CN107698795B - Preparation method and application of porous polymer microspheres with controllable structures - Google Patents

Abstract

A method for preparing porous polymer microspheres with controllable structure and application thereof. After mixing a cyclodextrin solution with a polyethylene glycol solution, adding it to a dichloromethane solution of PLGA, ultrasonically obtaining 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 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, porous polymer microspheres with different pore structures are prepared in only one step of reaction, the preparation method is simple, and the industrial production is easy to be realized. The invention has short reaction period and high yield, and can effectively improve work efficiency. The porous polymer microspheres can be used in the preparation of gel patches for skin repair, and have a good effect on skin repair.

Description

A method for preparing porous polymer microspheres with controllable structure and its application

technical field

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

Background technique

The application of biomolecules in clinical therapy is a major research hotspot in recent years. However, most biomolecules have short plasma half-lives and low bioavailability, which make it difficult to use them effectively in the clinic. The preparation of micro-scale or nano-scale polymer microspheres as biomolecular carriers can realize the efficient utilization of biomolecules. The carrier can realize the controlled release of biomolecules, and can protect the unstable bioactive molecules from their degradation. Degradable natural 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 can be biodegraded spontaneously, so it has obvious advantages for drug controlled release.

Single-component microspheres can achieve the delivery of a single or multiple biomolecules, and can achieve long-term controlled release of biomolecules, but their release curves for biomolecules are similar. This limits its application because 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 method for tuning the release profile of the microspheres. Controlling the degree of polymerization not only requires sophisticated synthesis techniques, but it is also difficult to significantly alter the release rate.

In recent years, porous polymer microspheres prepared by various emulsion methods have been widely used in the field of healthcare. 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 the two microspheres with different pore structures are complementary, combining the two to achieve the sequential release of different biomolecules is an effective method for tissue and organ regeneration. However, conventional methods of forming porous structures require the insertion and removal of hard (soft) templates (porogens) during the preparation of emulsions. This not only complicates the synthesis process, but also destroys the polymer matrix. Besides, it is difficult to directly prepare porous microspheres with closed pore structure due to the rapid aggregation of water inside and outside the emulsion. Therefore, it is usually necessary to perform heat treatment or solvent treatment on the porous microspheres with an open-pore structure to close the surface pores, thereby producing microspheres with a closed-pore structure. Therefore, the combination of open-cell and closed-cell porous microspheres usually requires multiple steps. 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.

SUMMARY OF THE INVENTION

In view of the above problems in the prior art, the purpose of the present invention is to provide a simple and one-step process for preparing porous polymer microspheres with controllable structure and application thereof. The method is simple and feasible, 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.

For achieving the above object, the present invention adopts the following technical scheme:

A method for preparing porous polymer microspheres with a controllable structure. After mixing a cyclodextrin solution with a polyethylene glycol solution, it is added to a dichloromethane solution of PLGA, ultrasonically obtains a water/oil emulsion, and the water/oil is mixed. The emulsion is added to the PVA solution, ultrasonicated 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 to 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;

The polyethylene glycol solution is prepared by adding polyethylene glycol to water, and the concentration of the polyethylene glycol solution is 50-150 mg/mL; the molar mass of the polyethylene glycol is 2000-50,000 g;

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

The PVA solution is prepared by adding PVA into 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 power of the ultrasonic is 10-100W, the time of the ultrasonic is 10-60s, and the stirring time is 1-48h;

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

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

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

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 with 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 the water/oil/oil emulsion. Water emulsion, the water/oil/water emulsion is stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain porous polymer microspheres with closed-cell structure;

After mixing the cyclodextrin solution with 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 the water/oil/oil emulsion. Water emulsion, after 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 taken and dispersed in sodium alginate solution, and then spread evenly on the glass slide, and then the calcium chloride solution was added dropwise, and another piece of The glass slide is pressed to obtain a gel patch; wherein, the ratio of the closed-pore structure porous polymer microspheres, the open-pore structure porous polymer microspheres and the sodium alginate solution is 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 with 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 the water/oil/oil emulsion. Water emulsion, the water/oil/water emulsion is stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain porous polymer microspheres with closed-cell structure;

