技术领域technical field
本发明涉及一种PLGA纤维-微球双载药复合支架，还涉及这种PLGA纤维-微球双载药复合支架的制备方法。The invention relates to a PLGA fiber-microsphere double drug-loaded composite support, and also relates to a preparation method of the PLGA fiber-microsphere double drug-loaded composite support.
背景技术Background technique
药物控制释放系统的应用具有巨大的商业潜力，其在美国的年销售额超过10亿美元。随着药用可降解高分子材料的广泛研究及应用，控释体系日益增多。可降解高分子凝胶，微球及纳米纤维都可以作为药物载体。近年来，具有生物相容性及可降解的高分子生物材料作为新型组织工程支架材料为组织的修复及重建带来了希望。但是，现有的组织修复及重建的支架功能单一，难以满足组织修复及重建中的复杂需要。The application of controlled drug release systems has enormous commercial potential, with annual sales in the United States exceeding $1 billion. With the extensive research and application of pharmaceutical degradable polymer materials, controlled release systems are increasing. Degradable polymer gels, microspheres and nanofibers can all be used as drug carriers. In recent years, biocompatible and degradable polymer biomaterials have brought hope for tissue repair and reconstruction as new tissue engineering scaffold materials. However, the existing scaffolds for tissue repair and reconstruction have single functions, and are difficult to meet the complex needs in tissue repair and reconstruction.
静电纺丝技术是一种制备连续纤维结构的简单高效的方法。由静电纺丝法制备的组织工程支架具有较大的比表面积，其三维多孔结构类似于细胞外基质，利于细胞的粘附，增殖和生长。同时纤维支架也可以作为药物或某些活性因子的缓释载体，满足组织工程的要求。静电喷射技术是一种有前景的制备微纳米微球的技术，其制备的微球可以作为负载药物的良好载体，微纳米颗粒分散性良好，粒径可控。组织工程构建的三要素包括种子细胞，支架材料和生物活性因子。其中生物活性因子在组织修复和重建过程中是不可或缺的，但存在稳定性差，半衰期短和易被体内酶作用而失活等特点。利用静电纺丝及静电喷射技术制备具有药物缓控释功能的纤维-微球复合支架，以期促进组织修复及重建，满足组织修复和重建中的复杂要求。Electrospinning technology is a simple and efficient method to prepare continuous fiber structures. The tissue engineering scaffold prepared by electrospinning has a large specific surface area, and its three-dimensional porous structure is similar to the extracellular matrix, which is beneficial to the adhesion, proliferation and growth of cells. At the same time, the fibrous scaffold can also be used as a slow-release carrier for drugs or certain active factors to meet the requirements of tissue engineering. Electrostatic spraying technology is a promising technology for preparing micro-nano microspheres. The prepared microspheres can be used as a good carrier for loading drugs. The micro-nano particles have good dispersion and controllable particle size. The three elements of tissue engineering construction include seed cells, scaffold materials and bioactive factors. Among them, bioactive factors are indispensable in the process of tissue repair and reconstruction, but they have the characteristics of poor stability, short half-life and easy inactivation by enzymes in vivo. Using electrospinning and electrostatic spraying technology to prepare fiber-microsphere composite scaffold with slow and controlled drug release function, in order to promote tissue repair and reconstruction, and meet the complex requirements in tissue repair and reconstruction.
文献1“专利公开号是CN 104448356 A”的中国发明专利，公开了一种空白PLGA微球及其制备方法。该空白微球由水-油-水乳胶法制备，PLGA溶于二氯甲烷中，添加乳化剂聚乙烯醇，磁力搅拌去除二氯甲烷，离心收集微球体，洗涤冻干得到PLGA微球，可在组织工程中充当良好的细胞载体。但单一的PLGA微球难以满足组织修复及重建中复杂的需求；而且制备方法中添加了乳化剂，后续操作中要通过搅拌作用除去有机溶剂，析出洗涤冻干得到微球，过程稍显复杂，限制了此方法的应用。Document 1, the Chinese invention patent of "Patent Publication No. CN 104448356 A", discloses a blank PLGA microsphere and its preparation method. The blank microspheres are prepared by the water-oil-water emulsion method, PLGA is dissolved in dichloromethane, the emulsifier polyvinyl alcohol is added, the dichloromethane is removed by magnetic stirring, the microspheres are collected by centrifugation, and the PLGA microspheres are obtained by washing and freeze-drying. Acts as a good cell carrier in tissue engineering. However, a single PLGA microsphere is difficult to meet the complex needs of tissue repair and reconstruction; moreover, an emulsifier is added in the preparation method. In the subsequent operation, the organic solvent must be removed by stirring, and the microspheres are obtained by precipitation, washing and freeze-drying. The process is a bit complicated. The application of this method is limited.
