A method for preparing absorbable block copolymer microspheres with controllable segment structure

Abstract

The application provides a preparation method of absorbable block copolymer microspheres with controllable segment structure, which comprises the following steps: synthesizing a prepolymer by two-step prepolymerization block technology through a small molecule initiator; and in-situ initiating ring-opening polymerization of lactide by using the prepolymer to obtain a polylactic acid caprolactone block copolymer (BPLCL) with accurate controllable segment structure. Meanwhile, the BPLCL absorbable microspheres with excellent biocompatibility are prepared by using a microemulsion method, the BPLCL absorbable microspheres have simple components, uniform particle size, good cell affinity and adhesion, and have potential application value in the fields of drug loading and medical cosmetology.

Description

Technical Field

[0001] This invention relates to the field of biofunctional polymer materials technology, and in particular to a method for preparing absorbable block copolymer microspheres with controllable chain segment structure. Background Technology

[0002] As biodegradable polymers approved by the U.S. Food and Drug Administration (FDA), polylactic acid (PLA) and polycaprolactone (PCL) have good biocompatibility. After implantation, they can be absorbed by the human body and degraded into low-molecular-weight water and carbon dioxide, which are eventually excreted. PLA has high strength but poor toughness, while PCL has good toughness but low strength. These different mechanical properties limit their application range.

[0003] Copolymerization is one method to improve polymer properties. Polylactic acid (PLA) and polycaprolactone (PCL) can achieve complementary properties through copolymerization. PCL random copolymers possess unique mechanical properties and degradation rates, but the monomer molar ratio cannot be adjusted during polymerization, making it impossible to precisely control the component content of the copolymer, hindering its further application in medical device manufacturing. Block copolymerization involves the ordered polymerization arrangement of two or more monomers on the macromolecular chain. Compared to random copolymers, PLA block copolymers have a narrow molecular weight distribution, and their segment lengths and compositions can be precisely controlled while maintaining the crystallinity and thermal properties of the two copolymer components.

[0004] Microspheres are particulate dispersion systems formed by the adsorption or dispersion of drugs in a polymer matrix. They possess diverse structures and functions, making them ideal tools for tissue engineering. Microspheres have a high specific surface area, which can promote cell adhesion, proliferation, and differentiation. Their rough surface morphology ensures good cell adhesion, thereby enabling cells to grow better on the surface. Polylactic acid caprolactone copolymer microspheres have demonstrated significant application value in many fields. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing absorbable polylactic acid caprolactone block copolymer microspheres with excellent cell compatibility and precise controllable chain segment structure.

[0006] To achieve the above objectives, this invention proposes a method for preparing absorbable block copolymer microspheres with controllable segmental structure. Using a small molecule initiator, a two-step prepolymer block copolymerization technique is employed to initiate the ring-opening polymerization of ε-caprolactone monomers to obtain a polycaprolactone prepolymer. The polycaprolactone prepolymer is then used to initiate the ring-opening polymerization of lactide in situ to obtain a polylactic acid-caprolactone block copolymer. Finally, using the polylactic acid-caprolactone block copolymer as a raw material, polylactic acid-caprolactone block copolymer microspheres are prepared via a microemulsion method.

[0007] Furthermore, the preparation method specifically includes the following steps:

[0008] S1. The initiator is mixed with a medical-grade monomer and reacted to obtain a polycaprolactone prepolymer;

[0009] S2. The polycaprolactone prepolymer is mixed with medical-grade monomers and polymerized under the action of a catalyst to obtain polylactic acid caprolactone block copolymer.

[0010] S3. Purify the polylactic acid caprolactone block copolymer, dry it, and then dissolve it in a solvent to prepare the oil phase;

[0011] S4. Prepare the aqueous phase and wait for the oil phase and the aqueous phase to dissolve evenly.

[0012] S5. Under mechanical stirring, the oil phase is added to the aqueous phase, and the emulsification and evaporation are carried out to obtain microspheres;

[0013] S6. Wash and collect the microspheres, and freeze-dry them to obtain absorbable block copolymer microspheres with controllable chain segment structure.

[0014] Furthermore, the monomer mentioned in step S1 is ε-caprolactone.