The porous polymer microspheres with closed-cell structure were dispersed in deionized water, interleukin was added, stirred for 24 h in an ice bath, and then freeze-dried to obtain microspheres loaded with interleukin; among them, the porous polymer microspheres with closed-pore structure, deionized The ratio of ionized water to interleukin is 20mg: 5mL: 10mg;

Take the microspheres loaded with interleukin and disperse them in sodium alginate solution, then spread evenly on a glass slide, add calcium chloride solution dropwise, and press another glass slide for 30 minutes to obtain a gel patch; The ratio of microspheres with interleukin to sodium alginate solution was 20 mg: 300 μL.

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

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

A further improvement of the present invention is that after mixing the cyclodextrin solution with the polyethylene glycol solution, adding it to the dichloromethane solution of PLGA, ultrasonically obtaining a water/oil emulsion, and adding the water/oil emulsion to the PVA solution , ultrasonication to obtain a water/oil/water emulsion, the water/oil/water emulsion was stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain porous polymer microspheres with closed-cell structure; The microspheres were dispersed in deionized water, interleukin was added, stirred in an ice bath for 24 hours, and then freeze-dried to obtain microspheres loaded with interleukin; among them, the ratio of porous polymer microspheres with closed-cell structure, deionized water and interleukin was 20 mg : 5mL: 10mg;

Melatonin was dissolved in the polyethylene glycol solution, mixed with the cyclodextrin solution, added to the dichloromethane solution of PLGA, sonicated to form a water/oil emulsion; the above water/oil emulsion was added to the solution. In the PVA solution, ultrasonication was used to obtain a water/oil/water emulsion, which was stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain melatonin-containing polymer microspheres with internal pores; The ratio of ethylene glycol is 10mg:7.25mg;

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

A further improvement of the present invention lies in the sum of the porous polymer microspheres of closed cell structure and the porous polymer microspheres of open cell structure, the microspheres loaded with interleukin and the internal porous polymer microspheres containing melatonin and seaweed The ratio of the sodium solution is 10 mg to 40 mg: 500 μL.

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

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

Compared with the prior art, the present invention has the following 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 the degradation product is non-toxic, suitable for the field of biomedicine, and the present invention uses PLGA as Porous microsphere substrate, combined with double emulsion method and sol-gel method, by controlling the aging time of the emulsion, porous microspheres with different pore structures are prepared through one-step reaction, and the particle size of the prepared porous polymer microspheres is micrometer. grade, with an average particle size of 2-5 μm, and good dispersion and sphericity. The length of the stirring time in the present invention can control the pore structure of the porous polymer microspheres. If the stirring time is short, the surface of the microsphere has no pores, and if the stirring time is long, the surface of the microsphere has pores.

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

3. The reaction cycle of the present invention is short, the yield is high, and the work efficiency can be effectively improved.

4. The present invention does not generate organic waste liquid in 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 with different structures prepared by the present invention have many uses, such as being used as biomolecule carrier for regeneration of tissues and organs, as antibacterial drug carrier to improve the antibacterial ability of antibacterial drugs and so on. 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

FIG. 1 is a porous microsphere with a closed pore structure provided in Example 2. FIG.

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

FIG. 3 is a porous microsphere with an open-pore structure provided in Example 4. FIG.

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

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

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

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

FIG. 8 is a graph showing the effect of migration and differentiation of phagocytes provided in Example 17. FIG.

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

FIG. 10 is a statistical value of the amount of vascular endothelial growth factor (VEGF) provided in Example 17. FIG.

