Drug targeting and sustained release method of posthypospadias repair degradable stent

Abstract

The application relates to the technical field of biological medicine, and particularly discloses a preparation method of a post-hypospadias surgery degradable stent with drug-targeting slow release. The product composition comprises a stent base body composed of left-handed polylactic acid and polycaprolactone, drug-loaded slow-release microspheres containing polylactic acid-hydroxyacetic acid copolymer, berberine hydrochloride, sirolimus and folic acid, sodium alginate and calcium chloride for fixing the microspheres, and a hyaluronic acid coating layer on the surface of the stent. The preparation method comprises the following steps: first, preparing the stent base body containing multiple levels of pores; second, preparing the drug slow-release microspheres; third, compounding the microspheres with the stent; fourth, performing surface lubricating coating treatment after cross-linking and fixing the microspheres; and fifth, sterilizing and packaging. The product can be used after hypospadias surgery, has the functions of urethral support and drug-targeting slow release, has the advantages that the degradation rate is adapted to the tissue repair period, and the multiple levels of pores are beneficial to cell climbing; and the preparation method has the advantages that the process controllability is high, each step cooperatively guarantees the product performance, and is suitable for medical implant production.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to a drug-targeted slow-release method of a post-hypospadias surgery degradable stent. BACKGROUND

[0002] Hypospadias is a common congenital urethral deformity in clinical practice. After surgery, a stent needs to be implanted to support the urethral cavity to avoid urethral stricture caused by tissue contraction during the healing process. At the same time, the stent also needs to be used to slowly release drugs to inhibit postoperative infection and tissue fibrosis, and to create a suitable environment for urethral mucosa tissue repair. Therefore, the drug-targeted slow-release method of the post-hypospadias surgery degradable stent is to prepare a stent with biodegradability, structural stability and cell compatibility. The stent not only needs to maintain the urethral support function for a long time, but also needs to provide conditions for mucosa cell growth, and the degradation rate needs to be adapted to the postoperative tissue repair period, which is of great significance to ensure the postoperative recovery effect and reduce the incidence of complications.

[0003] In the prior art, the degradable stent for post-hypospadias surgery has obvious defects in the design of the base. Some stents are prepared by using a single polymer material, which is difficult to balance the structural stability and degradability, and is prone to problems such as premature degradation and loss of support function or slow degradation and foreign body reaction. Some stents construct pores by using a single pore-forming agent, which can only form a single size of pore structure and cannot provide sufficient space for the growth of urethral mucosa cells, resulting in difficulty in cell adhesion and proliferation, which affects the repair process of mucosa tissue and ultimately restricts the clinical application effect of the stent. SUMMARY

[0004] In order to solve the problems of the post-hypospadias surgery degradable stent in the prior art, such as difficulty in balancing structural stability and degradability due to a single polymer material, and no multi-level pores due to a single pore-forming agent, resulting in poor adaptation to the repair period and limited mucosa cell proliferation, the application provides a drug-targeted slow-release method of a post-hypospadias surgery degradable stent.

[0005] A drug-targeted slow-release method of a post-hypospadias surgery degradable stent, comprising the following steps:

[0006] S1, stent base preparation: mixing polylactic acid and polycaprolactone, then dissolving in a mixed solvent of dichloromethane and N,N-dimethylformamide to prepare a spinning solution as a shell layer; spinning with a mixed solution of polylactic acid and polycaprolactone as the shell layer and a suspension containing a pore-forming agent as the core layer, and then performing leaching treatment to form a stent base with macroscopic through holes and microscopic fiber pores;

[0007] S2, drug microspheres preparation: polylactic acid-glycolic acid copolymer is dissolved in ethyl acetate to prepare a solution with a mass concentration of 2-5% as an oil phase; berberine hydrochloride and sirolimus are mixed according to the mass ratio, and a targeting molecule is added and dissolved in a phosphate buffer solution to prepare an aqueous phase with a mass concentration of 1-3%; then under high-speed shearing emulsification, the aqueous phase is slowly added to the oil phase at a volume ratio of 1:3 to 1:5 to form a primary emulsion; then the primary emulsion is added to an aqueous solution containing 1-3% polyvinyl alcohol (PVA), and after volatilization, the microspheres are collected by centrifugation, washed with deionized water three times, and freeze-dried to obtain drug-loaded sustained-release microspheres;

[0008] S3, microsphere stent complexation: the prepared sustained-release microspheres are dispersed in a sodium alginate solution to obtain a microsphere suspension, and then the stent matrix is completely immersed in the microsphere suspension, and the microsphere suspension is perfused into the macroscopic through-pore channels of the stent matrix by negative pressure perfusion;

[0009] S4, microsphere immobilization treatment: after the stent is complexed, it is immersed in a calcium chloride solution for cross-linking and immobilization, and a gel layer is formed inside the stent to immobilize the microspheres;

[0010] S5, surface functionalization coating: after the immobilization treatment, the stent is immersed in a hyaluronic acid solution, and a moisturizing and lubricating coating is formed on the outer surface of the stent by pulling and coating and drying treatment to obtain a functionalized stent;

[0011] S6, sterilization and packaging: the functionalized stent is placed in an ethylene oxide environment, sterilized at a temperature of 45-55℃ and a relative humidity of 40-60% for 4-6h; after sterilization, the residual gas is analyzed for 12-24h, and then packaged in a sterile environment under a nitrogen atmosphere.

[0012] By adopting the technical scheme, the polylactic acid and polycaprolactone are mixed in proportion and dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide in a specific ratio, a suspension containing a pore-forming agent is used as a core layer for spinning and subsequent leaching treatment, which plays a role in considering the degradability and structural stability of the scaffold matrix, constructing a multi-level structure of macro-porous channels and micro-fiber pores, and providing suitable climbing growth space for urethral mucosa cells; by using polylactic acid-glycolic acid copolymer as a drug microsphere encapsulating material, mixing berberine hydrochloride and sirolimus in proportion and adding a targeting molecule, and then performing high-speed shearing emulsification, temperature-controlled water bath stirring and solvent evaporation, followed by centrifugal washing and drying, the drug release rate is regulated and the drug is delivered to urethral repair cells in a targeted manner, thereby meeting the treatment needs of postoperative anti-infection and anti-fibrosis; by using negative pressure perfusion technology to perfuse the slow-release microspheres dispersed in the sodium alginate solution into the macro-porous channels of the scaffold matrix, the uniform distribution and full filling of the microspheres in the scaffold are ensured, thereby avoiding excessive or insufficient local drug concentration; by cross-linking the composite microsphere scaffold by immersing it in a calcium chloride solution, the microspheres inside the scaffold are fixed, and the microspheres are prevented from falling off in a dynamic urethral environment, thereby maintaining the stability of the drug release process; by coating the scaffold with a hyaluronic acid coating and drying by pulling and drying, the scaffold is provided with moisturizing and lubricating properties, thereby improving the compatibility of the scaffold with the urethral tissue and reducing the irritation to the urethral mucosa; after ethylene oxide sterilization, the residual gas is analyzed and then packaged in a nitrogen atmosphere, which ensures the sterility of the scaffold, eliminates the safety hazards of sterilization residual gas, and maintains the storage stability of the product, thereby meeting the sterile requirements and long-term storage needs of clinical use. Through the synergistic cooperation of the technical means in each step, the scaffold support function, drug targeted release function and biological adaptation function are integrated, thereby forming a complete technical scheme that adapts to the whole process of postoperative repair of hypospadias, avoiding the performance deficiency of the product caused by the lack of a single link technology, and ensuring the integrity and practicality of the technical scheme.

