用于药物输送的静电纺纳米管/聚乳酸—羟基乙酸复合纳米纤维的制备、表征、缓释行为及药物生物活性评价
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摘要
静电纺丝技术是一种简单、有效的制备超细纤维的方法。通常情况下,静电纺可生物降解和生物相容性的高聚物纳米纤维支架具有纤维尺寸可控、极大的比表面积、高孔隙率和三维网状结构等特点,能够从生物功能和结构方面很好地模拟天然细胞外基质,可以为细胞的粘附和生长提供良好的体外环境,从而为细胞-支架三维网络组织的构建提供了很好的条件。静电纺纳米纤维不仅可以用于组织工程支架,同时也是一种良好的药物载体。
本文旨在开发一种能够应用于组织工程和药剂科学的静电纺纳米纤维支架材料。针对传统的聚合物纳米纤维机械强度低,常见的纳米颗粒、脂质体、胶束等药物剂型以及某些静电纺纳米纤维载药剂型存在突释现象,以及纳米管载药体系非器件化等问题,首次提出了用纳米管载药后与静电纺丝技术相结合的方法,制备了静电纺纳米管/高聚物双载体纳米纤维药物缓释系统,可以很好地解决这几方面的问题。
本文主要以可生物降解和生物相容性的高聚物聚乳酸-羟基乙酸(PLGA)为静电纺纤维基质,埃洛石纳米管(HNTs)和多壁碳纳米管(MWCNTs)为药物载体,抗生素类药物盐酸四环素(TCH)和抗肿瘤药物盐酸阿霉素(DOX)为两种模型药。首先用HNTs和CNTs分别负载TCH和DOX,然后通过静电纺丝的方法将载药纳米管TCH/HNTs、 DOX/CNTs与PLGA溶液混合,制备静电纺TCH/HNTs/PLGA及DOX/CNTs/PLGA双载体纳米纤维载药体系。
对于静电纺TCH/HNTs/PLGA载药体系的研究,我们首先制备了静电纺PLGA纤维、HNTs/PLGA复合纳米纤维,分析研究了HNTs的加入对PLGA纤维的形貌、直径、孔隙率、机械性能、热学性能、内部结构的影响。结果表明,HNTs可以成功地负载到PLGA纤维的内部,HNTs的加入对纤维的形貌、纤维毡的孔隙率及PLGA的内部结构没有明显的影响。而纤维直径随HNTs含量的增加而增加。另外,少量HNTs(1%～3%)的加入使纤维的机械性能大大提高,并且PLGA的热稳定性也因HNTs的增加而有少量增加。因此,静电纺HNTs/PLGA复合纳米纤维支架具备的较细的纤维直径、高孔隙率、增强的机械性能,为构建组织工程支架奠定了很好的基础。
构建一个纳米纤维组织工程材料,评价材料的生物相容性是非常关键的一步。我们通过MTT法和SEM法检验了L929小鼠成纤维细胞在静电纺PLGA及HNTs/PLGA纳米纤维支架上粘附、增殖的情况以及细胞生长的形貌,从而评价材料的体外生物相容性。另外,通过模拟细胞培养的过程,研究了掺杂HNTs的静电纺PLGA支架对蛋白的吸附情况。结果表明,静电纺PLGA和HNTs/PLGA纳米纤维支架具有良好的体外生物相容性。L929小鼠成纤维细胞在PLGA基静电纺纤维支架上表现出与在ECM中相似的显型形状。另外,HNTs/PLGA纤维支架比PLGA有更好的吸附蛋白的能力,从而有利于细胞的粘附和增殖。综合静电纺HNTs/PLGA复合纳米纤维增强的机械性能,良好的生物相容性,该三维多孔纤维支架材料将会在组织工程和药剂科学方面具有很好的应用前景。
MTT比色法是一种评价材料生物相容性常用的方法。但有时会出现生物相容性良好的材料MTT甲躜OD值较低的现象,针对这一问题我们提出了MTT法表征静电纺PLGA基纤维支架材料的生物相容性时可能存在伪阴性偏差的假设。我们通过静电纺PLGA基支架材料吸附MTT甲臜染料的实验证实了这一假设,并对MTT材料生物相容性实验数据进行了校正。结果表明,PLGA基纤维支架存在很明显的吸附甲臜的能力,HNTs/PLGA及CNTs/PLGA复合纤维比PLGA纤维能更好地促进细胞的粘附、增殖。该实验结果可能对多种多孔支架材料的其它比色法实验也有一定的参考价值。
在静电纺HNTs/PLGA纤维的基本性能表征的基础上,我们制备了静电纺TCH/HNTs/PLGA双载体复合纳米纤维毡,研究了该载药纤维的形貌、直径、机械性能、体外生物相容性、药物释放行为以及药物的抑菌活性。结果表明,TCH/HNTs/PLGA复合载药纤维仍可以保持增强的机械性能,对L929小鼠成纤维细胞仍然具有良好的体外生物相容性。TCH/HNTs载药粉末和TCH/PLGA混纺载药纳米纤维都存在明显的初始突释现象。而TCH/HNTs/PLGA双载体纳米纤维载药体系具有很好的持续释放效果,可以很好地延缓TCH/HNTs粉末载药体系的药物释放。并且,TCH/HNTs/PLGA载药纤维毡对金黄色葡萄球菌(S. aureus)有很好的抑菌活性。因此,该载药纤维毡在伤口包覆、术后防粘连防感染方面有很好的应用价值。不仅可以用于药物缓释,同时又可以作为功能型组织工程支架。