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

Detailed ways

The present invention will be further described below with reference to the embodiments and the accompanying drawings, but is 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 a controllable structure. The cyclodextrin is added to water to obtain a cyclodextrin solution, and polyethylene glycol (PEG) is added to the water to obtain a polyethylene glycol solution. After the refined 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 ultrasound, and the water/oil emulsion is added to the polyvinyl alcohol (PVA) In the solution, ultrasonically sonicated for 10-60s at 10-100W to obtain a water/oil/water emulsion, stir the water/oil/water emulsion at room temperature for 1-48h, wash with water for 3-5 times, and freeze-dry 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 dichloromethane solution of the PLGA is 5% to 30%.

The molar mass of the PVA is 15,000-120,000 g, and the mass concentration of the polyvinyl alcohol solution is 0.5%-2%.

The particle size of the porous polymer microspheres prepared by the invention is in the micron order, and the average particle size is 2-5 μm. 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, the alcoholysis degree 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL of water to prepare a 1% (w/v) PVA solution.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and mix well with 50 μL of 145 mg/mL PEG solution, 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 to 10 mL of 1% (w/v) PVA solution, and ultrasonically obtain a water/oil/water emulsion. The emulsion was slowly stirred at room temperature at 200-600 r/min for 1 h, 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 have pores on the inside, with few internal pores and uneven pore dispersion.

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

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and mix well with 50 μL of 145 mg/mL PEG solution, 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 to 10 mL of 1% (w/v) PVA solution, and ultrasonically obtain a water/oil/water emulsion. The emulsion was slowly stirred at room temperature at 200-600 r/min for 3 hours, washed with water for 3 times, and freeze-dried to obtain porous polymer microspheres. The scanning electron microscopes of the microspheres are shown in Figure 1 and Figure 2. It can be seen from Figures 1 and 2 that the microspheres obtained by curing the emulsion for 3h have no pores on the surface but have pores on the inside, which 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL of water to prepare a 1% (w/v) solution of PVA in dichloromethane.

Mix 50 μL of 13.12 mg/mL α-cyclodextrin solution with 50 μL of 145 mg/mL PEG solution, add to 1 mL of 10% (w/v) PLGA solution, and sonicate at 15 W for 20 s to form a water/oil emulsion Add the above water/oil emulsion to 10 mL of 1% (w/v) PVA solution, and ultrasonically obtain a water/oil/water emulsion. The emulsion was slowly stirred at room temperature at 200-600 r/min for 12 h, 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 have pores on the inside, and the pore size 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL of water to prepare a 1% (w/v) solution of PVA in dichloromethane.

Mix 50 μL of 13.12 mg/mL α-cyclodextrin solution with 50 μL of 145 mg/mL PEG solution, add to 1 mL of 10% (w/v) PLGA solution, and sonicate at 15 W for 20 s to form a water/oil emulsion Add the above water/oil emulsion to 10 mL of 1% (w/v) PVA solution, and ultrasonically obtain a water/oil/water emulsion. 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 microscopes of the microspheres are shown in Figure 3 and Figure 4. It can be seen from Figure 3 and Figure 4 that the microspheres obtained by curing the emulsion for 24h 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL of water to prepare a 1% (w/v) solution of PVA in dichloromethane.

Mix 50 μL of 13.12 mg/mL α-cyclodextrin solution with 50 μL of 145 mg/mL PEG solution, add to 1 mL of 10% (w/v) PLGA solution, and sonicate at 15 W for 20 s to form a water/oil emulsion Add the above water/oil emulsion to 10 mL of 1% (w/v) PVA solution, and ultrasonically obtain a water/oil/water emulsion. The emulsion was slowly stirred at room temperature at 200-600 r/min for 36 h, 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL of water to prepare a 1% (w/v) solution of PVA in dichloromethane.

