CN107698795A - A kind of porous polymer microsphere preparation method and applications of structure-controllable - Google Patents

Abstract

A method for preparing porous polymer microspheres with controllable structure and its application. After mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, and obtain the water/oil emulsion by ultrasonication. The water/oil emulsion is added to the PVA solution, and the water/oil/water emulsion is obtained by ultrasonication. The water/oil/water emulsion is stirred at room temperature, washed with water, and freeze-dried to obtain porous polymer microspheres. By controlling the aging time of the emulsion, the porous polymer microspheres with different pore structures are prepared by only one-step reaction, the preparation method is simple, and the industrial production is easy to realize. The invention has short reaction cycle, high yield and can effectively improve working efficiency. The porous polymer microsphere can be applied in the preparation of a gel patch for repairing the skin, and has a good effect on skin repairing.

Description

A preparation method and application of porous polymer microspheres with controllable structure

technical field

The invention relates to the fields of polymer material technology and biomedical materials, in particular to a method for preparing porous polymer microspheres with controllable structure and its application.

Background technique

The application of biomolecules in clinical treatment is a major research hotspot in recent years. However, most biomolecules have a short plasma half-life and low bioavailability, which makes it difficult to be effectively used clinically. The preparation of micron-scale or nano-scale polymer microspheres as biomolecule carriers can realize the efficient utilization of biomolecules. Carriers can realize the controlled release of biomolecules and can protect unstable bioactive molecules from degradation. Both degradable natural polymers and synthetic polymers have been extensively studied in the field of controlled drug release. Among many polymers, polylactic-glycolic acid (PLGA) has good biocompatibility and spontaneous biodegradation, so it has obvious advantages in drug controlled release.

Single-component microspheres can deliver a class of single or multiple biomolecules, and can achieve long-term controlled release of biomolecules, but their release curves for biomolecules are similar. This limits its applications, since regeneration of tissues and organs is often driven by the synergistic effect of different biomolecules on cells at different growth and maturation stages. Therefore, in order to regenerate damaged, diseased and removed tissues, it is necessary to design and combine microspheres with different release properties to achieve the delivery of different biomolecules. Changing the degree of polymerization of the polymer is the most common way to adjust the release profile of microspheres. Controlling the degree of polymerization not only requires sophisticated synthesis techniques, but also makes it difficult to change the release rate significantly.

In recent years, porous polymer microspheres prepared by various emulsion methods have been widely used in the field of medical care. These methods can prepare polymer microspheres with open pores (fast release) and closed pores (slow release), which can effectively regulate the release rate of biomolecules. Since the release properties of these two microspheres with different pore structures are complementary, combining the two to realize the sequential release of different biomolecules is an effective method for tissue and organ regeneration. However, conventional methods for forming porous structures require the embedding and removal of hard (soft) templates (porogens) during the emulsion preparation process. This not only complicates the synthesis process but also damages the polymer matrix. In addition, due to the rapid accumulation of water inside and outside the emulsion, it is difficult to directly prepare porous microspheres with closed pore structure. Therefore, it is usually necessary to heat-treat or solvent-treat porous microspheres with an open-pore structure to close the surface pores, thereby obtaining microspheres with a closed-pore structure. Therefore, the combination of open-cell and closed-cell porous microspheres usually requires multiple steps to complete. In order to facilitate the preparation of a large number of porous microspheres with different pore structures in practical production and application, it is necessary to develop a simpler and more feasible method.

Contents of the invention

In view of the above problems in the prior art, the purpose of the present invention is to provide a simple one-step process for preparing porous polymer microspheres with controllable structure and its application. The method is simple and easy, and the reaction can be completed in one step. , can be used for large-scale production, and the prepared microspheres can be used to construct biomedical materials, such as gel patches, scaffold materials, and used for skin repair and bone tissue repair.

To achieve the above object, the present invention adopts the following technical solutions:

A method for preparing porous polymer microspheres with controllable structure. After mixing the cyclodextrin solution and the polyethylene glycol solution, adding it to the dichloromethane solution of PLGA, ultrasonically obtaining a water/oil emulsion, and mixing the water/oil The emulsion is added to the PVA solution, ultrasonically obtained to obtain a water/oil/water emulsion, and the water/oil/water emulsion is stirred at room temperature, washed with water, and freeze-dried to obtain porous polymer microspheres.

The further improvement of the present invention is that the cyclodextrin solution is prepared by adding cyclodextrin into water, and the concentration of the cyclodextrin solution is 3-15 mg/mL; the cyclodextrin is α-cyclodextrin, β-cyclodextrin , any one of γ-cyclodextrin;

Polyethylene glycol solution is prepared by adding polyethylene glycol into water, the concentration of polyethylene glycol solution is 50-150mg/mL; the molar mass of polyethylene glycol is 2000-50000g;

The methylene chloride solution of PLGA is prepared by adding PLGA to methylene chloride, and the mass concentration of the methylene chloride solution of PLGA is 50-300mg/mL;

The PVA solution is prepared by adding PVA to water, the mass concentration of the PVA solution is 5-20 mg/mL; the molar mass of the PVA is 15000-120000 g.