静电纺DOX/CNTs/PLGA纳米纤维载药体系是一种抗肿瘤药物剂型。我们研究了静电纺CNTs/PLGA纤维及DOX/CNTs/PLGA载药纤维的形貌、机械性能、内部结构等。另外,研究了DOX/CNTs/PLGA载药纤维的缓释行为及药物抗肿瘤活性等。结果表明,静电纺DOX(1,2%)/CNTs/PLGA纤维与CNTs/PLGA一样可以增强PLGA纤维的机械性能。缓释实验表明,DOX/CNTs/PLGA载药纳米纤维具有良好的持续释放效果,不仅可以延缓DOX/CNTs粉末载药体系的药物释放,同时又实现了DOX/CNTs载药体系的器件化。静电纺DOX/CNTs/PLGA及DOX/PLGA载药纤维释放的药物具有良好的抗肿瘤活性。总之,静电纺DOX/CNTs/PLGA复合纳米纤维抗肿瘤载药体系在药物控释领域具有巨大的应用潜质。
Electrospinning is a simple and effective method for fabricating ultrafine fibers. Generally, electrospun biodegradable and biocompatible polymeric nanofibrous scaffolds with controllable diameters, high specific surface and porosity and three dimensional network structures, have the ability to closely mimic the biological function and structure of nature extracellular matrix (ECM). And electrospun nanofibrous scaffolds could provide favorable environment for cell adhesion, migration and growth, thus a three dimensional fiber/cell integrated network could be constructed due to cell-cell and cell-matrix interaction. Beyond the application of electrospun nanofibers in tissue engineering, the application in drug delivery is another direction.
The objective of this research is to develop electrospun nanofiber-based scaffolding materials for both tissue engineering and pharmaceutical sciences. Traditional polymer nanofibers lack enough mechanical strength and often have a burst release profile when drug molecules are incorporated within the nanofibers. In addition, the formulation of drug-loaded nanotubes (multiwalled carbon nanotubes (MWCNTs) or halloysite nanotubes (HNTs)) is not in the status of device but often presented as powders. Therefore, we combined drug-loaded nanotubes and electrospinning to improve the mechanical properties of the fibers, to develop a double-container drug delivery system, and to avoid the burst release of the incorporated drugs.
In this study, poly(lactic-co-glycolic acid)(PLGA) is used as the electrospinning fibrous matix. Tetracycline hydrochloride (TCH) and Doxorubicine hydrochloride (DOX) used as model drugs were loaded into or onto HNTs and CNTs, respectively. Then, the drug-loaded TCH/HNTs and DOX/CNTs with optimized encapsulation efficiency were mixed with PLGA polymer solution for subsequent electrospinning to form nanotube/polymer double-container composite nanofibrous drug delivery system.
