A Universal Method Of Membrane Labeling With Trans-cyclooctene

Cell membrane plays a critical role in the biological processes and physiological activities of cells,including maintenance of the intracellular stability,selective transport,cell signaling,energy conversion and so on.In addition,some enveloped viruses integrate the cell membrane of host cells into envelope when released from the host cells.And the extracellular vesicles(EVs)could be enclosed by cell membrane to form the outer membrane and then release from or shed from the surface of cell.In order to better understand various complex physiological activities concerning with cell membrane and to take advantages of its properties to develop further biological applications,it is a common strategy to functionalize the cell membrane with chemical labels.This requires minimum impairment to the integrity of cell structure and normal physiological activities while modify the membrane.This thesis described the development and practical application of the novel labeling strategy by Diels-Alder reaction.The bioorthogonal label molecule trans-cyclooctene that reacts with tetrazine was first synthesized.Then it was linked to the phospholipid analogues,generating the trans-cyclooctene derivatives of cell membrane.Taking advantage of the membrane fluidity,the analogues were successfully incorporated into the cell membrane,and more than 90% of cells can be incorporated successully.Derivatived cell membrane shows inverse electron demand Diels-Alder reaction with functional probe that modified by tetrazine.So that it can bio-functionalize the cell membrane.This bioorthogonal reaction shows the advantages of high reaction speed,high regioselectivity,mild reaction conditions and good biocompatibility.It will not cause damages on the structure and function when modified cell membrane specifically.Meanwhile,this strategy was applied to the labeling of oncolytic virus.In virtue of the homology between envelope of virus and cell membrane,the envelopes of virus were modified with trans-cyclooctene from host membrane.The targeting enrichment and proliferation were observed around tumor cells,increasing the expansion of the oncolytic virus 1.3 times,which presented a new way to enhance tumor targeting of oncolytic virus.Furthermore,it achieved the trans-cyclooctene labeling of EVs through the EV generation process from cell membrane.85 persent of the trans-cyclooctene EVs were rapidly captured by magnetic nanoparticles modified with tetrazine,which developed novel method for exosome separation.Collectively,this labeling method of cell membrane is simple,efficient and universal to multiple types of cells,and it provides a complementary strategy for dissecting the cell membrane and related physiological activities.