电纺制备封装水溶性纳米粒子的复合纳米纤维及其在药物传输和生物成像领域的应用
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摘要
静电纺丝纳米纤维因其具有高比表面积、高孔隙率、小尺寸效应及表面效应，作为缓控释载体传递抗肿瘤药物用于癌症术后的局部化疗受到广泛的关注。随着静电纺丝技术的兴起和可降解生物相容性高分子材料的大量涌现，开发新型基于高分子材料的纳米纤维药物输送系统成为生物医学应用领域中的一个富有挑战性的课题。构建新型聚合物纳米纤维支架在肿瘤术后局部直接给予多种具有不同药理活性和作用机制的药物，控制药物时序性和差异性的释放，发挥药物联合应用的协同增效作用；构建具有抗肿瘤和生物成像多功能的复合纳米纤维支架，对组织工程支架和肿瘤细胞之间的相互作用及术后治疗的全过程进行实时示踪监测，这些对于肿瘤术后治疗具有重要的意义。
基于此，本文构建一种新型封装载药胶束的PLGA复合纳米纤维，同时装载两种疏水性药物(紫杉醇和布雷菲德菌素A)，实现在同一载体中有效地控制两种疏水性药物的梯度释放；构建一种新型掺杂水溶性富勒烯荧光纳米粒子的载药复合纳米纤维，研究载药复合纳米纤维的体外抗肿瘤活性及生物成像作用，以期为肿瘤的术后治疗提供全新的给药系统选择。
本论文开展的主要研究工作及其结果如下：
(1)以两亲性嵌段聚合物MePEG-PLLA为载体材料，制备负载抗肿瘤药物布雷菲德菌素A (BFA)的化学键合型和物理包埋型两类聚合物胶束。化学键合型胶束(MePEG-PLLA/BFA)的载药率为4.3%，平均粒径为120nm；物理包埋型胶束(BFA-PM)的载药率为4.8%，平均粒径为160nm。胶束呈壳-核结构的球形粒子，形态规整，分散均匀。在两类载药胶束的体外药物缓释曲线中，药物没有明显的突释现象，BFA可以长时间的控制释放，化学键合型胶束的释药速度较慢，呈现一级动力学释药。MTT法体外评价两类载药胶束对人肝癌细胞HepG-2的细胞毒性，结果说明药物从聚合物胶束缓释且没有失去其细胞毒性，有效抑制肿瘤细胞HepG-2的增殖。
(2)应用静电纺丝技术成功制备负载布雷菲德菌素A的PEG-PLLA纳米纤维缓释制剂(BFA/PEG-PLLA)。负载BFA纳米纤维的平均直径低于950nm，分布均匀，表面光滑，药物以无定形均匀分散在纳米纤维中。载药纳米纤维BFA/PEG-PLLA的体外药物缓释实验结果表明纳米纤维膜对药物BFA有很好的缓释效果，可以长时间的控制释放。MTT法体外评价负载3wt.%，6wt.%，9wt.%，12wt.%和15wt.%BFA的纳米纤维对人肝癌细胞HepG-2的细胞毒性，肿瘤细胞抑制率在72h分别为64%，77%，80%，81%和85%。
(3)通过“胶束乳液”静电纺丝制备新型封装载药胶束BFA-PM和紫杉醇的PLGA复合纳米纤维，载药胶束乳化后形成的乳滴通过静电纺丝被成功封装到PLGA纤维中。体外药物释放实验结果表明在电纺纳米纤维中药物释放速度由其所处位置决定，药物BFA的缓释需要受到胶束和纤维两层壁垒的阻碍，显现比较缓慢和长期持续的控制释放效果，而紫杉醇只受到PLGA纤维的影响，释放速度相对较快。体外细胞毒性实验研究表明，负载两种疏水性药物的复合纳米纤维能更有效地抑制HepG-2细胞增殖。封装载药胶束的PLGA复合纳米纤维实现了同一载体中有效地控制两种疏水性药物的梯度释放，适用于癌症的术后化疗。
(4)通过简单的共混静电纺丝技术制备新型封装水溶性富勒烯纳米粒子的PLLA荧光复合纳米纤维。纳米纤维分布均匀且表面光滑，平均直径为300~600nm。富勒烯纳米粒子封装到复合纳米纤维中形成壳-核结构，纤维表面显示出优异的亲水性。将富勒烯纳米粒子/PLLA复合纳米纤维作为底物与人肝癌细胞HepG-2共培养，进行复合纳米纤维的体外生物成像作用评价，富勒烯纳米粒子强烈的红色荧光信号显现在每一个HepG-2细胞的细胞核中。此类新型封装水溶性富勒烯纳米粒子的荧光纳米纤维在组织工程与生物成像领域有很好的应用前景。
(5)静电纺丝制备封装水溶性富勒烯荧光纳米粒子(C70-TEGs)和紫杉醇的PLLA复合纳米纤维(PTX/PLLA@C70-TEGs)。纳米纤维分布均匀且表面光滑，平均直径为350~750nm。复合纳米纤维封装水溶性富勒烯纳米粒子后，显示出优异的表面亲水性且力学性能较好。体外药物释放实验结果表明复合纳米纤维中药物紫杉醇的释放速度可通过纳米粒子的含量来调控，随着纳米粒子C70-TEGs含量的增加，药物的释放速度越快，释放总量越多。将PTX/PLLA@C70-TEGs作为底物与人肝癌细胞HepG-2共培养，进行复合纳米纤维的体外生物成像和细胞毒性作用评价，紫杉醇从纳米纤维缓释后能有效抑制肿瘤细胞的增殖，在HepG-2细胞中能明显检测到富勒烯纳米粒子C70-TEGs的信号，清晰反映细胞的生长状态。此类同时具有抗肿瘤和生物成像功能的新型复合纳米纤维，在组织工程、药物传输及生物成像等领域有潜在的应用。