[0013] Preferably, in step S1, the mass percentage of the polylactic acid is 80-95%, the mass percentage of the polycaprolactone is 5-20%, the volume ratio of dichloromethane to N,N-dimethylformamide is 7:3 to 9:1, and the solid content of the spinning solution is 5-15%; the pore-forming agent suspension is a mixed suspension of sodium chloride particles and polyethylene glycol, and the mass ratio of the two is 1:1 to 1:3.

[0014] By adopting the technical scheme, the rigidity of the left-handed polylactic acid can guarantee the early postoperative support of the stent, avoid the collapse of the urethra, and the flexibility of the polycaprolactone can buffer the friction impact in the physiological activity of the urethra. The ratio range can make the stent maintain structural stability and gradually degrade with tissue healing at different repair stages after surgery, avoiding premature failure or long-term residual foreign body reaction; the volume ratio of dichloromethane and N,N-dimethylformamide is selected to balance the solubility of the solvent to the polymer and the evaporation rate: dichloromethane can quickly take out part of the solvent to shape the fiber, and N,N-dimethylformamide can prolong the dissolution stability period to prevent the spinning solution from solidifying too early. The ratio can avoid broken filaments and fiber adhesion during spinning, ensuring the continuity of the micro-fiber structure; the control of the solid content of the spinning solution is to match the process requirements of coaxial electrospinning. Too low solid content will result in too thin and easily broken fibers, making it difficult to build a stable stent framework; too high solid content will make the spinning solution too thick, and the fibers will easily agglomerate and the porosity will decrease. The range can ensure uniform fiber diameter and provide suitable space for subsequent pore formation. The mass ratio of sodium chloride particles to polyethylene glycol is set to utilize the complementary pore-forming mechanisms of the two: the pores formed after the dissolution of sodium chloride are relatively large, which can guarantee nutrient transport and metabolic product discharge; polyethylene glycol can form small pores between the micro-fibers, assisting in adjusting the pore distribution density. The ratio can avoid uneven pore size or poor connectivity caused by single-component pore formation, ensuring that the overall pore structure of the stent meets the needs of cell adhesion and material exchange.

[0015] Preferably, in step S1, the leaching treatment is vacuum drying for 48-54h, followed by immersion in deionized water for 10-30min of ultrasonic treatment.

[0016] By adopting the technical scheme, first, 48-54h of vacuum drying is used to accelerate the volatilization of the mixed solvent of dichloromethane and N,N-dimethylformamide remaining in the stent after spinning, avoiding long-term retention of the solvent inside the stent; if the solvent remains, it may stimulate the mucosal tissue and cause an inflammatory response after subsequent implantation in the urethra. Long-term vacuum drying can also allow the fiber structure of the stent matrix to slowly set, preventing macroscopic pore collapse or micro-fiber breakage during subsequent processing, and ensuring the integrity of the overall morphology and pore structure of the stent; subsequent immersion in deionized water for 10-30min of ultrasonic treatment can produce high-frequency micro-amplitude fluctuations, promoting the penetration of deionized water into the macroscopic through-holes and micro-fiber gaps of the stent, more efficiently dissolving and removing sodium chloride particles and polyethylene glycol from the pore-forming agent; if the pore-forming agent remains, it will occupy the pore space of the stent, hindering subsequent cell adhesion and microsphere filling. The mildness of ultrasonic treatment can avoid damaging the already set stent structure, ensuring complete removal of the pore-forming agent while maintaining the connectivity of the multi-level pores, creating a suitable pore environment for subsequent urethral mucosal cell attachment and growth and uniform drug microsphere filling.

[0017] Preferably, in step S1, the macro-aperture of the stent base is 100-300 μm, the micro-fiber diameter is 0.5-2 μm, and the macro-porosity is 70-85%.

[0018] By adopting the above technical solution, the macro-aperture is in the range of 100-300 μm, which can not only adapt to uniform filling of the subsequent drug-loaded microspheres into the pores, avoid blockage of the pores by the microspheres due to too small aperture, and unable to fully contact the urethral tissue, but also prevent the stent from deforming and collapsing in the physiological activity of the urethra due to too large aperture, and at the same time, the aperture size can ensure smooth delivery of nutrients in the urethral cavity to the mucosal cells in the pores, and timely discharge of metabolic products, creating a good material exchange environment for cell growth; the micro-fiber diameter is 0.5-2 μm, the fiber surface with this diameter has a suitable specific surface area, which can provide sufficient adhesion sites for cells, and the fiber is moderately thick, which will not be too thin to cause insufficient mechanical strength and easy to break during cell proliferation, nor too thick to occupy too much pore space and squeeze the cell growth area, which can help maintain the microstructure stability and cell adaptability of the stent base; and the macro-porosity is in the high proportion range of 70-85%, which maximizes the space for cell growth and drug release on the premise of ensuring the overall mechanical support of the stent and avoiding postoperative collapse, which can not only allow mucosal cells to fully proliferate in the pores and gradually cover the surface of the stent to achieve tissue repair, but also provide sufficient channels for the diffusion of drugs released from the microspheres to the urethral tissue, avoiding drug retention, local concentration imbalance or limited cell growth space due to too low porosity.

[0019] Preferably, in step S2, the molecular weight of the polylactic acid-glycolic acid copolymer is 1000-5000; the mass ratio of berberine hydrochloride to sirolimus is 1:1 to 1:5; the targeting molecule is folic acid, and the addition amount is 0.5-2% of the total mass of the drug; and the pH value of the phosphate buffer is 7.3-7.5.

[0020] By adopting the above technical scheme, the molecular weight of the polylactic acid-glycolic acid copolymer is in the range of 1000-5000, which can form an oil phase with moderate viscosity in ethyl acetate, and can stably wrap the water phase drug during emulsification, and will not cause loose microsphere structure due to too short molecular chain, or too long molecular chain to affect the emulsification uniformity due to too high viscosity of the oil phase, thereby ensuring the regularity of the microsphere shape; the mass ratio of berberine hydrochloride to sirolimus is 1:1 to 1:5, considering the difference in diffusion characteristics of the two drugs in the body, the ratio can make the drugs orderly release with the gradual erosion of the carrier material during the degradation of the microspheres, avoid the drug efficacy gap at a certain stage due to the imbalance of the ratio, and better cover the treatment needs at different periods after the operation; the set amount of folic acid is 0.5-2%, which can embed the targeting molecules uniformly on the surface of the microspheres without interfering with the drug loading amount, ensure the specific binding efficiency with urethral repair cells, and will not cause mutual repulsion between molecules due to excessive amount, thereby affecting the stability of the microspheres; the range of the pH value of the phosphate buffer solution is 7.3-7.5, which can maintain the balance between the charged state and the solubility of the drug molecules, avoid the precipitation or structural change of the drug due to pH fluctuation, and ensure the activity of the drug during the whole preparation process of the microspheres, thereby laying a foundation for subsequent targeted release.

