Investigation On Electrospun Ultrafine Biodegradable Fibers As Potential Carriers Of Plasmid DNA

Electrospinning technology as a novel type of materials processing method,has many excellences,such as relative ease of use,adaptability,and the ability to fabricate fibers with diameters on the nanometer to micrometer scale.Furthermore, the electrospinning process affords the opportunity to engineer scaffolds with micro to nanoscale topography and high porosity similar to the natural extracellular matrix. The inherently high surface to volume ratio of electrospun scaffolds can enhance cell attachment and drug loading.In the current study,electrospun ultrafine fibers were investigated as carriers of plasmid DNA,and the loading efficiency and sustained release behaviors,the structural intergrity and transfection efficacy of entrapped DNA were examined in detail.The competent Escherichia coli bacteria cells were obtained by CaCl₂ method. pEGFP-N₂（4737 bp） encoding green fluorescent protein（GFP） was amplified in a transformant of competent bacteria and isolated and purified from the bacteria by Qiagen kit.The purified plasmid was verified by UV and gel electrophoresis.Poly（DL-lactide）-poly（ethylene glycol）（PELA） ultrafine fibers containing pEGFP-N₂ were prepared by emulsion electrosipnning method.The obtained DNA/PELA fibers indicated randomly arrayed with average diameter of 440±150 nm and DNA loading efficiency of 92.7±3.8%.The labeled fibers were examined with laser confocal scanning microscope to visualize the DNA distribution in the elctrospun fibers,indicating a core-sheath structure.The core-sheath composite fibers released DNA at a constant rate for 35 days with a burst release of 29.1%in initial 12h.Compared with free DNA,the plasmid encapsulated within the polymer shell retained much higher content of supercoiled structure and transfer efficiency after incubation into the media,indicating the protective effect of the core-sheath structure.Poly（ethylene imine）（PEI） was added in the oil and inner aqueous phase, repectively,to obtain ultrafine fibers of DNA/PELA-PEI and DNA-PEI/PELA by emulsion electrospinning.Compared with DNA/PELA fibers,the morphologies indicated no significant changes after PEI addition either in the oil or in the inner aquous phase.In vitro release of DNA from DNA/PELA-PEI andDNA/PELA indicated similar profiles,but that from DNA-PEI/PELA fibers showed substantial initial burst release followed by no signifcant sustained release.Cell attachment and proliferation assay illustrated that the addition of PEI had certain toxic effects on cells.Cell transfection experiments showed that transfected cells appeared different levels on DNA/PELA-PEI and DNA-PEI/PELA fibers,but not on DNA/PELA fibers. The transfection efficiency was related with some factors,such as the incorporated mode and amount of PEI,the release rates of DNA and PEI.In summary,core-sheath structured ultrafine fibers with encapsulation of plasmid DNA were successfully prepared by emulsion electrosipnning.The obtained results provided a basis for further exploration of the controlled release,structural integrity and bioactivity of the encapsulated DNA,which would find better use of functional electrospun fibers as drug carriers and tissue engineering scaffolds and offer a new way of thinking for the development of gene therapy.