Mix 50 μL of 13.12 mg/mL α-cyclodextrin solution with 50 μL of 145 mg/mL PEG solution, add to 1 mL of 10% (w/v) PLGA solution, and sonicate at 15 W for 20 s to form a water/oil emulsion Add the above water/oil emulsion to 10 mL of 1% (w/v) PVA solution, and ultrasonically obtain a water/oil/water emulsion. The emulsion was slowly stirred at room temperature at 200-600 r/min for 48 h, 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL of water to prepare a 1% (w/v) PVA solution.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and mix well with 50 μL of 145 mg/mL PEG solution, 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 to 10 mL of 1% PVA solution, and ultrasonically obtain a water/oil/water emulsion. The emulsion was slowly stirred at room temperature at 200-600 r/min for 3 hours, washed with water for 3 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL of water to prepare a 1% (w/v) PVA solution.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and mix well with 50 μL of 145 mg/mL PEG solution, 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 to 10 mL of 1% (w/v) PVA solution, and ultrasonically obtain a water/oil/water emulsion. 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL of water to prepare a 1% (w/v) PVA solution.

Mix 50 μL of 13.12 mg/mL α-cyclodextrin solution with 50 μL of 145 mg/mL PEG solution in dichloromethane, add it to 1 mL of 10% (w/v) PLGA solution, and sonicate at 50 W for 20 s to form Water/oil emulsion; add the above water/oil emulsion to 10 mL of 1% (w/v) PVA solution, and ultrasonically obtain a water/oil/water emulsion. The emulsion was slowly stirred at room temperature at 200-600 r/min for 3 hours, washed with water for 3 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL of water to prepare a 1% (w/v) PVA solution.

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

Example 11 Long-term release ability test of porous microspheres with 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL 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 it with 50 μL of 13.12 mg/mL α-cyclodextrin solution, and add it to 1 mL of 10% (w/v) in the dichloromethane solution of PLGA, sonicated at 15W for 20s to form a water/oil emulsion; the above water/oil emulsion was added to 10mL of a 1% (w/v) PVA solution, and ultrasonically obtained 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 the 0th, 1st, 3rd...30th day, 2mL was taken out, centrifuged, and the fluorescence intensity of the supernatant was measured. The test results from Figure 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.

Example 12 Long-term release ability test of porous microspheres with 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL of water to prepare a 1% (w/v) PVA solution.

Take 50 μL of 13.12 mg/mL α-cyclodextrin solution and mix well with 50 μL of 145 mg/mL PEG solution, 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 to 10 mL of 1% (w/v) PVA solution, and ultrasonically obtain a water/oil/water emulsion. The emulsion was slowly stirred for 24h at room temperature at 200-600r/min, washed with water 3 times, dispersed in 5mL of water, added with 5mg FITC-BSA (isothiocyanate-labeled bovine serum albumin), stirred for 24h, centrifuged, and redispersed In water, freeze-dried to obtain microspheres with an open-pore structure. Take 10 mg of microspheres with open pore structure and disperse them in 5 mL of pH=7.4 PBS buffer, take out 2 mL of centrifuge on the 0th, 1st, 3rd, 5th, and 7th day, and measure the supernatant for fluorescence. The liquid and centrifuged solids were sonicated and then put into PBS buffer. It can be seen from the analysis results in Fig. 5 that on the 5th day, the released amount of microspheres reached 90% of the total amount.

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

Example 13

Dissolve 6 mg of sodium alginate in 300 μL of deionized water, respectively take 10 mg of the microspheres prepared in Example 2 and Example 4, and disperse them in the sodium alginate solution. The above dispersion liquid was spread evenly on the glass slide, 300 μL of 0.5 mol/L calcium chloride solution was added dropwise on it, and another glass slide was gently pressed for 30 min to obtain gel patch 1 . This whole gel patch was cut into a circular patch with a diameter of about 50dm, for 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 interleukin-loaded microspheres for later use.

