A New FRET-based Ratiometric Zn²⁺ Fluorescent Probe And Its Application In Imaging And Flow Cytometry

As the second most abundant transition metal in the human body,most zinc cations are bound by proteins,yet there still many zinc pools for "labile" or "free”Zn2+.Zn2+ homeostasis is closely associated with many physiological processes such as neurotransmission,signal transduction,cell apoptosis,gene transcription.Zinc disorder is also involved in many diseases such as Parkinson's diseases,Alzheimer's disease,and cancer.The in situ monitoring of endogenous Zn2+is essential to understand the physiological and pathological roles of Zn2+,and fluorescence Zn2+imaging with Zn2+ probes has been developed for this purpose due to its high sensitivity and selectivity,quick response and non-invasiveness.However,Zn2+imaging in living systems is still suffering from the interference caused by photobleaching,specimen difference,and other deviated factors such as local probe concentration,microenvironment and measurement conditions.Therefore,ratiometric Zn2+ probes were proposed since their calibration effect in dual emission is able to reduce these interferences.However,the ratiometric Zn2+ probe suitable for endogenous Zn2+ detection is very few.In addition,Zn2+ bioinorganic study requires still the 3D spatial Zn2+distribution in tissue and animal model,which demands the probe of NIR or two-photon excited fluorescence(TPEF)to improve the imaging depth.Finally,the determination of the general endogenous Zn2+ fluctuation in batches cells needs a reliable method such as flow cytometry.In this study,a FRET-based ratiometric Zn2+probe,CPBT,was constructed by integrating a two-photon excitable coumarin with an ICT fluorophore,4-amine-7-sulfamoylbenzo[c][1,2,5]oxadiazole(ASBD).Its FRET efficiency can be altered via Zn2+ coordination binding,showing the emission shift from 565 to 485 nm.With this probe,the 2D confocal ratiometric Zn2+imaging in cells and zebra fish larvae was realized and the probe sensitivity was confirmed to be suitable for endogenous Zn2+detection.Then 3D ratiometric imaging for endogenous labile Zn2+in the head of zebra fish larvae was also realized with an imaging depth of 150 ?m.The 3D reconstruction of ratiometric images disclosed the high level of labile Zn2+in the ears,the upper front superficial of yolk sac,and the medium level in the pupil.Moreover,the TPEF ratiometric imaging for endogenous labile Zn2+ in the opaque frontal lobe of mouse brain was achieved with an imaging depth of?200 ?m.The imaging found that a kernel and the rest part in the detction region show the different depth-dependence of labile Zn2+level from 0-200 ?m.With CPBT,the ratiometric Zn2+flow cytometry was developed also to monitor the labile Zn2+fluctuation in a large number of cells,and a ? angle analysis for the cell number contour map in a coordinates of green and red channel fluorescence shows that the endogenous labile Zn2+level in cisplatin sensitive MCF-7 cells undergoes a distinct decrease,while that in cisplatin insensitive SKOV3 cells undergoes a gradual enhancement,in the initial 8 h of cisplatin incubation.All these imply that the new ratiometric protocols of flow cytometry and 3D imaging developed in study for endogenous Zn2+ tracking in diverse biological specimens should provide new impetus to explore the Zn2+-associated biological processes.The aforementioned study found cisplatin is able to induce the endogenous Zn2+fluctuation in different tumor cell lines.On the other hand,the cisplatin resistance-associated GSH can be regulated by intracellular Zn2+ level in certain cells.Therefore,exploring the association between cisplatin-induced GSH up-regulation,which might be the origin for the acquired resistance of cisplatin,and the cisplatin-induced Zn2+fluctuation was carried out in gastric cancer line SGC7901 and its cisplatin-resistant cell line SGC7901/DDP.Ratiometric Zn2+flow cytometric assay with CPBT found that the intracellular labile Zn2+ level in SGC7901/DDP cells(?=51.1°)is higher than that in SGC7901 cells(?=43.2°),and the intracellular GSH level in SGC7901/DDP cells(95.1±9.2 nmol/mg protein)is also distinctly higher than that in SGC7901 cells(48.4±2.4 nmol/mg protein).Moreover,cisplatin incubation(20?M)of SGC7901 leads to the up-regulation of intracellular labile Zn2+(?=50.9±0.1°)and GSH(68.0±5.5 nmol/mg protein),and this might be the origin for the higher level of labile Zn2+ and GSH in SGC7901/DDP cells,which are induced by cisplatin incubation with the gradually increased cisplatin concentration.Moreover,the loading of exogenous Zn2+into SGC7901 cells makes the intracellular GSH level increase to 72.2±6.2 nmol/mg protein,while incubation with the membrane permeable Zn2+scavenger BPEA(N?,N?