文献2“专利公开号是CN 103585635 A”的中国发明专利，公开了一种可保持蛋白、多肽类药物活性的缓释聚乳酸类微球及其制备方法。由聚乳酸类生物可降解材料PLGA或聚乳酸(PLA)，生物玻璃和主药经静电喷射工艺制备。首先配制三种不同溶液，反应制备溶胶，烘制得凝胶再干燥，球磨筛分，煅烧得到纳米生物活性玻璃。在此过程中需要的化学试剂较多，且步骤繁琐，耗时，对工艺要求较高；之后将生物活性玻璃与蛋白、多肽类药物混合于聚乳酸类的有机溶剂中，进行静电喷射，所得液球要进一步在液氮中冻结，之后于低温环境中去除有机溶剂，沉积固化微球，该过程需要液氮装置及低温环境；整个制备过程步骤繁琐复杂，制备条件也较为苛刻。Document 2, the Chinese invention patent of "Patent Publication No. CN 103585635 A", discloses a slow-release polylactic acid microsphere capable of maintaining the activity of protein and polypeptide drugs and its preparation method. It is prepared by electrostatic spraying process from polylactic acid biodegradable material PLGA or polylactic acid (PLA), bioglass and main drug. Firstly, three different solutions are prepared, reacted to prepare sol, dried to obtain gel, ball milled and screened, and calcined to obtain nano bioactive glass. In this process, many chemical reagents are required, and the steps are cumbersome, time-consuming, and have high requirements for the process; after that, the bioactive glass, protein and polypeptide drugs are mixed in the organic solvent of polylactic acid, and electrostatic spraying is carried out. The liquid sphere needs to be further frozen in liquid nitrogen, and then the organic solvent is removed in a low-temperature environment to deposit and solidify the microspheres. This process requires a liquid nitrogen device and a low-temperature environment; the entire preparation process is cumbersome and complicated, and the preparation conditions are relatively harsh.
PLGA(poly(lactic-co-glycolic acid))是已经美国FDA认证的可临床应用的可降解高分子，其无毒性，无抗原性，生物相容性好，力学性能优良，降解速率可控，被广泛用于药物或基因控释以及皮肤、血管、肌腱、软骨及骨等组织工程方面。他汀类药物(辛伐他汀、洛伐他汀、普伐他汀、美伐他汀、氟伐他汀、阿托伐他汀、洛伐他汀等)具有调节血脂的作用，近来发现他汀类药物对骨组织的修复及重建具有促进作用。但是，他汀类药物经口服后对肝脏具有高度选择性，被人体吸收后大部分随胆汁排出，在骨组织及周围组织中含量很低，因此直接将他汀类药物作用于修复部位有利于药物的高效利用。生物活性因子在组织修复及重建中具有不可或缺的作用，生长因子(血管内皮化生长因子(VEGF)、转化生长因子(TGF-β)、表皮生长因子(EGF)等)，活性蛋白(骨形态发生蛋白(BMP-2)、牛血清白蛋白(BSA)等)和DNA质粒已被成功负载于组织工程支架中以促进组织修复及重建。但这类生物活性因子多是水溶性的，在有机溶剂体系中难以保持活性，并且在体内半衰期短，需要较高的剂量，价格昂贵，因此研究其在体内的药物递送体系，达到控缓释，能够提高其促进组织修复及重建的效果。然而，如何将生物活性因子与支架材料良好结合以达到理想的控释效果是目前亟待解决的问题。PLGA (poly(lactic-co-glycolic acid)) is a clinically applicable biodegradable polymer certified by the US FDA. It has no toxicity, no antigenicity, good biocompatibility, excellent mechanical properties, and controllable degradation rate. It is widely used in drug or gene controlled release and tissue engineering such as skin, blood vessel, tendon, cartilage and bone. Statins (simvastatin, lovastatin, pravastatin, mevastatin, fluvastatin, atorvastatin, lovastatin, etc.) have the effect of regulating blood lipids, recently found that statins can repair bone tissue and reconstruction has a stimulating effect. However, statins are highly selective to the liver after oral administration, and most of them are excreted with bile after being absorbed by the human body, and the content in bone tissue and surrounding tissues is very low. Efficient use of. Bioactive factors play an indispensable role in tissue repair and reconstruction, growth factors (vascular endothelial growth factor (VEGF), transforming growth factor (TGF-β), epidermal growth factor (EGF), etc.), active proteins (bone Morphogenetic proteins (BMP-2, bovine serum albumin (BSA), etc.) and DNA plasmids have been successfully loaded in tissue engineering scaffolds to promote tissue repair and reconstruction. However, most of these biologically active factors are water-soluble, difficult to maintain activity in organic solvent systems, and have a short half-life in vivo, requiring high doses and expensive prices. Therefore, research on their drug delivery systems in vivo to achieve controlled and sustained release , can improve its effect of promoting tissue repair and reconstruction. However, how to combine bioactive factors with scaffold materials to achieve the ideal controlled release effect is an urgent problem to be solved.
发明内容Contents of the invention
为了克服现有制备PLGA纤维-微球双载药复合支架的方法复杂的不足，本发明提供一种PLGA纤维-微球双载药复合支架及其制备方法。该方法采用PLGA纤维与他汀类药物制备含有他汀类药物的静电纺丝液，再采用PLGA纤维与生物活性因子按质量比为20～600000:1配制均一的电喷溶液，最后将制备的静电纺丝液和电喷溶液同时电纺和电喷，将收集的支架材料置于真空干燥箱中干燥24～48h，得到他汀类药物和生物活性因子双负载的PLGA纤维-微球复合支架材料。由于采用乳液电喷的方法，形成一种油包水型乳液，避免药物与有机溶剂直接接触引起失活，方法简单。同时由于制备过程未使用表面活性剂和乳化剂，具有更高的生物安全性。In order to overcome the disadvantages of the complicated method for preparing the PLGA fiber-microsphere dual drug-loaded composite scaffold, the present invention provides a PLGA fiber-microsphere dual drug-loaded composite scaffold and a preparation method thereof. The method uses PLGA fibers and statins to prepare an electrospinning solution containing statins, then uses PLGA fibers and bioactive factors to prepare a uniform electrospray solution at a mass ratio of 20 to 600,000:1, and finally the prepared electrospinning solution The silk liquid and the electrospray solution were electrospun and electrosprayed at the same time, and the collected scaffold material was dried in a vacuum oven for 24-48 hours to obtain a PLGA fiber-microsphere composite scaffold material double-loaded with statins and bioactive factors. Due to the adoption of the emulsion electrospray method, a water-in-oil emulsion is formed to avoid inactivation caused by direct contact between the drug and the organic solvent, and the method is simple. At the same time, because no surfactant and emulsifier are used in the preparation process, it has higher biological safety.
本发明解决其技术问题所采用的技术方案是：一种PLGA纤维-微球双载药复合支架，其特点是：由电纺PLGA纤维及电喷微球构成，他汀类药物和生物活性因子分别被包埋于PLGA纤维和电喷微球中。其中PLGA纤维直径为200～700nm，载药量为12.77±0.12％；微球粒径为4～8μm，载药量为23.14±0.75％，包封率为92.55±3.01％。The technical scheme adopted by the present invention to solve the technical problem is: a PLGA fiber-microsphere dual drug-loaded composite scaffold, which is characterized in that it is composed of electrospun PLGA fibers and electrosprayed microspheres, and statins and bioactive factors are respectively Embedded in PLGA fibers and electrosprayed microspheres. Among them, the diameter of PLGA fiber is 200-700nm, the drug-loading capacity is 12.77±0.12%; the microsphere particle size is 4-8 μm, the drug-loading capacity is 23.14±0.75%, and the encapsulation efficiency is 92.55±3.01%.