Due to electrospun nanofibers’ high surface area, high porosity, small size effectand surface effect, it is widely used as controlled release carrier of anticancer drugsfor postoperative chemotherapy, which has obtained widespread attention. With thedevelopment of electrospinning techniques and the emergence of variousbiodegradable and biocompatible polymer materials, construction of novel drugdelivery system based on electrospun nanofibers remains a scientific challenge in thefield of biomedical applications. Construction of novel nanofiber scaffolds loadedvarious drugs with distinct characteristics and action mechanisms could be used inlocal tumor, to realize drug releasing in a sequential way and maximizing therapeuticefficacy. Construction of novel multifunctional composite scaffolds for antitumor andbioimaging application could be realized real-time tracing and monitoring theinteraction between tumor cells and the tissue engineering scaffolds, as well as thewhole process of postoperative therapy. These have important implications for tumortherapy after surgery.
In the paper, novel poly(lactic-co-glycolic acid)(PLGA) composite nanofibersencapsulated with drug-loading micelles was successfully fabricated. It couldencapsulate two hydrophobic drugs at the same time (paclitaxel and brefeldin A) andrealize controlled dual release of two hydrophobic drugs with distinct rates in avehicle. Novel composite nanofibers encapsulated with water-soluble fullerenenanoparticles and antitumor drug (paclitaxel) was fabricated via a simpleelectrospinning method. In vitro cytotoxicity and bioimaging of composite nanofiberswas studied in detail, the results strongly suggested that the electrospun compositenanofibers was able to provide a good alternative for cancer postoperativechemotherapy.