[0021] Preferably, in step S2, the rotation speed of the emulsification is 8000-12000 rpm, and the emulsification time is 2-5 min; the volatilization is achieved by stirring, specifically: stirring at a rotation speed of 300-600 rpm for 2-4 h in a water bath environment of 25-30℃.

[0022] By adopting the above technical scheme, the emulsification rotation speed of 8000-12000 rpm and the emulsification time of 2-5 min can uniformly disperse the water phase into small droplets and stably embed them in the oil phase through high-speed shearing to form primary emulsion with uniform particle size; if the rotation speed is insufficient or the time is too short, the water phase will not be dispersed sufficiently, which may lead to large differences in the size of the microspheres; if the rotation speed is too high or the time is too long, the drug molecule structure may be damaged or the oil phase may be prematurely demulsified due to severe shearing; the water bath environment of 25-30℃ can control the volatilization rate of ethyl acetate, avoid slow volatilization due to too low temperature, swelling and deformation of the microspheres due to long-time contact with the water phase, or uneven pore rough structure on the surface of the microspheres due to too high temperature; the stirring rotation speed of 300-600 rpm can uniformly suspend the primary emulsion in the PVA solution to prevent local accumulation and adhesion, and the stirring time of 2-4 h can ensure that the oil phase solvent is fully volatilized, so that the microspheres are solidified and formed with a dense internal structure, thereby laying a process foundation for subsequent stable drug encapsulation and sustained release.

[0023] Preferably, the particle size of the sustained-release microspheres prepared in step S2 is in the range of 10-30 μm.

[0024] By adopting the technical scheme, the particle size range of the slow-release microspheres is set to 10-30 pm, which is adapted to the pore structure of the stent base and the physiological environment of the urethra, and at the same time, the drug release efficiency is ensured; from the adaptability to the stent, the particle size is much smaller than the macro-pore diameter of the stent base of 100-300 pm, which can make the microspheres smoothly enter the pore channel during subsequent negative pressure perfusion, uniformly fill the pore gap, avoid uneven filling caused by blockage of the pore channel due to too large particle size, and also avoid leakage from the micro-fiber gap of the stent due to too small particle size, thereby ensuring the stable retention of the microspheres in the stent; from the drug release angle, the particle size of 10-30 pm can form a suitable specific surface area; if the particle size is too large, the drug inside the microspheres needs a longer time to diffuse to the surface, which is easy to cause release lag; if the particle size is too small, the specific surface area is too large, which may cause initial burst release of the drug, exceeding the required concentration in the early postoperative treatment; the particle size range can make the drug gradually degrade with the microspheres, so as to release at a stable rate, matching the drug demand at different stages after the operation. At the same time, the particle size is similar to the size of the urethral mucosa cells, which can reduce the frictional stimulation of the microspheres to the mucosa when moving in the urethra, reduce the risk of foreign body sensation and mucosal damage, and adapt to the physiological environment in the urethral cavity.

[0025] Preferably, in step S3, the mass concentration of the sodium alginate solution is 1-2%, and the viscosity is 200-800 mPa·s; the mass-volume ratio of the microspheres to sodium alginate is 100-200 mg / mL; and the negative pressure range of the negative pressure perfusion is-0.08 MPa to-0.05 MPa, and the duration is 10-20 min.

[0026] By adopting the technical scheme, the particle size range of the slow-release microspheres is set to 10-30 pm, which is adapted to the pore structure of the stent base and the physiological environment of the urethra, and at the same time, the drug release efficiency is ensured; from the adaptability to the stent, the particle size is much smaller than the macro-pore diameter of the stent base of 100-300 pm, which can make the microspheres smoothly enter the pore channel during subsequent negative pressure perfusion, uniformly fill the pore gap, avoid uneven filling caused by blockage of the pore channel due to too large particle size, and also avoid leakage from the micro-fiber gap of the stent due to too small particle size, thereby ensuring the stable retention of the microspheres in the stent; from the drug release angle, the particle size of 10-30 pm can form a suitable specific surface area; if the particle size is too large, the drug inside the microspheres needs a longer time to diffuse to the surface, which is easy to cause release lag; if the particle size is too small, the specific surface area is too large, which may cause initial burst release of the drug, exceeding the required concentration in the early postoperative treatment; the particle size range can make the drug gradually degrade with the microspheres, so as to release at a stable rate, matching the drug demand at different stages after the operation. At the same time, the particle size is similar to the size of the urethral mucosa cells, which can reduce the frictional stimulation of the microspheres to the mucosa when moving in the urethra, reduce the risk of foreign body sensation and mucosal damage, and adapt to the physiological environment in the urethral cavity.

[0027] Preferably, in step S4, the mass concentration of the calcium chloride solution is 2-4%, and the crosslinking time is 60-120s.

[0028] By adopting the above technical scheme, the crosslinking degree of sodium alginate is regulated, and then the balance between the stable fixation of microspheres and the function of the scaffold can be realized; the mass concentration range of 2-4% can provide a suitable calcium ion source for sodium alginate; when the concentration is too low, the crosslinking between sodium alginate molecules is insufficient due to the lack of calcium ions, the gel layer structure is loose, it is difficult to stably wrap the microspheres, and the microspheres are easy to fall off in the dynamic environment of the urethra; when the concentration is too high, the crosslinking reaction is too intense due to the excess of calcium ions, and the gel layer will be densified due to excessive shrinkage, which may block the drug release channel or make the scaffold locally too hard to stimulate the urethral mucosa; the crosslinking time of 60-120s is designed in cooperation with the concentration: if the time is too short, the crosslinking reaction is not fully carried out, and the strength of the gel layer is insufficient; if the time is too long, the gel layer formed may have increased brittleness due to continuous crosslinking, and is easy to crack under stress. The time range can ensure that the sodium alginate forms a gel layer with moderate strength and uniform structure in the scaffold channel, which can firmly fix the microspheres to prevent them from falling off, retain appropriate pores for slow drug release, and maintain the flexibility of the gel layer to adapt to the slight deformation in the physiological activity of the urethra.

[0029] Preferably, in step S5, the mass concentration of the hyaluronic acid solution is 0.5-1.5%, the molecular weight is 500-1500 million Da, and the drying temperature is 40-50℃.

[0030] By adopting the technical scheme, a 0.5-1.5% mass concentration range can make the hyaluronic acid form a coating with a moderate thickness on the surface of the stent, and a too low concentration can make the coating too thin, the moisturizing and lubricating effect lasts for a short time, and the coating is easily lost in the urethral friction; a too high concentration can make the coating thick, which can increase the hardness of the stent surface or cause the pores to be blocked, affecting the drug release and cell contact. The range can balance the durability of the coating and the biocompatibility of the stent; the selection of a 500,000-1,500,000 Da molecular weight is related to the moisturizing ability and viscoelasticity of the hyaluronic acid: when the molecular weight is too low, the molecular chain is short, the moisturizing and water-locking ability is weak, and the hyaluronic acid is easily degraded by the enzymes in the urethra; when the molecular weight is too high, the solution viscosity is large, the stent surface is difficult to uniformly cover during coating, and local accumulation can be caused; the range can make the hyaluronic acid form a continuous and moderately elastic film on the surface of the stent, which can not only long-term maintain the moisture of the stent surface, but also buffer the mechanical friction during the urethral peristalsis; the drying temperature of 40-50 DEG C can realize the stable formation of the coating: a too low temperature can prolong the drying time, which can cause the hyaluronic acid molecules to be unevenly aggregated, and cracks can be caused in the coating; a too high temperature can damage the molecular structure of the hyaluronic acid, so that the moisturizing activity is lost. The temperature range can retain the biological activity of the hyaluronic acid, promote the rapid evaporation of the solvent, form a uniform and dense surface coating, provide the stent with a continuous moisturizing and lubricating property, and reduce the irritation to the urethral mucosa.

