Aggregation-based Dual-Target Probe for Dual-Color Super-Resolution Monitoring Mitophagy and Evaluating Drugs Regulating Mitochondria

Developing single ﬂuorescent probe for simultaneously visualizing mitophagy ﬂux and oxidative stress with super-resolution is highly demanded yet quite challenging. Herein, we design and synthesize a ratiometric HClO probe AHOH , which is capable of simultaneously staining lysosomes and mitochondria with red and green color, respectively. AHOH could be selectivity oxidized by HClO, leading to a large emission blue shift (90 nm) and an over 1300-fold enhancement of the emission ratio of Fl 547 nm /Fl 637 nm . We apply AHOH in super-resolution microscopy and clearly visualize the dynamics of mitochondria-lysosomes interactions and the oxidative stress states upon diﬀerent stimuli. Mitochondria dysfunction triggered by diﬀerent drugs and genetic defect lead to elevated oxidative stress and higher levels of mitophagy. Moreover, AHOH could serve as a reliable tool for evaluating the eﬃcacy of drugs regulating mitochondria dysfunction. This work provides a powerful dual-color super-resolution imaging agent for real time monitoring the dynamics of organelle interactions and oxidative stress.


Introduction
The typical mitophagy inducers are reactive oxygen species (ROS), which are mainly produced in mitochondria via electron leakage from electron transport chain (ETC). [11]Simultaneously visualization of the subcellular redox imbalance and mitochondria-lysosomes interactions could help to understand the pathogenesis of mitochondrion-associated diseases, monitoring disease progression and optimizing therapies.Traditional methods used to assess the mitophagy are western blotting and transmission electron microscopy (TEM), however, they have to face problems such as financially costly, time-consuming, and not suitable for live samples. [12][15][16][17][18] However, the resolution of optical imaging (typically over 200 nm) is not enough to show the details of structural changes at the suborganelle level.[27] Although there are some dual-color/dual-target probes available, few of them showed response to ROS. [28] Therefore, developing super-resolution imaging probes that are capable of simultaneously monitoring the dynamic mitophagy process and evaluating cellular oxidative stress states is highly demanded and challenging.Herein, we report the construction and super-resolution imaging application of a ratiometric HClO probe AHOH (Scheme 1), which is capable of simultaneously staining lysosomes and mitochondria with red and green emission, respectively.Due to its near planar structure and the intermolecular electrostatic interaction, AHOH readily self-assembles to red emitting nanoparticles (˜20 nm) and locates in lysosomes via endocytosis.On the other hand, the monomeric form of AHOH with lipophilic cationic feature tends to accumulate in mitochondria, then oxidized by mitochondrial basal HClO and possesses green emission.Simultaneous dual-color monitoring mitochondria-lysosomes interactions and HClO levels are realized usingAHOH by super-resolution fluorescence imaging.Drug and genetic defect induced mitochondria dysfunction leading to elevated green emission, decreased red emission and enhanced overlap between lysosomes and mitochondria, suggesting elevated oxidative stress and higher levels of mitophagy.Finally, AHOH can be applied to evaluate the efficacy of drugs regulating mitochondria dysfunction.To the best of our knowledge, this is the first dual-color/dual-target probe that could simultaneously monitor mitophagy and oxidative stress under super-resolution fluorescent imaging.

Design and Synthesis of Probe AHOH
The olefinic C=C double bonds of cyanine dyes were susceptible to oxidative species, [29][30] the structural change would enable ratiometric detection of ROS and potentially imaging differential organelles. Probe AHOH was synthesized by aldol condensation reaction (Scheme 1), the detailed reaction procedures and proposed mechanism of the related intermediate is shown in Figure S1 (Supporting Information).AHOH was fully characterized by 1 H NMR, 13 C NMR, HRMS, and single-crystal X-ray diffraction analysis (see Figures S2-S5 of the Supporting Information and Figure 1a).