The main research contents and results of this thesis are as follows:
(1) Chemical bonding and physical embedding polymeric micelles withamphiphilic block copolymer MePEG-PLLA as carrier material were developed ascontrolled release systems for macrolide antibiotic drug brefeldin A (BFA). Chemicalbonding polymeric micelles (MePEG-PLLA/BFA) had average diameter of120nm and4.3%drug-loading efficiency of BFA. Meanwhile, physical embeddingpolymeric micelles (BFA-PM) had average diameter of160nm and4.8%drug-loading efficiency of BFA. All polymeric micelles had spherical and core-shellstructures, regular shape and uniform dispersion. The release profiles of BFA in PBSwere measured by HPLC, demonstrating that the drug had no obvious burst releaseand the controlled release of BFA could be gained for long time. The release profileof BFA in chemical bonding polymeric micelles was a first-order release, slower thanthat of physical embedding polymeric micelles. The in vitro antitumor activity ofpolymeric micelles against human liver carcinoma HepG-2cells was evaluated byMTT method, and the results showed that BFA could be released from the micelleswithout losing cytotoxicity and inhibited HepG-2cells proliferation effectively.
(2) PEG-PLLA electrospun nanofibers were developed as a new controlledrelease system for macrolide antibiotic drug brefeldin A (BFA). The average diameterof the BFA-loaded PEG-PLLA fibers was below950nm with smooth surfaces, andthe drug was well incorporated into the fibers in amorphous form. The release profilesof BFA in PBS were measured by HPLC, demonstrating that the fibers had a goodslow-release effect of the drug BFA and the controlled release of BFA could begained for long time. The in vitro antitumor activity against human liver carcinomaHepG-2cells of the fibers contained3wt.%,6wt.%,9wt.%,12wt.%and15wt.%BFA were examined by MTT method, and the results showed that cell growthinhibition rates at72h were64%,77%,80%,81%and85%, respectively.
(3) By means of “emulsion-electrospinning”, paclitaxel (PTX) and polymericmicelles containing BFA were successfully loaded into the electrospun PLGAcomposite nanofibers. The emulsion droplets containing drug-loaded polymericmicelles was produced by emulsification of micelles, and then successfullyencapsulated into PLGA fiber. The in vitro release results demonstrated that thelocation of the drugs in the electrospun fibers determined their release profiles. PTX,loaded in the PLGA polymer matrix directly, was released in a relatively rapid rate,while BFA showed a long-term and sustained release behavior due to hindrance fromMePEG-PLLA micelles and PLGA matrix. In vitro cytotoxicity studies revealed thatthe composite nanofibers with two drugs restrained HepG-2cells more efficiently.These results strongly suggested that the electrospun composite nanofibers containing polymeric micelles could realize controlled dual release of two hydrophobic drugs withdistinct rates in a vehicle and were suitable for postoperative chemotherapy of cancers.
(4) A novel fluorescent nanofibrous material, consisted of water-solublefullerene nanoparticles and poly(L-lactide)(PLLA) was fabricated via a simpleelectrospinning method. The nanofibers were uniform and their surfaces werereasonably smooth, with the average diameters of fibers ranging from300to600nm.The fullerene nanoparticles were encapsulated within the composite nanofibers,forming a core-shell structure. The nanofiber scaffolds showed excellent hydrophilicsurface due to the addition of water-soluble fullerene nanoparticles. The compositenanofibers used as substrates for bioimaging in vitro were evaluated with human livercarcinoma HepG-2cells, the fullerene nanoparticles’ intense red fluorescence signalalmost displayed in every HepG-2cell nucleus, implying the potential of fluorescentfullerene nanoparticles/PLLA nanofibers to be used as scaffolds for tissueengineering and bioimaging application.
(5) Novel PLLA composite nanofibers (PTX/PLLA@C70-TEGs) encapsulatedwith water-soluble fullerene nanoparticles C70-TEGs and paclitaxel (PTX) wassuccessfully fabricated. The nanofibers were uniform and their surfaces werereasonably smooth, with the average diameters of fibers ranging from350to750nm.The nanofiber scaffolds showed excellent hydrophilic surface and good mechanicalproperties. The in vitro release results demonstrated that the release rate of paclitaxelcould be controlled by the content of nanoparticles. With increasing the content ofC70-TEGs nanoparticles, the drug release rate was faster and the total release wasmore. The composite nanofibers used as substrates for cytotoxicity and bioimaging invitro were evaluated with human liver carcinoma HepG-2cells, and the resultsshowed that PTX inhibited HepG-2cells proliferation effectively after controlledrelease from the composite nanofibers. Meanwhile, the fluorescent signal of fullerenenanoparticles C70-TEGs could be detected in HepG-2cell nucleus, which reflected thegrowth state of cells clearly. These results strongly suggested that the potential ofcomposite nanofibers PTX/PLLA@C70-TEGs could be used as scaffolds for tissueengineering, drug delivery and bioimaging application.
