A preparation method of chitosan-polycaprolactone microspheres and the obtained product

Abstract

The present invention belongs to the technical field of surface modification, and specifically relates to a method for preparing chitosan-polycaprolactone microspheres and the resulting products. The present invention first pre-treats chitosan, and then utilizes electron beam radiation to cause the main chains of polycaprolactone and chitosan to break or the H of the side groups to fall off through primary processes such as ionization and excitation, stabilization of electrons by becoming hot electrons, neutralization of ions, and cleavage of free radicals, thereby obtaining polymer free radicals, and then inducing polymerization and graft polymerization reactions to obtain chitosan-polycaprolactone. The microspheres prepared by the present invention enhance the solubility of the material in water through surface grafting modification; on the other hand, the degradation rate of the material is regulated by utilizing different irradiation doses, and the chitosan after hydrolysis will produce weakly alkaline free NH2 — Neutralizes the acidic products of PCL breakdown, raising the pH of the surrounding tissue, thereby reducing the inflammatory incidence of its degradation process.

Description

Technical Field

[0001] The invention belongs to the technical field of surface modification, and particularly relates to a preparation method of chitosan-polycaprolactone microspheres and the obtained products. Background Art

[0002] At present, common surface modification methods include chemical reagent modification, ultraviolet radiation, plasma spraying, electron beam irradiation, etc. Among them, irradiation technology has no strict requirements on the material form and irradiation temperature, and has fast reaction, high product purity, easy control, and can be operated continuously. It is a new processing technology that saves energy and is pollution-free or less polluting. Radiation grafting refers to the process in which polymers generate active points on the macromolecular skeleton under the action of high-energy rays to initiate copolymerization of grafted monomers, or monomers are connected and polymerized on the polymer macromolecular chain to form side chains through high-energy rays.

[0003] Polycaprolactone is a synthetic polyester biopolymer material that is biodegradable and biocompatible. It is a hydrophobic polymer with a long-term degradation mechanism. Its final degradation products will be absorbed and excreted by the body. It is widely used in sustained-release drug carriers or facial fillers. However, polycaprolactone has strong crystallinity, poor hydrophilicity, slow biodegradation, and the degradation process is prone to nonspecific sterile inflammatory reactions due to low local pH values, which limits its wider application in the biomedical field.

[0004] Chitosan has a structure similar to plant cellulose and a structure similar to human collagen tissue. This dual molecular structure gives chitosan excellent biomedical properties, such as excellent compatibility with human tissue and can be decomposed and absorbed by lysozyme in the human body. It has antibacterial, anti-inflammatory, hemostatic, analgesic, and wound healing functions. This property has laid the foundation for the wide application of chitosan and its derivatives in the medical field.

[0005] In the prior art, when chitosan is used as a grafting material for polycaprolactone, its hydrophilicity and biocompatibility still need to be improved. Summary of the invention

[0006] In view of the problems in the prior art that polycaprolactone has poor hydrophilicity, slow biodegradation rate, and low local pH during degradation causing inflammation, the present invention provides a method for preparing chitosan-polycaprolactone microspheres, in which polycaprolactone is surface grafted to prepare chitosan-polycaprolactone microspheres with uniform size distribution and controllable particle size. The hydrophilicity of the modified microspheres is significantly improved, and the solution is weakly acidic and neutral after hydrolysis.

[0007] The present invention also provides chitosan-polycaprolactone microspheres prepared by the above preparation method.