[0015] Furthermore, the small molecule alcohol initiator mentioned in step S1 is one or more of ethylene glycol, benzyl alcohol, pentaerythritol, isopropanol, polyethylene glycol, etc.

[0016] Furthermore, the block copolymer described in step S1 comprises, in molar ratio: ε-caprolactone: 10-100 parts; initiator: 0.1-50 parts.

[0017] Further, in step S1, a certain amount of initiator and caprolactone are weighed into a flask, and under anhydrous and oxygen-free conditions, the mixture is heated to 100-180°C and reacted for 6-30 hours. The product is then dissolved in dichloromethane, purified by precipitation with ethanol, and the purified polymer is dried and collected to obtain the polycaprolactone prepolymer.

[0018] Furthermore, in step S2, the pharmaceutical grade monomer is L-lactide; the catalyst is stannous octoate.

[0019] Furthermore, the block copolymer mentioned in step S2 comprises, in molar ratio: L-lactide: 10-100 parts; polycaprolactone prepolymer: 10-50 parts; catalyst: 0.01-10 parts;

[0020] Furthermore, in step S2, the polycaprolactone prepolymer, medical-grade monomer, and catalyst are mixed and polymerized in an anhydrous and oxygen-free environment at a temperature of 100-180°C for 20-30 hours.

[0021] Furthermore, the oil phase mentioned in step S3 is prepared by dissolving polylactic acid caprolactone block copolymer in dichloromethane. The concentration of the oil phase is 15%-50% (w / v).

[0022] Furthermore, in step S4, the aqueous phase solution is polyvinyl alcohol (PVA). The concentration of the aqueous phase is 0.1%-1% (w / v), and the volume is 100-300 mL.

[0023] Furthermore, in step S5, the ratio of oil phase to water phase is 1:5-1:20 (v / v). Under mechanical stirring at a speed of 150-450 rpm, the oil phase is added dropwise to the water phase. After full emulsification, the cap is removed and the mixture is allowed to evaporate for 16-20 hours.

[0024] Furthermore, the polylactic acid caprolactone block microspheres dried in step S6 are subjected to vacuum freeze-drying for 24-72 hours until completely lyophilized. Compared with the prior art, the advantages of the present invention are:

[0025] 1. This invention employs a two-step prepolymer block copolymerization method, which significantly improves the yield of polylactic acid caprolactone block copolymers and effectively controls the molecular weight of the block copolymers, resulting in block copolymers with a narrower molecular weight distribution index. While ensuring high crystallinity and thermal stability, it can maintain the crystallinity and thermal properties of the copolymer components.

[0026] 2. This invention greatly improves the tensile properties of polylactic acid caprolactone block copolymer by adding a small amount of polycaprolactone prepolymer.

[0027] 3. This invention uses the traditional emulsion method to prepare absorbable polylactic acid caprolactone block copolymer microspheres. The surface of the prepared microspheres exhibits a wrinkled state similar to that of dried jujube skin, and has excellent cell adhesion. After being implanted into the human body, it can be completely degraded. The degradation products are non-toxic and can be excreted from the body through human metabolism.

[0028] 4. The microspheres prepared by this invention have a simple composition and do not introduce a third component, which is conducive to clinical approval and translational application. Attached Figure Description

[0029] Figure 1 The 1H NMR spectra of polycaprolactone prepolymers (PCL and PCL8 prepolymers) with initiator to caprolactone monomer molar ratios of 0:100 and 8:100 are shown.

[0030] Figure 2 Fourier transform infrared spectra of L-polylactic acid, polycaprolactone, random copolymer of polylactic acid and caprolactone, and block copolymer of polylactic acid and caprolactone (PLLA, PCL, PLCL5050 and BPLCL5050).

[0031] Figure 3Scanning electron microscope images of polylactic acid caprolactone random copolymer solid microspheres (a, b) and polylactic acid caprolactone block copolymer solid microspheres (c, d).

[0032] Figure 4 Laser confocal images of L929 cells cultured with polylactic-caprolactone random copolymer microspheres (PM) and polylactic-caprolactone block copolymer microspheres (BM) for 3 days. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0034] Comparative Example 1

[0035] Synthesis of polylactic-caprolactone random copolymer (PLCL)

[0036] mole ratio is n L-LA :n ε-CL Weigh medical-grade L-lactide and ε-caprolactone in a 50:50 ratio.

