电纺PLGA/明胶组织工程支架和药物载体的制备与性能研究
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摘要
静电纺丝法是制备微纳米级连续纤维的有效手段，其独特优势在于，所制备出的纤维膜比表面积大、孔隙小且孔隙率高，不但与细胞外基质的结构相似，而且能够装载药物，因此在生物医学领域具有广泛的应用前景。本论文采用静电纺丝法制备了聚乙丙交酯（PLGA）/明胶复合纳米纤维，并对其在组织工程支架、表面矿化、表面修饰及药物控释等方面开展系统研究工作。具体研究内容包括以下几个方面：
首先，利用静电纺丝法成功制备出具有非定向和定向结构的PLGA/明胶纳米纤维膜，通过扫描电子显微镜、衰减全反射傅立叶变换红外光谱、吸水率实验、拉伸实验对戊二醛交联前后的纤维的表面形貌、化学结构、亲水性及力学性能等进行表征。研究发现，随着明胶含量的增加，非定向纤维的直径呈现先降后升的趋势，直径分布范围变宽，纤维膜亲水性提高，纤维膜的弹性模量和拉伸强度呈现出先升后降的趋势。交联后纤维膜亲水性下降，但拉伸性能有所提高。与非定向纤维膜相比，定向纤维膜的纤维直径、孔隙率和吸水率略低，但拉伸强度显著提高。神经干细胞培养结果表明，明胶的加入显著改善了纤维膜的细胞相容性，纤维的定向结构更有利于细胞的黏附与增殖。
其次，采用浓缩模拟体液法、过饱和法和交替矿化法在PLGA和PLGA/明胶9/1电纺纤维的表面进行矿化沉积。三种方法都使矿化速率显著提高，其中浓缩模拟体液法和交替矿化法都在短时间内将大量矿化物沉积到纤维表面，而过饱和法的矿化物产量相对较低，但纤维膜中的大量孔隙却得以保留。由于明胶对Ca2+离子具有吸附作用，因此会促进矿化物的形核过程，导致矿化物在PLGA/明胶9/1纤维表面沉积速度大于PLGA纤维。由三种矿化方法得到的钙磷矿化物都包含透钙磷灰石（DCPD）、磷酸八钙（OCP）和羟基磷灰石（HA），浓缩模拟体液法形成矿化物以DCPD为主，而过饱和法和交替矿化法中的HA含量有所增加。细胞培养结果显示，矿化后的纤维膜对骨细胞的黏附、增殖和分化均有良好的促进作用，因此矿化纤维在骨组织培养及修复方面具有潜在应用价值。
再次，采用表面截留法对PLGA电纺纤维支架进行表面修饰。通过考察三氟乙醇水溶液浓度、溶胀时间和截留大分子溶液浓度对截留效果的影响，确定了截留的最佳工艺条件，获得表面截留明胶的PLGA电纺纤维以及表面截留接枝海藻酸钠/明胶的PLGA电纺纤维。表面形貌与红外光谱的结果证实，天然大分子是通过截留而非黏附固定在PLGA纤维表面。经过截留修饰的PLGA纤维膜产生的收缩现象增加了纤维膜单位面积上的纤维密度，从而使纤维膜的力学性能提高。经过表面修饰的PLGA电纺纤维的亲水性和细胞相容性大大提高，其中海藻酸钠/明胶截留接枝的PLGA纤维的亲水性和细胞相容性要高于明胶截留的PLGA纤维，显示了双层大分子修饰的优势。采用表面截留方法不仅改善PLGA电纺纤维的亲水性和细胞相容性，而且提高其力学性能，充分拓展了其在生物医学领域中的用途。
最后，分别采用静电纺丝法和溶液浇注法制备装载药物芬布芬的PLGA/明胶电纺纤维膜和浇注薄膜。芬布芬在纤维中呈无定形态，而在薄膜中以结晶态存在，从而导致纤维膜的药物释放速率高于薄膜。载药体中明胶含量增加导致载体溶胀率增大，从而使药物释放速率增大，而交联处理降低了纤维膜的溶胀率，从而使药物释放速率随交联时间延长而降低。与非定向纤维膜相比，定向纤维膜由于纤维排列紧密阻碍了药物的扩散，从而使药物释放速率略有降低。在不同pH值缓冲液中，PLGA基体的玻璃化转变温度发生变化，导致药物释放速率随pH值增加而增大。通过研究甲硝唑/芬布芬双药载体的药物释放性能，发现双层结构载药纤维膜适合作双药载体，因为每种药物可以独立装载与释放，两种药物的释放不产生相互干扰，而且其含量可由载药纤维的层厚进行调节。在药物释放过程中，采用外裹PLGA纤维的方法可以有效缓解药物释放初期的突释现象。
Electrospining is an effective method to yield continues fiber of nanometer tomicrometer scale. The fibrous membrane with large surface to volume ratio, interconnectedpores and high porosity, is similar to extracellular matrix and can carry drugs, thus has broadapplication in biomedical fields. In this paper, the electrospun nanofibers consisted ofsynthetic polymer poly(D,L-lactide-co-glycolide)(PLGA) and natural polymer gelatin areinvestigated systematically in tissue engineering, surface mineralization, surface entrapmentand drug release. The main contents are as followed：
Firstly, randomly-oriented and aligned nanofibers with different ratios of PLGA/gelatinare produced through electrospinning. The surface morphology, chemical structure,hydrophilicity and mechanical property of PLGA/gelatin nanofibers before and aftercrosslinking are revealed by scanning electron microscope (SEM), attenuated total reflectionFourier transform infrared (ATR-FT-IR), swelling ratio and tensile test. It is found that thefiber diameter first increases and then decreases with broader fiber diameter distribution whenthe gelatin content increases. The hydrophilicity of fibrous scaffolds increases with increasingof gelatin content. The increasing of gelatin makes the elastic modulus and tensile stress offibrous scaffolds first increase and then decrease sharply. The crosslinking by glutaraldehydedecreases the hydrophilicity, but improves the elastic modulus and tensile stress of fibrousscaffolds. Compared with randomly-oriented PLGA/gelatin nanofibrous scaffolds, the fiberdiameter, porosity and swelling ratio of aligned nanofibrous scaffolds are lower, but theelastic modulus and tensile stress are higher than that of randomly-oriented nanofibrousscaffolds. Moreover, the addition of gelatin improves the biocompatibility of the nanofibrousscaffold, and aligned nanofibrous scaffold reveals better performance for cell adhesion andproliferation.