一种上述PLGA纤维-微球双载药复合支架的制备方法，其特点是包括以下步骤：A preparation method of the above-mentioned PLGA fiber-microsphere dual drug-loaded composite scaffold is characterized in that it comprises the following steps:
步骤一、将电纺PLGA纤维与他汀类药物按质量比为4～20:1溶于有机溶剂中，配制混合溶液，密封，0～4℃静置12h，磁力搅拌4～6h，涡旋10～15min，超声30～60s消除气泡，得到均一的含有他汀类药物的静电纺丝液。Step 1. Dissolve electrospun PLGA fibers and statins in an organic solvent at a mass ratio of 4-20:1, prepare a mixed solution, seal it, let it stand at 0-4°C for 12 hours, stir it magnetically for 4-6 hours, and vortex for 10 ～15min, ultrasonication for 30～60s to eliminate air bubbles, and obtain a uniform electrospinning solution containing statins.
步骤二、将电纺PLGA纤维溶解于二氯甲烷中，密封，0～4℃静置12h，磁力搅拌4～6h，涡旋10～15min，再超声30～60s消除气泡，得到PLGA溶液；将生物活性因子溶解于PBS缓冲溶液中；按所述质量比20～600000:1将含生物活性因子的PBS溶液加入到PLGA溶液中，在冰浴条件下，超声乳化，得到均一的含有生物活性因子的电喷溶液。Step 2. Dissolve the electrospun PLGA fiber in dichloromethane, seal it, let it stand at 0-4°C for 12 hours, stir it magnetically for 4-6 hours, vortex for 10-15 minutes, and then sonicate for 30-60 seconds to eliminate air bubbles to obtain a PLGA solution; The biologically active factor is dissolved in PBS buffer solution; the PBS solution containing the biologically active factor is added to the PLGA solution according to the mass ratio of 20 to 600000:1, and is ultrasonically emulsified under ice bath conditions to obtain a uniform biologically active factor. electrospray solution.
步骤三、将步骤一制备的静电纺丝液和步骤二制备的电喷溶液同时电纺和电喷，形成支架材料，将收集的支架材料置于真空干燥箱中干燥24～48h，得到他汀类药物和生物活性因子双负载的PLGA纤维-微球复合支架材料。Step 3: Electrospinning and electrospraying the electrospinning liquid prepared in step 1 and the electrospraying solution prepared in step 2 at the same time to form a scaffold material, and drying the collected scaffold material in a vacuum drying oven for 24 to 48 hours to obtain statins PLGA fiber-microsphere composite scaffold material loaded with drugs and bioactive factors.
所述电纺PLGA纤维用PLGA的相对分子质量为100～120kDa。The relative molecular weight of the PLGA used for the electrospun PLGA fiber is 100-120 kDa.
所述电纺PLGA纤维用PLGA中乳酸:乙醇酸的摩尔百分比为75:25。The molar percentage of lactic acid in the PLGA used for the electrospinning PLGA fiber: glycolic acid is 75:25.
所述电喷微球用PLGA的相对分子质量为50～60kDa。The relative molecular mass of the PLGA used for the electrospray microspheres is 50-60 kDa.
所述电喷微球用PLGA中乳酸:乙醇酸的摩尔百分比为50:50。The molar percentage of lactic acid in the PLGA for the electrospray microspheres: glycolic acid is 50:50.
所述他汀类药物是辛伐他汀、洛伐他汀、普伐他汀、氟伐他汀、美伐他汀或者阿托伐他汀的任一种。The statin drug is any one of simvastatin, lovastatin, pravastatin, fluvastatin, mevastatin or atorvastatin.
所述生物活性因子是VEGF、TGF-β、EGF、BMP-2、BSA或者DNA质粒的任一种。The bioactive factor is any one of VEGF, TGF-β, EGF, BMP-2, BSA or DNA plasmid.
所述有机溶剂为二氯甲烷与N，N-二甲基甲酰胺的混合溶剂。The organic solvent is a mixed solvent of dichloromethane and N,N-dimethylformamide.
