Investigation On Core-Sheath Structured Electrospun Fibers For Controlled Release Of Bioactive Macromoleculs

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.