Two-Photon Fluorescence Probe For Detection Of Changes In Lysosomal Polarity Based On Naphthalimide

In recent years,there has been a great improvement in vivo fluorescence imaging due to the recent advances in bioimaging devices and molecular imaging probes.Currently in all kinds of technologies,the fluorescence quantitative method can perform real-time in situ detection in vivo by virtue of its high sensitivity,non-destructive rapid analysis and the like.They have good cell permeability and rapid response to biological targets,which are important for in vivo detection and cell imaging.In biochemical research,two-photon technology has become a novel and creative tool.the two-photon technique has good application prospects by reducing background fluorescence in living cells and tissues,reducing light damage in biological samples,and photobleaching,providing better three-dimensional spatial positioning and increasing penetration depth,Especially in the detection of biological micro-environment have unique advantages.compared to the single-photon and other detection methods.The microenvironment of the organism mainly includes polarity,viscosity,temperature,pH,oxygen content,acidic medium and the like.In many micro-environmental parameters,polarity occupies a very important position.On the cellular level,polarity is a form of feedback from a series of complex mechanisms that operate long enough to maintain its normal function in a particular area.These areas include the spatial arrangement of functional proteins,the protein compositional processes such as differentiation,local membrane growth,immune activation reactions,directed cell migration and vector trafficking.Soabnormal changes of polarity are closely related to the disease.For example,the occurrence of diabetes and cirrhosis are directly related to abnormal changesof polarity.Therefore,it is very practical to try to detect the polarity by using the two-photon technique.Lysosomes are garbage disposal "factories" of cells,because of its acidic environment inside,which contains more than 50 kinds of hydrolases,so cells can be generated in the metabolism of waste degradation.At present,the academic community has a new understanding of lysosomes.Now it is generally accepted that lysosomes not only function as degrading cells,but also are key regulators of cell homeostasis and stress adaptation.Recent evidence suggests that the intracellular distribution of lysosomes affects the regulation of cellular responses to various stimuli and that this altered regulatory mechanism is strongly associated with the development of various disorders.polarity has great influence on its function.at present,there are not many studies on the polarity of lysosome in academia.It is imperative to dig out the scientific value behind it. naphthalimide was selected as the fluorophore and two-photon groups were introduced into it.Two novel two-photon polarity fluorescent probes,Lyso-NA and Lyso-NB,were designed according to their solvatochromic effect.The probes were all generated by Sonogashira coupling reaction.and their structures were confirmed by 1HNMR,13CNMR and HR-MS(ESI).According to the structural characteristics of the probe,the related design experiments of the behavior of the probe in various solvents were first carried out.Then simulate the internal environment of the organism in vitro experiments,adopt the way is to study the behavior of the probe in the mixed changed solvent system,the results perfectly fit with the design idea.Further experiments on the application of probes in living organisms were carried out.A series of experiments such as cytotoxicity test,lysosomal co-localization,biological fluorescence imaging and so on were carried out in line with the expected experimental results.From the conclusion,we affirm that the correctness of the principle of the design and enrich the polarity of the lysosomal microenvironment factors,further filling the gap in this aspect.The probe Lyso-NA was synthesized by introducing the diethylamino morpholine into the amide nitrogen atom of naphthalene diimide and inserting the strong electron donating group 4-methoxyphenylacetylene at position 4.In themolecular structureof probe Lyso-NA,4-methoxyphenylacetylene as donor(D)and diethylamino morpholine as acceptor(A)cooperate to form a typical D-π-A structure.The probe Lyso-NA was tested by the same experimental method as above and found to be an excellent two-photon probe.It is worth mentioning that the use of naphthalimide as a fluorophore,mainly due to its high fluorescence quantum yield,low toxicity,with good two-photon properties,and more conducive to the design of excellent two-photon polarity probe,easy to apply in the study of organisms.By introducing the diethylamino morpholine into the amide nitrogen atom of naphthalene diimide,4-hydroxyphenylacetylene is donated to the electron donating group at position 4,to increase the conjugated area of the system to form a classic push-pull electronic system,designed a molecules of probe Lyso-NB.in the molecular structure of probe Lyso-NB,4-hydroxy-phenylacetylene as donor(D),diethylamino morpholine as acceptor(A),together form a typical D-π-A structure,It has two photon-induced fluorescence properties and is confirmed by the corresponding experiments.The analysis of the optical data in different polar solvent and the mixed system of methanol-tetrahydrofuran and further experiments,such as lysosomal co-localization,demonstrated that the probe Lyso-NB molecule can be well localized in lysosomes and has a good response to changes in the lysosome polarity and can be achieved change tracking.