Preparation method of surface-modified degradable polymer microspheres

Abstract

The invention discloses a preparation method of surface-modified degradable polymer microspheres, which comprises the following steps: (1) preparing dry microsphere powder: putting an oil-phase solution and a water-phase solution into emulsifying equipment for mixing and emulsifying to obtain emulsion; carrying out temperature rise and pressure reduction curing on the emulsion to obtain biodegradable microsphere dry powder; (2) dissolving the modification material: dissolving the modification material containing the amino group to obtain a solution containing the modification material; (3) swelling: dispersing the biodegradable microsphere dry powder in a third solvent and stirring to swell the surface structure of the biodegradable microsphere; (4) activation: adding a carbodiimide compound and an N-hydroxyl activator into the swollen biodegradable microsphere solution for activation, so that an O-acyl isourea intermediate is formed on the surface of the microsphere, and a surface-activated microsphere precipitate is obtained; and (5) modification: mixing the microsphere precipitate with a solution containing a modification material, and stirring to obtain the surface-modified degradable polymer microsphere. Compared with the prior art, the hydrogel has good hydrophilicity and biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and more specifically to a method for preparing surface-modified biodegradable polymer microspheres. Background Technology

[0002] Microsphere products are a very important type of medical device. In recent years, with the rapid development of materials science, biodegradable materials such as polylactic acid (PLA), polyhydroxyalkanoate (PHA), and polycaprolactone (PCL) have been used more and more widely in these products. However, they also face more challenges, such as insufficient biocompatibility, long degradation cycle, and lack of functionality, which still restrict their further development.

[0003] The carbodiimide reaction is a key technique for constructing amide bonds. It involves first activating the carboxyl group of the substrate to generate an O-acylisourea intermediate, then using N-hydroxy activators such as N-hydroxysuccinimide to generate an activated ester intermediate. Finally, this activated ester is coupled with an amino-containing compound to form an amide bond, completing the reaction. This method is commonly used in biochemistry, materials science, drug development, and diagnostic techniques, offering advantages such as simplicity, speed, high efficiency, mildness, and high specificity, while also possessing strong modification capabilities.

[0004] However, in current technologies, biodegradable materials such as polylactic acid, polyhydroxyalkanoates, and polycaprolactone are difficult to modify using the methods described above. This is because these biodegradable materials have very limited carboxyl groups and are mostly hydrophobic, meaning they can only dissolve in a limited number of organic solvents. However, many materials used for functional modification may not dissolve well in these organic solvents. Therefore, due to the difference in solubility between biodegradable materials and functional modified materials, it is difficult to introduce the carbodiimide reaction under solvent conditions. Even if the carbodiimide reaction can proceed, the modified portion is difficult to distribute uniformly in the overall structure after processing due to factors such as processing technology and grafting rate.

[0005] Therefore, existing surface modification methods for biodegradable materials need to be improved. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing surface-modified biodegradable polymer microspheres with good hydrophilicity and biocompatibility, in light of the above-mentioned technical status.

[0007] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a method for preparing surface-modified biodegradable polymer microspheres, characterized by comprising the following steps: ① Preparation of microsphere dry powder: Dissolve the biodegradable material in a first solvent to obtain an oil phase solution, wherein the first solvent is an organic solvent; dissolve the surfactant in an aqueous solution to obtain an aqueous phase solution; mix and emulsify the oil phase solution and the aqueous phase solution in an emulsification device to obtain an emulsion; solidify the emulsion to obtain biodegradable microsphere dry powder; ② Dissolving the modified material: Dissolving the amino-containing modified material in the second solvent to obtain a solution containing the modified material, wherein the aforementioned biodegradable material is insoluble in the second solvent; ③ Swelling: The biodegradable microsphere dry powder is dispersed in a third solvent and stirred to cause the surface structure of the biodegradable microspheres to swell, resulting in a swollen biodegradable microsphere solution; the third solvent is a poor solvent that can initiate the swelling of the biodegradable microspheres; ④ Activation: After adding carbodiimide compound and N-hydroxy activator to the swollen biodegradable microsphere solution, an O-acyl isourea intermediate is formed on the surface of the microspheres. After centrifugation and washing, the surface-activated microsphere precipitate is obtained. ⑤ Modification: After mixing the microsphere precipitate with a solution containing the modifying material and stirring, the mixture is centrifuged, washed, and dried to obtain surface-modified biodegradable polymer microspheres.

[0008] The emulsification equipment is one of the emulsification equipment such as high-shear emulsifier, homogenizer and membrane emulsification device; the O-acyl isourea intermediate generated by activation on the surface of microspheres undergoes a coupling reaction with the amino groups on the modifying material to form amide bonds, and the modifying material is chemically fixed on the surface of microspheres to complete the modification.