[0037] Based on the total mass of the two monomers, calculate and weigh a certain amount of stannous octoate, where the mass of stannous octoate is 0.1% of the total mass of the two monomers.

[0038] Place the weighed monomers and catalyst into a round-bottom flask and seal it.

[0039] A round-bottom flask containing the monomer and catalyst was magnetically stirred at room temperature, argon gas was introduced, and the temperature was raised to 140°C for 24 hours.

[0040] After the reaction was complete, the product was dissolved in dichloromethane and purified by precipitation with anhydrous ethanol. This process was repeated twice.

[0041] The purified polymer was placed in a vacuum drying oven and dried at a temperature of 40°C. After drying, the PLCL sample was collected.

[0042] Comparative Example 2:

[0043] Preparation of PLCL microspheres

[0044] Prepare 200 mL of a 0.5% polyvinyl alcohol (PVA) aqueous solution as the aqueous phase.

[0045] Weigh 3g of the self-made PLCL and dissolve it in 20mL of dichloromethane to prepare the oil phase.

[0046] After the aqueous phase and oil phase have dissolved evenly, the oil phase is added dropwise to the aqueous phase under mechanical stirring at 200 rpm. After full emulsification, the cap is removed and the mixture is allowed to evaporate for 18 hours before the mechanical stirring is turned off.

[0047] The microspheres were rinsed with ultrapure water and filtered until no foam was generated. The microspheres were collected and freeze-dried to obtain solid PLCL microspheres.

[0048] Comparative Example 3

[0049] Synthesis of polycaprolactone (PCL) with a molar ratio of ethylene glycol to caprolactone monomers of 0:100

[0050] Weigh a certain amount of medical-grade caprolactone into a round-bottom flask and add a certain amount of catalyst.

[0051] Under anhydrous and oxygen-free conditions, the system temperature was raised to 140℃ and the reaction was carried out for 24 hours.

[0052] After the reaction was complete, the product was dissolved in dichloromethane and purified by precipitation with anhydrous ethanol. This process was repeated twice.

[0053] The purified polymer was placed in a vacuum drying oven and dried at a temperature of 40°C. After drying, the PCL sample was collected.

[0054] Example 1

[0055] Synthesis of polylactic acid caprolactone block copolymer (BPLCL)

[0056] Weigh a certain amount of ethylene glycol and caprolactone into a flask according to a molar ratio of 1:100.

[0057] Under anhydrous and oxygen-free conditions, the mixture is heated to 140°C and reacted for 24 hours.

[0058] After the reaction is complete, the product is dissolved, precipitated, and purified. This process is repeated twice.

[0059] The purified polymer was placed in a vacuum drying oven and dried at a temperature of 40°C. After drying, the PCL prepolymer sample was collected.

[0060] mole ratio is n L-LA :n PCL Weigh out medical-grade L-lactide and PCL prepolymer in a 50:50 ratio.

[0061] Weigh out a certain amount of stannous octoate, the mass of which is 0.1% of the total mass of the two monomers.

[0062] The weighed raw materials were transferred to a flask, the flask was placed at room temperature and magnetically stirred, argon gas was introduced and the temperature was raised to 140°C, and the reaction was carried out for 24 hours.

[0063] After the reaction was complete, the product was dissolved in dichloromethane and purified by precipitation with ethanol. This process was repeated twice.

[0064] The purified product was placed in a vacuum drying oven and dried at a temperature of 40°C. After drying, the BPLCL sample was obtained.

[0065] Preparation of BPLCL microspheres

[0066] Weigh 3g of self-made BPLCL and dissolve it in 20mL of dichloromethane to prepare the oil phase. Prepare 200mL of 0.5% PVA aqueous solution as the aqueous phase.

[0067] After the oil phase and the aqueous phase have dissolved evenly, the oil phase is added dropwise to the aqueous phase under mechanical stirring at a speed of 200 rpm. After sufficient emulsification, the cap is removed and the mixture is allowed to evaporate for 18 hours to complete the reaction.

[0068] The microspheres were washed with ultrapure water and filtered until no foam was generated. The microspheres were then collected and freeze-dried to obtain solid BPLCL microspheres.