A further improvement of the present invention is that the cyclodextrin is α-cyclodextrin.

The further improvement of the present invention is that the ultrasonic power is 10-100W, the ultrasonic time is 10-60s; the stirring time is 1-48h;

The mass ratio of cyclodextrin, polyethylene glycol, PLGA and PVA is 0.656:7.25:100:100.

A further improvement of the present invention is that the stirring time is 3 hours.

A further improvement of the present invention is that the stirring time is 24 hours.

Application of a porous polymer microsphere in the preparation of a gel patch for repairing skin.

A further improvement of the present invention is that the preparation method of the gel patch for repairing the skin is as follows:

After mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, ultrasonically obtain the water/oil emulsion, add the water/oil emulsion to the PVA solution, and ultrasonically obtain the water/oil/oil emulsion. Water emulsion, stirring the water/oil/water emulsion at room temperature for 3 hours, washing with water, and freeze-drying to obtain porous polymer microspheres with closed-cell structure;

After mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, ultrasonically obtain the water/oil emulsion, add the water/oil emulsion to the PVA solution, and ultrasonically obtain the water/oil/oil emulsion. Water emulsion, stirring the water/oil/water emulsion at room temperature for 24 hours, washing with water, and freeze-drying to obtain porous polymer microspheres with an open-pore structure;

The porous polymer microspheres with closed-pore structure and the porous polymer microspheres with open-pore structure were respectively dispersed in sodium alginate solution, and then evenly spread on the glass slide, and then the calcium chloride solution was added dropwise, and another slide was used. The glass slide was pressed to obtain a gel patch; wherein, the ratio of the closed-pore porous polymer microspheres, the open-pore porous polymer microspheres to the sodium alginate solution was 10 mg: 10 mg: 300 μL.

A further improvement of the present invention is that the preparation method of the gel patch for repairing the skin is as follows:

After mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, ultrasonically obtain the water/oil emulsion, add the water/oil emulsion to the PVA solution, and ultrasonically obtain the water/oil/oil emulsion. Water emulsion, stirring the water/oil/water emulsion at room temperature for 3 hours, washing with water, and freeze-drying to obtain porous polymer microspheres with closed-cell structure;

Disperse the porous polymer microspheres with closed-cell structure in deionized water, add interleukin, stir in an ice bath for 24 hours, and freeze-dry to obtain microspheres loaded with interleukin; wherein, the porous polymer microspheres with closed-cell structure, deionized The ratio of ionized water to interleukin is 20mg: 5mL: 10mg;

Disperse the above-mentioned microspheres loaded with interleukin in sodium alginate solution, spread evenly on a glass slide, then add calcium chloride solution dropwise, and press another piece of glass slide for 30 minutes to obtain a gel patch; The ratio of interleukin-containing microspheres to sodium alginate solution is 20mg: 300μL.

The further improvement of the present invention is that melatonin is dissolved in polyethylene glycol solution, then mixed with cyclodextrin solution evenly, added to the dichloromethane solution of PLGA, and ultrasonicated to form a water/oil emulsion; The water/oil emulsion is added to the PVA solution, and the water/oil/water emulsion is obtained by ultrasonication. The emulsion is stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain internally porous polymer microspheres containing melatonin; Among them, the ratio of melatonin to polyethylene glycol is 10mg: 7.25mg;

Disperse the internal porous polymer microspheres containing melatonin in sodium alginate solution, spread evenly on a glass slide, then add calcium chloride solution dropwise, and press with another glass slide to obtain a gel patch ; Wherein, the ratio of the internal porous polymer microspheres containing melatonin to the sodium alginate solution is 20 mg: 300 μL.

The further improvement of the present invention is that, after mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, ultrasonically obtain the water/oil emulsion, and add the water/oil emulsion to the PVA solution , ultrasonically obtained water/oil/water emulsion, after stirring the water/oil/water emulsion at room temperature for 3h, washing with water, and freeze-drying to obtain porous polymer microspheres with closed-cell structure; the porous polymer microspheres with closed-cell structure Microspheres are dispersed in deionized water, interleukin is added, stirred in an ice bath for 24 hours, and then freeze-dried to obtain microspheres loaded with interleukin; wherein, the ratio of closed-cell porous polymer microspheres, deionized water to interleukin is 20mg : 5mL: 10mg;

Dissolve melatonin in polyethylene glycol solution, mix evenly with cyclodextrin solution, add it to the dichloromethane solution of PLGA, and ultrasonicate to form a water/oil emulsion; add the above water/oil emulsion to In the PVA solution, the water/oil/water emulsion was obtained by ultrasound, and the emulsion was stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain internal porous polymer microspheres containing melatonin; wherein, melatonin and poly The ratio of ethylene glycol is 10mg: 7.25mg;

Disperse the interleukin-loaded microspheres and melatonin-containing internal porous polymer microspheres in sodium alginate solution, spread evenly on a glass slide, then add calcium chloride solution dropwise, and use another glass slide The tablet was compressed to obtain a gel patch; wherein, the ratio of interleukin-loaded microspheres, melatonin-containing internal porous polymer microspheres, and sodium alginate solution was 10 mg: 10 mg: 300 μL.