Firstly, the main research is about the electrospun TCH/HNTs/PLGA nanofibers. Electrospun PLGA nanofibers and HNTs/PLGA composite nanofibers were fabricated, and the affection of the doped HNTs into PLGA on the morphology, structure, fiber diameter, porosity, mechanical and thermal properties was investigated. The results showed that HNTs were successfully incorporated into PLGA fibers, and the incorporation of HNTs did not significantly influence the morphology, structure and porosity of PLGA fibers. While the fiber diameters were increased with the quantity of the doped HNTs. Importantly, the mechanical properties of PLGA fibrous mats were significantly improved and the thermal properties were slightly enhanced with the incorporation of HNTs. All these properties give the priority for the application in tissue engineering.
A key step to develop a nanofibrous scaffold for tissue engineering is to evaualte the biocompatibility of the materials. The adhesion and proliferation of L929mouse fibroblast cells cultured on both PLGA and HNT-doped PLGA fibrous scaffolds were compared through MTT assay of cell viability and SEM observation of cell morphology. And protein adsorption behavior on nanofibrous scaffolds was investigated. Similar to electrospun PLGA nanofibers, HNTs-doped PLGA nanofibers were able to promote cell attachment and proliferation and fibroblasts displayed a phenotypic shape, suggesting that the incorporation of HNTs within PLGA nanofibers does not compromise the biocompatibility of the PLGA nanofibers. In addition, compared with PLGA, HNTs-doped PLGA scaffolds allow more protein adsorption, which is favorable for cell adhesion and proliferation. The developed electrospun HNT-doped composite fibrous scaffold may find applications in tissue engineering and pharmaceutical sciences.
As a popular method, MTT colorimetric assay is often used to evaluate the viability of cells cultured onto various fibrous tissue engineering scaffolds. While sometimes the cell viability determined from MTT assay is relatively lower than other characterization method, which probably due to the strong dye sorption capability of porous scaffolding materials. Therefore, we proposed that the OD values obtained from MTT assay likely has a false negative result. In this study, the adsorption of MTT formazan onto porous electrospun PLGA based nanofibrous mats was investigated. We show that PLGA, and the HNTs-or CNTs-doped PLGA nanofibers display appreciable MTT formazan dye sorption, corresponding to35.58～50.23%deviation from the real cell viability assay data. By quantifying the MTT formazan dye sorption amount, the MTT assay data could be rectified to reflect the real cell viability. Our study provides a general insight into the deviation and rectification of MTT cell viability assay, which is likely applicable to other colorimetric assays of different porous scaffolding materials for various biomedical applications.
On the basis of previous characterization of electrospun PLGA and HNTs/PLGA nanofibers, electrospun TCH/HNTs/PLGA double container drug delivery systems were fabricated. The morphology, structure, fiber diameter, mechanical properties, in-vitro biocompatibility, drug release behavior and antibacterial activity of the drug-loaded composite nanofibrous mats were investigated. The results showed that the mechanical properties of TCH/HNTs/PLGA fibers were also improved by comparing with PLGA fibers. And drug-loaded fibrous mats had good compatibility for the adhesion and proliferation of L929mouse fibroblasts. Compared with drug-loaded TCH/HNTs and TCH/PLGA nanofibers with obvious burst release, electrospun TCH/HNTs/PLGA double container nanofibrous drug delivery system prolonged the release rate of the drug and appreciably eliminated the initial burst release, and sustained release was obtained for more than one month. The TCH-medicated HNTs/PLGA scaffolds displayed effective activity to inhibit the growth of S. aureus both in liquid medium and on solid medium in vitro. These medicated PLGA-based electrospun scaffolds hold a promising potential in wound dressing applications and in preventing in vivo postsurgical adhesions and infections.
Electrospun DOX/CNTs/PLGA drug-loaded nanofibrous mat is an antitumer formulation. For this formulation, the morphology, structure, and mechanical properties of electrospun CNTs/PLGA composite nanofibers and DOX/CNTs/PLGA drug-loaded nanofibers were investigated. Similar to electrospun CNTs/PLGA composite nanofibrous mats, DOX (1,2%)/CNTs/PLGA drug-loaded nanofibrous mats could improve the mechanical properties of PLGA fibers. In addition, DOX/CNTs/PLGA double container nanofibrous drug delivery system prolonged the release rate of drug-loaded DOX/CNTs and appreciably eliminated the initial burst release, and sustained release was obtained. The DOX/CNTs/PLGA and DOX/PLGA drug-loaded scaffolds displayed effective anticancer bioactivity on KB cells. In summary, this drug delivery system with controllable release will find various applications in cancer treatment area.