本发明的有益效果是：该方法采用PLGA纤维与他汀类药物制备含有他汀类药物的静电纺丝液，再采用PLGA纤维与生物活性因子按质量比为20～600000:1配制均一的电喷溶液，最后将制备的静电纺丝液和电喷溶液同时电纺和电喷，将收集的支架材料置于真空干燥箱中干燥24～48h，得到他汀类药物和生物活性因子双负载的PLGA纤维-微球复合支架材料。由于采用乳液电喷的方法，形成一种油包水型乳液，避免药物与有机溶剂直接接触引起失活，方法简单。同时由于制备过程未使用表面活性剂和乳化剂，具有更高的生物安全性。The beneficial effects of the present invention are: the method adopts PLGA fibers and statins to prepare electrospinning solution containing statins, and then uses PLGA fibers and bioactive factors to prepare a uniform electrospray solution at a mass ratio of 20 to 600,000:1 Finally, the prepared electrospinning solution and electrospray solution were electrospun and electrosprayed at the same time, and the collected scaffold materials were placed in a vacuum drying oven to dry for 24-48 hours to obtain PLGA fibers double-loaded with statins and bioactive factors- Microsphere composite scaffold material. Due to the adoption of the emulsion electrospray method, a water-in-oil emulsion is formed to avoid inactivation caused by direct contact between the drug and the organic solvent, and the method is simple. At the same time, because no surfactant and emulsifier are used in the preparation process, it has higher biological safety.
以下结合附图和实施例详细说明本发明。The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.
附图说明Description of drawings
图1是本发明PLGA纤维-微球双载药复合支架制备过程示意图。Fig. 1 is a schematic diagram of the preparation process of the PLGA fiber-microsphere dual drug-loaded composite scaffold of the present invention.
具体实施方式detailed description
以下实施例参照图1。The following examples refer to FIG. 1 .
实施例1：将PLGA(100kDa，75/25)称取200mg，与50mg辛伐他汀溶于二氯甲烷与N，N-二甲基甲酰胺的混合溶剂(2:1，v/v)中，密封，4℃静置一夜，磁力搅拌4h，涡旋10min，超声30s消除气泡，得到均一的含有辛伐他汀的静电纺丝溶液；将PLGA(50kDa，50/50)与BMP-2配制均一的电喷溶液。具体做法如下：将PLGA(50kDa，50/50)60mg溶解于二氯甲烷中，密封，4℃静置一夜，磁力搅拌4h，涡旋10min，再超声45s消除气泡，得到PLGA溶液；将BMP-2 200ug溶解于4mLPBS缓冲溶液中；将100μL含BMP-2的PBS溶液加入到PLGA溶液中，在冰浴条件下，超声5s，停止5s，再超声5s，重复10次，乳化得到均一的含有BMP-2的电喷溶液；将上述得到的电纺溶液和电喷溶液同时电纺和电喷。高压静电纺丝电压为15kV，纺丝液流速为1.0mL/h，纺丝溶液浓度为0.15g/mL；高压静电喷射电压为12kV，电喷液流速为0.4mL/h，电喷液浓度为0.08g/mL。所收集的支架材料置于真空干燥箱中干燥24h，得到辛伐他汀和BMP-2双负载的PLGA纤维-微球复合支架材料。Example 1: 200 mg of PLGA (100 kDa, 75/25) was weighed, and 50 mg of simvastatin was dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide (2:1, v/v) , sealed, let stand overnight at 4°C, magnetically stirred for 4h, vortexed for 10min, and ultrasonicated for 30s to eliminate air bubbles to obtain a uniform electrospinning solution containing simvastatin; PLGA (50kDa, 50/50) and BMP-2 were prepared uniformly electrospray solution. The specific method is as follows: dissolve 60 mg of PLGA (50kDa, 50/50) in dichloromethane, seal it, let it stand overnight at 4°C, stir it with magnetic force for 4 hours, vortex for 10 minutes, and then sonicate for 45 seconds to eliminate bubbles to obtain a PLGA solution; 2 200ug was dissolved in 4mL PBS buffer solution; 100μL of BMP-2-containing PBS solution was added to the PLGA solution, under ice bath conditions, ultrasonicated for 5s, stopped for 5s, and then ultrasonicated for 5s, repeated 10 times, emulsified to obtain a uniform BMP-containing -2 electrospray solution; electrospinning and electrospraying the above obtained electrospinning solution and electrospray solution simultaneously. The voltage of high-voltage electrospinning is 15kV, the flow rate of spinning solution is 1.0mL/h, and the concentration of spinning solution is 0.15g/mL; 0.08g/mL. The collected scaffold materials were dried in a vacuum oven for 24 hours to obtain a PLGA fiber-microsphere composite scaffold material double-loaded with simvastatin and BMP-2.
