| In the past decade,DNA nanotechnology has experienced rapid development,which provides great promise for developing smart drug delivery systems with DNA nanostructures.DNA is inherently biocompatible,soluble and biodegradable.Highly specific base-pairing rule allows bottom-up self-assembly of DNA strands into structures with predictable size,shape and dimension.Importantly,these nanostructures can be functionalized with chemical drugs and biomolecules with precise control within the nanometer scale.Therefore,DNA nanostructures possess incomparable advantages over other nanomaterials for targeted drug delivery in vivo.Herein,My work focus on the cellar uptake,membrane transport process and regulatory mechanism of nanoparticles.Cell imaging was used as the main research method.Laser scanning confocal microscopy,flow cytometry,total internal reflection fluorescence microscopy and dark field microscopy were used to systematically qualitatively and quantitatively analyzed the process of endocytosis,intracellular distribution and vesicle transport of DNA nanostructures.The complete endocytosis and transport process were revealed intuitively.The main results are as follows:(1)We investigated the effects of serum(FBS)on cellular uptake behaviors of DNA tetrahedron(TDNs).Fluorescence labeled TDNs were synthesized by selfassembly and purified with HPLC to achieve a purity of 95%.By means of flow cytometry and confocal microscopy,the kinetics of TDNs endocytosis in HeLa cells in the presence or absence of FBS was compared.The results showed that DNA tetrahedron remained intact in complete medium and cell lysate for more than 12 hours.FBS increased the amount of internalized TDNs in He La cells,whereas it did not change the route of caveolin-dependent endocytosis.Herein,our study provides new insight into the effects of biomolecules on the interaction between cells and DNA nanostructures,which helps future development of DNA-based intracellular nanocarriers.(2)Systematic and systematic study the cell distribution and intracellular dynamic transport process of a tetrahedron-based high-order DNA structure.A dendrimer superstructures G1 was assembled by five DNA tetrahedra as a central tetrahedron linked with four surrounded ones by Single stranded hybridization.G1 accumulated in the cells over time,eventually most of them were distributed in lysosomes,Whereas exocytosis occured.G1 entered the cell through the caveolindependent endocytosis pathway,through the early endosome,the late endosome,and eventually accumulated in the lysosome.G1 transported along the microtubules in the cells.Our work systemic analyzed the movement of G1 with single-particles tracking and entire vesicle system.(3)We investigated DNA nanostructures interaction with cell membrane during cellular internalization by combining molecular simulations with experimental verifications.Tetrahedral DNA nanostructures(TDNs)of three different sizes(with 13,20 or 32 base pairs in each edge,referred as TDN13,TDN20 and TDN32 respectively)were designed and fabricated.Two TDN20 structures were connected via hybridization of complementary single strand DNA to form a linear dimeric structure(L-TDN20).A 6-helix DNA tubular nanostructure with 20 base pairs(6-helix20)was also synthesized.The TDN20 entered He La cells through caveolae-mediated endocytosis.Since the way of point-attacking and the specific-region choice was very similar among these TDNs,there may existed little difference of uptake efficiency between different sizes TDNs.The uptake efficiency of 6helix-20 and L-TDN20 would become a bit lower when compared with TDN20.Our experimental results perfectly match the DPD simulation results.(4)Nanomaterials got new properties with cancer cell membrane-coated by two ways of sonic and extruded.New materials were homogeneously by characterized with TEM,DLS,and western blot.Membrane specific proteins were involved.Cancer cell membrane coated AuNPs(CCP)had a faster uptake efficiency compare to bare AuNPs,with different endocytosis ways. |
PDF Full Text Request