Dissolve 6 mg of sodium alginate in 300 μL of deionized water, and disperse 20 mg of the interleukin-loaded microspheres in this sodium alginate solution. The dispersion was spread evenly on a glass slide, 300 μL of 0.5 mol/L calcium chloride solution was added dropwise on it, and another glass slide was gently pressed for 30 min to obtain gel patch 2 . This whole gel patch was cut into a circular patch with a diameter of about 50dm, for 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 13.12 mg of α-cyclodextrin and dissolve it in 1 mL of water to prepare a 13.12 mg/mL α-cyclodextrin solution; weigh 145 mg of PEG-2000 and dissolve it in 1 mL of water to prepare a 145 mg/mL PEG solution. Dissolve 2 g of PVA in 200 mL 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 it with 50 μL of 13.12 mg/mL α-cyclodextrin solution, and add it to 1 mL of 10% (w/v) PLGA in dichloromethane solution , ultrasonic for 20s at 15W power to form a water/oil emulsion; add the above water/oil emulsion to 10 mL of 1% (w/v) PVA solution, and ultrasonically obtain 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 melatonin-containing polymer microspheres with internal pores.

Dissolve 6 mg of sodium alginate in 300 μL of deionized water, and disperse 20 mg of melatonin-containing polymer microspheres with internal pores in the sodium alginate solution. The dispersion was evenly spread on the glass slide, and then a certain volume of 0.5 mol/L calcium chloride solution was added dropwise thereon, and another glass slide was gently pressed for 30 min to obtain gel patch 3. This whole gel patch was cut into a circular patch with a diameter of about 50dm, for use.

Example 16

Dissolve 6 mg of sodium alginate in 300 μL of deionized water, respectively take 10 mg of the microspheres containing interleukin and melatonin in Example 14 and Example 15, and disperse them all in the sodium alginate solution. The above dispersion liquid was spread evenly on the glass slide, and then a certain volume of 0.5 mol/L calcium chloride solution was added dropwise thereon, and another glass slide was gently pressed for 30 min to obtain gel patch 4. This whole gel patch was cut into a circular patch with a diameter of about 50dm, for use.

Example 17

Gel patch 1, gel patch 2, gel patch 3 and gel patch 4 in Examples 13, 14, 15, and 16 were taken to treat the mouse wounds, and the wound healing of the mice was recorded by taking pictures. And calculate its healing rate. The results are shown in Figures 6 and 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 combined action of IL-4 and melatonin, the wound healing effect of the mouse is the best. After 7 days of treating the mouse wound, the number of M2-positive macrophages was detected, and the detection results are shown in Figure 8 and Figure 9 . The results showed that IL-4 played a major role in promoting the differentiation of macrophages into M2-positive macrophages. And 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 Figure 10 and Figure 11. The results indicated that melatonin played a major role in promoting angiogenesis.

The porous polymer microspheres with different pore structures prepared by the present invention can be used for loading different kinds of biomolecules. In Example 16, these two microspheres were combined as biomolecule carriers, which could not only protect them, but also selectively release biomolecules required for 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 3 mg/mL; wherein, the cyclodextrin is γ-cyclodextrin;

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

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

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

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 to the PVA solution, and ultrasonically obtain water for 60s at 10W. /oil/water emulsion, the water/oil/water emulsion was stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain porous polymer microspheres; wherein, the mass ratio of cyclodextrin, polyethylene glycol, PLGA and PVA 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;

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

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

PVA solution is prepared by adding PVA to water, the mass concentration of PVA solution is 20mg/mL; the molar mass of 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 to 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 and PVA 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;

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

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

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

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 to the PVA solution, and ultrasonically obtain water at 50W for 30s. /oil/water emulsion, the water/oil/water emulsion was stirred at room temperature for 8 hours, washed with water, and freeze-dried to obtain porous polymer microspheres; wherein, the mass ratio of cyclodextrin, polyethylene glycol, PLGA and PVA is 0.656:7.25:100:100.