The further improvement of the present invention is that the sum of the porous polymer microspheres of the closed-pore structure and the porous polymer microspheres of the open-pore structure, the microspheres loaded with interleukin and the internal porous polymer microspheres containing melatonin and seaweed The ratio of sodium bicarbonate solution is 10mg ~ 40mg: 500μL.

The further improvement of the present invention lies in that the mass concentration of sodium alginate is 10-30 mg/mL.

The further improvement of the present invention lies in that the calcium chloride concentration is 0.2-0.8 mol/L.

Compared with the prior art, the present invention has the beneficial effects:

1. The polymer microsphere matrix PLGA (polylactic acid-glycolic acid) that the present invention adopts has a wide range of sources, good biocompatibility, degradable, and degradation products are nontoxic, and is applicable to the field of biomedicine. The present invention uses PLGA as The porous microsphere substrate, combined with the double emulsion method and the sol-gel method, controlled the aging time of the emulsion, and prepared porous microspheres with different pore structures through a one-step reaction, and the particle size of the prepared porous polymer microspheres was micron grade, with an average particle size of 2-5 μm, good dispersion and sphericity. The length of stirring time in the present invention can control the pore structure of the porous polymer microsphere. If the stirring time is short, the surface of the microsphere has no pores; if the stirring time is long, the surface of the microsphere has pores.

2. The present invention adopts the method of combining double emulsification method and sol-gel method. By controlling the aging time of the emulsion, porous polymer microspheres with different pore structures are prepared in only one step reaction. The preparation method is simple and easy to realize industrialization Production.

3. The present invention has short reaction cycle and high yield, and can effectively improve work efficiency.

4. The present invention does not generate organic waste liquid during the operation process, and is a green and environment-friendly preparation method.

5. The present invention does not need to use large and expensive instruments and equipment, and the production cost is low.

6. The porous microspheres of different structures prepared by the present invention have many uses, such as being used as a biomolecular carrier for the regeneration of tissues and organs, as an antibacterial drug carrier to improve the antibacterial ability of antibacterial drugs, and the like. The application in the construction of biomedical materials, such as the preparation of gel patches for skin repair, has a good effect on skin repair.

Description of drawings

Figure 1 is the porous microsphere with closed pore structure provided by Example 2.

FIG. 2 is a partially enlarged view of FIG. 1 .

Fig. 3 is the porous microsphere with open-pore structure provided by Example 4.

FIG. 4 is a partially enlarged view of FIG. 3 .

Fig. 5 is the release curve of FITC-BSA by the porous microspheres with closed pore structure and porous microspheres with open pore structure provided in Example 11 and Example 12.

Fig. 6 is a diagram of wound healing in mice provided in Example 17.

FIG. 7 is a statistical chart of the wound healing rate of mice provided in Example 17. FIG.

FIG. 8 is an effect diagram of the migration and differentiation of phagocytic cells provided in Example 17.

FIG. 9 is a statistical diagram of the number of CD206-positive macrophages provided in Example 17. FIG.

Figure 10 is the statistical value of vascular endothelial growth factor (VEGF) provided by Example 17.

FIG. 11 is the statistical value of angiotensin II (Ang II) provided in Example 17.

detailed description

The present invention will be further described below in conjunction with the embodiments and drawings, but not limited thereto.

The invention relates to one-step preparation of polymer microspheres with different structures and the construction and application of biomedical materials based on the polymer microspheres.

A method for preparing porous polymer microspheres with controllable structure, adding cyclodextrin to water to obtain cyclodextrin solution, adding polyethylene glycol (PEG) to water to obtain polyethylene glycol solution, and cyclodextrin After the fine solution is mixed with the polyethylene glycol solution, it is added to the dichloromethane solution of PLGA (polylactic acid-glycolic acid), and the water/oil emulsion is obtained by ultrasonication, and the water/oil emulsion is added to polyvinyl alcohol (PVA) In the solution of 10-100W, ultrasonication is performed for 10-60s to obtain water/oil/water emulsion, and the water/oil/water emulsion is stirred at room temperature for 1-48h, washed with water for 3-5 times, and freeze-dried to obtain Porous polymer microspheres.

The cyclodextrin is any one of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin.

Preferably, the cyclodextrin is α-cyclodextrin. The concentration of the cyclodextrin solution is 3-10 mg/mL.

The molar mass of the polyethylene glycol (PEG) is 2000-50000 g, and the concentration of the polyethylene glycol solution is 50-90 mg/mL.

The mass concentration of the PLGA dichloromethane solution is 5%-30%.

The molar mass of the PVA is 15000-120000g, and the mass concentration of the polyvinyl alcohol solution is 0.5%-2%.

The particle size of the porous polymer microsphere prepared by the invention is in the order of microns, and the average particle size is 2-5 μm. Both dispersion and sphericity are good.

The molar mass of PLGA used in Examples 1-17 of the present invention is 64000g, lactic acid/glycolic acid=50/50; the molar mass of PVA used is 25000g, and the degree of alcoholysis is 88%; the molar mass of PEG is 2000g, cyclodextrin For α-cyclodextrin.