Secondly, calcium phosphate apatite mineralization on the PLGA and PLGA/gelatin9/1nanofibrous scaffolds is prepared by three methods: concentrated simulated body fluid(10SBF), supersaturated calcification solution and alternate soaking. The carboxyl groups ofgelatin could lead to the enrichment of Ca2+ion, which could induce the nucleation ofcrystallites and result in the higher weight increase of mineralized PLGA/gelatin9/1nanofibers than PLGA nanofibers. The results of EDS, FT-IR and XRD depicts the apatiteyielded by three methods all contains DCPD, OCP and HA. The main component in10SBF isDCPD, and HA content is increasing in the other two methods. The efficiency of mineralization is higher, and large apatite coats on the nanofibers. It is also found that themineralized nanofibers show positive influence on adhesion, proliferation and differentiationof cells, which has a potential application in bone tissue engineering.
Thirdly, the entrapment technology is used for surface modification of PLGA electrospunnanofibers with natural polymers. The influence of concentration of2,2,2-thifluoroethanolaqueous solution, swelling time and concentration of entrapment polymer on entrapment areinvestigated to confirm the best conditions and obtain the gelatin entrapment PLGAnanofibers and sodium alginate/gelatin entrapment-graft PLGA nanofibers. The results ofSEM and FT-IR shows the natural polymer is entrapping on the surface of PLGA nanofibers.The water contact angle test shows the higher hydrophilicity of modified PLGA nanofibers,and the hydrophilicity of sodium alginate/gelatin entrapment-graft PLGA nanofibers is betterthan that of gelatin entrapment PLGA nanofibers. The mechanical property of PLGAnanofibers is improved through entrapment. The MTT results shows that the entrapmentenhances the biocompatibility of the PLGA elecrospun nanofibers, and the biocompatibility ofsodium alginate/gelatin entrapment-graft PLGA nanofibers is better than that of gelatinentrapment PLGA nanofibers. The entrapment not only improves the hydrophilicity but alsoenhances the mechanical property, thus broader the application as tissue engineeringscaffolds.
Fourthly, fenbufen (FBF)-loaded electrospun PLGA and PLGA/gelatin nanofibrousscaffolds as well as FBF-loaded solvent-cast films are produced and their drug releasecharacteristics are further investigated. It is found that FBF is in amorphous condition withinthe electrospun nanofibers but form crystal within the films, which lead to the higher drugrelease rate of nanofibers than that of films. The increasing of gelatin in the drug carrierenhances the swelling ratio, resulting in the large drug release. But after crosslinking, the drugrelease decreases with the increasing of crosslinking time. The structure of alignednanofibrous membrane is more compact than that of randomly-oriented nanofibrousmembrane, which leads to the lower drug release. In addition, the pH value of the buffersolution could change the Tgof the polymer, which affects the drug release rate. The furtherinvestigation finds that multi-layer structure is suitable as the two drug carrier ofmetronidazole/fenbufen, and the drug content depends on the thickness of the layer. Finally,the outer protected PLGA nanofibrous layer of the drug-loaded nanofibrous membrane couldinhibit the burst release of the drug effectively.