基于此，本文构建一种新型封装载药胶束的PLGA复合纳米纤维，同时装载两种疏水性药物(紫杉醇和布雷菲德菌素A)，实现在同一载体中有效地控制两种疏水性药物的梯度释放；构建一种新型掺杂水溶性富勒烯荧光纳米粒子的载药复合纳米纤维，研究载药复合纳米纤维的体外抗肿瘤活性及生物成像作用，以期为肿瘤的术后治疗提供全新的给药系统选择。
本论文开展的主要研究工作及其结果如下：
(1)以两亲性嵌段聚合物MePEG-PLLA为载体材料，制备负载抗肿瘤药物布雷菲德菌素A (BFA)的化学键合型和物理包埋型两类聚合物胶束。化学键合型胶束(MePEG-PLLA/BFA)的载药率为4.3%，平均粒径为120nm；物理包埋型胶束(BFA-PM)的载药率为4.8%，平均粒径为160nm。胶束呈壳-核结构的球形粒子，形态规整，分散均匀。在两类载药胶束的体外药物缓释曲线中，药物没有明显的突释现象，BFA可以长时间的控制释放，化学键合型胶束的释药速度较慢，呈现一级动力学释药。MTT法体外评价两类载药胶束对人肝癌细胞HepG-2的细胞毒性，结果说明药物从聚合物胶束缓释且没有失去其细胞毒性，有效抑制肿瘤细胞HepG-2的增殖。
(2)应用静电纺丝技术成功制备负载布雷菲德菌素A的PEG-PLLA纳米纤维缓释制剂(BFA/PEG-PLLA)。负载BFA纳米纤维的平均直径低于950nm，分布均匀，表面光滑，药物以无定形均匀分散在纳米纤维中。载药纳米纤维BFA/PEG-PLLA的体外药物缓释实验结果表明纳米纤维膜对药物BFA有很好的缓释效果，可以长时间的控制释放。MTT法体外评价负载3wt.%，6wt.%，9wt.%，12wt.%和15wt.%BFA的纳米纤维对人肝癌细胞HepG-2的细胞毒性，肿瘤细胞抑制率在72h分别为64%，77%，80%，81%和85%。
(3)通过“胶束乳液”静电纺丝制备新型封装载药胶束BFA-PM和紫杉醇的PLGA复合纳米纤维，载药胶束乳化后形成的乳滴通过静电纺丝被成功封装到PLGA纤维中。体外药物释放实验结果表明在电纺纳米纤维中药物释放速度由其所处位置决定，药物BFA的缓释需要受到胶束和纤维两层壁垒的阻碍，显现比较缓慢和长期持续的控制释放效果，而紫杉醇只受到PLGA纤维的影响，释放速度相对较快。体外细胞毒性实验研究表明，负载两种疏水性药物的复合纳米纤维能更有效地抑制HepG-2细胞增殖。封装载药胶束的PLGA复合纳米纤维实现了同一载体中有效地控制两种疏水性药物的梯度释放，适用于癌症的术后化疗。
(4)通过简单的共混静电纺丝技术制备新型封装水溶性富勒烯纳米粒子的PLLA荧光复合纳米纤维。纳米纤维分布均匀且表面光滑，平均直径为300~600nm。富勒烯纳米粒子封装到复合纳米纤维中形成壳-核结构，纤维表面显示出优异的亲水性。将富勒烯纳米粒子/PLLA复合纳米纤维作为底物与人肝癌细胞HepG-2共培养，进行复合纳米纤维的体外生物成像作用评价，富勒烯纳米粒子强烈的红色荧光信号显现在每一个HepG-2细胞的细胞核中。此类新型封装水溶性富勒烯纳米粒子的荧光纳米纤维在组织工程与生物成像领域有很好的应用前景。
(5)静电纺丝制备封装水溶性富勒烯荧光纳米粒子(C70-TEGs)和紫杉醇的PLLA复合纳米纤维(PTX/PLLA@C70-TEGs)。纳米纤维分布均匀且表面光滑，平均直径为350~750nm。复合纳米纤维封装水溶性富勒烯纳米粒子后，显示出优异的表面亲水性且力学性能较好。体外药物释放实验结果表明复合纳米纤维中药物紫杉醇的释放速度可通过纳米粒子的含量来调控，随着纳米粒子C70-TEGs含量的增加，药物的释放速度越快，释放总量越多。将PTX/PLLA@C70-TEGs作为底物与人肝癌细胞HepG-2共培养，进行复合纳米纤维的体外生物成像和细胞毒性作用评价，紫杉醇从纳米纤维缓释后能有效抑制肿瘤细胞的增殖，在HepG-2细胞中能明显检测到富勒烯纳米粒子C70-TEGs的信号，清晰反映细胞的生长状态。此类同时具有抗肿瘤和生物成像功能的新型复合纳米纤维，在组织工程、药物传输及生物成像等领域有潜在的应用。
Due to electrospun nanofibers’ high surface area, high porosity, small size effectand surface effect, it is widely used as controlled release carrier of anticancer drugsfor postoperative chemotherapy, which has obtained widespread attention. With thedevelopment of electrospinning techniques and the emergence of variousbiodegradable and biocompatible polymer materials, construction of novel drugdelivery system based on electrospun nanofibers remains a scientific challenge in thefield of biomedical applications. Construction of novel nanofiber scaffolds loadedvarious drugs with distinct characteristics and action mechanisms could be used inlocal tumor, to