[0031] In summary, the present application has the following beneficial effects:

[0032] 1. Since the present application adopts the left-handed polylactic acid and the polycaprolactone mixed as the stent base material according to a specific ratio, is dissolved in a specified ratio of dichloromethane and N,N-dimethylformamide mixed solvent, and is matched with sodium chloride particles and polyethylene glycol mixed pore-forming agents for spinning and leaching treatment, the mixed material has the degradability and structural stability, the mixed pore-forming agent can form a multi-level structure of macro-penetrating channels and micro-fiber pores, and the stent base effect of providing a climbing space for urethral mucosa cells and adapting the degradation process to the postoperative tissue repair period is obtained.

[0033] 2. In the present application, the polylactic acid-glycolic acid copolymer is preferably used as the drug microsphere encapsulating material, and is matched with the mixed drug system of the folic acid targeting molecule, the berberine hydrochloride and the sirolimus, and is emulsified, stirred and volatilized by controlling the temperature of the water bath for a specific speed and time, since the polylactic acid-glycolic acid copolymer can regulate the drug release rate, the folic acid can guide the targeted delivery of the drug to the urethral repair cells, and the mixed drugs can have a synergistic effect, and the effect of good drug targeting, stable release process, and matching the postoperative anti-infection and anti-fibrosis requirements is obtained.

[0034] 3、The method of the application uniformly perfuses the microsphere suspension into the stent base body pore by negative pressure perfusion technology, crosslinks and fixes the microspheres in a specific concentration of calcium chloride solution, and then immerses the stent in a hyaluronic acid solution to form a coating. The negative pressure perfusion ensures sufficient filling of the microspheres, the crosslinking improves the stability of the combination of the microspheres and the stent, and the hyaluronic acid coating provides moisturizing and lubricating properties, so that the effect of the microspheres not being easy to fall off, the stent surface adapting to the physiological environment of the urethra, and the overall use performance being stable is obtained. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a flow chart of a drug targeting and sustained release method of a post-hypospadias surgery degradable stent provided by the application. DETAILED DESCRIPTION

[0036] The application will be further described in detail below in combination with examples and comparative examples.

[0037] Technical ideas:

[0038] In the existing technology related to post-hypospadias surgery degradable stents, the core problem is concentrated on the material adaptability and pore structure design of the stent base body: when a single polymer material is used, if the structural stability is emphasized, the degradation rate is likely to be too slow, which does not match the postoperative tissue repair period, and if the degradability is emphasized, the support function is likely to be lost too early; at the same time, the pores constructed by a single pore-forming agent are mostly of a single size, and cannot form a multi-level structure of macroscopic through-pores and microscopic fibers, which leads to a lack of suitable climbing growth space for urethral mucosal cells, and the root cause lies in the fact that the composite ratio optimization of the material and the synergistic design of the pore-forming system are not taken into account, and it is difficult to balance the multiple requirements of support, degradation and cell adaptation.

[0039] The technical solution is aimed at the above problems and solves them synergistically through multi-dimensional technical means: first, polylactic acid and poly-caprolactone are mixed in a mass ratio of 80%-95%:5%-20% to be used as the stent base body material, and dichloromethane and N,N-dimethylformamide are mixed in a ratio of 7:3 to 9:1 to be used as the mixed solvent, so as to take into account the structural stability and controllable degradability of the material; second, sodium chloride particles and polyethylene glycol are mixed in a mass ratio of 1:1 to 1:3 to be used as the mixed pore-forming agent, and coaxial electrospinning and 48-54h vacuum drying and 10-30min deionized water ultrasonic leaching treatment are combined to construct a multi-level pore structure with a macroscopic pore size of 100-300μm and a microscopic fiber diameter of 0.5-2μm; through the synergistic design of material composition and pore-forming system, the stent support function and the postoperative repair period are matched, and sufficient climbing space is provided for mucosal cells.

[0040] Preparation Example 1

[0041] The preparation method of polylactic acid-glycolic acid copolymer is as follows:

[0042] The lactic acid monomer and the glycolic acid monomer were added into a three-necked reaction bottle with a stirring paddle, a thermometer and a vacuum interface at a molar ratio of 3:1, 0.3% of stannous octoate based on the total mass of the monomers was added as a catalyst, and nitrogen was introduced to remove air after stirring and mixing; the reaction bottle was heated to 180°C, the vacuum system was opened to maintain a vacuum degree of-0.08 MPa, and the ring-opening polymerization reaction was carried out under constant temperature and stirring, and the reaction time was controlled for 6 h; after the reaction was completed, the product was dissolved in dichloromethane, and the solution was slowly dropped into 5 times the volume of anhydrous ethanol for precipitation, and the white solid was collected by suction filtration after standing for 1.5 h; the solid was dried in a vacuum drying oven at 45°C for 10 h to remove residual solvents and unreacted monomers, and a polylactic acid-glycolic acid copolymer with a molecular weight of 1000-5000 was obtained.

[0043] The main raw materials and reagents used in the following examples and comparative examples are as follows, and the reagents not specifically described are commercially available analytical pure or higher grade products:

[0044] 1. L-polylactic acid was purchased from Shanghai Qincheng Biological Technology Co., Ltd., CAS: 26811-96-1;

[0045] 2. Polycaprolactone was purchased from Shanghai Yuan Ye Biological Technology Co., Ltd., with a product code of S26795;

[0046] 3. Dichloromethane was purchased from Shandong Jin Yu Chemical Technology Co., Ltd., CAS: 75-09-2;

[0047] 4. N,N-dimethylformamide was purchased from Yangzhou Phoenix Island Chemical Co., Ltd., with product codes 007;

[0048] 5. Polyethylene glycol was purchased from Shanghai Yuan Ye Biological Technology Co., Ltd., with a product code of V32174;

[0049] 6. Berberine hydrochloride was purchased from Chengdu Pusai Biological Technology Co., Ltd., with a product code of PS0118;

[0050] 7. Sirolimus was purchased from Jiangsu Congzhong Chemical Co., Ltd., with a product code of 11663;

[0051] 8. Folic acid was purchased from Shenzhen Fis Biological Technology Co., Ltd., with a product code of F25708;

[0052] 9. Polyvinyl alcohol was purchased from Shanghai Yuan Ye Biological Technology Co., Ltd., with a product code of S30196;

[0053] 10. Sodium alginate was purchased from Shanghai Yuan Ye Biological Technology Co., Ltd., with a product code of S11053;

[0054] 11. Hyaluronic acid was purchased from Shaanxi Angxi Biological Technology Co., Ltd., CAS: 9004-61-9;

[0055] 12Ethyl acetate was purchased from Shandong Langcheng Chemical Co., Ltd., CAS: 141-78-6.