[0008] The present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for preparing chitosan-polycaprolactone microspheres, comprising the following steps:

[0010] (1) treating chitosan with a gradient acetic acid solution, freeze-drying it in a vacuum after the treatment, and then adding water again to obtain a chitosan aqueous solution; adding the chitosan aqueous solution to polycaprolactone powder, dispersing it evenly, repeatedly filling it with nitrogen to deoxygenate it, and then sealing it;

[0011] (2) irradiating the sealed polycaprolactone and chitosan aqueous solution at room temperature, centrifuging after the reaction, washing with ethanol to remove unreacted monomers, and vacuum drying to obtain chitosan-polycaprolactone;

[0012] (3) dissolving chitosan-polycaprolactone in dichloromethane to obtain an oil phase solution;

[0013] (4) dissolving polyvinyl alcohol powder in water to obtain a polyvinyl alcohol aqueous solution;

[0014] (5) shearing the polyvinyl alcohol aqueous solution, then injecting the oil phase solution into the aqueous solution at a uniform speed, and continuing the shearing to obtain an emulsion;

[0015] (6) The emulsion is stirred to evaporate the dichloromethane solvent to obtain solidified microspheres. After solidification is completed, the emulsion is sieved, collected, and dried under reduced pressure to obtain chitosan-polycaprolactone microspheres.

[0016] Furthermore, in step (1), the specific process of treating with gradient acetic acid solution is as follows: first, chitosan is added to 0.5% acetic acid solution to obtain a chitosan-acetic acid solution with a concentration of 2 mg / mL, and stirred for 1.5 hours; then, acetic acid is added until the mass concentration of acetic acid is 1%, and stirring is continued for 1.5 hours; then, sodium bicarbonate is added to pH 9-9.5, and the precipitate is filtered, washed, and freeze-dried.

[0017] Furthermore, in step (1), the weight average molecular weight of the polycaprolactone is 8 kDa-40 kDa; the concentration of the chitosan solution is 0.5-2 g / mL; and the ratio of the polycaprolactone powder to the chitosan aqueous solution is 1:6.

[0018] Furthermore, in step (2), the irradiation conditions are 10 MeV / 15 KW electron accelerator, ambient temperature 25-30° C., irradiation dose 10-15 KGy, and beam velocity 1.0 m / min.

[0019] Furthermore, in step (3), the concentration of the oil phase solution is 0.05-0.25 g / mL.

[0020] Furthermore, in step (4), the mass percentage concentration of the polyvinyl alcohol aqueous solution is 0.5-1.5%.

[0021] Furthermore, in step (5), the rate at which the oil phase solution is uniformly injected into the aqueous phase solution is 2-8 mL / s; the volume ratio of the oil phase solution to the polyvinyl alcohol aqueous phase solution is 1:15-30; the shearing rate is 1000-2500 rpm, and the shearing time is 8-13 s.

[0022] Furthermore, in step (6), the stirring rate is 150-400 rpm and the stirring time is 10-15 h.

[0023] The present invention also provides chitosan-polycaprolactone microspheres prepared by the above preparation method.

[0024] The polyvinyl alcohol used in the present invention has one or two types of polyvinyl alcohol 17-88, 17-92 and 17-99.

[0025] The present invention first pre-treats chitosan, and then utilizes electron beam radiation to cause ionization and excitation, stabilization of electrons by converting electrons into hot electrons, neutralization of ions, and cleavage of free radicals, thereby causing the main chains of polycaprolactone and chitosan to break or the H of the side groups to fall off, thereby obtaining polymer free radicals (free radicals·R).·R acts as an initiator for free radical polymerization of monomers, causing polymerization and graft polymerization reactions, and obtaining chitosan-polycaprolactone. The reaction structure is shown in Figure 1 .

[0026] The beneficial effects of the present invention are:

[0027] (1) The present invention first pre-treats chitosan, and then uses the treated chitosan as a modifier to modify polycaprolactone to prepare chitosan-polycaprolactone with controllable particle size and uniform size;

[0028] (2) The chitosan-polycaprolactone microspheres prepared by the present invention enhance the solubility of the material in water by surface grafting of polycaprolactone. On the other hand, the grafting rate is changed by different irradiation doses, thereby regulating the degradation rate of the material. The hydrolyzed chitosan produces weakly alkaline free NH2 — Neutralizes the acidic products of PCL breakdown, raising the pH of the surrounding tissue, thereby reducing the inflammatory incidence of its degradation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the chitosan-polycaprolactone preparation reaction of the present invention;