realize drug releasing in a sequential way and maximizing therapeuticefficacy. Construction of novel multifunctional composite scaffolds for antitumor andbioimaging application could be realized real-time tracing and monitoring theinteraction between tumor cells and the tissue engineering scaffolds, as well as thewhole process of postoperative therapy. These have important implications for tumortherapy after surgery.
In the paper, novel poly(lactic-co-glycolic acid)(PLGA) composite nanofibersencapsulated with drug-loading micelles was successfully fabricated. It couldencapsulate two hydrophobic drugs at the same time (paclitaxel and brefeldin A) andrealize controlled dual release of two hydrophobic drugs with distinct rates in avehicle. Novel composite nanofibers encapsulated with water-soluble fullerenenanoparticles and antitumor drug (paclitaxel) was fabricated via a simpleelectrospinning method. In vitro cytotoxicity and bioimaging of composite nanofiberswas studied in detail, the results strongly suggested that the electrospun compositenanofibers was able to provide a good alternative for cancer postoperativechemotherapy.
The main research contents and results of this thesis are as follows:
(1) Chemical bonding and physical embedding polymeric micelles withamphiphilic block copolymer MePEG-PLLA as carrier material were developed ascontrolled release systems for macrolide antibiotic drug brefeldin A (BFA). Chemicalbonding polymeric micelles (MePEG-PLLA/BFA) had average diameter of120nm and4.3%drug-loading efficiency of BFA. Meanwhile, physical embeddingpolymeric micelles (BFA-PM) had average diameter of160nm and4.8%drug-loading efficiency of BFA. All polymeric micelles had spherical and core-shellstructures, regular shape and uniform dispersion. The release profiles of BFA in PBSwere measured by HPLC, demonstrating that the drug had no obvious burst releaseand the controlled release of BFA could be gained for long time. The release profileof BFA in chemical bonding polymeric micelles was a first-order release, slower thanthat of physical embedding polymeric micelles. The in vitro antitumor activity ofpolymeric micelles against human liver carcinoma HepG-2cells was evaluated byMTT method, and the results showed that BFA could be released from the micelleswithout losing cytotoxicity and inhibited HepG-2cells proliferation effectively.
