembled microsphere gel.

Fig. 6 is a graph showing the results of cell viability 24 hours after cell culture using the injectable self-assembled microsphere gel of the present invention as a matrix. As shown in fig. 6, after culturing the injectable self-assembled microsphere gel of example 2 for 24 hours, the cell survival rates of the human adipose-derived stem cells, schwann cells and fibroblasts were all greater than 90%, wherein the cell survival rate of the human adipose-derived stem cells was the best, and the injectable self-assembled microsphere gel of the present invention has excellent cell survival performance compared to comparative examples 1 and 2, which shows that the injectable self-assembled microsphere gel of the present invention has excellent biocompatibility and can be used to prepare biomedical materials for promoting tissue repair.

2. Test of cell proliferation-promoting ability of injectable self-assembled microsphere gels of the present invention

The ability of the injectable self-assembled microsphere gels of the present invention to promote cell proliferation was tested using the injectable self-assembled microsphere gels of example 2 described above and comparative example 3, which contained a conventional non-porous hydrogel.

Human adipose stem cells, Schwann cells and fibroblasts were experimentally cultured in the injectable self-assembled microsphere gel of example 2 and the non-porous hydrogel of comparative example 3 for different periods of time, treated with 3.7% formaldehyde for 15 minutes, then added with 0.1% Triton-X100 and soaked for 10 minutes, then added with fluorescently labeled Phalloidin (Phalloidin) diluted at a ratio of 1:300 for 1 hour, then added with DAPI diluted at a ratio of 1:500 for 5 minutes, rinsed with physiological saline, and observed under a fluorescence microscope, wherein the cell culture periods of example 2 were 3 hours, 2 days and 6 days, respectively, and the cell culture periods of comparative example 3 were 3 hours and 4 days, respectively.

Fig. 7 is a graph showing the staining results of cells cultured for different time periods with the injectable self-assembled microsphere gel of the present invention as a matrix. In addition, in order to make the immunofluorescent staining result of FIG. 7 clear, appendix 1 is the color drawing of FIG. 7, in which the blue-fluorescent portion is the nucleus and the green-fluorescent portion is the cytoskeleton. As shown in fig. 7, human adipose-derived stem cells, schwann cells and fibroblasts were attached to the surface of the injectable self-assembled microsphere gel of example 2 after 3 hours of culture without adding additional bioactive factors such as adhesion proteins, and proliferated with the increase of culture time. After continuous culture for 6 days, the human adipose stem cells, schwann cells and fibroblasts all formed compact growth in the injectable self-assembled microsphere gel of example 2, in contrast to the non-porous hydrogel of comparative example 3, the human adipose stem cells, schwann cells and fibroblasts could not be effectively attached to the surface of the non-porous hydrogel after 3 hours of culture and could not grow thereon after 6 days of continuous culture.

In addition, in this experiment, the number of human adipose stem cells, schwann cells and fibroblasts proliferating with the culture time after culturing the injectable self-assembled microsphere gel of the present invention for different time periods was analyzed by flow cytometry respectively to further evaluate the biocompatibility of the injectable self-assembled microsphere gel of the present invention.

Fig. 8 is a graph showing the results of cell proliferation after cell culture for different time periods using the injectable self-assembled microsphere gel of the present invention as a matrix. As shown in fig. 8, the human adipose stem cells, schwann cells and fibroblasts all showed stable and sustained proliferation within 6 days when cultured in the injectable self-assembled microsphere gel of example 2, wherein the proliferation of schwann cells and fibroblasts is the best, and the Doubling Time (DT) is 2.5 days and 1.5 days, respectively.

By combining the experimental results, the injectable self-assembled microsphere gel disclosed by the invention has excellent biocompatibility and excellent average porosity, and is beneficial to cell adhesion and proliferation in the microsphere gel, so that the injectable self-assembled microsphere gel disclosed by the invention can be used for preparing a biomedical material for promoting tissue repair, and is suitable for related technical fields such as tissue engineering or regenerative medicine.