Example 1

100 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 10% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. Dissolve 2g of PVA in 200mL of water to prepare a 1% (w/v) PVA solution.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and 50 μL of 145 mg/mL PEG solution, mix evenly, add to 1 mL of 10% (w/v) PLGA in dichloromethane solution, and sonicate at 15W for 20s to form Water/oil emulsion: the above water/oil emulsion was added to 10 mL of 1% (w/v) PVA solution, and the water/oil/water emulsion was obtained by ultrasonication. The emulsion was slowly stirred at room temperature at 200-600 r/min for 1 hour, washed with water three times, and freeze-dried to obtain porous polymer microspheres. The microspheres obtained under this condition have no pores on the surface but internal pores, and the internal pores are less and the pores are not uniformly dispersed.

Example 2

100 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 10% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. Dissolve 2g of PVA in 200mL of water to prepare a 1% (w/v) PVA solution.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and 50 μL of 145 mg/mL PEG solution, mix evenly, add to 1 mL of 10% (w/v) PLGA in dichloromethane solution, and sonicate at 15W for 20s to form Water/oil emulsion: the above water/oil emulsion was added to 10 mL of 1% (w/v) PVA solution, and the water/oil/water emulsion was obtained by ultrasonication. The emulsion was slowly stirred at room temperature at 200-600 r/min for 3 hours, washed with water three times, and freeze-dried to obtain porous polymer microspheres. The scanning electron microscope of the microspheres is shown in Figure 1 and Figure 2. It can be seen from Figure 1 and Figure 2 that the microspheres obtained by aging the emulsion for 3 hours have no pores on the surface and pores inside, that is, a closed-cell structure, and the particle size of the microspheres is about 5 μm.

Example 3

100 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 10% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. 2 g of PVA was dissolved in 200 mL of water to prepare a 1% (w/v) solution of PVA in dichloromethane.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and 50 μL of 145 mg/mL PEG solution, mix evenly, add to 1 mL of 10% (w/v) PLGA solution, and ultrasonicate for 20 seconds at 15W to form a water/oil emulsion Liquid; the above-mentioned water/oil emulsion was added to 10 mL of 1% (w/v) PVA solution, and the water/oil/water emulsion was obtained by ultrasonication. The emulsion was slowly stirred at room temperature at 200-600 r/min for 12 hours, washed with water three times, and freeze-dried to obtain porous polymer microspheres. The microspheres obtained under this condition have no pores on the surface and pores inside, and the pore diameter is relatively large.

Example 4

100 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 10% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. 2 g of PVA was dissolved in 200 mL of water to prepare a 1% (w/v) solution of PVA in dichloromethane.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and 50 μL of 145 mg/mL PEG solution, mix evenly, add to 1 mL of 10% (w/v) PLGA solution, and ultrasonicate for 20 seconds at 15W to form a water/oil emulsion Liquid; the above-mentioned water/oil emulsion was added to 10 mL of 1% (w/v) PVA solution, and the water/oil/water emulsion was obtained by ultrasonication. The emulsion was slowly stirred at room temperature at 200-600 r/min for 24 hours, washed with water three times, and freeze-dried to obtain porous polymer microspheres. The scanning electron microscope of the microspheres is shown in Figure 3 and Figure 4. It can be seen from Figure 3 and Figure 4 that the microspheres obtained by aging the emulsion for 24 hours have an open-pore structure, that is, there are pores on the surface and inside of the microspheres, and the particle size of the microspheres is about 5 μm.

Example 5

100 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 10% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. 2 g of PVA was dissolved in 200 mL of water to prepare a 1% (w/v) solution of PVA in dichloromethane.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and 50 μL of 145 mg/mL PEG solution, mix evenly, add to 1 mL of 10% (w/v) PLGA solution, and ultrasonicate for 20 seconds at 15W to form a water/oil emulsion Liquid; the above-mentioned water/oil emulsion was added to 10 mL of 1% (w/v) PVA solution, and the water/oil/water emulsion was obtained by ultrasonication. The emulsion was slowly stirred at room temperature at 200-600 r/min for 36 hours, washed with water three times, and freeze-dried to obtain porous polymer microspheres.

Example 6

100 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 10% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. 2 g of PVA was dissolved in 200 mL of water to prepare a 1% (w/v) solution of PVA in dichloromethane.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and 50 μL of 145 mg/mL PEG solution, mix evenly, add to 1 mL of 10% (w/v) PLGA solution, and ultrasonicate for 20 seconds at 15W to form a water/oil emulsion Liquid; the above-mentioned water/oil emulsion was added to 10 mL of 1% (w/v) PVA solution, and the water/oil/water emulsion was obtained by ultrasonication. The emulsion was slowly stirred at room temperature at 200-600 r/min for 48 hours, washed with water three times, and freeze-dried to obtain porous polymer microspheres.

