Construction Of Carbon Dot-based Lysosome Targeting Fluorescent Probe System And Research On Its Biological Application

The lysosome is an important subcellular organelle,which plays an important role in intracellular signal transduction,maintaining homeostasis and degrading biological macromolecules.Abnormal lysosomal function can easily lead to lysosomal storage diseases and tumor invasion and metastasis.For example,pancreatic ductal adenocarcinoma(PDAC)tumors show high lysosomal activity,and low lysosome expression can inhibit the proliferation and metastasis of PDAC cells.Therefore,it’s important significance for monitoring the activity of lysosomes,the diagnosis in vivo and treatment of related diseases that the development of visually tracing lysosome targeting probes.The resported visual lysosomal fluorescent probes are easily photodegradable,strong biological toxicity,lysosomal diffusion,etc.,which are problems that need to be solved urgently in live cell imaging and visualized lysosomes.Carbon dots(CDs)have become a new star in bioimaging due to their good photostability,biocompatibility,and photocatalysis.At present,carbon dots used for biological imaging are concentrated in the blue region due to their short emission wavelengths,which interfere with cell imaging and cause optical radiation damage to living tissues and cells.Therefore,constructing a fluorescent carbon dot with stable optical properties and longer wavelength emission is the key to biological imaging and expanding its biological applications.In this article,based on the presence of more acid hydrolases in lysosomes and the characteristics of greater polarity,this paper designed an electron-donating nitrogen-containing functional group with strong polarity and interaction with lysosomes,and biologically endogenous materials used as precursors,then a one-step hydrothermal method is used to synthesize multi-color fluorescent carbon dots to construct a carbon dot-based fluorescent probe system with high selectivity,high fluorescence quantum yield,strong optical stability and high loading capacity.And application research on these systems,such as lysosomal labeling,bioimaging and drug delivery,exploring the cell internalization mechanism of multi-color fluorescent carbon dots,and the system realized self-targeted lysosomal imaging,tumor targeting,in vivo imaging and integrated monitoring for drug delivery.The specific research content is as follows:(1)Construction of glutathione fluorescent probe system and its application in lysosomal targeting,cell imaging,and drug delivery1)The endogenous substance glutathione,which has an isoelectric point in the acidic range,is selected as the precursor,and is self-passivated by hydrothermal method to form a multicolor fluorescent carbon doped with foreign elements(GSH-CDs),good light stability,and low toxicity.The average particle size of the carbon dots is 2 nm,and the surface is rich in functional groups such as hydroxyl,carboxyl,six-membered nitrogen heterocycles,etc.,which provides a targeting group for lysosomal imaging,and has low cytotoxicity,good blood compatibility and good photobleaching resistance and salt stability.GSH-CDs are excitation wavelength-dependent and can be applied to multicolor cell imaging: fluorescence is distributed in the cytoplasm in normal cells,while in tumor cells,it is distributed throughout the cell.Therefore,it can identify normal and tumor cells.Further studies have shown that GSH-CDs and Lyso Red-Tracker have good fluorescence overlap.The GSH-CDs can target lysosomes with certain versatility.Cell internalization mechanism experiments show that GSH-CDs enter cells through caveolin/clathrin-mediated endocytosis,and achieve lysosomal targeted imaging through lysosomal steramine.Simultaneously,it have been successfully applied to zebrafish in vivo imaging.It can provide research directions for future drug targeting research,the determination of disease and tumor targets.2)Based on the fluorescence characteristics of GSH-CDs,the GSH-CDs-DOX drug-delivery system with fluorescence tracing capability was constructed.Experiments show that the rate of the drug-loading system is as high as 88.65%,and the drug-loading is 44.3 mg/g.The drug was released for 72 hours,and the cumulative release rate was75.43% at p H=5.0,which was 5.2 times than that of p H 7.4 in vitro.It is attributed to the protonation of carboxyl on the surface of GSH-CDs and the weakening of the electrostatic adsorption with adriamycin,indicating that the drug-delivery system is p H sensitive.Compared with the control group,the drug-delivery system has a strong inhibitory effect on tumor cells,and the cell survival rate is as low as 69.9%,and it has no obvious killing effect on normal cells(survival rate is 94%).At the same time,the drug-delivery system can deliver doxorubicin in the nucleus and inhibit cell growth.It can be seen that the drug-delivery system has tumor cell targeting and can be used for tumor targeted therapy and drug tracking in vivo.(2)Construction of Maltose-Tryptophan fluorescence probe system and research on its biological application1)Selecting maltose as the carbon source,the isoelectric point is in the acidic range,the electron-donating group,and the more biologically active tryptophan as the doped nitrogen source,then the nitrogen-doped fluorescent carbon is prepared by a gentle and green hydrothermal synthesis method(MS-CDs).Compared with GSH-CDs,this carbon dot introduces a nitrogen heterocyclic electron donating group,which makes the emission wavelength redshift to 45 nm,the surface nitrogen content increases by0.54%,the fluorescence quantum yield increases by 7.78%,and the fluorescence deacy lifetime increases by 0.563 ns.The heterocyclic nitrogen group improves the photoluminescence properties of the carbon dots.Lower concentrations of MS-CDs are distributed in the cytoplasm of normal cells and tumor cells,and the cell morphology is intact.It can be used as a probe for multi-color cell imaging and in vivo cytoplasmic imaging with a broad spectrum.Normal cells and tumor cells overlap perfectly with the fluorescence of the lysosomal dye,and the co-localized lysosomes are clearly observed.MS-CDs enter the cells mainly through the endocytic pathway mediated by tubulin/cell membrane cavernous depressions.Based on its excellent optical properties,zebrafish live imaging is realized.2)Based on the C-N bond covalent bonding and electrostatic adsorption,the MS-CDs-DOX drug delivery system was constructed.The drug loading rate is as high as 90.62%,and the drug-loading amount is 45.31 mg/g.At p H 5.0,the cumulative release of the drug was the highest,reaching 79.22% within 72 hours,which was 5.08 times than that of p H 7.4,indicating its p H responsiveness.The tumor cell survival rate of the drug-delivery system was 78%,which was lower than that of the control group(only doxorubicin group,84%),and the normal cell survival rate was 95%,indicating that the drug-delivery system has high drug-loading rate and low toxic side effects.MS-CDs-DOX drug delivery system targeted delivery of DOX in the cytoplasm,less distributed in the nucleus,and blurred cell morphological boundaries.Therefore,the nitrogen-doped MS-CDs-DOX drug delivery system is more conducive to tumor tracing and targeted therapy.3)Based on the strong graphitization of MS-CDs and the strong reducibility of oxygen-containing functional groups,MS-CDs/AuNPs with low-toxicity and emitting green fluorescent were prepared.The average particle size of the nanoparticles is 28.56 nm,the UV absorption peak is at 548 nm,and the surface is rich in oxygen-containing functional groups such as amino and hydroxyl groups.CCK8 experiment results show that the nanoparticles have low cytotoxicity with survival rate of nearly 100%.Compared with MS-CDs,the fluorescence deacy lifetime of MS-CDs/AuNPs is increased by 3.8 ns,and the quantum yield is increased by 4.1%.At the same time,it has a large stokes shift and strong anti-interference ability.It is distributed in tumor cells throughout the cell.Based on the distribution of fluorescence signalnormal,so it can be used to identify normal cells and tumor cells.