Fourth, the capability evaluation of the injectable self-assembled microsphere gel of the invention for promoting nerve cell repair

The Nerve conduit of example 4, which encapsulates Nerve Growth Factor (NGF) in the first and second gel microspheres of the injectable self-assembled microsphere gel of the invention and forms a Nerve conduit with a Nerve Growth Factor concentration gradient, was implanted into a damaged area of the hind limb sciatic Nerve of a rat (i.e., the gap connecting sciatic Nerve injuries using the Nerve conduit of example 4), and the sciatic Nerve strength and function of the regenerated sciatic Nerve axon section were evaluated at 7 days, 30 days and 60 days of implantation to evaluate the ability of the Nerve conduit comprising the injectable self-assembled microsphere gel of the invention to contribute to Nerve cell repair.

In addition, in this experiment, comparative example 4, which contained a conventional nerve conduit, was further used to perform an experiment to further illustrate the ability of the injectable self-assembled microsphere gel of the present invention to promote nerve cell repair.

1. Evaluation of nerve fiber strength and growth of regenerated sciatic nerve

The ability of nerve conduits comprising the injectable self-assembled microsphere gels of the present invention to facilitate nerve cell repair was evaluated in this test for nerve fiber strength and growth status in regenerating sciatic nerves. The regenerated sciatic nerve is further divided into four sections of axon stump (i.e. sciatic nerve amputation), axon proximal end, axon middle and axon remote according to the distance from the regenerated sciatic nerve to the rat trunk in the test, so that the nerve fiber strength and growth condition can be evaluated according to the growth condition of different regenerated sciatic nerve axon sections in the test.

Referring to FIG. 9A, it is a graph showing the results of nerve strength evaluation of regenerated sciatic nerve at different axon segments 7 days after the nerve conduit of example 4 of the present invention was implanted into the hindlimb of rat. As shown in fig. 9A, the strength of nerve fibers of the regenerated sciatic nerve gradually decreased from the axon stump section to the axon distal section of the sciatic nerve 7 days after the implantation of the nerve conduit of example 4, showing that the sciatic nerve can stably grow in the nerve conduit of example 4, and the strength of nerve fibers of the regenerated sciatic nerve in the nerve conduit of example 4 at each axon section is significantly superior to that of the nerve conduit of comparative example 4.

Referring again to fig. 9B, there is shown an analysis chart showing the results of the growth of the axon remote section of the regenerated sciatic nerve into the nerve conduit 7 days after the implantation of the nerve conduit of example 4 of the present invention into the hindlimb of the rat. As shown in fig. 9B, the ratio of axon remote sections of the regenerated sciatic nerve in the nerve conduit of example 4 was significantly increased in the nerve conduit 7 days after the nerve conduit was implanted in the injured region of the sciatic nerve of the hind limb of the rat compared to comparative example 4, showing that the nerve conduit of example 4 is more favorable for the regeneration of the sciatic nerve compared to comparative example 4.

2. Nerve function, nerve conduction velocity and compound action potential analysis of regenerated sciatic nerve

Nerve function analysis assay for regenerating sciatic nerve the nerve conduit of example 4 was analyzed for sciatic nerve function index (SFI) of regenerated sciatic nerve after implantation into the injured area of rat hindlimb sciatic nerve to assess the ability of the nerve conduit comprising the injectable self-assembled microsphere gel of the present invention to repair nerve cells. In addition, control group 1, which was transplanted using autologous nerves, was further included in the test to further illustrate the ability of the nerve conduit comprising the injectable self-assembled microsphere gel of the present invention to contribute to nerve cell repair.