[0009] Preferably, the biodegradable material is a polymer with carboxyl groups at the end; the molecular weight of the biodegradable material is 50,000 to 200,000.

[0010] Preferably, the biodegradable material is one or more of poly(L-lactic acid) PLLA, poly(racemic lactic acid) PDLLA, polycaprolactone PCL, polyhydroxyalkanoate PHA, polylactic acid-glycolic acid copolymer PLGA, and polyethylene glycol block copolymer HOOC-PEG-PLA / HOOC-PEG-PCL. The surfactant is one or more of polyvinyl alcohol, Span 20, Span 60, Span 80, Tween 20, Tween 60, Tween 80, sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, and hexadecyltrimethylammonium chloride; The first solvent is a good solvent for biodegradable materials and is one or more of dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1,4-dioxane, anisole, and acetone.

[0011] Preferably, in step ①, the mass fraction of biodegradable material in the oil phase solution is 1% to 20%; the mass fraction of surfactant in the aqueous phase solution is 0.05% to 10%; and the volume ratio of the aqueous phase solution to the oil phase solution in step ① is 1 to 30:1.

[0012] Preferably, the curing pressure of the heating and depressurization curing in step ① is 0.02-0.1 MPa, the temperature after heating is 30-45℃, and the curing time is 12-24 h; the drying is freeze drying, and the drying temperature is -20 to -80℃.

[0013] The amino-containing modifying material is a synthetic or natural polymer rich in amino groups, thereby improving biocompatibility. Preferably, the amino-containing modifying material in step ② is one or more of polyurethane, polyurethane derivatives, aminated polycaprolactone, chitosan, and gelatin. The second solvent is one or more of water, dimethyl sulfoxide, methanol, tetrahydrofuran, and n-hexane, and the mass fraction of the modifying material in the solution containing the modifying material is 5-20%.

[0014] Preferably, the third solvent in step ③ includes one or more solvents such as dimethyl sulfoxide, methanol, ethanol, propanol, ethyl acetate, and petroleum ether; the weight ratio of the biodegradable microspheres to the third solvent is 2-20%.

[0015] Preferably, the stirring rate in step ③ is 800-2000 rpm, and the stirring time is 8-12 h.

[0016] In order to generate an O-acylisourea intermediate during the carboxyl activation process, preferably, the carbodiimide compound in step ④ is one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and its hydrochloride EDC·HCl, N,N'-diisopropylcarbodiimide DIC, N,N'-dicyclohexylcarbodiimide DCC, and N,N'-di-tert-butylcarbodiimide DBC; the stirring rate for activation is 500-1000 rpm, and the stirring time is 8-12 h.

[0017] Preferably, the centrifuge speed for centrifugal washing is 3000-8000 rpm, the centrifugation time is 5-10 min, and the washing is performed with deionized water 3-4 times.

[0018] Compared with existing technologies, the advantages of this invention are as follows: It employs a poor solvent for the biodegradable microsphere material. This poor solvent can dissolve the carbodiimide compound and N-hydroxyl activator in the carbodiimide reaction, but only induces swelling of the microsphere material upon contact. Therefore, the structure of the microsphere is not destroyed during carboxyl group activation. Simultaneously, the swelling of the microsphere exposes more carboxyl groups, which helps to introduce the carbodiimide reaction to activate the carboxyl groups and generate the O-acylisourea intermediate. The generated O-acylisourea intermediate is amphiphilic, thus the microsphere has good dispersibility in aqueous solutions or organic solvents, thereby improving the contact efficiency with the modified material and achieving a high modification success rate. The modified biodegradable microsphere surface exhibits improved hydrophilicity and biocompatibility, while the negative charge of the biodegradable material is reduced. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the contact angle test for Example 1 and Comparative Example 1; Figure 2 These are scanning electron microscope images of Example 1 and Comparative Example 1; Figure 3 This is a schematic diagram of the Zeta potential test for Example 1 and Comparative Example 1. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Example 1