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

Claims (4)

1. the application of a porous polymer microsphere in the preparation of the gel patch for repairing skin, wherein, the preparation method of the gel patch for repairing skin is as follows: After mixing the cyclodextrin solution with 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 the water/oil/oil emulsion. Water emulsion, the water/oil/water emulsion is stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain porous polymer microspheres with closed-cell structure; After mixing the cyclodextrin solution with 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 the water/oil/oil emulsion. Water emulsion, after 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 taken and dispersed in sodium alginate solution, and then spread evenly on the glass slide, and then the calcium chloride solution was added dropwise, and another glass slide was used. The tablet is pressed to obtain a gel patch; wherein, the ratio of the closed-pore structure porous polymer microspheres, the open-pore structure porous polymer microspheres and the sodium alginate solution is 10 mg: 10 mg: 300 μL. 2. the application of a porous polymer microsphere in the preparation of the gel patch for repairing skin, wherein, the preparation method of the gel patch for repairing skin is as follows: After mixing the cyclodextrin solution with 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 the water/oil/oil emulsion. Water emulsion, the water/oil/water emulsion is stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain porous polymer microspheres with closed-cell structure; The porous polymer microspheres with closed-cell structure were dispersed in deionized water, interleukin was added, stirred for 24 h in an ice bath, and then freeze-dried to obtain microspheres loaded with interleukin; among them, the porous polymer microspheres with closed-pore structure, deionized The ratio of ionized water to interleukin is 20mg: 5mL: 10mg; Take the microspheres loaded with interleukin and disperse them in sodium alginate solution, then spread evenly on a glass slide, add calcium chloride solution dropwise, and press another glass slide for 30 minutes to obtain a gel patch; The ratio of microspheres with interleukin to sodium alginate solution was 20 mg: 300 μL. 3. the application of a porous polymer microsphere in the preparation of the gel patch for repairing skin, wherein, the preparation method of the gel patch for repairing skin is as follows: Melatonin was dissolved in the polyethylene glycol solution, mixed with the cyclodextrin solution, added to the dichloromethane solution of PLGA, sonicated to form a water/oil emulsion; the above water/oil emulsion was added to the solution. In the PVA solution, ultrasonication was used to obtain a water/oil/water emulsion, which was stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain melatonin-containing polymer microspheres with internal pores; The ratio of ethylene glycol is 10mg:7.25mg; Take the internal porous polymer microspheres containing melatonin and disperse them in the sodium alginate solution, then spread them evenly on the glass slide, add the calcium chloride solution dropwise, and press it with another glass slide to obtain a gel patch ; wherein, the ratio of the inner porous polymer microspheres containing melatonin to the sodium alginate solution is 20 mg: 300 μL. 4. the application of a porous polymer microsphere in the preparation of a gel patch for repairing skin, wherein the preparation method for the gel patch for repairing skin is as follows: After mixing the cyclodextrin solution with 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 the water/oil/oil emulsion. Water emulsion, the water/oil/water emulsion was stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain porous polymer microspheres with closed-cell structure; the porous polymer microspheres with closed-cell structure were dispersed in deionized water , adding interleukin, stirring in an ice bath for 24 hours, and then freeze-drying to obtain microspheres loaded with interleukin; wherein, the ratio of the porous polymer microspheres with closed-cell structure, deionized water and interleukin is 20mg: 5mL: 10mg; Melatonin was dissolved in the polyethylene glycol solution, mixed with the cyclodextrin solution, added to the dichloromethane solution of PLGA, sonicated to form a water/oil emulsion; the above water/oil emulsion was added to the solution. In the PVA solution, ultrasonication was used to obtain a water/oil/water emulsion, which was stirred at room temperature for 3 hours, washed with water, and freeze-dried to obtain melatonin-containing polymer microspheres with internal pores; The ratio of ethylene glycol is 10mg:7.25mg; The interleukin-loaded microspheres and the melatonin-containing internal porous polymer microspheres were dispersed in the sodium alginate solution, and then spread evenly on the glass slide, and then the calcium chloride solution was added dropwise, and another glass slide was used. The tablet was pressed to obtain a gel patch; wherein, the ratio of the interleukin-loaded microspheres, the melatonin-containing polymer microspheres with internal porous and the sodium alginate solution was 10 mg: 10 mg: 300 μL.

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