【作者】 杨晔；

【导师】 李孝红；

【作者基本信息】 西南交通大学 ， 材料学， 2010， 博士

【摘要】 电纺丝是一种使带电荷聚合物溶液或熔体在静电场中射流来制备聚合物超细纤维的技术,纤维直径可控制在几个纳米到数十微米之间。电纺纤维由于具有高比表面积和类似于天然细胞外基质的纳米尺度结构,在药物释放载体及组织工支架方面显示出很大的应用潜力。将携载生物活性大分子的电纺纤维作为组织工程支架,在为细胞增殖和迁移提供支撑的同时,可通过释放生物活性物质调控细胞功能、促进细胞外基质分泌以及加快组织再生。然而,由于多数生物活性物质,例如生长因子和核酸等,并不具有可纺性,同时稳定性低,因此制备携载生长因子和核酸的纤维支架就需要在制备及使用过程中有效保护其结构与生物活性、调节其释放行为。据此,本论文以乳液静电纺丝技术制备具有芯壳结构的载蛋白质或基因纤维,研究蛋白质或基因的释放行为与纤维基质材料组成以及其蛋白质或基因在纤维中存在状态之间的关系,以期得到可持续、可控并有效释放蛋白质或基因的纤维释放体系。最后,将乳液电纺法制备的包裹碱性成纤维细胞生长因子(bFGF)和其表达质粒(pbFGF)的电纺纤维膜,应用于大鼠糖尿病皮肤损伤的治疗。以溶菌酶为模型蛋白,使用乳液静电纺丝法制备了载蛋白质的聚乳酸(PDLLA)纤维。经扫描电子显微镜、透射电子显微镜以及激光共聚焦微镜观察,纤维呈现芯壳结构,溶菌酶被作为芯层包裹于聚合物纤维内部。通过电泳、高效液相、红外光谱、酶活检测以及体外释放实验研究表明,芯壳纤维结构可以抑制突释,并且在释放过程中保护溶菌酶的结构完整性以及生物学活性。在载溶菌酶电纺纤维中,以提高包裹效率、结构完整性和生物活性保持率为目标,优化了乳液电纺体系参数,如聚合物组成、水油相体积比与蛋白质稳定剂等,溶菌酶包裹效率达94.0%,酶比活力保持率为64.6%。载溶菌酶纤维在体外释放过程中,释放速率受纤维结构塌陷加速释放、与纤维粘连减少有效释放面积减慢释放的竞争性作用影响。以优化参数制备的包裹量为0.46%的载溶菌酶聚乳酸-聚乙二醇嵌段共聚物(PELA-10, PEG含量为10%)纤维,在体外释放过程中的突释量仅有近6%,且在约5周时间内持续释放。根据糖尿病皮肤溃疡治疗时生长因子的用量要求,制备包裹量为0.079±0.015‰的载bFGF电纺纤维,体外释放中突释量为14.0±2.2%,在25天内释放持续。通过细胞增殖检测、细胞伊红染色、分泌胶原的天狼猩红染色和蛋白质免疫印迹法检测,结果表明与浸润自由bFGF的细胞培养板和空白纤维膜相比,载bFGF纤维对鼠胚胎成纤维细胞(MEF)的粘附、增殖和Ⅰ型胶原分泌起到持续的促进作用。以绿色荧光蛋白表达质粒(pEGFP-N2)为模型,分别以裸质粒或与阳离子聚合物聚乙烯亚胺(PEI)形成复合粒子的方式,通过乳液静电法包裹于PELA-10纤维或PELA-10与PEI的共混纤维中。载质粒或复合粒子的纤维具有芯壳结构,电泳和细胞转染结果显示,纤维的芯核结构能够保护质粒或复合粒子不被降解。携载裸pDNA电纺纤维由于缺乏转染载体,不能对接种于纤维膜上的细胞进行有效转染；以PEI和聚合物共混物作为纤维基质材料携载裸pDNA时,显示出较高转染效率,但是对细胞的生长具有明显的毒性；包裹PEI-pDNA复合粒子的电纺纤维释放速率缓慢,但是可以有效转染接种于纤维膜上细胞,并且对细胞的毒性较小。为调节携载pDNA-PEI复合粒子电纺纤维中复合粒子的释放行为,在纤维基质材料中加入聚乙二醇(PEG)制备复合纤维。随着PEG分子量和含量的增加,复合粒子的突释量增加,持续释放时间相应减短,通过调节PEG的分子量和用量,释放持续时间可控制在1周到1月的范围内。虽然加入聚合物纤维的PEG改善了纤维的亲疏水性,但纤维释放的复合粒子转染细胞对细胞的粘附与增殖都有影响。与未添加PEG电纺纤维的低转染水平相比,含PEG电纺纤维能持续转染细胞并表达绿色荧光蛋白。根据糖尿病皮肤溃疡治疗的时间要求,在优化条件下制备了携载pbFGF-PEI复合粒子的PELA和PEG复合纤维,复合粒子可以在4周内持续释放,接种于纤维膜上的MEF细胞持续表达bFGF。和浸润pbFGF复合粒子的细胞培养板以及空白纤维膜相比,接种于包裹复合粒子纤维膜上的细胞,其增殖受转染引起的细胞毒性、bFGF表达引起的促进增殖的争竞性影响。在建立糖尿病大鼠皮肤溃疡模型的基础上,将载bFGF电纺纤维、载pbFGF-PEI复合粒子电纺纤维分别覆盖于糖尿病大鼠的皮肤缺损处时,研究对组织修复的促进作用。纤维膜在创伤愈合中主要起三方面作用：抑菌敷料、细胞生长支架以及生长因子或其质粒的释放载体。包裹bFGF的方式可以避免组织液中蛋白水解酶对生长因子的降解作用,而通过持续释放pbFGF-PEI复合粒子,可在创面持续表达bFGF。从新生微血管形成、细胞增殖、胶原分泌量及结构、创面再上皮化速度、皮肤附属器管生长等方面分别研究了皮肤修复过程,结果表明加快了创面各修复阶段的进程,显著缩短了创面愈合时间,新生上皮具有与正常皮肤类似的结构特征。综上所述,本论文首次系统地研究了以乳液静电纺丝法构建载蛋白质或基因等生物活性大分子的芯壳结构纤维,并成功应用于糖尿病皮肤溃疡的治疗。通过对制备体系、释放机制及与细胞相互作用的深入研究,得到具有包裹效率高、结构完整性和生物学活性保持率好、释放和转染可控的载生物活性大分子电纺纤维,显著提高了组织缺损的愈合速度和质量,为进一步应用提供了理论及实验研究基础。更多还原

【Abstract】 Electrospinning is a novel processing technique for the production of polymer fibers with diameter of several nanometers and tens micrometers from electrically charged liquid jets of polymer solutions or melts under static electric field. Electrospun nanofibers have gained widespread interests for drug deliver carriers and tissue engineered scaffolds because their high specific surface area and nanoscale stucture are beneficial to drug release. Integrated with the biomimicity to the morphology of extracellular matrix (ECM) and the loading capacity of bioactive substances, electrospun fibers show potentials as inductive tissue engineering scaffolds. The scaffold mediated delivery could maintain a relatively higher concentration of bioactive substances around the cell surface, which can not only support the cell proliferation and migration, but also maintain the cell functions and phenotype-specific activities, enhance the ECM secretion, and promote the tissue regeneration. However, due to the nonelectrospinnable and low stability of most bioactive