The Application Research Of Nuclear Pore Membrane In Inducing Lipid Vesicle Membrane Fusion And Intracellular Delivery

Nuclear track membranes are membrane filter with uniform pore size and cylindrical pore shape,are extensively used in precision filtration.Additionally,nuclear track membrane(100-300 nm)-based extrusion is used for controlling the particle size of liposomes.Herein,we explore whether the extrusion induces fusion of FRET pairs-labeled liposomes and blank liposomes,furthermore,whether extrusion induces fusion of FRET pairs-labeled liposomes and exosomes.Besides,based on the progress of physical methods for intracellular delivery,we assumed that nuclear track membrane filter(7-11 μm)-based extrusion can induce cell membrane permeabilization and be used for intracellular delivery of macromolecules.FITC labeled dextran was delivered into CT26 and K562 by extrusion,and the influence of pore size and the molecular size of dextran on delivery efficiency were studied.Further,whether dextran of different molecular weight was trafficked into the nucleus and trafficking pathway were investigated.The methods and results are as following:1)Membrane fusion of fluorescent liposomes and blank liposomes by extrusionBlank liposomes and FRET pairs-labeled liposomes were prepared,mixed,and treated by extrusion and freeze-thawing.Particle size was assayed by dynamic light scattering(DLS),the phenomenon of lipid bilayer fusion was observed by CLSM,and membrane fusion rate was calculated.In addition,the effects of extrusion times,pressure,and temperature on membrane fusion rate were investigated.It’s shown that extrusion induced membrane fusion of the liposomes mixture,which was confirmed by enhancement of NBD fluorescence intensity in CLSM images,and membrane fusion rate was 26%for 75 times extrusion.By contrast,membrane fusion rate was 26%and 45%for two freeze-thawing protocols.However,liposomes treated by extrusion has a more uniform particle size than by freeze-thawing.Also,low extrusion pressures,37℃,and more extrusion times are beneficial to improve membrane fusion rate.2)Engineering hybrid exosomes by extrusionA mixture of FRET pair-labeled liposomes and exosomes isolated from the supernatant of K562 by supercentrifugation was extruded.Exosomes and hybrid exosomes were observed by transmission electron microscopy(TEM),membrane fusion phenomenon was observed by CLSM and quantified by fluorescence spectrometer,and particle size was detected by DLS.Membrane fusion of liposomes and exosomes was confirmed by CLSM,and hybrid exosomes retained classical saucer-like morphology under TEM,The lipid dilution ratio was 0.74 by extrusion,and lower than that by freeze-thawing.However,hybrid exosomes produced by extrusion showed a more uniform particle size than by freeze-thawing.3)Intracellular delivery FITC-dextran into CT26 by extrusionNuclear track membrane filter(8-11 μm)-based extrusion was used to induce CT26 cell deformation and transient membrane perforation to introduce dextran of different molecular weight.The delivery efficiency was analyzed by flow cytometry.Intracellular localization and subcellular distribution of FITC-dextran were observed by CLSM.The effects of pore size and MW of dextran on delivery efficiency were investigated.It’s confirmed that dextran(3 kDa,70 kDa,2 MDa)was introduced into CT26 by extrusion.The pore size of nuclear track membrane and MW of dextran have a negative correlation with delivery efficiency.Dextran of 3 kDa or 70 kDa were more evenly distributed than dextran of 2 MDa between cytoplasm and nucleus.4)Intracellular delivery FITC-dextran into K562 by extrusionNuclear track membrane filter(7-10 μm)-based extrusion was used to induce K562 cell deformation and transient membrane perforation to introduce dextran of different molecular weight.The delivery efficiency was analyzed by flow cytometry,the subcellular distribution of FITC-dextran was observed by CLSM and cytoplasmic fluorescence intensity/nuclear fluorescence intensity ratio(Fcyt/Fnuc ratio)was analyzed by ImageJ.The effects of pore size of nuclear track membrane and MW of dextran on delivery efficiency were investigated.The pore size of nuclear track membrane and MW of dextran have a negative correlation with delivery efficiency.Dextran of 2 MDa was trafficked into the nucleus,which suggests extrusion induced rupture of nuclear envelope and 2 MDa dextran whosehydrodynamic radii is larger than the upper limit of diffusion channel of nuclear pore complex(NPC)was allowed to penetrate NPC.In summary,nuclear track membrane(100-300 nm)-based extrusion induced membrane fusion of lipid vesicles and engineered hybrid exosomes;also,nuclear track membrane(7-11 μm)-based extrusion induced cell membrane perforation and introduced dextran of different molecular weight into CT26 and K562.