[0021] The method for preparing surface-modified biodegradable polymer microspheres includes the following steps: ① Preparation of microsphere dry powder: Dissolve polyhydroxybutyrate (PHB) with a molecular weight of 100,000 in dichloromethane as the first solvent to obtain 200 mL of an oil phase solution with a mass fraction of 10% of the biodegradable material; The surfactant polyvinyl alcohol was dissolved in an aqueous solution and heated to 90°C with stirring until completely dissolved to obtain an aqueous solution with a polyvinyl alcohol mass fraction of 1%. The aqueous solution and the oil solution were added to a high-shear emulsifier at a volume ratio of 5:1 and an emulsion was prepared at 1200 rpm. The emulsion was then cured under reduced pressure at 0.1 MPa and 30°C for 12 h. After curing, the emulsion was centrifuged at 5000 rpm for 10 min, washed, and dried. The centrifuge was washed three times with deionized water, and the precipitate was collected. The collected precipitate was freeze-dried at -80°C for 12 h to obtain polyhydroxybutyrate microsphere powder. ② Dissolving the modified material: Dissolve the amino-containing modified material chitosan in a 2% acetic acid weak acid aqueous solution to prepare 2000 mL of a 2% (w / w) chitosan solution, and obtain a solution containing the modified material. The aforementioned biodegradable material is insoluble in a 2% acetic acid weak acid aqueous solution. ③ Swelling: Add the dry powder of polyhydroxybutyrate microspheres to 500 ml of dimethyl sulfoxide (DMSO) solution at 800 rpm and stir for 10 h to swell the surface structure of the biodegradable microspheres and obtain a swollen biodegradable microsphere solution. ④ Activation: Add 2g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2g of N-hydroxysuccinimide sulfonate to the swollen biodegradable microsphere solution and stir at 800 rpm for 12 h to form an O-acyl isourea intermediate on the surface of the microspheres. After centrifugation and washing, the surface-activated microsphere precipitate is obtained. ⑤ Modification: The microsphere precipitate was mixed with a solution containing the modifying material and stirred at 1200 rpm for 12 h. After centrifugation to collect the precipitate, it was washed three times with 2% acetic acid aqueous solution to remove uncoupled chitosan and other organic residues on the surface. Then, it was washed three times with deionized water to restore the pH of the microspheres to neutral. The obtained microsphere precipitate was freeze-dried at -80℃ for 12 h to obtain polyhydroxybutyrate microspheres with chitosan modified on the surface. Example 2

[0022] The method for preparing surface-modified biodegradable polymer microspheres includes the following steps: ① Preparation of microsphere dry powder: Dissolve polylactic acid (PLLA), a biodegradable material with a molecular weight of 120,000, in dichloromethane as the first solvent to obtain 200 mL of an oil phase solution with a biodegradable material mass fraction of 10%; dissolve polyvinyl alcohol, a surfactant, in an aqueous solution, heat to 90℃ and stir until completely dissolved to obtain 1000 mL of an aqueous phase solution with a surfactant mass fraction of 1%; add the aqueous phase solution and the oil phase solution at a volume ratio of 5:1 to a high-shear emulsifier and prepare an emulsion at 500 rpm; The emulsion was solidified under reduced pressure at 0.1 MPa and 30℃ for 12 h. The solidified microspheres were centrifuged at 5000 rpm for 10 min, and washed three times. The precipitate was collected and freeze-dried at -80℃ for 12 h to obtain polylactic acid microsphere powder, i.e., biodegradable microsphere powder. ② Dissolving the modified material: Dissolve the amino-containing modified material chitosan in a 2% acetic acid weakly acidic aqueous solution to prepare 2000 mL of a 1% (w / w) chitosan solution, and obtain a solution containing the modified material. The aforementioned biodegradable material is insoluble in a 2% acetic acid weakly acidic aqueous solution. ③ Swelling: The biodegradable microsphere dry powder was added to 500 ml of DMSO solution at 800 rpm to swell the surface structure of the biodegradable microspheres, resulting in a swollen biodegradable microsphere solution; the stirring time was 8 h.

[0023] ④ Activation: Add 2g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2g of N-hydroxysuccinimide sulfonate to the swollen biodegradable microsphere solution and stir for 12h to complete the activation of the carboxyl group of the L-polylactic acid microspheres; centrifuge the activated microspheres at 5000rpm for 10min and repeat centrifugation and washing three times to obtain the surface-activated microsphere precipitate; ⑤ Modification: The microsphere precipitate was mixed with the chitosan solution and stirred at 1200 rpm for 9 h to complete the coupling reaction between the activated carboxyl groups on the microsphere surface and the chitosan. The precipitate was collected by centrifugation and washed three times with 2% acetic acid aqueous solution to remove uncoupled chitosan on the surface. Then, it was washed three times with deionized water to restore the pH of the microspheres to neutral. The obtained microsphere precipitate was freeze-dried at -80℃ for 24 h to obtain L-type polylactic acid microspheres with chitosan modified on the surface. Example 3