agents, such as growth factor and nucleic acid, the challenges for growth factor or nucleic acid loaded fibrous scaffold are to maintain the sturcture integrity and bioactivity, and regulate the release profile. In this thesis, core-shell structured fibers with protein or gene encapsulated were prepared by emulsion electrospinning. To achieve highly sustainable, controllable, and effective protein or gene releasing, the relationships were closely determined between the release profiles and matrix polymer components and the existing status of proteins or genes within fibers. Finally, fibrous mats with the encapsulation of basic fibroblast growth factor (bFGF) or bFGF eukaryotic expression plasmid (pbFGF) were prepared and evaluated as inductive skin tissue engineering scaffolds.Lysozyme was chosen as model protein and encapluated within poly(DL-lactide acid) (PDLLA) ultrafine fibers by emulsion electrospinning. Images of scanning electron microscope (SEM), transmission electron microscope (TEM), and laser scanning confocal microscope (LSCM) showed that the obtained fibers were core-shell-structured, and the lysozyme was indeed encapsulated within the polymer shell. Through the analyses of sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE), high performance liquid chromatography (HPLC), fourier transfor infrared spectrum (FTIR), enzyme bioactivity, and in vitro release, the core-shell-structured ultrafine fibers exhibited inhibitive effects on burst release and protective effects on proteins from structure rearrangement and inactiviation.The process parameters of emulsion electrospinning, such as the matrix polymer, the emulsion components, the volume ratio of emulsion phases and the addition of protein stabilizers, were evaluated to enhance the encapsulation efficiency, structure integrity, and bioactivity retention of lysozyme encapsulated. With the optimization of process parameters, the lysozyme encapsulation efficiency of 94.0% and the spectific activity retention of 64.6% were achieved. A gradual release, which was determined by a competition of fiber collapse leading to accelerated release and fiber fusion leading to decelerated release, was determined for the optimized fibers. Only around 6% of the burst release was detected from poly(ethylene glycol)-poly(DL-lactie) (PELA) fibers with 0.46% of the lysozyme loading, followed by sustained releasing for over 5 weeks.Based on the bFGF dosage for diabetic ulcler treatment, fibrous scaffolds containning 0.079±0.015‰of bFGF were prepared by emulsion electrospinning. A burst release of 14.0±2.2% was detected during initial 12 hours, followed by a sustained release for 25 days. Compared with the tissue culture plate (TCP) and blank PELA-10 fibrous mats containing free bFGF, bFGF loaded fibrous mats exhibited much more sustainable promotions on the adhension, proliferation and type Icollagen secretion of mouse embryo fibroblast (MEF).Green fluorescent protein (GFP) eukaryotic expression plasmid (pEGFP-N2) was encapsulated as naked or condensed state into polymer ultrafine fibers of PELA-10 or mixture of PELA-10 and polyethyleneimine (PEI) by emulsion electrospinning. The pDNA or pDNA-PEI polyplexes loaded fibers were core-sheath structured. With the analyses of agarose gel electrophoresis (AGE) and cell transfection, it exhibited that the core-shell structure of