[0056] Example 1

[0057] The embodiment of the present application provides a drug targeting and sustained release method of a posthypospadias surgery degradable stent, comprising the following steps:

[0058] S1, stent base preparation: mixing polylactic acid and polycaprolactone, and then dissolving in a mixed solvent of dichloromethane and N,N-dimethylformamide to prepare a spinning solution as a shell layer; spinning with a mixed solution of polylactic acid and polycaprolactone as the shell layer and a suspension containing a porogen as the core layer, and then performing leaching treatment to form a stent base with macroscopic through pores and microscopic fiber pores;

[0059] In the formula, the mass percentage of polylactic acid is 87.5%, the mass percentage of polycaprolactone is 12.5%, the volume ratio of dichloromethane to N,N-dimethylformamide is 8:2, the mass ratio of sodium chloride particles to polyethylene glycol is 1:2, and the solid content of the spinning solution is 10%; the porogen suspension is a mixed suspension of sodium chloride particles and polyethylene glycol, and the mass ratio of the two is 1:2.

[0060] In the formula, the leaching treatment is vacuum drying for 51h, followed by ultrasonic treatment in deionized water for 20min.

[0061] In the formula, the macroscopic pore size of the stent base is 200μm, the microscopic fiber diameter is 1μm, and the macroscopic porosity is 77%.

[0062] S2, drug microsphere preparation: dissolving polylactic acid-glycolic acid copolymer in ethyl acetate to prepare a solution with a mass concentration of 3% as an oil phase; mixing berberine hydrochloride and sirolimus according to a mass ratio, adding a targeting molecule, and dissolving in a phosphate buffer to prepare an aqueous phase with a mass concentration of 2%; then under high-speed shearing emulsification, the aqueous phase is slowly added to the oil phase at a volume ratio of 1:4 to form a primary emulsion; then the primary emulsion is added to an aqueous solution containing 2% polyvinyl alcohol (PVA), and after volatilization, the microspheres are collected by centrifugation, washed with deionized water for three times, and then freeze-dried to obtain drug-loaded sustained release microspheres;

[0063] In the formula, the molecular weight of polylactic acid-glycolic acid copolymer is 3000, the mass ratio of berberine hydrochloride to sirolimus is 1:3, the targeting molecule is folic acid, and the addition amount of the targeting molecule accounts for 1.25% of the total mass of the drugs; the pH value of the phosphate buffer is 7.4.

[0064] In the formula, the rotation speed of emulsification is 10000rpm, and the emulsification time is 3.5min; volatilization is achieved by stirring, specifically: stirring at a rotation speed of 450rpm for 3h in a water bath environment at 27.5℃;

[0065] The particle size of the slow-release microspheres prepared in step S2 ranges from 20 μm;

[0066] S3, microsphere scaffold complexing: dispersing the prepared slow-release microspheres in a sodium alginate solution to obtain a microsphere suspension, and then completely immersing the scaffold matrix in the microsphere suspension, and filling the microsphere suspension into the macroscopic through-pore channels of the scaffold matrix by negative pressure perfusion;

[0067] The mass concentration of the sodium alginate solution is 1%, and the viscosity is 500 mPa・s; the mass-volume ratio of the microspheres to the sodium alginate is 150 mg / mL; the negative pressure of the negative pressure perfusion is-0.065 MPa, and the duration is 15 min;

[0068] S4, microsphere immobilization treatment: immersing the complexed scaffold in a calcium chloride solution for cross-linking and immobilization, and forming a gel layer inside the scaffold to immobilize the microspheres;

[0069] The mass concentration of the calcium chloride solution is 3%, and the cross-linking time is 90 s;

[0070] S5, surface functionalization coating: immersing the immobilized scaffold in a hyaluronic acid solution, and performing pull-coating and drying treatment to form a moisturizing and lubricating coating on the outer surface of the scaffold, thereby obtaining a functionalized scaffold;

[0071] The mass concentration of the hyaluronic acid solution is 1%, the molecular weight is 1 million Da, and the drying temperature is 45℃;

[0072] S6, sterilization and packaging: placing the functionalized scaffold in an ethylene oxide environment, sterilizing for 5 h under the condition of a temperature of 50℃ and a relative humidity of 50%, and then analyzing the residual gas for 18 h after sterilization, and then packaging in a nitrogen atmosphere in a sterile environment.

[0073] Example 2

[0074] The application embodiment provides a drug-targeting slow-release method of a postoperative degradable scaffold for hypospadias, which comprises the following steps:

[0075] S1, scaffold matrix preparation: mixing polylactic acid and polycaprolactone, and then dissolving them in a mixed solvent of dichloromethane and N,N-dimethylformamide to prepare a spinning solution as a shell layer; spinning with a mixed solution of polylactic acid and polycaprolactone as the shell layer and a suspension containing a porogen as the core layer, and then performing leaching treatment to form a scaffold matrix with macroscopic through-pore channels and microscopic fiber pores;

[0076] The left-handed polylactic acid has a mass percentage of 80%, and the polycaprolactone has a mass percentage of 5%; the volume ratio of dichloromethane to N,N-dimethylformamide is 7:3; the mass ratio of sodium chloride particles to polyethylene glycol is 1:1, and the solid content of the spinning solution is 5%; the porogen suspension is a mixed suspension of sodium chloride particles and polyethylene glycol, and the mass ratio of the two is 1:1;

[0077] The leaching treatment is vacuum drying for 48 hours, followed by ultrasonic treatment in deionized water for 10 minutes.

[0078] The macro-pore diameter of the scaffold matrix is 100 microns, the micro-fiber diameter is 0.5 microns, and the macro-porosity is 70%.

[0079] S2, drug microsphere preparation: dissolve polylactic acid-glycolic acid copolymer in ethyl acetate to prepare a solution with a mass concentration of 2% as an oil phase; mix hydrochloric acid berberine and sirolimus according to a mass ratio, and add a targeting molecule, and dissolve them in a phosphate buffer solution to prepare an aqueous phase with a mass concentration of 1-3%; then under high-speed shearing emulsification, the aqueous phase is slowly added to the oil phase at a volume ratio of 1:3 to form a primary emulsion; then the primary emulsion is added to an aqueous solution containing 1% polyvinyl alcohol (PVA), and after volatilization, the microspheres are collected by centrifugation, washed with deionized water three times, and then freeze-dried to obtain drug-loaded sustained-release microspheres;

[0080] The molecular weight of the polylactic acid-glycolic acid copolymer is 1000; the mass ratio of hydrochloric acid berberine to sirolimus is 1:1; the targeting molecule is folic acid, and the addition amount is 0.5% of the total mass of the drug; and the pH value of the phosphate buffer solution is 7.3.

[0081] The rotation speed of emulsification is 8000 rpm, and the emulsification time is 2 minutes; volatilization is achieved by stirring, specifically: stirring at a rotation speed of 300 rpm for 2 hours in a water bath environment at 25°C;

[0082] The particle size range of the sustained-release microspheres prepared in step S2 is 10 microns.