首先，利用静电纺丝法成功制备出具有非定向和定向结构的PLGA/明胶纳米纤维膜，通过扫描电子显微镜、衰减全反射傅立叶变换红外光谱、吸水率实验、拉伸实验对戊二醛交联前后的纤维的表面形貌、化学结构、亲水性及力学性能等进行表征。研究发现，随着明胶含量的增加，非定向纤维的直径呈现先降后升的趋势，直径分布范围变宽，纤维膜亲水性提高，纤维膜的弹性模量和拉伸强度呈现出先升后降的趋势。交联后纤维膜亲水性下降，但拉伸性能有所提高。与非定向纤维膜相比，定向纤维膜的纤维直径、孔隙率和吸水率略低，但拉伸强度显著提高。神经干细胞培养结果表明，明胶的加入显著改善了纤维膜的细胞相容性，纤维的定向结构更有利于细胞的黏附与增殖。
其次，采用浓缩模拟体液法、过饱和法和交替矿化法在PLGA和PLGA/明胶9/1电纺纤维的表面进行矿化沉积。三种方法都使矿化速率显著提高，其中浓缩模拟体液法和交替矿化法都在短时间内将大量矿化物沉积到纤维表面，而过饱和法的矿化物产量相对较低，但纤维膜中的大量孔隙却得以保留。由于明胶对Ca2+离子具有吸附作用，因此会促进矿化物的形核过程，导致矿化物在PLGA/明胶9/1纤维表面沉积速度大于PLGA纤维。由三种矿化方法得到的钙磷矿化物都包含透钙磷灰石（DCPD）、磷酸八钙（OCP）和羟基磷灰石（HA），浓缩模拟体液法形成矿化物以DCPD为主，而过饱和法和交替矿化法中的HA含量有所增加。细胞培养结果显示，矿化后的纤维膜对骨细胞的黏附、增殖和分化均有良好的促进作用，因此矿化纤维在骨组织培养及修复方面具有潜在应用价值。
再次，采用表面截留法对PLGA电纺纤维支架进行表面修饰。通过考察三氟乙醇水溶液浓度、溶胀时间和截留大分子溶液浓度对截留效果的影响，确定了截留的最佳工艺条件，获得表面截留明胶的PLGA电纺纤维以及表面截留接枝海藻酸钠/明胶的PLGA电纺纤维。表面形貌与红外光谱的结果证实，天然大分子是通过截留而非黏附固定在PLGA纤维表面。经过截留修饰的PLGA纤维膜产生的收缩现象增加了纤维膜单位面积上的纤维密度，从而使纤维膜的力学性能提高。经过表面修饰的PLGA电纺纤维的亲水性和细胞相容性大大提高，其中海藻酸钠/明胶截留接枝的PLGA纤维的亲水性和细胞相容性要高于明胶截留的PLGA纤维，显示了双层大分子修饰的优势。采用表面截留方法不仅改善PLGA电纺纤维的亲水性和细胞相容性，而且提高其力学性能，充分拓展了其在生物医学领域中的用途。
最后，分别采用静电纺丝法和溶液浇注法制备装载药物芬布芬的PLGA/明胶电纺纤维膜和浇注薄膜。芬布芬在纤维中呈无定形态，而在薄膜中以结晶态存在，从而导致纤维膜的药物释放速率高于薄膜。载药体中明胶含量增加导致载体溶胀率增大，从而使药物释放速率增大，而交联处理降低了纤维膜的溶胀率，从而使药物释放速率随交联时间延长而降低。与非定向纤维膜相比，定向纤维膜由于纤维排列紧密阻碍了药物的扩散，从而使药物释放速率略有降低。在不同pH值缓冲液中，PLGA基体的玻璃化转变温度发生变化，导致药物释放速率随pH值增加而增大。通过研究甲硝唑/芬布芬双药载体的药物释放性能，发现双层结构载药纤维膜适合作双药载体，因为每种药物可以独立装载与释放，两种药物的释放不产生相互干扰，而且其含量可由载药纤维的层厚进行调节。在药物释放过程中，采用外裹PLGA纤维的方法可以有效缓解药物释放初期的突释现象。
Electrospining is an effective method to yield continues fiber of nanometer tomicrometer scale. The fibrous membrane with large surface to volume ratio, interconnectedpores and high porosity, is similar to extracellular matrix and can carry drugs, thus has broadapplication in biomedical fields. In this paper, the electrospun nanofibers consisted ofsynthetic polymer poly(D,L-lactide-co-glycolide)(PLGA) and natural polymer gelatin areinvestigated systematically in tissue engineering, surface mineralization, surface entrapmentand drug release. The main contents are as followed：
Firstly, randomly-oriented and aligned nanofibers with different ratios of PLGA/gelatinare produced through electrospinning. The surface morphology, chemical structure,hydrophilicity and mechanical property of PLGA/gelatin nanofibers before and aftercrosslinking are revealed by scanning electron microscope (SEM), attenuated total reflectionFourier transform infrared (ATR-FT-IR), swelling ratio and tensile test. It is found that thefiber diameter first increases and then decreases with broader fiber diameter distribution whenthe gelatin content increases. The hydrophilicity of fibrous scaffolds increases with increasingof gelatin content. The increasing of gelatin makes the elastic modulus and tensile stress offibrous scaffolds first increase and then decrease sharply. The crosslinking by glutaraldehydedecreases the hydrophilicity, but improves the elastic modulus and tensile stress of fibrousscaffolds. Compared with randomly-oriented PLGA/gelatin nanofibrous scaffolds, the fiberdiameter, porosity and swelling ratio of aligned nanofibrous scaffolds are lower, but theelastic modulus and tensile stress are higher than that of randomly-oriented nanofibrousscaffolds. Moreover, the addition of gelatin improves the biocompatibility of the nanofibrousscaffold, and aligned nanofibrous scaffold reveals better performance for cell adhesion andproliferation.