polymer fiber could protect the pDNA or pDNA polyplexes from digestion, and the condensation with PEI can promote the cellular entry and transfection efficiency. It indicated that the addition of hydrophilic PEI into matrix material could accelerate the release of pDNA from pDNA/PELA-10-PEI fibers, leading high cytotoxicity. While, the slow release rate of pDNA-PEI polyplexes from pDNA-PEI/PELA-10 fibrous mats led a balance of transfection efficiency and cell viability.In order to regulate the release rate of pDNA polyplexes from PELA-10 fibers, PEG was incorporated into the matrix materials. The effective release lifetime of pDNA polyplexes from fibers could be controlled between 6 and 25 d after incubation, dependent on the loading amount and molecular weights of PEG. Although the PEG addition enhanced the surface wettability of electrospun fibers, the invasive transfection of pDNA polyplexes released from fibers affected the attachment and proliferation abilities of cells during initial incubation. Compared with the relatively low transfection level of fibers without PEG addition, the sustained release of pDNA polyplexes from fibers with PEG inoculations led a persistent and increasing target protein expression. The bFGF eukaryotic expression plasmid (pbFGF) was condensed by PEI, and entrapped into core-sheath structured PELA-10 fibers with PEG blended. Based on the duration for diabetic ulcer healing process, pbFGF-PEI/PELA-PEG fibrous mat, which exhibit susutained release profile of pDNA polyplexes for nearly 1 month, were investigated on the transfection effeciency on MEF cells. Compared with the free pbFGF polyplexes infiltrated TCP or blank fibrous mats, the proliferation of cells seeded on the pbFGF-PEI/PELA-PEG fibrous mats were determined by a competition of invasive transfection leading cytotoxicity and bFGF expression leading promotive enhancement.In vivo examination had been made to guide the dermal regeneration after the covery of pbFGF-PEI/PELA-PEG fibrous scaffold or bFGF-CD/PELA-10 fibrous scaffold on the diabetic ulcer wounds. The bFGF or pbFGF-PEI polyplex loaded fibrous scaffold might play a triple role as an antimicrobial dressing, cell cultrue substrate, and delivery vehicle of bFGF or pbFGF-PEI polyplexes. Compared with free bFGF infiltrated blank PELA-10 fibrous scaffolds, bFGF-CD/PELA-10 fibrous scaffold led a much quicker wound healing process, through the promotion of inflammatory cell infiltration, angiogenesis, extracellular matrix secretion, and re-epithelialization. The persistent bFGF expression from fibrous scaffolds pbFGF-PEI/PELA-PEG indicated similar wound healing results.In conclusion, emulsion electrospun core-shell-structured ultrafine fibers were firstly investigated as carriers of proteins or genes, and the obtained scaffolds were successfully applied for treatment of diabetic ulcers. With the optimization of fabrication process and release profile, the growth factor and its eukaryotic expression plasmid could be loaded in polymer fibers with high ecapsulation efficiency, improved structure integrity and bioactivity retention, and contollable release rate and transfection efficiency. These results should provide solid theoretic and experimental bases for further investigations on fibrous scaffolds with the loading of bioactive substances for biomedical applications.更多还原