Example 7

25 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 2.5% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. Dissolve 2g of PVA in 200mL of water to prepare a 1% (w/v) PVA solution.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and 50 μL of 145 mg/mL PEG solution, mix evenly, add to 1 mL of 10% (w/v) PLGA in dichloromethane solution, and sonicate at 15W for 20s to form Water/oil emulsion: Add the above water/oil emulsion into 10mL of 1% PVA solution, and ultrasonically obtain the water/oil/water emulsion. The emulsion was slowly stirred at room temperature at 200-600 r/min for 3 hours, washed with water three times, and freeze-dried to obtain porous polymer microspheres.

Example 8

25 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 2.5% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. Dissolve 2g of PVA in 200mL of water to prepare a 1% (w/v) PVA solution.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and 50 μL of 145 mg/mL PEG solution, mix evenly, add to 1 mL of 10% (w/v) PLGA in dichloromethane solution, and sonicate at 15W for 20s to form Water/oil emulsion: the above water/oil emulsion was added to 10 mL of 1% (w/v) PVA solution, and the water/oil/water emulsion was obtained by ultrasonication. The emulsion was slowly stirred at room temperature at 200-600 r/min for 24 hours, washed with water three times, and freeze-dried to obtain porous polymer microspheres.

Example 9

100 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 10% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. Dissolve 2g of PVA in 200mL of water to prepare a 1% (w/v) PVA solution.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and 50 μL of 145 mg/mL PEG solution in dichloromethane, mix evenly, add to 1 mL of 10% (w/v) PLGA solution, and sonicate at 50 W for 20 seconds to form Water/oil emulsion: the above water/oil emulsion was added to 10 mL of 1% (w/v) PVA solution, and the water/oil/water emulsion was obtained by ultrasonication. The emulsion was slowly stirred at room temperature at 200-600 r/min for 3 hours, washed with water three times, and freeze-dried to obtain porous polymer microspheres.

Example 10

100 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 10% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. Dissolve 2g of PVA in 200mL of water to prepare a 1% (w/v) PVA solution.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and 50 μL of 145 mg/mL PEG solution, mix evenly, add to 1 mL of 10% (w/v) PLGA in dichloromethane solution, and sonicate at 50 W for 20 seconds to form Water/oil emulsion: the above water/oil emulsion was added to 10 mL of 1% (w/v) PVA solution, and the water/oil/water emulsion was obtained by ultrasonication. The emulsion was slowly stirred at room temperature at 200-600 r/min for 24 hours, washed with water three times, and freeze-dried to obtain porous polymer microspheres.

The long-term release ability test of the porous microsphere of embodiment 11 closed cell structure:

100 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 10% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. Dissolve 2g of PVA in 200mL of water to prepare a 1% (w/v) PVA solution.

Dissolve 5 mg of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) in 50 μL of 145 mg/mL PEG solution, mix with 50 μL of 13.12 mg/mL α-cyclodextrin solution, and add to 1 mL of 10% (w/v) in the dichloromethane solution of PLGA, ultrasonic 20s with 15W power, form water/oil emulsion; Add above-mentioned water/oil emulsion in the PVA solution of 10mL1% (w/v), obtain by ultrasonic Water/oil/water emulsion. The emulsion was slowly stirred at room temperature for 3 hours, washed with water three times, and freeze-dried to obtain porous polymer microspheres.

Take 10 mg of microspheres and disperse them in 5 mL of phosphate buffered saline (PBS) with pH=7.4. On days 0, 1, 3...30, take out 2 mL, centrifuge, and measure the fluorescence intensity of the supernatant. The test results from Fig. 5 show that at day 25, the microspheres released 90% of the total amount of FITC-BSA. This shows that the porous microspheres with closed-cell structure prepared by this method have long-term release ability.

The long-term release ability test of the porous microsphere of embodiment 12 open-pore structure:

100 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 10% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. Dissolve 2g of PVA in 200mL of water to prepare a 1% (w/v) PVA solution.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and 50 μL of 145 mg/mL PEG solution, mix evenly, add to 1 mL of 10% (w/v) PLGA in dichloromethane solution, and sonicate at 15W for 20s to form Water/oil emulsion: the above water/oil emulsion was added to 10 mL of 1% (w/v) PVA solution, and the water/oil/water emulsion was obtained by ultrasonication. Stir the emulsion slowly at room temperature at 200-600r/min for 24h, wash it with water three times, disperse it in 5mL water, add 5mg FITC-BSA (bovine serum albumin labeled with isothiocyanate), stir for 24h, centrifuge, and redisperse Freeze-dried in water to obtain microspheres with open-pore structure. Disperse 10 mg of microspheres with an open pore structure in 5 mL of PBS buffer at pH = 7.4, take out 2 mL of microspheres on days 0, 1, 3, 5, and 7, and measure the fluorescence of the supernatant. After the test, the supernatant The solution and the centrifuged solid were sonicated and then put into PBS buffer. From the analysis results in Fig. 5, it can be known that on the 5th day, the released amount of microspheres reached 90% of the total amount.

It can be seen that the porous microspheres with two different pore structures prepared by this method have different release properties.