[0083] S3, microsphere scaffold compounding: disperse the prepared sustained-release microspheres in a sodium alginate solution to obtain a microsphere suspension, and then immerse the scaffold matrix completely in the microsphere suspension, and fill the microsphere suspension into the macro-penetrating channels of the scaffold matrix through negative pressure perfusion;

[0084] The mass concentration of the sodium alginate solution is 1%, and the viscosity is 200 mPa·s; the mass-volume ratio of the microspheres to sodium alginate is 100 mg / mL; and the negative pressure of the negative pressure perfusion is -0.08 MPa, and the duration is 10 minutes.

[0085] S4, microsphere immobilization treatment: after compounding, the stent is immersed in a calcium chloride solution for cross-linking fixation, a gel layer is formed inside the stent to fix the microspheres;

[0086] The mass concentration of the calcium chloride solution is 2%, and the cross-linking time is 60 s.

[0087] S5, surface functionalization coating: after the immobilization treatment, the stent is immersed in a hyaluronic acid solution, and after pulling and coating and drying treatment, a moisturizing and lubricating coating is formed on the outer surface of the stent to obtain a functionalized stent.

[0088] The mass concentration of the hyaluronic acid solution is 0.5%, the molecular weight is 500,000 Da, and the drying temperature is 40°C.

[0089] S6, sterilization and packaging: the functionalized stent is placed in an ethylene oxide environment, sterilized at a temperature of 45°C and a relative humidity of 40% for 4 h; after sterilization, the residual gas is analyzed for 12 h, and then packaged in a sterile environment under a nitrogen atmosphere.

[0090] Example 3

[0091] The embodiment of the present application provides a drug targeting and sustained release method of a postoperative degradable stent for hypospadias, which comprises the following steps:

[0092] S1, stent base preparation: mixing polylactic acid and polycaprolactone, then dissolving in a mixed solvent of dichloromethane and N,N-dimethylformamide to prepare a spinning solution as a shell layer; spinning with a mixed solution of polylactic acid and polycaprolactone as a shell layer and a suspension containing a porogen as a core layer, and then leaching to form a stent base with macroscopic through pores and microscopic fiber pores;

[0093] The mass percentage of polylactic acid is 95%, the mass percentage of polycaprolactone is 20%, the volume ratio of dichloromethane to N,N-dimethylformamide is 9:1, the mass ratio of sodium chloride particles to polyethylene glycol is 1:3, the solid content of the spinning solution is 15%, and the porogen suspension is a mixed suspension of sodium chloride particles and polyethylene glycol with a mass ratio of 1:3.

[0094] The leaching treatment is vacuum drying for 54 h, followed by immersion in deionized water and ultrasonic treatment for 30 min.

[0095] The macroscopic pore size of the stent base is 300 μm, the microscopic fiber diameter is 2 μm, and the macroscopic porosity is 85%.

[0096] S2, drug microsphere preparation: polylactic acid-glycolic acid copolymer is dissolved in ethyl acetate to prepare a solution with a mass concentration of 5% as an oil phase; berberine hydrochloride and sirolimus are mixed according to a mass ratio, and a targeting molecule is added and dissolved in a phosphate buffer solution to prepare an aqueous phase with a mass concentration of 3%; then the aqueous phase is slowly added to the oil phase at a volume ratio of 1:5 under high-speed shearing emulsification to form a primary emulsion; then the primary emulsion is added to an aqueous solution containing 3% polyvinyl alcohol (PVA), and after volatilization, the microspheres are collected by centrifugation, washed with deionized water three times, and freeze-dried to obtain drug-loaded sustained-release microspheres;

[0097] In the formula, the molecular weight of the polylactic acid-glycolic acid copolymer is 5000; the mass ratio of berberine hydrochloride to sirolimus is 1:5; the targeting molecule is folic acid, and the addition amount is 2% of the total mass of the drug; and the pH value of the phosphate buffer solution is 7.5;

[0098] In the formula, the rotation speed of emulsification is 12000 rpm, and the emulsification time is 5 min; volatilization is achieved by stirring, specifically: stirring at a rotation speed of 600 rpm for 4 h in a water bath environment at 30°C;

[0099] In the formula, the particle size range of the sustained-release microspheres prepared in step S2 is 30 μm;

[0100] S3, microsphere scaffold complexing: dispersing the prepared sustained-release microspheres in a sodium alginate solution to obtain a microsphere suspension, and then completely immersing the scaffold matrix in the microsphere suspension, and filling the microsphere suspension into the macroscopic through-pore channels of the scaffold matrix by negative pressure perfusion;

[0101] In the formula, the mass concentration of the sodium alginate solution is 2%, and the viscosity is 800 mPa·s; the mass-volume ratio of the microspheres to the sodium alginate is 200 mg / mL; and the negative pressure of the negative pressure perfusion is -0.05 MPa, and the duration is 20 min;

[0102] S4, microsphere immobilization treatment: immersing the complexed scaffold in a calcium chloride solution for cross-linking and immobilization, and forming a gel layer inside the scaffold to immobilize the microspheres;

[0103] In the formula, the mass concentration of the calcium chloride solution is 4%, and the cross-linking time is 120 s;

[0104] S5, surface functionalization coating: immersing the immobilized scaffold in a hyaluronic acid solution, and performing pull-coating and drying treatment to form a moisturizing and lubricating coating on the outer surface of the scaffold to obtain a functionalized scaffold;

[0105] In the formula, the mass concentration of the hyaluronic acid solution is 1.5%, the molecular weight is 1.5 million Da, and the drying temperature is 50°C;

[0106] S6, sterilization and packaging: the functionalized scaffold is placed in an ethylene oxide environment, sterilized for 6h at a temperature of 55℃ and a relative humidity of 60%, and then analyzed for residual gas for 24h after sterilization, and then packaged in a sterile environment under a nitrogen atmosphere.

[0107] Comparative Example 1

[0108] The difference between this comparative example and Example 1 is only that in step S2, no folic acid targeting molecule is added, and only berberine hydrochloride and sirolimus are dissolved together in the phosphate buffer.

[0109] Comparative Example 2

[0110] The difference between this comparative example and Example 1 is only that in step S2, only berberine hydrochloride is used as the drug, and no sirolimus is added, and the total mass of the drug is equal to the total mass of the two drugs in Example 1.

[0111] Comparative Example 3

[0112] The difference between this comparative example and Example 1 is only that in step S1, a single pore-forming agent is used, and only sodium chloride particles are used to prepare the core layer suspension, without adding polyethylene glycol, and the mass of sodium chloride is equal to the total mass of sodium chloride particles and polyethylene glycol in Example 1.

[0113] Comparative Example 4

[0114] The difference between this comparative example and Example 1 is only that in step S2, instead of using polylactic acid-glycolic acid copolymer, an equal amount of gelatin is used as the encapsulating material for the microspheres; specifically, gelatin is dissolved in warm water at 50℃ to prepare a solution with a mass concentration of 3% as the water phase, and the subsequent emulsification and solidification steps are the same as in Example 1.

[0115] Comparative Example 5

[0116] The difference between this comparative example and Example 1 is only that step S4, the microsphere immobilization process, is completely omitted; after step S3, the negative pressure infusion, step S5, the surface functionalization coating, is performed directly.