[0069] Experimental data characterization:

[0070] Structural characterization of prepolymer PCL

[0071] The self-made PCL was characterized using a hydrogen nuclear magnetic resonance spectrometer (Ascend 600, Bruker).

[0072] Figure 1 The 1H NMR spectra of polycaprolactone prepolymers (PCL and PCL8) with monomer molar ratios of 0:100 and 8:100 are shown. A certain mass of the two PCL prepolymers with different monomer molar ratios was placed at the bottom of an NMR tube, dissolved in deuterated chloroform (CDCL3), and the PCL prepolymers were obtained using tetramethylsilane (TMS) as an internal standard.

[0073] At a chemical shift δ = 4.27 ppm, PCL8 has an additional characteristic peak g compared to pure PCL. This peak is the characteristic peak of the methylene group on ethylene glycol when ethylene glycol is linked to the caprolactone segment (-CH2-CH2-). This indicates that ethylene glycol successfully initiated the ring-opening polymerization of the caprolactone monomer, and the PCL prepolymer was successfully prepared.

[0074] Structural characterization of polymer BPLCL

[0075] Figure 2 Fourier transform infrared spectra of polylactic acid (PLLA), polycaprolactone (PCL), random copolymer of polylactic acid and caprolactone, and block copolymer of polylactic acid and caprolactone.

[0076] The structure of the self-made BPLCL5050 was characterized using a Fourier transform infrared spectroscopy (FT-IR, Nicolet 6700, Thermo Fisher Scientific). The potassium bromide pellet method was employed for testing. BPLCL, purified and dried by melting and heating, was uniformly coated onto a potassium bromide pellet. The Fourier transform infrared spectroscopy measurements were performed in the wavelength range of 4000-500 cm⁻¹. -1 .

[0077] PLCL5050 and BPLCL5050 at 935cm -1 The absence of -CH characteristic peaks on the cyclic backbone at all locations indicates that the lactide and caprolactone monomers in the copolymer have reacted completely, and there are no residual monomers in the product. PLCL5050 and BPLCL5050 show peaks at 1725-1756 cm⁻¹. -1 The C=O stretching vibration peaks originated from the PLLA polymer chain (1756 cm⁻¹). -1 ) and PCL segment (1725cm) -1 Furthermore, the methyl (-CH3) bending vibration peak of the copolymer is located at 2866-2993 cm⁻¹. -1 The presence of a distinct triplet indicates that the random copolymer of PLCL and the block copolymer of BPLCL have been successfully synthesized.

[0078] Surface morphology analysis of BPLCL microspheres

[0079] The microstructure of the PLCL microspheres was characterized using a scanning electron microscope (SEM, S-3400N, Hitachi).

[0080] Figure 3 The surface morphology of PLCL5050 microspheres (PM) and BPLCL5050 (BM) microspheres;

[0081] Solid microspheres (PM) made from the random copolymer of polylactic acid caprolactone (PLCL5050) are nearly spherical with a smooth surface, while solid microspheres (BM) made from the block copolymer of polylactic acid caprolactone (BPLCL5050) exhibit numerous wrinkles. Despite the same preparation method, the surface morphology of the microspheres differs significantly. The main reason is that block copolymerization better preserves the segmental structure of each component of the copolymer; that is, the polylactic acid caprolactone block copolymer better retains the characteristics of both polylactic acid and polycaprolactone segments. During microsphere formation, the rigid PLLA blocks provide good support, resulting in a smooth surface. However, the PCL segments in the copolymer are flexible, and their support is weaker. When the solvent evaporates, wrinkles form on the microsphere surface.

[0082] Cell compatibility evaluation of PLCL microspheres

[0083] Figure 4Laser confocal images of L929 cells cultured with polylactic-caprolactone random copolymer microspheres (PM) and polylactic-caprolactone block copolymer microspheres (BM) for 3 days.

[0084] The cell compatibility of PLCL microspheres was characterized using laser confocal microscopy (A1R, Nikon).

[0085] PLCL microspheres and BPLCL microspheres were co-cultured with L929 cells for 3 days. The results showed that fewer cells adhered to the solid PLCL microspheres. This was mainly due to the smooth surface morphology and poor hydrophilicity of the PLCL microspheres, which hindered cell adhesion. BPLCL microspheres, prepared using the same method, exhibited a unique wrinkled texture, effectively improving cell affinity on the microsphere surface and significantly increasing cell adhesion.