[0030] Figure 2 This is a microscope image (100×) of chitosan-polycaprolactone microspheres prepared in Example 1;

[0031] Figure 3 This is a microscope image (100×) of chitosan-polycaprolactone microspheres prepared in Example 2;

[0032] Figure 4 This is a microscope image (100×) of chitosan-polycaprolactone microspheres prepared in Example 3;

[0033] Figure 5 This is a microscope image (100×) of the polycaprolactone microspheres prepared in Comparative Example 1;

[0034] Figure 6 A microscope image (100×) of polycaprolactone microspheres prepared in Comparative Example 2;

[0035] Figure 7 The particle size distribution diagram of chitosan-polycaprolactone microspheres prepared in Examples 1 to 3; wherein (a) is Example 1; (b) is Example 2; (c) is Example 3;

[0036] Figure 8 The particle size distribution diagram of chitosan-polycaprolactone microspheres prepared in Comparative Examples 1 and 2; wherein (a) is Comparative Example 1; (b) is Comparative Example 2;

[0037] Fig. 9 The diagram is a comparison of the re-dissolution of the chitosan-polycaprolactone microspheres prepared in Example 2 and the polycaprolactone microspheres prepared in Comparative Example 1; wherein (a) is Example 2; and (b) is Comparative Example 1. DETAILED DESCRIPTION

[0038] The specific embodiments of the present invention are further described below to further describe the technical solution of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] (1) Treat chitosan with a gradient acetic acid solution: first, add chitosan to a 0.5% (mass concentration) acetic acid solution to obtain a chitosan-acetic acid solution with a concentration of 2 mg / mL, and stir for 1.5 hours; then continue to add acetic acid until the mass concentration of acetic acid is 1%, and continue to stir for 1.5 hours; then add sodium bicarbonate to pH 9.1, filter and wash the precipitate, and then freeze-dry it. Then add water again to obtain a 0.5g / mL chitosan aqueous solution; add 60mL of chitosan aqueous solution to 10g of polycaprolactone (10kDa) powder, transfer it to a sealed tube, disperse it evenly, repeatedly fill it with nitrogen to deoxygenate it, and then seal it;

[0041] (2) irradiating the polycaprolactone and chitosan aqueous solution in step (1) at room temperature (irradiation conditions are 10 MeV / 15 KW electron accelerator, irradiation dose of 10 KGy, beam speed of 1.0 m / min, ambient temperature of 26° C.), centrifuging after the reaction, washing with ethanol to remove unreacted monomers, and vacuum drying to obtain chitosan-polycaprolactone;

[0042] (3) The chitosan-polycaprolactone modified in step (2) is dissolved in dichloromethane to obtain a 0.2 g / mL polycaprolactone oil phase solution;

[0043] (4) dissolving polyvinyl alcohol 17-88 powder in water to obtain a polyvinyl alcohol aqueous solution with a mass percentage concentration of 0.5%;

[0044] (5) 200 mL of the polyvinyl alcohol aqueous solution was sheared at a rate of 1200 rpm, and then 9 mL of the polycaprolactone oil phase solution was uniformly injected into the polyvinyl alcohol aqueous solution at a rate of 3 mL / s, and the shearing was continued for 10 s to obtain an emulsion;

[0045] (6) Stirring the emulsion in step (5) at a stirring rate of 210 rpm for 11 h to evaporate the dichloromethane solvent to obtain solidified microspheres, sieving through wet sieving, collecting and drying overnight, and then transferring to a vacuum drying oven for vacuum drying for 24 h to obtain surface-modified polycaprolactone microspheres.