Referring to fig. 10A and 10B, fig. 10A is a diagram showing images of toes of a rat after a nerve conduit of example 4 of the present invention is implanted for 30 days, and fig. 10B is a diagram showing analysis results of sciatic nerve functional indexes of a nerve conduit of example 4 of the present invention after a rat hindlimb is implanted for 30 days and 60 days. As shown in fig. 10A, the nerve conduit of example 4 and the nerve conduit of comparative example 4 were implanted into the injured left hind paw (i.e., hind limb paw near the right side in the figure), respectively, of the rat, wherein the injured hind paw of the rat had a greater degree of toe expansion than the injured hind paw of the nerve conduit of comparative example 4 after the nerve conduit of example 4 was implanted for 30 days, whereas as shown in fig. 10B, the sciatic nerve function index of the nerve conduit of example 4 was similar to and significantly better than that of control group 1 after the nerve conduit of example 4 was implanted for 30 days and 60 days, showing that the nerve conduit comprising the injectable self-assembled microsphere gel of the present invention has an excellent effect on the repair of the injured sciatic nerve.

The nerve conduit of example 4 was analyzed in terms of nerve conduction velocity and complex action potential analysis of regenerated sciatic nerves, and after being implanted into hind limbs of rats, it regenerated the nerve conduction velocity and action potential of sciatic nerves to evaluate the ability of the nerve conduit comprising the injectable self-assembled microsphere gel of the present invention to contribute to nerve cell repair. In addition, the control group 1 and the control group 2 containing rats with undamaged sciatic nerves were included in the test to further illustrate the ability of the nerve conduit containing the injectable self-assembled microsphere gel of the present invention to contribute to nerve cell repair.

Referring to fig. 11A and 11B, fig. 11A is a graph showing the results of a nerve conduction velocity test of sciatic nerves of a nerve conduit according to example 4 of the present invention after being implanted in a rat hindlimb for 60 days, and fig. 11B is a graph showing the results of a compound action potential test of sciatic nerves of a nerve conduit according to example 4 of the present invention after being implanted in a rat hindlimb for 60 days. As shown in fig. 11A, the nerve conduction velocity of the sciatic nerve of the nerve catheter of example 4 was significantly superior to the nerve conduction velocity of comparative example 4 and control group 1 and similar to the nerve conduction velocity of control group 2 60 days after implantation of the hind limb of the rat. As shown in fig. 11B, the nerve conduit of example 4 had a significantly higher combined action potential of sciatic nerve than comparative example 4 60 days after implantation into rat hindlimb.

From the above results, it can be seen that the nerve conduit comprising the injectable self-assembled microsphere gel of the present invention will help nerve cells to repair, making the injectable self-assembled microsphere gel of the present invention potentially useful for preparing pharmaceutical compositions for promoting nerve cell growth.

In summary, the injectable self-assembled microsphere gel and the preparation method thereof of the invention blend the first gel microsphere and the second gel microsphere with opposite electric properties, so that the injectable self-assembled microsphere gel can be directly administered by injection and has excellent self-assembly capability. In the preparation method of the injectable self-assembled microsphere gel, the acrylic acid or the derivative thereof and the base material with high biocompatibility are used as the raw materials of the first gel microsphere and the second gel microsphere, so that the toxicity of the raw materials to cells can be avoided, the mass production can be effectively carried out, and the use safety and the application range of the injectable self-assembled microsphere gel prepared by the method of the injectable self-assembled microsphere gel are improved. Moreover, the injectable self-assembled microsphere gel provided by the invention has high biocompatibility and an effect of promoting cell growth, so that the injectable self-assembled microsphere gel can be applied to a biomedical material for promoting cell proliferation and tissue repair, has the potential to be further applied to preparation of a pharmaceutical composition for promoting nerve cell growth, and further achieves the aim of providing efficient and safe adjuvant therapy for a human body.