[0117] Comparative Example 6

[0118] The difference between this comparative example and Example 1 is only that in step S4, instead of using calcium chloride solution for crosslinking, the next coating process is performed directly after negative pressure infusion.

[0119] I. Take three scaffolds each from Examples 1-3 and Comparative Examples 1-6, for a total of nine groups, with three parallel samples per group. Place each sample in a sealed centrifuge tube containing 50 mL of pH 7.4 phosphate buffer (which simulates the urethral fluid environment). Place the centrifuge tubes in a 37°C constant-temperature water bath and shake at 100 rpm. At 0.5 h, 1 h, 4 h, 1 d, 3 d, 7 d, 14 d, 21 d, and 28 d, respectively, aspirate 2 mL of supernatant and simultaneously add 2 mL of fresh phosphate buffer to maintain a constant system volume. Use high-performance liquid chromatography (HPLC) to detect the concentrations of the two drugs in the supernatant. The detection wavelengths are set to 265 nm for berberine hydrochloride and 278 nm for sirolimus. Calculate the cumulative release rate, initial burst release rate, and total release rate at each time point for each group of samples. Separately, take one sample from each group and co-culture it with rabbit urethral repair cells cultured in vitro in a 24-well plate. Adjust the cell concentration to 1 × 10⁻⁶ cells / well. 5 Cells / mL were incubated in 24-well plates at 37°C with 5% CO2 for 24 hours. Cell lysis was then collected, and the supernatant was collected by centrifugation at 12000 rpm for 10 min. The intracellular concentrations of the two drugs were detected using high-performance liquid chromatography (HPLC), and the drug targeting enrichment efficiency was calculated using the following formula:

[0120] .

[0121] II. Take three stents each from Examples 1-3 and Comparative Examples 1-6, weigh them precisely using an electronic balance with an accuracy of 0.1 mg and record the weight as m0. Fix the stents in a custom-made dynamic device simulating urethral peristalsis. Inject 100 mL of pH 7.4 phosphate buffer into the device and maintain a constant temperature of 37°C. Simulate physiological urethral peristalsis at a frequency of 5 times / min and an amplitude of ±2 mm. After 1 day, 3 days, 7 days, 14 days, and 21 days of dynamic simulation, remove the stents, blot off the surface liquid with filter paper, weigh them again and record the weight as m1. Calculate the stent mass change rate using the following formula:

[0122] ;

[0123] Simultaneously, the buffer solution within the device was collected and centrifuged at 3000 rpm for 10 min. The number of precipitated microspheres was counted under a 10x objective microscope and recorded as n1. Using the initial total amount of microspheres loaded onto the scaffold (n0), and pre-calculated using the microsphere suspension concentration and perfusion volume, the microsphere detachment rate was calculated. The calculation formula is as follows: Additionally, 2 mL of buffer solution was aspirated at each of the above time points, and the drug concentration was detected using high-performance liquid chromatography (HPLC). The drug release rate (in μg / h) for each sample group at each time point was calculated, and the fluctuation range of the release rate was statistically analyzed. The calculation formula is as follows:

[0124] .

[0125] 3. Take three scaffolds each from Examples 1-3 and Comparative Examples 1-6, sterilize them with ethylene oxide, cut them to 1cm × 1cm size, and place them in 24-well plates, one scaffold per well. Pre-treat them by soaking them in DMEM / F12 medium containing 10% fetal bovine serum for 24 hours. Take rabbit urethral mucosal epithelial cells in the logarithmic growth phase and adjust the cell concentration to 5 × 10⁻⁶ cells using the above medium. 4 Cells were cultured at a concentration of 1 mL / mL, with 1 mL of cell suspension added to each well. The 24-well plate was incubated at 37°C in a 5% CO2 incubator. After 1, 3, 5, and 7 days of culture, the culture medium was removed from the wells, and the cells were gently washed three times with PBS. 200 μL of MTT solution (5 mg / mL) was added to each well, and the cells were cultured for another 4 hours. The MTT solution was then removed, and 150 μL of dimethyl sulfoxide was added to each well. The plates were shaken for 10 minutes to dissolve the purple crystals. The absorbance (OD) of each well was measured at 490 nm using a microplate reader. The relative cell proliferation rate was calculated using the following formula:

[0126]

[0127] The blank control group consisted of cell culture wells without scaffolds. After 7 days of culture, the cells on the scaffolds were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 10 min, stained with FITC-labeled phalloidin for 20 min, and stained with DAPI for 5 min. The cell morphology was observed under a fluorescence microscope with a 20x objective to determine whether the cells were polygonal and tightly connected. Five fields of view were randomly selected to count the number of cells and observe the adhesion density.

[0128] The results of in vitro drug release and targeted enrichment performance measurements are shown in Table 1.

[0129] Table 1:

[0130]

[0131] The results of the stent stability and microsphere detachment rate determination under dynamic simulation environment are shown in Table 2:

[0132] Table 2:

[0133]

[0134] Cell compatibility and cell adhesion growth are shown in Table 3.

[0135] Table 3:

[0136]

[0137] Summarize:

[0138] It can be seen from Examples 1-3 and Comparative Example 1 in combination with Table 1 that the initial burst release rate and 28-day cumulative release rate of Comparative Example 1 are basically close to those of Example 1, but the drug targeting enrichment efficiency is much lower than that of Example 1 and is also significantly lower than those of Examples 2 and 3, which indicates that the folate targeting molecule has a direct impact on the targeting enrichment ability of the drug, and after the folate is absent, the drug is difficult to be delivered to the urethral repair cells in a targeted manner, resulting in a significant reduction in the intracellular drug enrichment amount, while the initial release speed of the drug and the long-term cumulative release total amount are basically not affected.

[0139] It can be seen from Examples 1-3 and Comparative Example 2 in combination with Table 3 that the relative proliferation rate and cell adhesion density of Comparative Example 2 are much lower than those of Examples 1, 2 and 3, while the drug release related parameters of Comparative Example 2 in Table 1 are close to those of Example 1, which indicates that sirolimus plays an important role in the proliferation and adhesion growth of urethral mucosa cells, and after the sirolimus is absent, the cells are difficult to proliferate normally and adhere to the surface of the stent, while the drug release process is basically not affected.

[0140] It can be seen from Examples 1-3 and Comparative Example 3 in combination with Tables 1 and 2 that the initial burst release rate of Comparative Example 3 is higher than those of Examples 1, 2 and 3; the mass change rate, microsphere shedding rate and drug release rate fluctuation amplitude of Comparative Example 3 are all higher than those of Example 1, which indicates that the mixed porogen composed of sodium chloride and polyethylene glycol plays an important role in the structural stability of the stent matrix, and the stent matrix prepared by a single porogen has poor uniformity of pore structure, resulting in an accelerated initial release speed of the drug, a decreased stability of the microsphere and stent combination, a greater mass change of the stent in a dynamic environment, and also affecting the consistency of the drug release.