[0046] Example 2

[0047] (1) Treat chitosan with a gradient acetic acid solution: first, add chitosan to a 0.5% acetic acid solution to obtain a chitosan-acetic acid solution with a concentration of 2 mg / mL, and stir for 1.5 hours; then continue to add acetic acid until the mass concentration of acetic acid is 1%, and continue to stir for 1.5 hours; then add sodium bicarbonate to pH 9.2, filter and wash the precipitate, and then freeze-dry it. Then add water again to obtain a 1g / mL chitosan aqueous solution; add 60mL of chitosan aqueous solution to 10g of polycaprolactone (15kDa) powder, transfer it to a sealed tube, disperse it evenly, repeatedly fill it with nitrogen to deoxygenate it, and then seal it;

[0048] (2) irradiating the polycaprolactone and chitosan aqueous solution in step (1) at room temperature (irradiation conditions are 10MeV / 15KW electron accelerator, irradiation dose of 15KGy, beam speed of 1.0 m / min, ambient temperature of 25°C), centrifuging after the reaction, washing with ethanol to remove unreacted monomers, and vacuum drying to obtain chitosan-polycaprolactone;

[0049] (3) The chitosan-polycaprolactone modified in step (2) is dissolved in dichloromethane to obtain a 0.125 g / mL polycaprolactone oil phase solution;

[0050] (4) dissolving polyvinyl alcohol 17-92 powder in water to obtain a polyvinyl alcohol aqueous solution with a mass percentage concentration of 0.75%;

[0051] (5) 200 mL of the polyvinyl alcohol aqueous solution was sheared at a rate of 1500 rpm, and then 8 mL of the polycaprolactone oil phase solution was uniformly injected into the polyvinyl alcohol aqueous solution at a rate of 4 mL / s, and the shearing was continued for 9 seconds to obtain an emulsion;

[0052] (6) Stirring the emulsion in step (5) at a stirring rate of 180 rpm for 15 h to evaporate the dichloromethane solvent to obtain solidified microspheres, sieving through wet sieving, collecting and drying overnight, and then transferring to a vacuum drying oven for vacuum drying for 24 h to obtain surface-modified polycaprolactone microspheres.

[0053] Example 3

[0054] (1) Treat chitosan with a gradient acetic acid solution: first, add chitosan to a 0.5% acetic acid solution to obtain a chitosan-acetic acid solution with a concentration of 2 mg / mL, and stir for 1.5 hours; then continue to add acetic acid until the mass concentration of acetic acid is 1%, and continue to stir for 1.5 hours; then add sodium bicarbonate to pH 9.5, filter and wash the precipitate, and then freeze-dry it. Then add water again to obtain a 1.5g / mL chitosan aqueous solution; add 60mL of chitosan aqueous solution to 10g of polycaprolactone (20kDa) powder, transfer it to a sealed tube, disperse it evenly, repeatedly fill it with nitrogen to deoxygenate it, and then seal it;

[0055] (2) irradiating the polycaprolactone and chitosan aqueous solution in step (1) at room temperature (irradiation conditions are 10MeV / 15KW electron accelerator, irradiation dose of 12KGy, beam speed of 1.0 m / min, ambient temperature of 28°C), centrifuging after reaction, washing with ethanol to remove unreacted monomers, and vacuum drying to obtain chitosan-polycaprolactone;

[0056] (3) The chitosan-polycaprolactone modified in step (2) is dissolved in dichloromethane to obtain a 0.15 g / mL polycaprolactone oil phase solution;

[0057] (4) dissolving polyvinyl alcohol 17-99 powder in water to obtain a polyvinyl alcohol aqueous solution with a mass percentage concentration of 1%;

[0058] (5) 200 mL of the polyvinyl alcohol aqueous solution was sheared at a rate of 1700 rpm, and then 10 mL of the polycaprolactone oil phase solution was uniformly injected into the polyvinyl alcohol aqueous solution at a rate of 5 mL / s, and the shearing was continued for 12 seconds to obtain an emulsion;

[0059] (6) Stirring the emulsion in step (5) at a stirring rate of 160 rpm for 13 h to evaporate the dichloromethane solvent to obtain solidified microspheres, sieving through wet sieving, collecting and drying overnight, and then transferring to a vacuum drying oven for vacuum drying for 24 h to obtain surface-modified polycaprolactone microspheres.