[0024] The method for preparing surface-modified biodegradable polymer microspheres includes the following steps: ① Preparation of microsphere dry powder: Dissolve a biodegradable material with a molecular weight of 80,000 in the first solvent to obtain 200 mL of an oil phase solution with a biodegradable material mass fraction of 10%; dissolve the surfactant polyvinyl alcohol in an aqueous solution, heat to 90℃ and stir until completely dissolved to obtain 1000 mL of an aqueous phase solution with a surfactant mass fraction of 1%; add the aqueous phase solution and the oil phase solution at a volume ratio of 5:1 to a high-shear emulsifier and prepare an emulsion at a speed of 700 rpm; The emulsion was cured under reduced pressure at 0.1 MPa and 30℃ for 12 h. The cured microspheres were centrifuged at 5000 rpm for 10 min, and washed repeatedly by centrifugation three times. The precipitate was collected and freeze-dried at -80℃ for 12 h to obtain polyhydroxyvalerate microsphere powder, i.e., biodegradable microsphere powder. ② Dissolving the modified material: Dissolve the amino-containing modified material chitosan in a 2% acetic acid weakly acidic aqueous solution to prepare 2000 mL of a 1% (w / w) chitosan solution, and obtain a solution containing the modified material. The aforementioned biodegradable material is insoluble in a 2% acetic acid weakly acidic aqueous solution. ③ Swelling: Add the biodegradable microsphere dry powder to 500 ml of the third solvent DMSO solution at 800 rpm and stir to swell the surface structure of the biodegradable microspheres, and obtain a swollen biodegradable microsphere solution; the stirring time is 10 h.

[0025] ④ Activation: Add 2g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2g of N-hydroxysuccinimide sulfonate to the swollen biodegradable microsphere solution and stir for 15h to form an O-acylisourea intermediate on the surface of the microspheres. Centrifuge the activated microspheres at 5000rpm for 10min, repeat centrifugation and washing three times, collect the microsphere precipitate, and obtain the surface-activated microsphere precipitate. ⑤ Modification: The microsphere precipitate was mixed with the chitosan solution and stirred at 1200 rpm for 18 h to complete the coupling reaction between the activated carboxyl groups on the microsphere surface and the chitosan. The precipitate was collected by centrifugation and washed three times with 2% acetic acid aqueous solution to remove uncoupled chitosan on the surface. Then, it was washed three times with deionized water to restore the pH of the microspheres to neutral. The obtained microsphere precipitate was freeze-dried at -80℃ for 24 h to obtain polyhydroxyvalerate microspheres with chitosan modified on the surface.

[0026] Comparative Example 1 The preparation method of biodegradable polymer microspheres is as follows: Preparation of microsphere dry powder: Dissolve polyhydroxybutyrate (PHB) with a molecular weight of 100,000 in dichloromethane as the first solvent to obtain 200 mL of an oil phase solution with a mass fraction of 10% of the biodegradable material. The surfactant polyvinyl alcohol was dissolved in an aqueous solution and heated to 90°C with stirring until completely dissolved to obtain an aqueous solution with a polyvinyl alcohol mass fraction of 1%. The aqueous solution and the oil solution were added to a high-shear emulsifier at a volume ratio of 5:1 and an emulsion was prepared at 700 rpm. The emulsion was then cured under reduced pressure at 0.1 MPa and 30°C for 12 h. After curing, the emulsion was centrifuged at 5000 rpm for 10 min, washed, and dried. The centrifuge was used to wash the emulsion three times with deionized water. The precipitate was collected and freeze-dried at -80°C for 12 h to obtain polyhydroxybutyrate microsphere powder.

[0027] Contact angle testing, scanning electron microscopy (SEM) analysis, and zeta potential testing were performed on Example 1 and Comparative Example 1.

[0028] like Figure 1 The image shows a contact angle test. Figure 1 (a) is the polyhydroxybutyrate microspheres of Comparative Example 1. Figure 1 (b) shows the chitosan-modified polyhydroxybutyrate microspheres prepared in Example 1. The results show that the unmodified polyhydroxybutyrate microspheres have strong hydrophobicity, while the polyhydroxybutyrate microspheres in Example 1 show significantly enhanced hydrophilicity after surface coupling modification with chitosan. This indicates that the polyhydroxybutyrate microspheres were successfully modified and their hydrophilicity was enhanced. Furthermore, the change in the hydrophilicity and hydrophobicity of the microsphere surface will help enhance the biocompatibility of the microspheres.

[0029] like Figure 2 The image shown is a scanning electron microscope (SEM) image. Figure 2 (a) is the polyhydroxybutyrate microspheres of Comparative Example 1. Figure 2 (b) shows the chitosan-modified polyhydroxybutyrate microspheres prepared in Example 1. The image results show that the surface morphology of the modified microspheres is intact and no structural damage occurred during the modification process. The method of the present invention can achieve the modification of microspheres under safe and reliable conditions.