实施例2：将PLGA(100kDa，75/25)称取200mg，与20mg洛伐他汀溶于二氯甲烷与N，N-二甲基甲酰胺的混合溶剂(2:1，v/v)中，密封，0℃静置一夜，磁力搅拌5.5h，涡旋15min，超声45s消除气泡，得到均一的含有洛伐他汀的静电纺丝液；将PLGA(55kDa，50/50)与EGF配制均一的电喷溶液。具体做法如下：将PLGA(55kDa，50/50)60mg溶解于二氯甲烷中，密封，0℃静置一夜，磁力搅拌5h，涡旋10min，再超声60s消除气泡，得到PLGA溶液；将EGF 10ug溶解于10mLPBS缓冲溶液中；将200μL含EGF的PBS溶液加入PLGA溶液中，在冰浴条件下，超声5s，停止5s，再超声5s，重复8次，乳化得到均一的含有EGF的电喷溶液；将上述得到的电纺溶液和电喷溶液同时电纺和电喷。高压静电纺丝电压为12kV，纺丝液流速为0.8mL/h，纺丝溶液浓度为0.10g/mL；高压静电喷射电压为15kV，电喷液流速为0.6mL/h，电喷液浓度为0.10g/mL。所收集的支架材料置于真空干燥箱中干燥36h，得到洛伐他汀和EGF双负载的PLGA纤维-微球复合支架材料。Example 2: 200 mg of PLGA (100 kDa, 75/25) was weighed, and 20 mg of lovastatin was dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide (2:1, v/v) , sealed, left standing overnight at 0°C, magnetically stirred for 5.5h, vortexed for 15min, and ultrasonicated for 45s to eliminate air bubbles to obtain a uniform electrospinning solution containing lovastatin; PLGA (55kDa, 50/50) and EGF were prepared into a uniform EFI solution. The specific method is as follows: Dissolve 60 mg of PLGA (55kDa, 50/50) in dichloromethane, seal it, let it stand overnight at 0°C, stir magnetically for 5 hours, vortex for 10 minutes, and then sonicate for 60 seconds to eliminate bubbles to obtain a PLGA solution; EGF 10ug Dissolve in 10mL of PBS buffer solution; add 200μL of EGF-containing PBS solution to PLGA solution, in ice bath condition, sonicate for 5s, stop for 5s, and then sonicate for 5s, repeat 8 times, and emulsify to obtain a uniform electrospray solution containing EGF; The electrospinning solution and the electrospray solution obtained above were simultaneously electrospun and electrosprayed. The voltage of high-voltage electrospinning is 12kV, the flow rate of spinning solution is 0.8mL/h, and the concentration of spinning solution is 0.10g/mL; 0.10g/mL. The collected scaffold material was dried in a vacuum oven for 36 hours to obtain a PLGA fiber-microsphere composite scaffold material loaded with lovastatin and EGF.
