Fabrication Of Photothermal Coaxial Electrospun Nanofibers For Application In Gene Delivery

Gene delivery refers to the delivery of functional molecules(e.g.plasmid DNA,pDNA)to target cells,which is of vital importance in regulation of cell behavior and gene therapy.In recent years,coaxial electrospun nanofibers have received much attention due to their core-sheath structure and physical and chemical characteristics,which is beneficial for to loading and delivering biomolecules to target cells.However,most of the currently reported gene delivery based on coaxial electrospun nanofibers relies on cell endocytosis,resulting in low delivery efficiency.In addition,it is also difficult to achieve the release of pDNA on demand,restricting the widespread application of coaxial electrospun nanofibers.Upon exposure to near-infrared(NIR)irradiation of appropriate energy,photothermally-responsive materials convert the absorbed light energy to heat,resulting in an increase in local temperature.The permeability of the membranes of exposed cells is thereby enhanced,and the entry of biomolecules such as proteins and pDNA into the cytoplasm is facilitated.The thesis mainly focuses on the design,preparation of coaxial electrospun nanofibers for efficient gene transfection and wound healing.The detailed researches are as follows:(1)Preparation of electrospun nanofibers,optimization of conditions and study of gene delivery mechanism based on photothermal membrane disruption.The sheath layer material is composed of gelatin/poly(L-lactic acid)(named PG for short),and gold nanorods(GNRs)with excellent photothermal properties.The core layer material is composed of composite particles of polyethyleneimine(PEI)and pDNA.Firstly,the concentration of GNR in the electrospun nanofibers and the ratio of N/P of PEI and pDNA(N/P:the molar ratio of nitrogen in PEI to phosphorus in pDNA)were optimized.It was found that under a certain condition:GNR concentration is 0.7 mg/mL and laser intensity is 0.45 W/cm2,the electrospun nanofibers can obtain appropriate light-to-heat conversion capacity as well as keep mechanical strength.In addition,the gene complexes with N/P=30 can achieve the highest transfection efficiency(87%)while maintaining high cell viability(92%).Then,the coaxial electrospun fibers(PG@GNR/pDNA)were prepared under the optimal conditions,and scanning electronic microscopy,transmission electron microscopy,fluorescent microscope,thermal camera and water contact angle goniometer were utilized to characterize the PG@GNR/pDNA electrospun nanofibers.Finally,we investigated the mechanism of photothermal membrane disruption mediated gene delivery.(2)PG@GNR/pDNA electrospun nanofiber was used to transfect certain cells,thereby characterize the proliferation and migration behavior of cells in vitro and the application in wound healing in vivo.For the gene delivery in vitro,pGFP-bFGF(encoding green fluorescent protein(GFP)and fibroblast growth factor(bFGF))and NIH-3T3 fibroblasts were used as model pDNA and cells,respectively.The results of fluorescence staining,flow cytometry,and reverse transcription-polymerase chain reaction(RT-PCR)showed that only PG@GNR/pDNA electrospun nanofibers can achieve high transfection efficiency(>85%)and gene expression after NIR light irradiation.Then,the effect of bFGF gene expression on the proliferation of NIH-3T3 fibroblasts was characterized by fluorescence staining,and the effects on cell migration rate were characterized by Transwell assay,scratch experiments,and live-content imaging.It was demonstrated that the proliferation and migration rate of NIH-3T3 fibroblasts cultured on PG@GNR/pDNA electrospun nanofibers can be significantly accelerated after NIR irradiation.For in vivo studies,a full-thickness skin defect model in rats was established to determine the effectiveness of PG@GNR/pDNA electrospun nanofibers in promoting wound healing.Through photographic observation and the characterization of hematoxylin-eosin staining(H&E)and Masson's trichrome staining,it was found that rat wounds treated with PG@GNR/pDNA electrospun nanofibers after NIR irradiation had a faster recovery rate and the damaged skin could be fully recovered in 12 days.In summary,based on the advantages of the photothermal membrane disruption method,we prepared a coaxial electrospun nanofibers for efficient gene delivery.Coaxial electrospun nanofibers play a role in loading and protecting pDNA,and the photothermal membrane disruption method can achieve efficient delivery of genes to target cells.Under appropriate NIR irradiation,the GNR embedded in the sheath layer of the electrospun nanofibers generated heat to increase the local temperature.On the one hand,the permeability of the electrospun nanofibers was enhanced and pDNA was released from the core layer of the electrospun nanofibers.On the other hand,the increase of temperature creating transient pores on the membranes of attached cells to facilitate intracellular delivery and transfection with pDNA.The electrospun nanofiber,which can achieve a simple loading method and high transfection efficiency,and have great potential in tissue engineering and cell-based therapy.