(2) PEG-PLLA electrospun nanofibers were developed as a new controlledrelease system for macrolide antibiotic drug brefeldin A (BFA). The average diameterof the BFA-loaded PEG-PLLA fibers was below950nm with smooth surfaces, andthe drug was well incorporated into the fibers in amorphous form. The release profilesof BFA in PBS were measured by HPLC, demonstrating that the fibers had a goodslow-release effect of the drug BFA and the controlled release of BFA could begained for long time. The in vitro antitumor activity against human liver carcinomaHepG-2cells of the fibers contained3wt.%,6wt.%,9wt.%,12wt.%and15wt.%BFA were examined by MTT method, and the results showed that cell growthinhibition rates at72h were64%,77%,80%,81%and85%, respectively.
(3) By means of “emulsion-electrospinning”, paclitaxel (PTX) and polymericmicelles containing BFA were successfully loaded into the electrospun PLGAcomposite nanofibers. The emulsion droplets containing drug-loaded polymericmicelles was produced by emulsification of micelles, and then successfullyencapsulated into PLGA fiber. The in vitro release results demonstrated that thelocation of the drugs in the electrospun fibers determined their release profiles. PTX,loaded in the PLGA polymer matrix directly, was released in a relatively rapid rate,while BFA showed a long-term and sustained release behavior due to hindrance fromMePEG-PLLA micelles and PLGA matrix. In vitro cytotoxicity studies revealed thatthe composite nanofibers with two drugs restrained HepG-2cells more efficiently.These results strongly suggested that the electrospun composite nanofibers containing polymeric micelles could realize controlled dual release of two hydrophobic drugs withdistinct rates in a vehicle and were suitable for postoperative chemotherapy of cancers.
(4) A novel fluorescent nanofibrous material, consisted of water-solublefullerene nanoparticles and poly(L-lactide)(PLLA) was fabricated via a simpleelectrospinning method. The nanofibers were uniform and their surfaces werereasonably smooth, with the average diameters of fibers ranging from300to600nm.The fullerene nanoparticles were encapsulated within the composite nanofibers,forming a core-shell structure. The nanofiber scaffolds showed excellent hydrophilicsurface due to the addition of water-soluble fullerene nanoparticles. The compositenanofibers used as substrates for bioimaging in vitro were evaluated with human livercarcinoma HepG-2cells, the fullerene nanoparticles’ intense red fluorescence signalalmost displayed in every HepG-2cell nucleus, implying the potential of fluorescentfullerene nanoparticles/PLLA nanofibers to be used as scaffolds for tissueengineering and bioimaging application.
(5) Novel PLLA composite nanofibers (PTX/PLLA@C70-TEGs) encapsulatedwith water-soluble fullerene nanoparticles C70-TEGs and paclitaxel (PTX) wassuccessfully fabricated. The nanofibers were uniform and their surfaces werereasonably smooth, with the average diameters of fibers ranging from350to750nm.The nanofiber scaffolds showed excellent hydrophilic surface and good mechanicalproperties. The in vitro release results demonstrated that the release rate of paclitaxelcould be controlled by the content of nanoparticles. With increasing the content ofC70-TEGs nanoparticles, the drug release rate was faster and the total release wasmore. The composite nanofibers used as substrates for cytotoxicity and bioimaging invitro were evaluated with human liver carcinoma HepG-2cells, and the resultsshowed that PTX inhibited HepG-2cells proliferation effectively after controlledrelease from the composite nanofibers. Meanwhile, the fluorescent signal of fullerenenanoparticles C70-TEGs could be detected in HepG-2cell nucleus, which reflected thegrowth state of cells clearly. These results strongly suggested that the potential ofcomposite nanofibers PTX/PLLA@C70-TEGs could be used as scaffolds for tissueengineering, drug delivery and bioimaging application.
引文
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