实施例3：将PLGA(120kDa，75/25)称取200mg，与10mg普伐他汀溶于二氯甲烷与N，N-二甲基甲酰胺的混合溶剂(2:1，v/v)中，密封，0℃静置一夜，磁力搅拌5h，涡旋15min，超声60s消除气泡，得到均一的含有普伐他汀的静电纺丝溶液；将PLGA(60kDa，50/50)与TGF-β配制均一的电喷溶液。具体做法如下：将PLGA(60kDa，50/50)60mg溶解于二氯甲烷中，密封，0℃静置一夜，磁力搅拌4.5h，涡旋15min，再超声30s消除气泡，得到PLGA溶液；将TGF-β10ug溶解于10mLPBS缓冲溶液中；将100μL含TGF-β的PBS溶液加入到PLGA溶液中，在冰浴条件下，超声5s，停止5s，再超声5s，重复10次，乳化得到均一的含有TGF-β的电喷溶液；将上述得到的电纺溶液和电喷溶液同时电纺和电喷。高压静电纺丝电压为18kV，纺丝液流速为1.2mL/h，纺丝溶液浓度为0.12g/mL；高压静电喷射电压为12kV，电喷液流速为0.6mL/h，电喷液浓度为0.06g/mL。所收集的支架材料置于真空干燥箱中干燥32h，得到普伐他汀和TGF-β双负载的PLGA纤维-微球复合支架材料。Example 3: 200 mg of PLGA (120 kDa, 75/25) was weighed, and 10 mg of pravastatin was dissolved in a mixed solvent of dichloromethane and N, N-dimethylformamide (2:1, v/v) , sealed, left standing overnight at 0°C, magnetically stirred for 5h, vortexed for 15min, and ultrasonicated for 60s to eliminate air bubbles to obtain a uniform electrospinning solution containing pravastatin; PLGA (60kDa, 50/50) and TGF-β were prepared uniformly electrospray solution. The specific method is as follows: dissolve 60 mg of PLGA (60kDa, 50/50) in dichloromethane, seal it, let stand at 0°C overnight, stir magnetically for 4.5 hours, vortex for 15 minutes, and then sonicate for 30 seconds to eliminate air bubbles to obtain a PLGA solution; -β10ug was dissolved in 10mL of PBS buffer solution; 100μL of TGF-β-containing PBS solution was added to the PLGA solution, in an ice bath, ultrasonicated for 5s, stopped for 5s, and then ultrasonicated for 5s, repeated 10 times, emulsified to obtain a uniform TGF-containing - the electrospray solution of β; the electrospinning solution and the electrospray solution obtained above are electrospun and electrosprayed at the same time. The voltage of high-voltage electrospinning is 18kV, the flow rate of spinning solution is 1.2mL/h, and the concentration of spinning solution is 0.12g/mL; 0.06g/mL. The collected scaffold materials were dried in a vacuum oven for 32 hours to obtain a PLGA fiber-microsphere composite scaffold material loaded with pravastatin and TGF-β.
实施例4：将PLGA(120kDa，75/25)称取200mg，与40mg氟伐他汀溶于二氯甲烷与N，N-二甲基甲酰胺的混合溶剂(2:1，v/v)中，密封，4℃静置一夜，磁力搅拌6h，涡旋15min，超声30s消除气泡，得到均一的含有氟伐他汀的静电纺丝溶液；将PLGA(60kDa，50/50)与BSA配制均一的电喷溶液。具体做法如下：将PLGA(60kDa，50/50)60mg溶解于二氯甲烷中，密封，0℃静置一夜，磁力搅拌6h，涡旋10min，再超声45s消除气泡，得到PLGA溶液；将BSA 300mg溶解于10mLPBS缓冲溶液中；将100μL含BSA的PBS溶液加入到PLGA溶液中，在冰浴条件下，超声5s，停止5s，再超声5s，重复10次，乳化得到均一的含有BSA的电喷溶液；将上述得到的电纺溶液和电喷溶液同时电纺和电喷。高压静电纺丝电压为15kV，纺丝液流速为0.8mL/h，纺丝溶液浓度为0.12g/mL；高压静电喷射电压为15kV，电喷液流速为0.6mL/h，电喷液浓度为0.06g/mL。所收集的支架材料置于真空干燥箱中干燥48h，得到氟伐他汀和BSA双负载的PLGA纤维-微球复合支架材料。Example 4: 200 mg of PLGA (120 kDa, 75/25) was weighed, and 40 mg of fluvastatin was dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide (2:1, v/v) , sealed, left standing overnight at 4°C, magnetically stirred for 6h, vortexed for 15min, and ultrasonicated for 30s to eliminate air bubbles to obtain a uniform electrospinning solution containing fluvastatin; PLGA (60kDa, 50/50) and BSA were used to prepare a uniform electrospinning solution Spray solution. The specific method is as follows: Dissolve 60 mg of PLGA (60kDa, 50/50) in dichloromethane, seal it, let stand overnight at 0°C, stir magnetically for 6 hours, vortex for 10 minutes, and then sonicate for 45 seconds to eliminate bubbles to obtain a PLGA solution; Dissolve in 10mL of PBS buffer solution; add 100μL of BSA-containing PBS solution to the PLGA solution, in an ice bath, sonicate for 5s, stop for 5s, repeat for 10 times, and emulsify to obtain a uniform electrospray solution containing BSA ; Electrospinning and electrospraying the electrospinning solution and the electrospraying solution obtained above at the same time. The voltage of high-voltage electrospinning is 15kV, the flow rate of spinning solution is 0.8mL/h, and the concentration of spinning solution is 0.12g/mL; 0.06g/mL. The collected scaffold material was dried in a vacuum oven for 48 hours to obtain a PLGA fiber-microsphere composite scaffold material double-loaded with fluvastatin and BSA.