Example 13

6 mg of sodium alginate was dissolved in 300 μL of deionized water, and 10 mg of the microspheres prepared in Example 2 and Example 4 were respectively taken and dispersed in the sodium alginate solution. The above dispersion was evenly spread on a glass slide, and then 300 μL of 0.5 mol/L calcium chloride solution was added dropwise on it, and another glass slide was used to gently press for 30 minutes to obtain a gel patch 1. Cut the entire piece of gel patch into a circular patch with a diameter of about 50dm for later use.

Example 14

The microspheres prepared in Example 2 were dispersed in 5 mL of deionized water, 10 mg of interleukin (IL-4) was added, stirred in an ice bath for 24 h, and then freeze-dried to obtain microspheres loaded with interleukin for future use.

6 mg of sodium alginate was dissolved in 300 μL of deionized water, and 20 mg of the above-mentioned interleukin-loaded microspheres were dispersed in the sodium alginate solution. The dispersion was evenly spread on a glass slide, and then 300 μL of 0.5 mol/L calcium chloride solution was added dropwise on it, and another glass slide was gently pressed for 30 minutes to obtain gel patch 2. Cut the entire piece of gel patch into a circular patch with a diameter of about 50dm for later use.

Example 15

100 mg of PLGA was weighed and dissolved in 1 mL of dichloromethane to prepare a 10% (w/v) solution of PLGA in dichloromethane. Weigh 13.12mg α-cyclodextrin and dissolve it in 1mL water to prepare 13.12mg/mL α-cyclodextrin solution; weigh 145mg PEG-2000 and dissolve it in 1mL water to prepare 145mg/mL PEG solution. Dissolve 2g of PVA in 200mL of water to prepare a 1% (w/v) PVA solution.

Dissolve 10 mg of melatonin in 50 μL of 145 mg/mL PEG solution, mix well with 50 μL of 13.12 mg/mL α-cyclodextrin solution, and add to 1 mL of 10% (w/v) PLGA in dichloromethane , ultrasonication with 15W power for 20s to form a water/oil emulsion; the above water/oil emulsion was added to 10mL of 1% (w/v) PVA solution, and ultrasonically obtained a water/oil/water emulsion. The emulsion was slowly stirred at room temperature at 200-600 r/min for 3 hours, washed with water three times, and freeze-dried to obtain internally porous polymer microspheres containing melatonin.

6 mg of sodium alginate was dissolved in 300 μL of deionized water, and 20 mg of internally porous polymer microspheres containing melatonin were dispersed in the sodium alginate solution. The dispersion was evenly spread on a glass slide, and a certain volume of 0.5 mol/L calcium chloride solution was added dropwise on it, and another glass slide was used to gently press for 30 minutes to obtain a gel patch 3 . Cut the entire piece of gel patch into a circular patch with a diameter of about 50dm for later use.

Example 16

Dissolve 6 mg of sodium alginate in 300 μL of deionized water, take 10 mg of microspheres containing interleukin and melatonin in Example 14 and Example 15, and disperse them all in the sodium alginate solution. Spread the dispersion above evenly on a glass slide, then drop a certain volume of 0.5 mol/L calcium chloride solution on it, and gently press it with another glass slide for 30 minutes to obtain the gel patch 4 . Cut the entire piece of gel patch into a circular patch with a diameter of about 50dm for later use.

Example 17

Take the gel patch 1, gel patch 2, gel patch 3 and gel patch 4 in Examples 13, 14, 15, and 16 respectively to treat mouse wounds, take pictures and record the wound healing of the mice, And calculate its healing rate. The results are shown in Figure 6 and Figure 7. The results in Fig. 4 show that after treating the mouse wound with the gel patch of Example 16, the wound healing of the mouse is the best, that is, under the joint action of IL-4 and melatonin, the wound healing effect of the mouse is the best. After treating the mouse wound for 7 days, the number of M2-positive macrophages was detected, and the detection results are shown in Fig. 8 and Fig. 9 . The results showed that IL-4 played a major role in promoting the differentiation of macrophages into M2-positive macrophages. After 7 days, the contents of vascular endothelial growth factor (VEGF) and angiotensin II (Ang II) were detected, and the detection results are shown in Fig. 10 and Fig. 11 . The results suggest that melatonin plays a major role in promoting angiogenesis.

The porous polymer microspheres with different pore structures prepared by the present invention can be used to load different types of biomolecules. In Example 16, these two kinds of microspheres were combined as biomolecule carriers, which can not only protect them, but also selectively release biomolecules required in different stages of tissue and organ regeneration.

Example 18

The cyclodextrin solution is prepared by adding cyclodextrin to water, and the concentration of the cyclodextrin solution is 3mg/mL; wherein, the cyclodextrin is γ-cyclodextrin;

Polyethylene glycol solution is prepared by adding polyethylene glycol into water, the concentration of polyethylene glycol solution is 50mg/mL; the molar mass of polyethylene glycol is 2000g;

The dichloromethane solution of PLGA is prepared by adding PLGA into dichloromethane, and the concentration of the PLGA dichloromethane solution is 50 mg/mL.

The PVA solution is prepared by adding PVA to water, the mass concentration of the PVA solution is 5mg/mL; the molar mass of the PVA is 120000g.

After mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, ultrasonically obtain the water/oil emulsion, add the water/oil emulsion into the PVA solution, and ultrasonicate for 60 seconds at 10W to obtain water /oil/water emulsion, the water/oil/water emulsion was stirred at room temperature for 3h, washed with water, and freeze-dried to obtain porous polymer microspheres; wherein, the mass ratio of cyclodextrin, polyethylene glycol, PLGA to PVA It is 0.656:7.25:100:100.

Example 19

The cyclodextrin solution is prepared by adding cyclodextrin to water, and the concentration of the cyclodextrin solution is 15 mg/mL; wherein, the cyclodextrin is β-cyclodextrin;

Polyethylene glycol solution is prepared by adding polyethylene glycol into water, the concentration of polyethylene glycol solution is 150mg/mL; the molar mass of polyethylene glycol is 10000g;

The dichloromethane solution of PLGA is prepared by adding PLGA into dichloromethane, and the concentration of the PLGA dichloromethane solution is 200 mg/mL.

The PVA solution is prepared by adding PVA to water, the mass concentration of the PVA solution is 20mg/mL; the molar mass of the PVA is 75000g.

After mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, ultrasonically obtain a water/oil emulsion, add the water/oil emulsion into the PVA solution, and ultrasonically obtain water at 100W for 10s /oil/water emulsion, the water/oil/water emulsion was stirred at room temperature for 24h, washed with water, and freeze-dried to obtain porous polymer microspheres; wherein, the mass ratio of cyclodextrin, polyethylene glycol, PLGA to PVA It is 0.656:7.25:100:100.

Example 20

The cyclodextrin solution is prepared by adding cyclodextrin to water, and the concentration of the cyclodextrin solution is 8 mg/mL; wherein, the cyclodextrin is γ-cyclodextrin;

Polyethylene glycol solution is prepared by adding polyethylene glycol into water, the concentration of polyethylene glycol solution is 100mg/mL; the molar mass of polyethylene glycol is 50000g;

The dichloromethane solution of PLGA is prepared by adding PLGA into dichloromethane, and the concentration of the PLGA dichloromethane solution is 300 mg/mL.

The PVA solution is prepared by adding PVA to water, the mass concentration of the PVA solution is 15mg/mL; the molar mass of the PVA is 15000g.

After mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, ultrasonically obtain the water/oil emulsion, add the water/oil emulsion into the PVA solution, and ultrasonicate for 30 seconds at 50W to obtain water /oil/water emulsion, the water/oil/water emulsion was stirred at room temperature for 8h, washed with water, and freeze-dried to obtain porous polymer microspheres; wherein, the mass ratio of cyclodextrin, polyethylene glycol, PLGA to PVA It is 0.656:7.25:100:100.

In the present invention, the sum of the porous polymer microspheres of the closed-cell structure and the porous polymer microspheres of the open-pore structure, the microspheres loaded with interleukin and the internal porous polymer microspheres containing melatonin and the sodium alginate solution The ratio can be 10 mg-40 mg:500 μL, and the present invention only uses 10 mg:500 μL as an example for illustration. The concentration of sodium alginate in the present invention can be 10-30 mg/mL, and the present invention only takes 20 mg/mL as an example for illustration. The concentration of calcium chloride in the present invention can be 0.2-0.8 mol/L, and only 0.5 mol/L is taken as an example in the present invention for illustration.

Claims (10)