Claims

1. The preparation method of the injectable self-assembled microsphere gel is characterized by comprising the following steps:

carrying out a first gel microsphere preparation step;

carrying out a second gel microsphere preparation step;

the blending step is carried out, and the blending step,

the preparation step of the first gel microsphere comprises the following steps:

providing a first solution, the first solution being used as an aqueous phase and comprising a first component and a second component, wherein the first component comprises acrylic acid or a derivative thereof and the second component comprises chitosan or silk;

providing a first oil phase solution;

performing a first water-in-oil emulsion reaction, mixing the first solution with the first oil phase solution to form a plurality of first gel microspheres,

the preparation step of the second gel microsphere comprises the following steps:

providing a second solution, wherein the second solution is used as an aqueous phase and comprises a third component and a fourth component, the third component comprises acrylic acid or derivatives thereof, and the fourth component comprises gelatin, hyaluronic acid or algin;

providing a second oil phase solution;

performing a second water-in-oil emulsion reaction, mixing the second solution with the second oil phase solution to form a plurality of second gel microspheres,

the blending step mixes the first gel microsphere, the second gel microsphere and an aqueous solution to obtain the injectable self-assembled microsphere gel,

each first gel microsphere has a first charge, each second gel microsphere has a second charge, and the first charge and the second charge are opposite in electrical property;

wherein the acrylic acid or derivative thereof comprises methacrylic acid, methacrylic anhydride and glycidyl methacrylate;

wherein the average particle size of the first gel microspheres is 30-500 μm, the average particle size of the second gel microspheres is 30-500 μm, and the average pore size of the injectable self-assembled microsphere gel is 30-90 μm.

2. The method of preparing an injectable self-assembled microsphere gel of claim 1, wherein the volume percentage of the first gel microspheres to the second gel microspheres in the injectable self-assembled microsphere gel is 1: 0.5 to 1: 6.

3. the method of claim 1, wherein the step of preparing the injectable self-assembled microsphere gel is carried out in a micro flow channel system.

4. The method of preparing an injectable self-assembled microsphere gel of claim 1, wherein the injectable self-assembled microsphere gel has a colloidal strength of 30 to 3100 Pa.

5. The method of preparing an injectable self-assembled microsphere gel of claim 1, wherein the injectable self-assembled microsphere gel has an average porosity of from 35% to 50%.

6. An injectable self-assembled microsphere gel, comprising:

a first microgel having a first charge, wherein the first microgel comprises a plurality of first gel microspheres, and the first gel microspheres have an average particle size of 30 μm to 500 μm; and

a second microgel having a second charge, wherein the second microgel comprises a plurality of second gel microspheres, and the average particle size of the second gel microspheres is 30 μm to 500 μm;

wherein the average pore diameter of the injectable self-assembled microsphere gel is 30-90 μm;

wherein the first and second charges are opposite in electrical property, the average particle diameter of the first gel microspheres of the injectable self-assembled microsphere gel is equal to the average particle diameter of the second gel microspheres, and each of the first gel microspheres comprises an acrylic chitosan polymer, an acrylic silk polymer, or a combination thereof, and each of the second gel microspheres comprises an acrylic gelatin polymer, an acrylic hyaluronic acid polymer, an acrylic alginate polymer, or a combination thereof.

7. The injectable self-assembled microsphere gel of claim 6, wherein each of the first gel microspheres comprises methacrylate chitosan, methacrylate silk, or a combination thereof, and each of the second gel microspheres comprises methacrylate gelatin, methacrylate hyaluronic acid, methacrylate algin, or a combination thereof.

8. The injectable self-assembled microsphere gel of claim 6, wherein the first microgel and the second microgel are present in a volume percentage of 1: 0.5 to 1: 6.

9. the injectable self-assembled microsphere gel of claim 6, wherein the injectable self-assembled microsphere gel has a colloidal strength of from 30Pa to 3100 Pa.

10. The injectable self-assembled microsphere gel of claim 6, wherein the injectable self-assembled microsphere gel has an average porosity of from 35% to 50%.

11. Use of the injectable self-assembled microsphere gel according to claim 6 for the preparation of a biomedical material for promoting tissue repair.

12. Use of the injectable self-assembling microgel of claim 6 for the preparation of a pharmaceutical composition for promoting nerve cell growth.