本文旨在开发一种能够应用于组织工程和药剂科学的静电纺纳米纤维支架材料。针对传统的聚合物纳米纤维机械强度低,常见的纳米颗粒、脂质体、胶束等药物剂型以及某些静电纺纳米纤维载药剂型存在突释现象,以及纳米管载药体系非器件化等问题,首次提出了用纳米管载药后与静电纺丝技术相结合的方法,制备了静电纺纳米管/高聚物双载体纳米纤维药物缓释系统,可以很好地解决这几方面的问题。
本文主要以可生物降解和生物相容性的高聚物聚乳酸-羟基乙酸(PLGA)为静电纺纤维基质,埃洛石纳米管(HNTs)和多壁碳纳米管(MWCNTs)为药物载体,抗生素类药物盐酸四环素(TCH)和抗肿瘤药物盐酸阿霉素(DOX)为两种模型药。首先用HNTs和CNTs分别负载TCH和DOX,然后通过静电纺丝的方法将载药纳米管TCH/HNTs、 DOX/CNTs与PLGA溶液混合,制备静电纺TCH/HNTs/PLGA及DOX/CNTs/PLGA双载体纳米纤维载药体系。
对于静电纺TCH/HNTs/PLGA载药体系的研究,我们首先制备了静电纺PLGA纤维、HNTs/PLGA复合纳米纤维,分析研究了HNTs的加入对PLGA纤维的形貌、直径、孔隙率、机械性能、热学性能、内部结构的影响。结果表明,HNTs可以成功地负载到PLGA纤维的内部,HNTs的加入对纤维的形貌、纤维毡的孔隙率及PLGA的内部结构没有明显的影响。而纤维直径随HNTs含量的增加而增加。另外,少量HNTs(1%～3%)的加入使纤维的机械性能大大提高,并且PLGA的热稳定性也因HNTs的增加而有少量增加。因此,静电纺HNTs/PLGA复合纳米纤维支架具备的较细的纤维直径、高孔隙率、增强的机械性能,为构建组织工程支架奠定了很好的基础。
构建一个纳米纤维组织工程材料,评价材料的生物相容性是非常关键的一步。我们通过MTT法和SEM法检验了L929小鼠成纤维细胞在静电纺PLGA及HNTs/PLGA纳米纤维支架上粘附、增殖的情况以及细胞生长的形貌,从而评价材料的体外生物相容性。另外,通过模拟细胞培养的过程,研究了掺杂HNTs的静电纺PLGA支架对蛋白的吸附情况。结果表明,静电纺PLGA和HNTs/PLGA纳米纤维支架具有良好的体外生物相容性。L929小鼠成纤维细胞在PLGA基静电纺纤维支架上表现出与在ECM中相似的显型形状。另外,HNTs/PLGA纤维支架比PLGA有更好的吸附蛋白的能力,从而有利于细胞的粘附和增殖。综合静电纺HNTs/PLGA复合纳米纤维增强的机械性能,良好的生物相容性,该三维多孔纤维支架材料将会在组织工程和药剂科学方面具有很好的应用前景。
MTT比色法是一种评价材料生物相容性常用的方法。但有时会出现生物相容性良好的材料MTT甲躜OD值较低的现象,针对这一问题我们提出了MTT法表征静电纺PLGA基纤维支架材料的生物相容性时可能存在伪阴性偏差的假设。我们通过静电纺PLGA基支架材料吸附MTT甲臜染料的实验证实了这一假设,并对MTT材料生物相容性实验数据进行了校正。结果表明,PLGA基纤维支架存在很明显的吸附甲臜的能力,HNTs/PLGA及CNTs/PLGA复合纤维比PLGA纤维能更好地促进细胞的粘附、增殖。该实验结果可能对多种多孔支架材料的其它比色法实验也有一定的参考价值。
在静电纺HNTs/PLGA纤维的基本性能表征的基础上,我们制备了静电纺TCH/HNTs/PLGA双载体复合纳米纤维毡,研究了该载药纤维的形貌、直径、机械性能、体外生物相容性、药物释放行为以及药物的抑菌活性。结果表明,TCH/HNTs/PLGA复合载药纤维仍可以保持增强的机械性能,对L929小鼠成纤维细胞仍然具有良好的体外生物相容性。TCH/HNTs载药粉末和TCH/PLGA混纺载药纳米纤维都存在明显的初始突释现象。而TCH/HNTs/PLGA双载体纳米纤维载药体系具有很好的持续释放效果,可以很好地延缓TCH/HNTs粉末载药体系的药物释放。并且,TCH/HNTs/PLGA载药纤维毡对金黄色葡萄球菌(S. aureus)有很好的抑菌活性。因此,该载药纤维毡在伤口包覆、术后防粘连防感染方面有很好的应用价值。不仅可以用于药物缓释,同时又可以作为功能型组织工程支架。
静电纺DOX/CNTs/PLGA纳米纤维载药体系是一种抗肿瘤药物剂型。我们研究了静电纺CNTs/PLGA纤维及DOX/CNTs/PLGA载药纤维的形貌、机械性能、内部结构等。另外,研究了DOX/CNTs/PLGA载药纤维的缓释行为及药物抗肿瘤活性等。结果表明,静电纺DOX(1,2%)/CNTs/PLGA纤维与CNTs/PLGA一样可以增强PLGA纤维的机械性能。缓释实验表明,DOX/CNTs/PLGA载药纳米纤维具有良好的持续释放效果,不仅可以延缓DOX/CNTs粉末载药体系的药物释放,同时又实现了DOX/CNTs载药体系的器件化。静电纺DOX/CNTs/PLGA及DOX/PLGA载药纤维释放的药物具有良好的抗肿瘤活性。总之,静电纺DOX/CNTs/PLGA复合纳米纤维抗肿瘤载药体系在药物控释领域具有巨大的应用潜质。