1. A method for preparing porous polymer microspheres with controllable structure, characterized in that, after cyclodextrin solution is mixed with polyethylene glycol solution, it is added in the dichloromethane solution of PLGA, and ultrasonically obtains water/oil emulsification The water/oil emulsion is added to the PVA solution, and the water/oil/water emulsion is obtained by ultrasonication. The water/oil/water emulsion is stirred at room temperature, washed with water, and freeze-dried to obtain porous polymer microspheres. 2. a kind of structure controllable porous polymer microsphere preparation method according to claim 1 is characterized in that, cyclodextrin solution is that cyclodextrin is added into water and makes, and the concentration of cyclodextrin solution is 3 ～15mg/mL; cyclodextrin is any one of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin; Polyethylene glycol solution is prepared by adding polyethylene glycol into water, the concentration of polyethylene glycol solution is 50-150mg/mL; the molar mass of polyethylene glycol is 2000-50000g; The methylene chloride solution of PLGA is prepared by adding PLGA to methylene chloride, and the mass concentration of the methylene chloride solution of PLGA is 50-300 mg/mL; PVA solution is prepared by adding PVA to water, the mass concentration of PVA solution is 5-20g/mL; the molar mass of PVA is 15000-120000g. 3. The method for preparing porous polymer microspheres with controllable structure according to claim 1, characterized in that the ultrasonic power is 10-100W, the ultrasonic time is 10-60s; the stirring time is 1-48h ; The mass ratio of cyclodextrin, polyethylene glycol, PLGA and PVA is 0.656:7.25:100:100. 4. The method for preparing a structure-controllable porous polymer microsphere according to claim 1, wherein the stirring time is 3 hours. 5. The method for preparing a structure-controllable porous polymer microsphere according to claim 1, wherein the stirring time is 24 hours. 6. The application of a porous polymer microsphere prepared by the method according to any one of claims 1-5 in the preparation of a gel patch for repairing skin. 7. A kind of application according to claim 6, is characterized in that, the preparation method of the gel patch for repairing skin is as follows: After mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, ultrasonically obtain the water/oil emulsion, add the water/oil emulsion to the PVA solution, and ultrasonically obtain the water/oil/oil emulsion. Water emulsion, stirring the water/oil/water emulsion at room temperature for 3 hours, washing with water, and freeze-drying to obtain porous polymer microspheres with closed-cell structure; After mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, ultrasonically obtain the water/oil emulsion, add the water/oil emulsion to the PVA solution, and ultrasonically obtain the water/oil/oil emulsion. Water emulsion, stirring the water/oil/water emulsion at room temperature for 24 hours, washing with water, and freeze-drying to obtain porous polymer microspheres with an open-pore structure; The porous polymer microspheres with closed-pore structure and the porous polymer microspheres with open-pore structure were respectively dispersed in sodium alginate solution, and then evenly spread on the glass slide, and then the calcium chloride solution was added dropwise, and another slide was used. The glass slide was pressed to obtain a gel patch; wherein, the ratio of the closed-pore porous polymer microspheres, the open-pore porous polymer microspheres to the sodium alginate solution was 10 mg: 10 mg: 300 μL. 8. A kind of application according to claim 6, is characterized in that, the preparation method of the gel patch for repairing skin is as follows: After mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, ultrasonically obtain the water/oil emulsion, add the water/oil emulsion to the PVA solution, and ultrasonically obtain the water/oil/oil emulsion. Water emulsion, stirring the water/oil/water emulsion at room temperature for 3 hours, washing with water, and freeze-drying to obtain porous polymer microspheres with closed-cell structure; Disperse the porous polymer microspheres with closed-cell structure in deionized water, add interleukin, stir in an ice bath for 24 hours, and freeze-dry to obtain microspheres loaded with interleukin; wherein, the porous polymer microspheres with closed-cell structure, deionized The ratio of ionized water to interleukin is 20mg: 5mL: 10mg; Disperse the above-mentioned microspheres loaded with interleukin in sodium alginate solution, spread evenly on a glass slide, then add calcium chloride solution dropwise, and press another piece of glass slide for 30 minutes to obtain a gel patch; The ratio of interleukin-containing microspheres to sodium alginate solution is 20mg: 300μL. 9. A kind of application according to claim 6, is characterized in that, the preparation method of the gel patch for repairing skin is as follows: Dissolve melatonin in polyethylene glycol solution, mix evenly with cyclodextrin solution, add it to the dichloromethane solution of PLGA, and ultrasonicate to form a water/oil emulsion; add the above water/oil emulsion to In the PVA solution, the water/oil/water emulsion was obtained by ultrasound, and the emulsion was stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain internal porous polymer microspheres containing melatonin; wherein, melatonin and poly The ratio of ethylene glycol is 10mg: 7.25mg; Disperse the internal porous polymer microspheres containing melatonin in sodium alginate solution, spread evenly on a glass slide, then add calcium chloride solution dropwise, and press with another glass slide to obtain a gel patch ; Wherein, the ratio of the internal porous polymer microspheres containing melatonin to the sodium alginate solution is 20 mg: 300 μL. 10. A kind of application according to claim 6, is characterized in that, the preparation method of the gel patch for repairing skin is as follows: After mixing the cyclodextrin solution and the polyethylene glycol solution, add it to the dichloromethane solution of PLGA, ultrasonically obtain the water/oil emulsion, add the water/oil emulsion to the PVA solution, and ultrasonically obtain the water/oil/oil emulsion. Water emulsion, stir the water/oil/water emulsion at room temperature for 3 hours, wash with water, and freeze-dry to obtain porous polymer microspheres with closed-pore structure; disperse the porous polymer microspheres with closed-pore structure in deionized water , adding interleukin, stirring in an ice bath for 24 hours, and then freeze-drying to obtain interleukin-loaded microspheres; wherein, the ratio of closed-cell porous polymer microspheres, deionized water, and interleukin is 20mg: 5mL: 10mg; Dissolve melatonin in polyethylene glycol solution, mix evenly with cyclodextrin solution, add it to the dichloromethane solution of PLGA, and ultrasonicate to form a water/oil emulsion; add the above water/oil emulsion to In the PVA solution, the water/oil/water emulsion was obtained by ultrasound, and the emulsion was stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain internal porous polymer microspheres containing melatonin; wherein, melatonin and poly The ratio of ethylene glycol is 10mg: 7.25mg; Disperse the interleukin-loaded microspheres and melatonin-containing internal porous polymer microspheres in sodium alginate solution, spread evenly on a glass slide, then add calcium chloride solution dropwise, and use another glass slide The tablet was compressed to obtain a gel patch; wherein, the ratio of interleukin-loaded microspheres, melatonin-containing internal porous polymer microspheres, and sodium alginate solution was 10 mg: 10 mg: 300 μL.