[0060] Comparative Example 1

[0061] (1) Dissolve 5 g of polycaprolactone (15 kDa) in 40 mL of dichloromethane to obtain a 0.125 g / mL polycaprolactone oil phase solution;

[0062] (2) dissolving polyvinyl alcohol 17-92 powder in water to obtain a polyvinyl alcohol aqueous solution with a mass percentage concentration of 0.75%;

[0063] (3) 200 mL of the polyvinyl alcohol aqueous solution was sheared at a rate of 1500 rpm, and then 8 mL of the polycaprolactone oil phase solution was uniformly injected into the polyvinyl alcohol aqueous solution at a rate of 4 mL / s, and the shearing was continued for 9 seconds to obtain an emulsion;

[0064] (4) Stirring the emulsion in step (5) at a stirring rate of 180 rpm for 15 h to evaporate the dichloromethane solvent to obtain solidified microspheres, sieving through wet sieving, collecting and drying overnight, and then transferring to a vacuum drying oven for vacuum drying for 24 h to obtain surface-modified polycaprolactone microspheres.

[0065] Comparative Example 2

[0066] (1) Treat chitosan with an acid solution: add chitosan to a 1% acetic acid solution to obtain a chitosan-acetic acid solution with a concentration of 2 mg / mL, and stir for 3 hours; then add sodium bicarbonate to pH 9.1, filter and wash the precipitate, and then freeze-dry it. Then add water again to obtain a 0.5 g / mL chitosan aqueous solution; add 60 mL of chitosan aqueous solution to 10 g of polycaprolactone (10 kDa) powder, transfer it to a sealed tube, disperse it evenly, repeatedly fill it with nitrogen to deoxygenate it, and then seal it;

[0067] The other steps are the same as in Example 1.

[0068] Effect Example

[0069] Microscope images (100×) of chitosan-polycaprolactone microspheres prepared in Examples 1 to 3 are as follows: Figure 2-Figure 4 As shown in FIG. 1 , a perfect round sphere with smooth edges can be observed under a microscope, and the size of the microspheres is evenly distributed. The microscopic image (100×) of the polycaprolactone microspheres prepared in Comparative Examples 1 and 2 is shown in FIG. Figure 5-Figure 6 As shown, under a microscope, it can be observed that the edges of the microspheres in Comparative Example 1 are smooth and perfectly round, while the edges of the microspheres in Comparative Example 2 are relatively rough, the size distribution range is relatively wide, and the uniformity is relatively poor.

[0070] The particle size distribution diagrams of chitosan-polycaprolactone microspheres prepared in Examples 1 to 3 are as follows: Figure 7 As shown in (a), (b), and (c), the main particle size distribution of the surface-modified polycaprolactone microspheres is between 20 and 71 μm, and the distribution is relatively concentrated. The particle size distribution of the polycaprolactone microspheres prepared in Comparative Examples 1 and 2 is shown in Figure 8 As shown in (a) and (b), the main particle size distribution of the microspheres is between 20 and 71 μm, but the size distribution is wide and the uniformity is poor.

[0071] The re-dissolution comparison of chitosan-polycaprolactone microspheres prepared in Example 2 and polycaprolactone microspheres prepared in Comparative Example 1 is shown in FIG. Fig. 9 Take 0.3 g of chitosan-polycaprolactone microspheres prepared in Example 2 and polycaprolactone microspheres prepared in Comparative Example 1, add 4 mL of purified water, and ultrasonicate for 1 min to obtain a suspension. Fig. 9 Among them, (a) is the suspension of Example 2 after being ultrasonicated and left to stand for 3 minutes; Fig. 9 In the figure, (b) is the suspension of comparative example 1 after ultrasonication and standing for 3 minutes. It can be clearly seen that the microspheres obtained in example 2 are more evenly dispersed in water without obvious sedimentation, and the hydrophilicity of the modified microspheres is significantly improved.