Electrospinning is a simple and effective method for fabricating ultrafine fibers. Generally, electrospun biodegradable and biocompatible polymeric nanofibrous scaffolds with controllable diameters, high specific surface and porosity and three dimensional network structures, have the ability to closely mimic the biological function and structure of nature extracellular matrix (ECM). And electrospun nanofibrous scaffolds could provide favorable environment for cell adhesion, migration and growth, thus a three dimensional fiber/cell integrated network could be constructed due to cell-cell and cell-matrix interaction. Beyond the application of electrospun nanofibers in tissue engineering, the application in drug delivery is another direction.
The objective of this research is to develop electrospun nanofiber-based scaffolding materials for both tissue engineering and pharmaceutical sciences. Traditional polymer nanofibers lack enough mechanical strength and often have a burst release profile when drug molecules are incorporated within the nanofibers. In addition, the formulation of drug-loaded nanotubes (multiwalled carbon nanotubes (MWCNTs) or halloysite nanotubes (HNTs)) is not in the status of device but often presented as powders. Therefore, we combined drug-loaded nanotubes and electrospinning to improve the mechanical properties of the fibers, to develop a double-container drug delivery system, and to avoid the burst release of the incorporated drugs.
In this study, poly(lactic-co-glycolic acid)(PLGA) is used as the electrospinning fibrous matix. Tetracycline hydrochloride (TCH) and Doxorubicine hydrochloride (DOX) used as model drugs were loaded into or onto HNTs and CNTs, respectively. Then, the drug-loaded TCH/HNTs and DOX/CNTs with optimized encapsulation efficiency were mixed with PLGA polymer solution for subsequent electrospinning to form nanotube/polymer double-container composite nanofibrous drug delivery system.
Firstly, the main research is about the electrospun TCH/HNTs/PLGA nanofibers. Electrospun PLGA nanofibers and HNTs/PLGA composite nanofibers were fabricated, and the affection of the doped HNTs into PLGA on the morphology, structure, fiber diameter, porosity, mechanical and thermal properties was investigated. The results showed that HNTs were successfully incorporated into PLGA fibers, and the incorporation of HNTs did not significantly influence the morphology, structure and porosity of PLGA fibers. While the fiber diameters were increased with the quantity of the doped HNTs. Importantly, the mechanical properties of PLGA fibrous mats were significantly improved and the thermal properties were slightly enhanced with the incorporation of HNTs. All these properties give the priority for the application in tissue engineering.