Secondly, calcium phosphate apatite mineralization on the PLGA and PLGA/gelatin9/1nanofibrous scaffolds is prepared by three methods: concentrated simulated body fluid(10SBF), supersaturated calcification solution and alternate soaking. The carboxyl groups ofgelatin could lead to the enrichment of Ca2+ion, which could induce the nucleation ofcrystallites and result in the higher weight increase of mineralized PLGA/gelatin9/1nanofibers than PLGA nanofibers. The results of EDS, FT-IR and XRD depicts the apatiteyielded by three methods all contains DCPD, OCP and HA. The main component in10SBF isDCPD, and HA content is increasing in the other two methods. The efficiency of mineralization is higher, and large apatite coats on the nanofibers. It is also found that themineralized nanofibers show positive influence on adhesion, proliferation and differentiationof cells, which has a potential application in bone tissue engineering.
Thirdly, the entrapment technology is used for surface modification of PLGA electrospunnanofibers with natural polymers. The influence of concentration of2,2,2-thifluoroethanolaqueous solution, swelling time and concentration of entrapment polymer on entrapment areinvestigated to confirm the best conditions and obtain the gelatin entrapment PLGAnanofibers and sodium alginate/gelatin entrapment-graft PLGA nanofibers. The results ofSEM and FT-IR shows the natural polymer is entrapping on the surface of PLGA nanofibers.The water contact angle test shows the higher hydrophilicity of modified PLGA nanofibers,and the hydrophilicity of sodium alginate/gelatin entrapment-graft PLGA nanofibers is betterthan that of gelatin entrapment PLGA nanofibers. The mechanical property of PLGAnanofibers is improved through entrapment. The MTT results shows that the entrapmentenhances the biocompatibility of the PLGA elecrospun nanofibers, and the biocompatibility ofsodium alginate/gelatin entrapment-graft PLGA nanofibers is better than that of gelatinentrapment PLGA nanofibers. The entrapment not only improves the hydrophilicity but alsoenhances the mechanical property, thus broader the application as tissue engineeringscaffolds.
Fourthly, fenbufen (FBF)-loaded electrospun PLGA and PLGA/gelatin nanofibrousscaffolds as well as FBF-loaded solvent-cast films are produced and their drug releasecharacteristics are further investigated. It is found that FBF is in amorphous condition withinthe electrospun nanofibers but form crystal within the films, which lead to the higher drugrelease rate of nanofibers than that of films. The increasing of gelatin in the drug carrierenhances the swelling ratio, resulting in the large drug release. But after crosslinking, the drugrelease decreases with the increasing of crosslinking time. The structure of alignednanofibrous membrane is more compact than that of randomly-oriented nanofibrousmembrane, which leads to the lower drug release. In addition, the pH value of the buffersolution could change the Tgof the polymer, which affects the drug release rate. The furtherinvestigation finds that multi-layer structure is suitable as the two drug carrier ofmetronidazole/fenbufen, and the drug content depends on the thickness of the layer. Finally,the outer protected PLGA nanofibrous layer of the drug-loaded nanofibrous membrane couldinhibit the burst release of the drug effectively.
引文
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