[0141] It can be seen from Examples 1-3 and Comparative Example 4 in combination with Tables 1 and 2 that the initial burst release rate of Comparative Example 4 is much higher than those of Examples 1-3, and the 28-day cumulative release rate is also slightly higher than those of Examples 1-3; the mass change rate, microsphere shedding rate and drug release rate fluctuation amplitude of Comparative Example 4 are all much higher than those of Examples 1-3, which indicates that the stability of polylactic acid-glycolic acid copolymer as a microsphere encapsulation material is better than that of gelatin, and the microsphere prepared by gelatin is easily degraded or swelled in a simulated body fluid environment, resulting in a serious initial burst release of the drug, a large amount of microsphere shedding, a dramatic change in the mass of the stent, and also making it difficult to maintain the stability of the drug release rate.

[0142] It can be seen from the combination of Examples 1-3 and Comparative Example 5 and in combination with Tables 1 and 2 that the initial burst rate of Comparative Example 5 is higher than that of Examples 1-3; the mass change rate, microsphere shedding rate and drug release rate fluctuation amplitude of Comparative Example 5 are all higher than those of Examples 1-3, which shows that the microsphere immobilization treatment is crucial for the combination of the microspheres and the stent, and after omitting this step, the microspheres cannot be stably fixed inside the stent and are prone to shedding in the dynamic simulation environment, which not only leads to an increase in the mass change of the stent, but also makes the drug release out of control, showing an accelerated initial burst and a larger release rate fluctuation.

[0143] It can be seen from the combination of Examples 1-3 and Comparative Example 6 and in combination with Tables 1 and 2 that the initial burst rate of Comparative Example 6 is higher than that of Examples 1-3; the mass change rate, microsphere shedding rate and drug release rate fluctuation amplitude of Comparative Example 6 are all higher than those of Examples 1-3, which shows that the calcium chloride solution crosslinking is a key link of the microsphere immobilization treatment, and after lacking crosslinking, the sodium alginate cannot form a stable gel layer to fix the microspheres, which leads to the microspheres being prone to shedding from the stent pores in the dynamic environment, thereby causing problems of an increased mass change of the stent, an accelerated initial burst of the drug and a larger release rate fluctuation.

[0144] The specific embodiments are merely an explanation of the present application, which is not a limitation of the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the Patent Law.

Claims

1. A method for preparing a biodegradable stent with targeted drug release after hypospadias surgery, characterized in that: Includes the following steps: S1. Scaffold matrix preparation: Polylactic acid (PLA) and polycaprolactone (PVC) are mixed and then dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide to prepare a spinning solution as the shell layer; using the mixed solution of PLA and PVC as the shell layer and a mixed suspension of sodium chloride particles and polyethylene glycol as the pore-forming agent core layer for spinning, and then leaching treatment to form a scaffold matrix with macroscopic through-pores and micro-fiber pores; S2. Preparation of drug microspheres: Polylactic acid-glycolic acid copolymer was dissolved in ethyl acetate to prepare a 2-5% (w / w) solution as the oil phase; berberine hydrochloride and sirolimus were mixed in a certain mass ratio, and folic acid was added as a targeting molecule, and dissolved together in phosphate buffer to prepare a 1-3% (w / w) aqueous phase. Then, under high-speed shear emulsification, the aqueous phase was slowly added to the oil phase at a volume ratio of 1:3 to 1:5 to form a pre-emulsion; subsequently, the pre-emulsion was added to an aqueous solution containing 1-3% polyvinyl alcohol (PVA), and after evaporation, the microspheres were collected by centrifugation, washed three times with deionized water, and then freeze-dried to obtain drug-loaded sustained-release microspheres; S3, Microsphere scaffold composite: The prepared sustained-release microspheres are dispersed in sodium alginate solution to obtain a microsphere suspension. Then, the scaffold matrix is ​​completely immersed in the microsphere suspension, and the microsphere suspension is injected into the macroscopic through-holes of the scaffold matrix through negative pressure perfusion. S4. Microsphere immobilization treatment: The composite scaffold is immersed in calcium chloride solution for cross-linking and immobilization, forming a gel layer inside the scaffold to fix the microspheres; S5. Surface functional coating: The immobilized scaffold is immersed in hyaluronic acid solution, and after lifting coating and drying, a moisturizing and lubricating coating is formed on the outer surface of the scaffold to obtain a functional scaffold. S6. Sterilization and Encapsulation: The functionalized scaffold is placed in an ethylene oxide environment and sterilized for 4-6 hours at a temperature of 45-55℃ and a relative humidity of 40-60%. After sterilization, residual gases are released for 12-24 hours, and then the scaffold is encapsulated in a nitrogen atmosphere under sterile conditions.

2. The method for preparing a biodegradable stent with targeted drug release after hypospadias surgery according to claim 1, characterized in that: In step S1, the mass percentage of L-polylactic acid is 80-95%, the mass percentage of polycaprolactone is 5-20%, the volume ratio of dichloromethane to N,N-dimethylformamide is 7:3 to 9:1, and the solid content of the spinning solution is 5-15%; the mass ratio of sodium chloride particles to polyethylene glycol in the mixed suspension is 1:1 to 1:

3.

3. The method for preparing a biodegradable stent with targeted drug release after hypospadias surgery according to claim 1, characterized in that: In step S1, the leaching process is as follows: vacuum drying for 48-54 hours, followed by immersion in deionized water and ultrasonic treatment for 10-30 minutes.

4. The method for preparing a biodegradable stent with targeted drug release after hypospadias surgery according to claim 1, characterized in that: Step S1: The scaffold matrix has a macroscopic pore size of 100-300 μm, a microfiber diameter of 0.5-2 μm, and a macroscopic porosity of 70-85%.

5. The method for preparing a biodegradable stent with targeted drug release after hypospadias surgery according to claim 1, characterized in that: In step S2, the molecular weight of the polylactic acid-glycolic acid copolymer is 1000-5000; the mass ratio of berberine hydrochloride to sirolimus is 1:1 to 1:5; the amount of folic acid added accounts for 0.5-2% of the total mass of the drug; and the pH value of the phosphate buffer is 7.3-7.

5.

6. The method for preparing a biodegradable stent with targeted drug release after hypospadias surgery according to claim 1, characterized in that: In step S2, the emulsification speed is 8000-12000 rpm and the emulsification time is 2-5 min; the evaporation is achieved by stirring, specifically: stirring at 300-600 rpm for 2-4 h in a water bath environment at 25-30℃.

7. The method for preparing a biodegradable stent with targeted drug release after hypospadias surgery according to claim 1, characterized in that: The sustained-release microspheres obtained in step S2 have a particle size range of 10-30 μm.

8. The method for preparing a biodegradable stent with targeted drug release after hypospadias surgery according to claim 1, characterized in that: In step S3, the sodium alginate solution has a mass concentration of 1-2% and a viscosity of 200-800 mPa·s; the mass-to-volume ratio of microspheres to sodium alginate is 100-200 mg / mL; the negative pressure range of the negative pressure perfusion is -0.08 MPa to -0.05 MPa, and the duration is 10-20 min.

9. The method for preparing a biodegradable stent with targeted drug release after hypospadias surgery according to claim 1, characterized in that: In step S4, the mass concentration of the calcium chloride solution is 2-4%, and the crosslinking time is 60-120 s.

10. The method for preparing a biodegradable stent with targeted drug release after hypospadias surgery according to claim 1, characterized in that: In step S5, the hyaluronic acid solution has a mass concentration of 0.5-1.5%, a molecular weight of 500,000-1,500,000 Da, and a drying temperature of 40-50℃.

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