实施例5：将PLGA(100kDa，75/25)称取200mg，与30mg美伐他汀溶于二氯甲烷与N，N-二甲基甲酰胺的混合溶剂(2:1，v/v)中，密封，0℃静置一夜，磁力搅拌5h，涡旋15min，超声60s消除气泡，得到均一的含美伐他汀的纺丝溶液；将PLGA(50kDa，50/50)与DNA质粒配制均一的电喷液。具体做法如下：将PLGA(50kDa，50/50)60mg溶解于二氯甲烷中，密封，0℃静置一夜，磁力搅拌5h，涡旋15min，再超声45s消除气泡，得到PLGA溶液；将DNA质粒100ug溶解于1mLPBS缓冲溶液中；将100μL含DNA质粒的PBS溶液加入到PLGA溶液中，在冰浴条件下，超声5s，停止5s，再超声5s，重复6次，乳化得到均一的含有DNA质粒的电喷溶液；将上述得到的电纺溶液和电喷溶液同时电纺和电喷。高压静电纺丝电压为12kV，纺丝液流速为1.0mL/h，纺丝溶液浓度为0.15g/mL；高压静电喷射电压为12kV，电喷液流速为0.4mL/h，电喷液浓度为0.08g/mL。所收集的支架材料置于真空干燥箱中干燥48h，得到美伐他汀和DNA质粒双负载的PLGA纤维-微球复合支架材料。Example 5: Weigh 200 mg of PLGA (100 kDa, 75/25), and dissolve 30 mg of mevastatin in a mixed solvent of dichloromethane and N, N-dimethylformamide (2:1, v/v) , sealed, left standing overnight at 0°C, magnetically stirred for 5h, vortexed for 15min, and ultrasonicated for 60s to eliminate air bubbles to obtain a uniform spinning solution containing mevastatin; PLGA (50kDa, 50/50) and DNA plasmids were used to prepare a uniform electrolytic spray liquid. The specific method is as follows: Dissolve 60 mg of PLGA (50kDa, 50/50) in dichloromethane, seal it, let it stand at 0°C overnight, stir magnetically for 5 hours, vortex for 15 minutes, and then sonicate for 45 seconds to eliminate air bubbles to obtain a PLGA solution; 100ug was dissolved in 1mL of PBS buffer solution; 100μL of PBS solution containing DNA plasmid was added to PLGA solution, under ice bath conditions, sonicated for 5s, stopped for 5s, and then sonicated for 5s, repeated 6 times, and emulsified to obtain a uniform DNA plasmid containing Electrospray solution: electrospin and electrospray the electrospinning solution and electrospray solution obtained above. The voltage of high-voltage electrospinning is 12kV, the flow rate of spinning solution is 1.0mL/h, and the concentration of spinning solution is 0.15g/mL; 0.08g/mL. The collected scaffold materials were dried in a vacuum oven for 48 hours to obtain a PLGA fiber-microsphere composite scaffold material double-loaded with mevastatin and DNA plasmids.
按上述实施例得到具有不同投药比的他汀类药物和生物活性因子双负载的PLGA纤维-微球复合支架。A PLGA fiber-microsphere composite scaffold double-loaded with statins and bioactive factors with different dosage ratios was obtained according to the above-mentioned examples.
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