[0072] The microspheres obtained in Example 2 and Comparative Example 1 were taken, vacuum dried (40°C) for 48h to constant weight, and then soaked in a container of 0.9% sodium chloride injection. The amount of degradation solution in the container was 100ml. The container was sealed and placed in a 37°C thermostat. No intervention measures were taken during the degradation process to keep the degradation system normal. The pH value of the soaking solution was measured at 4, 8, 12, and 24 weeks of soaking time, and a pH value change table was drawn. The pH value of the microspheres in Example 2 increased from 6.52 to 7.05 within 12 weeks of soaking, and was maintained at about 7.0 within the next 24 weeks; the pH value of the microspheres in Comparative Example 1 continued to decrease with time during soaking for 12 weeks, and the pH was 3.72 at the end of 12 weeks, and then maintained at this base.

[0073] Table 1. pH value of microspheres after hydrolysis in Example 2 and Comparative Example 1

[0074]

Claims

1. A method for preparing chitosan-polycaprolactone microspheres, characterized in that: The following steps are involved: (1) treating chitosan with a gradient acetic acid solution, freeze-drying it in a vacuum after the treatment, and then adding water again to obtain a chitosan aqueous solution; adding the chitosan aqueous solution to polycaprolactone powder, dispersing it evenly, repeatedly filling it with nitrogen to deoxygenate it, and then sealing it; The weight average molecular weight of the polycaprolactone is 8kDa-40kDa; the concentration of the chitosan solution is 0.5-2g / mL; the ratio of the polycaprolactone powder to the chitosan aqueous solution is 1g:6mL; (2) irradiating the sealed polycaprolactone and chitosan aqueous solution at room temperature, centrifuging after the reaction, washing with ethanol to remove unreacted monomers, and vacuum drying to obtain chitosan-polycaprolactone; The irradiation conditions were 10MeV / 15KW electron accelerator, ambient temperature 25-30°C, irradiation dose 10-15KGy, and beam velocity 1.0 m / min; (3) dissolving chitosan-polycaprolactone in dichloromethane to obtain an oil phase solution; (4) dissolving polyvinyl alcohol powder in water to obtain a polyvinyl alcohol aqueous solution; (5) shearing the polyvinyl alcohol aqueous solution, then injecting the oil phase solution into the aqueous solution at a uniform speed, and continuing the shearing to obtain an emulsion; (6) The emulsion is stirred to evaporate the dichloromethane solvent to obtain solidified microspheres. After solidification is completed, the emulsion is sieved, collected, and dried under reduced pressure to obtain chitosan-polycaprolactone microspheres.

2. The preparation method according to claim 1, characterized in that: In step (1), the specific process of treating with gradient acetic acid solution is as follows: first, chitosan is added to 0.5% acetic acid solution to obtain a chitosan-acetic acid solution with a concentration of 2 mg / mL, and stirred for 1.5 hours; then, acetic acid is added until the mass concentration of acetic acid is 1%, and stirring is continued for 1.5 hours; then, sodium bicarbonate is added to pH 9-9.5, and the precipitate is filtered, washed, and freeze-dried.

3. The preparation method according to claim 1, characterized in that: In step (3), the concentration of the oil phase solution is 0.05-0.25 g / mL.

4. The preparation method according to claim 1, characterized in that: In step (4), the mass percentage concentration of the polyvinyl alcohol aqueous solution is 0.5-1.5%.

5. The preparation method according to claim 1, characterized in that: In step (5), the rate at which the oil phase solution is uniformly injected into the aqueous phase solution is 2-8 mL / s; the volume ratio of the oil phase solution to the polyvinyl alcohol aqueous phase solution is 1:15-30; the shearing rate is 1000-2500 rpm, and the shearing time is 8-13 s.

6. The preparation method according to claim 1, characterized in that: In step (6), the stirring rate is 150-400 rpm and the stirring time is 10-15 h.

7. Chitosan-polycaprolactone microspheres prepared by the preparation method according to any one of claims 1 to 6.

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