[0086] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. A method for preparing absorbable block copolymer microspheres with controllable chain segment structure, characterized in that, Using a small molecule initiator, a two-step prepolymer block copolymerization technique was employed to initiate the ring-opening polymerization of ε-caprolactone monomers to obtain a polycaprolactone prepolymer. The polycaprolactone prepolymer was then used to initiate the in-situ ring-opening polymerization of lactide to obtain a polylactic acid-caprolactone block copolymer. Finally, using the prepared polylactic acid-caprolactone block copolymer as a raw material, polylactic acid-caprolactone block copolymer microspheres were prepared via a microemulsion method. The surface of the absorbable block copolymer microspheres exhibits wrinkles resembling the skin of a dried jujube. The preparation method specifically includes the following steps: S1. The initiator is mixed with a medical-grade monomer and reacted to obtain a polycaprolactone prepolymer; S2. The polycaprolactone prepolymer is mixed with a medical-grade monomer and polymerized under the action of a catalyst to obtain a polylactic acid caprolactone block copolymer; the medical-grade monomer in step S2 is L-lactide; the catalyst is stannous octoate. S3. Purify the polylactic acid caprolactone block copolymer, dry it, and then dissolve it in a solvent to prepare the oil phase; S4. Prepare the aqueous phase and wait for the oil phase and the aqueous phase to dissolve evenly. S5. Under mechanical stirring, the oil phase is added to the aqueous phase, and the emulsification and evaporation are carried out to obtain microspheres; S6. Wash and collect the microspheres, and freeze-dry them to obtain absorbable block copolymer microspheres with controllable chain segment structure. The monomer mentioned in step S1 is ε-caprolactone; In step S1, by molar fraction: ε-caprolactone: 10-100 parts; initiator: 0.1-50 parts; In step S2, the following components are included by molar percentage: L-lactide: 10-100 parts; polycaprolactone prepolymer: 10-50 parts; catalyst: 0.01-10 parts.

2. The method for preparing absorbable block copolymer microspheres with controllable chain segment structure according to claim 1, characterized in that, The small molecule initiator mentioned in step S1 is one or more of ethylene glycol, benzyl alcohol, pentaerythritol, isopropanol, and polyethylene glycol.

3. The method for preparing absorbable block copolymer microspheres with controllable chain segment structure according to claim 1, characterized in that, In step S1, a certain amount of initiator and caprolactone are weighed into a flask. Under anhydrous and oxygen-free conditions, the mixture is heated to 100-180°C and reacted for 6-30 hours. The product is then dissolved in dichloromethane, purified by precipitation with ethanol, and the purified polymer is dried and collected to obtain the polycaprolactone prepolymer.

4. The method for preparing absorbable block copolymer microspheres with controllable chain segment structure according to claim 1, characterized in that, In step S2, the polycaprolactone prepolymer, medical-grade monomer, and catalyst are mixed and polymerized in an anhydrous and oxygen-free environment at a temperature of 100-180°C for 20-30 hours.

5. The method for preparing absorbable block copolymer microspheres with controllable chain segment structure according to claim 1, characterized in that, The oil phase in step S3 is prepared by dissolving polylactic acid caprolactone block copolymer in dichloromethane, and the concentration of the oil phase is 15%-50% (w / v).

6. The method for preparing absorbable block copolymer microspheres with controllable chain segment structure according to claim 1, characterized in that, In step S4, the aqueous phase solution is polyvinyl alcohol (PVA); the concentration of the aqueous phase is 0.1%-1% (w / v), and the volume is 100-300 mL.

7. The method for preparing absorbable block copolymer microspheres with controllable chain segment structure according to claim 1, characterized in that, In step S5, the ratio of oil phase to water phase is 1:5-1:20 (v / v). Under mechanical stirring at a speed of 150-450 rpm, the oil phase is added dropwise to the water phase. After full emulsification, the cap is removed and the mixture is allowed to evaporate for 16-20 hours.

8. The method for preparing absorbable block copolymer microspheres with controllable chain segment structure according to claim 1, characterized in that, In step S6, the freeze-drying is vacuum freeze-drying for 24-72 hours until completely freeze-dried.

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