[0030] like Figure 3 As shown, the Zeta potential test results show that before modification, the surface charge of the polyhydroxybutyrate microspheres is negative, with an average Zeta potential of -22.07 mV. After surface modification with chitosan, the average Zeta potential is -17.17 MV. This is because chitosan itself is a cationic polymer with a positive charge. When modified on the surface of polyhydroxybutyrate microspheres, the overall negative charge of the microspheres is reduced, thereby improving compatibility and stability.

Claims

1. A method for preparing surface-modified biodegradable polymer microspheres, characterized in that: Includes the following steps: ① Preparation of microsphere dry powder: Dissolve the biodegradable material in a first solvent to obtain an oil phase solution, wherein the first solvent is an organic solvent; dissolve the surfactant in an aqueous solution to obtain an aqueous phase solution; mix and emulsify the oil phase solution and the aqueous phase solution to obtain an emulsion; Emulsion solidification yields biodegradable microsphere powder; ② Dissolving the modified material: Dissolving the amino-containing modified material in the second solvent to obtain a solution containing the modified material, wherein the aforementioned biodegradable material is insoluble in the second solvent; ③ Swelling: The biodegradable microsphere dry powder is dispersed in a third solvent and stirred to cause the surface of the biodegradable microspheres to swell, resulting in a swollen biodegradable microsphere solution; The third solvent is a poor solvent that can induce swelling of biodegradable microspheres; ④ Activation: After adding carbodiimide compound and N-hydroxy activator to the swollen biodegradable microsphere solution, O-acyl isourea intermediate is formed on the surface of the biodegradable microspheres, resulting in surface-activated microsphere precipitate; ⑤ Modification: The microsphere precipitate is mixed with a solution containing the modifying material and stirred to obtain surface-modified biodegradable polymer microspheres.

2. The preparation method according to claim 1, characterized in that: The biodegradable material is a polymer with carboxyl groups at the end; the molecular weight of the biodegradable material is 50,000 to 200,000.

3. The preparation method according to claim 2, characterized in that: The biodegradable material is one or more of poly-L-lactic acid, polyracemic lactic acid, polycaprolactone, polyhydroxyalkanoate, polylactic acid-glycolic acid copolymer, and polyethylene glycol block copolymer. The surfactant is one or more of polyvinyl alcohol, Span 20, Span 60, Span 80, Tween 20, Tween 60, Tween 80, sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, and hexadecyltrimethylammonium chloride; The first solvent is a good solvent for biodegradable materials and is one or more of dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1,4-dioxane, anisole, and acetone.

4. The preparation method according to claim 1, characterized in that: In step ①, the mass fraction of biodegradable material in the oil phase solution is 1%~20%; the mass fraction of surfactant in the aqueous phase solution is 0.05~10%; and the volume ratio of the aqueous phase solution to the oil phase solution in step ① is 1~30:

1.

5. The preparation method according to claim 1, characterized in that: The curing process in step ① is carried out under the following conditions: pressure 0.02-0.1MPa and temperature 30-45℃ for 12-24 hours.

6. The preparation method according to claim 1, characterized in that: The amino-containing modifying material in step ② is one or more of polyurethane, polyurethane derivatives, amino-modified polycaprolactone, chitosan, and gelatin. The second solvent is one or more of water, dimethyl sulfoxide, methanol, tetrahydrofuran, and n-hexane, and the modifying material in the solution containing the modifying material has a mass fraction of 5-20%.

7. The preparation method according to claim 1, characterized in that: The third solvent in step ③ includes one or more of dimethyl sulfoxide, methanol, ethanol, propanol, ethyl acetate, and petroleum ether; the weight ratio of the biodegradable microspheres to the third solvent is 2-20%.

8. The preparation method according to claim 7, characterized in that: The stirring rate in step ③ is 800-2000 rpm, and the stirring time is 8-12 h.

9. The preparation method according to claim 1, characterized in that: The carbodiimide compound in step ④ is one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, N,N'-dicyclohexylcarbodiimide, and N,N'-di-tert-butylcarbodiimide; the activation stirring rate is 500-1000 rpm, and the stirring time is 8-12 h.

10. The preparation method according to claim 1, characterized in that: In step ①, after the emulsion is solidified, it is centrifuged, washed, and dried in sequence; the centrifugation speed is 3000-8000 rpm, and the centrifugation time is 5-10 min; the drying is freeze drying, and the drying temperature is -20~-80℃.

Images