Research And Application Of Responsive DNA In Regulating Liposome Membrane Fusion

Membrane fusion plays a key role in the process of life.Many common membrane fusion processes like cell-cell fusion,fertilization,and intracellular transport.Inspired by soluble N-ethyl maleimide sensitive factor attachment protein receptors(SNARE),liposome is used as a model platform,different triggers such as DNA and peptides are the driving force to regulate liposome fusion.However,most of the membrane fusion processes are spontaneous and uncontrolled.Therefore,the establishment of one or more external stimuli spatiotemporal controllable membrane fusion process is significant.In this paper,liposomes are used as a model,combined with responsive DNA nanotechnology,to study one and two external stimuli to regulate the fusion of liposome-liposome and their applications.The research content is as follows:First,using o-nitrobenzyl phosphate functionalized-cholesterol tethered nucleic acid-modified liposomes as functional photoresponsive units for inducing,by near infrared light(NIR)irradiation,spatiotemporal liposome-liposome or liposomemembrane fusion processes.The liposomes are loaded with upconversion nanoparticles(UCNPs)and their NIR irradiation(980 nm)yields luminescence at 365 nm,providing a localized light-source to deprotect the o-nitrobenzyl phosphate groups and resulting in the fragmentation of the nucleic acid structures.In one system,the NIR-triggered fusion of two liposomes,L1 and L2,is exemplified.Liposome L1 is loaded with UCNPs and Tb3+ ions,and the liposome boundary is functionalized with a cholesteroltethered,o-nitrobenzyl phosphate caged hairpin nucleic acid structure.Liposome L2 is loaded with 2,6-pyridinedicarboxylic acid,DPA,and its boundary is modified with a cholesterol-tethered nucleic acid,complementary to a part of the caged hairpin,associated with L1.NIR-irradiation of the L1/L2 mixture resulted in the photocleavage of the hairpin structure,associated with L1,and the resulting fragmented nucleic acid associated with L1 hybridized with the nucleic acid linked to L2,leading to the fusion of the two liposomes.The fusion process was followed by dynamic light scattering,and by monitoring the fluorescence of the Tb3+-DPA complex generated upon the fusion of the liposomes and their exchange of contents(fusion efficiency 30%).Then,the fusion of the liposomes L1,loaded with UCNPs and doxorubicin(DOX),with He La cancer cells functionalized with nucleic acid tethers,complementary to the hairpin units associated with the boundary of L1,and linked to the MUC-1 receptor sites associated with the He La cells,through a MUC-1 aptamer unit is exemplified.The effect of DOXloaded L1/He La cell fusion on the cytotoxicity towards He La cells is addressed.The NIR UCNP-stimulated cleavage of the o-nitrobenzyl phosphate caged hairpin units associated with L1 leads to the fragmentation of the hairpin units and the resulting nucleic acid tethers hybridize with the nucleic acid-modified He La cells,resulting in the liposome-He La cell fusion and the release of DOX into the He La cells.Selective spatiotemporal cytotoxicity towards He La cells is demonstrated(ca.40% cell killing within two days).The study presents a comprehensive stepwise set of experiments directed towards the development of NIR-driven liposome-liposome or liposomemembrane fusion processes.Second,the hairpin DNA containing light-responsive group PC-Linker and p Hresponsive DNA are introduced,and the membrane fusion of liposome-liposome or liposome-giant vesicl can be controlled by UV light and adjusting the p H of the solution or changing the p H and UV light solution.The liposome L1 is functionalized with hairpin DNA,part of the nucleic acid complementary to the hairpin DNA is modified on the liposome L2,and liposome L3 is functionalized with p H-responsive DNA.Mixing the above-mentioned three types of liposomes functionalized with DNA together.The PC-Linker group is cleaved under UV light irradiate,resulting the hairpin DNA activated.DNA strand replacement reaction is occurred between activated hairpin DNA and DNA on the surface of liposome L2,making liposome L1 and L2 fused.Changing the p H of the liposome solution,triplex DNA and duplex strand are formed between the p H-responsive DNA and the product of strand replacement;simultaneously,the above DNA accomplishes the complementary reaction,causing the liposome L3 fused with the liposomes L1/L2.Changing the p H of the solution,under the effects of proton and base pairing,p H-responsive DNA and hairpin DNA form new DNA,leading to fuse between liposomes L1 and liposomes L3;the hairpin DNA is activated by UV light,and the activated hairpin reacts with the related DNA,allowing liposomes L2 is fused with liposomes L1/L3.The fusion of liposomes-liposomes is monitored using the fluorescence change.Irradiating with UV light and altering the p H of the solution or varying p H of solution and UV irradiation,loaded high concentration of sulforhodamine B(SRB)liposomes are fused with the empty liposomes,leading to the change in fluorescence.The fusion between liposomes is also characterized by DLS and SEM.Light-responsive hairpin DNA,DNA that part of complementary to the hairpin DNA,Exonuclease ?(EXO III)are loaded in liposomes,and the liposomes are functioned with DNA.The liposomes-liposomes are fused under UV light irradiated and changing the p H of the solution or altering p H of solution and UV irradiation,the DNA loaded in the liposome reacts a strand displacement and a signal amplification reaction with EXO III.Also,the fusion of liposomes-liposomes allows the cascade reaction occurred in glucose,glucose oxidase(GOx)and horse radish peroxidase(HRP).Similarly,the membrane fusion is taken place between liposomes functioned with DNA and giant vesicles(GUVs)decorated with DNA under UV light and changing the p H of the solution.Membrane fusion allows the strand displacement reaction is produced between hairpin DNA and single DNA loaded liposomes/giant vesicles,and the products of displacement reaction generats a signal amplification reaction with EXO III.In a similar way,the membrane fusion among of liposomes-giant vesicles causes a cascade reaction of glucose,GOx and HRP.