A key step to develop a nanofibrous scaffold for tissue engineering is to evaualte the biocompatibility of the materials. The adhesion and proliferation of L929mouse fibroblast cells cultured on both PLGA and HNT-doped PLGA fibrous scaffolds were compared through MTT assay of cell viability and SEM observation of cell morphology. And protein adsorption behavior on nanofibrous scaffolds was investigated. Similar to electrospun PLGA nanofibers, HNTs-doped PLGA nanofibers were able to promote cell attachment and proliferation and fibroblasts displayed a phenotypic shape, suggesting that the incorporation of HNTs within PLGA nanofibers does not compromise the biocompatibility of the PLGA nanofibers. In addition, compared with PLGA, HNTs-doped PLGA scaffolds allow more protein adsorption, which is favorable for cell adhesion and proliferation. The developed electrospun HNT-doped composite fibrous scaffold may find applications in tissue engineering and pharmaceutical sciences.
As a popular method, MTT colorimetric assay is often used to evaluate the viability of cells cultured onto various fibrous tissue engineering scaffolds. While sometimes the cell viability determined from MTT assay is relatively lower than other characterization method, which probably due to the strong dye sorption capability of porous scaffolding materials. Therefore, we proposed that the OD values obtained from MTT assay likely has a false negative result. In this study, the adsorption of MTT formazan onto porous electrospun PLGA based nanofibrous mats was investigated. We show that PLGA, and the HNTs-or CNTs-doped PLGA nanofibers display appreciable MTT formazan dye sorption, corresponding to35.58～50.23%deviation from the real cell viability assay data. By quantifying the MTT formazan dye sorption amount, the MTT assay data could be rectified to reflect the real cell viability. Our study provides a general insight into the deviation and rectification of MTT cell viability assay, which is likely applicable to other colorimetric assays of different porous scaffolding materials for various biomedical applications.
On the basis of previous characterization of electrospun PLGA and HNTs/PLGA nanofibers, electrospun TCH/HNTs/PLGA double container drug delivery systems were fabricated. The morphology, structure, fiber diameter, mechanical properties, in-vitro biocompatibility, drug release behavior and antibacterial activity of the drug-loaded composite nanofibrous mats were investigated. The results showed that the mechanical properties of TCH/HNTs/PLGA fibers were also improved by comparing with PLGA fibers. And drug-loaded fibrous mats had good compatibility for the adhesion and proliferation of L929mouse fibroblasts. Compared with drug-loaded TCH/HNTs and TCH/PLGA nanofibers with obvious burst release, electrospun TCH/HNTs/PLGA double container nanofibrous drug delivery system prolonged the release rate of the drug and appreciably eliminated the initial burst release, and sustained release was obtained for more than one month. The TCH-medicated HNTs/PLGA scaffolds displayed effective activity to inhibit the growth of S. aureus both in liquid medium and on solid medium in vitro. These medicated PLGA-based electrospun scaffolds hold a promising potential in wound dressing applications and in preventing in vivo postsurgical adhesions and infections.
Electrospun DOX/CNTs/PLGA drug-loaded nanofibrous mat is an antitumer formulation. For this formulation, the morphology, structure, and mechanical properties of electrospun CNTs/PLGA composite nanofibers and DOX/CNTs/PLGA drug-loaded nanofibers were investigated. Similar to electrospun CNTs/PLGA composite nanofibrous mats, DOX (1,2%)/CNTs/PLGA drug-loaded nanofibrous mats could improve the mechanical properties of PLGA fibers. In addition, DOX/CNTs/PLGA double container nanofibrous drug delivery system prolonged the release rate of drug-loaded DOX/CNTs and appreciably eliminated the initial burst release, and sustained release was obtained. The DOX/CNTs/PLGA and DOX/PLGA drug-loaded scaffolds displayed effective anticancer bioactivity on KB cells. In summary, this drug delivery system with controllable release will find various applications in cancer treatment area.
引文
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