-bis(pyridin-2-ylmethyl)ethane-1,2-diamine)makes GSH level in SGC7901 cells decrease to 23.2±2.0 nmol/mg protein.Western blot assay discloses that the exogenous Zn2+ introduction up-regulates the level of GCLC and GS,which are the rate-limiting enzymes for GSH synthesis,while Zn2+scavenging by BPEA leads to the down-regulation of the two enzymes.All these indicate that the intracellular GSH level could be regulated by labile Zn2+ in SGC7901 cells via regulating the expression of GCLC and GS.Therefore,the cisplatin-induced GSH enhancement can be ascribed to the cisplatin-induced up-regulation of labile Zn2+.In fact Zn2+scavenging via BEPA pretreatment not only decreases the cisplatin-induced GSH enhancement but also decreases the cisplatin-induced up-regulation of GCLC and GS.Our experiment also found the cisplatin-induced labile Zn2+enhancement in SGC7901 cells is accompanied by the intracellular ROS enhancement.Our study found that ROS scavenger N-acetylcysteine decreases distinctly the cisplatin-induced Zn2+ enhancement,implying cisplatin-induced labile Zn2+ enhancement might be mediated by the cisplatin-triggered ROS in cells.ICP-MS determination found the cisplatin incubation does not change the total Zn2+ amount in cells,implying the cisplatin-induced labile Zn2+ enhancement comes from Zn2+ release from "zinc pool"or zinc buffer proteins in cells,and ROS is the trigger of the release.In fact,cisplatin incubation of SGC7901 cells leads to the continue enhancement of ROS,which is not affected by BPEA pretreatment,yet the cisplatin-induced enhancement of labile Zn2+and GSH can be distinctly concealed by Zn2+scavenging with BPEA pretreatment.All the current results indicated that cisplatin is able to trigger the ROS production in SGC7901 cells,and ROS induces the intracellular labile Zn2+release and finally results in the enhancement of GSH,which is closely associated with the cisplatin resistance.The results in the second part of this study disclosed that Zn2+scavenging is an effective method to reduce the cisplatin-induced GSH enhancement,therefore,the antitumor activity of cisplatin might be promoted by Zn2+ scavenging via reducing the GSH-related resistance.A series of cell membrane permeable Zn2+ chelators of different Zn2+coordination numbers and Zn2+ affinity,BPA(bis(pyri din-2-ylmethyl)amine),BPEA,and TPEN(N,N,N',N'-tetrakis(pyridin-2-ylmethyl)ethane-1,2-diamine)were utilized to scavenging the cisplatin-enhanced labile Zn2+in SGC7901 cells at lower concentration to guarantee the cell viability is higher than 90%after 12 h of incubation.Therefore IC50 values of cisplatin against SGC7901 cells in the presence of Zn2+ chelators were determined,and the IC50 values after 12 h of cisplatin incubation are 25.5±1.2 ?M(blank control with no chelator pre-incubation),23.1±0.8?M(BPA),15.8±0.5 ?M(BPEA),11.0±0.4 ?M(TPEN),the related IC50 values against SGC7901/DDP cells are?100 ?M(blank control with no chelator pre-incubation),79.9±6.1 ?M(BPA),45.9±3.4 pM(BPEA),and 27.3±2.6 ?M(TPEN).Both cases indicated that higher Zn2+ affinity of the pre-incubation Zn2+scavenger leads to the higher cytotoxicity,and TPEN of the highest Zn2+affinity among all the three chelators displays the lowest cisplatin IC50 against SGC7901 cells.In fact,TPEN even makes IC50 value of cisplatin against SGC7901/DDP cells be comparable to that of cisplatin against SGC7901 cells in the absence of any chelator.Moreover,the ratiometric Zn2+ flow cytometry found that Zn2+ chelator especially those of higher Zn2+affinity is able to reverse the cisplatin-induced labile Zn2+enhancement in SGC7901 cells,while for SGC7901/DDP cells,the chelators of high Zn2+affinity is able to reduce the cisplatin-induced labile Zn2+enhancement,although the cisplatin-induced ROS production ability is promoted distinctly especially by those of higher Zn2+affinity.Moreover,both the intracellular GSH and the related GSH synthase GCLC and GS were down-regulated especially in the presences of chelator of high Zn2+ affinity.All these suggested that the Zn2+ chelator especially those of high Zn2+affinity is able to promote the antitumor cytotoxicity of cisplatin against SGC7901 and SGC7901/DDP cells via scavenging the labile Zn2+.Moreover,the presence of Zn2+chelator is able to retard the cisplatin resistance enhancement in SGC7901/DDP cells.Therefore,with the CPBT-favored ratiometric Zn2+flow cytometry,the specific Zn2+ roles in antitumor behavior of cisplatin against SGC7901 cells was disclosed in this study,and a promising clue to promote the antitumor activity of cisplatin was also demonstrated.