Spectroscopic Properties and Response to HClO in Solution
X-ray single crystal structure analysis demonstrated the highly planar conformation of AHOH (Figure 1a).Multiple intermolecular interactions, such as π-π σταςκινγ εφφεςτ ανδ ιντερμολεςυλαρ ηψδρογεν-βονδινγ ιντεραςτιονς ωερε οβσερ εδ, ωηιςη ςουλδ φαςιλιτατε αγγρεγατες φορματιον (Figure S6).TEM data showed that AHOH in water solution displayed well dispersed nanoparticles with average diameter of ˜15 nm (Figure 1b), which was consistent with the average hydrodynamic size (˜20 nm) tested by dynamic light scattering (DLS) (Figure 1c).The emission intensity of AHOH in MeOH-H 2 O or DMSO-H 2 O binary solution gradually decreased with increasing water content, while the emission maximum remaining unchanged (Figure S7).The above data showed that AHOH could readily form nano-aggregates in water solution, which might help for targeting lysosomes.The HClO titration of absorption and emission spectra of AHOH (10 μM) in water solution were conducted.
As the concentration of HClO increased (0-30 μM), an absorption peak at 462 nm was enhanced accompanied by a dramatic decrease in the absorption peak at 583 nm (Figure 1d).Upon 470 nm excitation, the fluorescence emission at 637 nm dropped gradually while a new fluorescence emission peak centered at 547 nm appeared and enhanced with the increase of HClO (0-30 μM) (Figure 1e), indicating a distinct ratiometric response of the probeAHOH to HClO.The ratio of the fluorescence intensity at 547 and 637 nm (Fl 547 nm /Fl 637 nm ) enhanced over 1300-fold from 0.004 to 5.3 upon addition of 30 μM HClO.The probe's emission response to HClO was rapid, occurring in less than 5 min from AHOH to intermediate and final product (Figure S8).The limit of detection (LOD) was calculated to be 35.2nM (Figure S9), suggesting that AHOH could response to HClO with high sensitivity.
[35] AHOH experienced addition with HClO on the conjugated double bond near the indole ring to form compound 5 , then quickly generated epoxide compound 6 through dehydrochlorination, followed by hydrolysis and generated compounds 7 and 8 eventually (Figure S11a).The MS detection of the reaction mixture showed MS peaks of 450.1868, 414.2153, 241.0964, 190.1218, which could be attributed to reaction intermediates 5 , 6 and products 7 , 8 , respectively (Figure S11b).The density functional theory (DFT) calculations demonstrated that the energy gap between HOMO-LUMO of Oxidized-AHOH (3.48 eV) was distinctly higher than that of AHOH (2.60 eV), which was in accordance with the experimental data that HClO induces large blue-shifted absorption/emission. [36] 2.3 Dual-color Super-resolution Imaging Performance of AHOH Encouraged by the results in solution, the application of AHOH for HClO imaging in living cells was explored by Olympus SpinSR10 super resolution microscope.The organelle imaging potential of AHOH in live HeLa cells was investigated with excitation at 488 nm and 561 nm, respectively.Interestingly, probe AHOH displayed fluorescence both in green channel (500-550 nm, excited at 488 nm) and red channel (580-610 nm, excited at 561 nm), signals from the two channels showed completely different locations in the subcellular zone (Figure 2a).The red fluorescence could be assigned to the characteristic red emission from probe aggregates, while the green color should be originated from the HClO oxidation product.The resolution was determined up to 176 nm, suggesting that the probe was capable to perform super-resolution imaging.To validate the specific subcellular organelle location, co-localization with commercially available organelle markers were performed (Figure 2b).The Pearson correlation coefficient (PCC) between the green channel image of AHOH with that of Mito-Tracker DeepRed (MTDR) was determined to be 0.833, while the PCC between the red channel image with that of Lyso-Tracker DeepRed (LTDR) was 0.896, indicating that the green channel overlapped well with mitochondria and the red channel overlaps with lysosomes.Colocalization analysis with other organelle-targeting dyes revealed that red and green fluorescence signals were exclusively present in lysosomes and mitochondria, respectively.The lysosomes-targeting and red emitting feature could be probably due to the endocytosis pathway and the lower HClO level in lysosomes, while the mitochondria-targeting and green emission ability could be attributed to the lipophilic cationic character of monomeric AHOH and the higher ROS level in mitochondria.All images were acquired in the blue channel (420-450 nm, excited at 405 nm), green channel (500-550 nm, excited at 488 nm), red channel (580-610 nm, excited at 561 nm), and deep red channel (650-700 nm, excited at 640 nm).Scale bars: 10 μm.
The cell uptake pathways of AHOH entering lysosomes was further validate by using different cell uptake pathway inhibitors.Compared with the fluorescence intensity of AHOH stained cells collected under 37 °C, those under 4 °C, 2-deoxy-Dglucose + oligomycin (MI, metabolic inhibitor) treatment, and NH 4 Cl (endocytic inhibitor) treatment decreased by 72.1%, 55.5%, 71.9%, respectively (Figure S12), suggesting that AHOH entered lysosomes mainly via the energy-dependent endocytosis pathway.The above result demonstrates that AHOH could target both lysosome and mitochondria with red and green emission, respectively.
The fluorescence responses of the probe AHOH to both exogenous and endogenous HClO in cells were investigated (Figure 3).HeLa cells were pre-incubated with HClO (5 μM) for 1 h, or lipopolysaccharide (LPS,2 μγ/μΛ ) for 4 h and phorbol myristate acetate (PMA, 2 μγ/μΛ ) for 2 h to trigger oxidative stress in cells, [37] then stained with AHOH (5 μM) for 10 min.As a control experiment, HeLa cells were incubated with only AHOH (5 μM) for 10 min.Compared with the control group, enhanced fluorescence in the green channel (1.90-fold for HClO group, 1.44-fold for LPS + PMA group) and decreased fluorescence in the red channel (29.24% left for HClO group, 31.75% left for LPS + PMA group) were observed.Then, the cells were pre-treated with 4aminobenzohydrazide (ABH, a ROS scavenger) or N-Acetylcysteine (NAC, a ROS scavenger) to reduce intracellular HClO, [38][39] followed by incubation of LPS and PMA.Intensive red fluorescence stronger than the control group and decreased green fluorescence weaker than the control group were observed, suggesting that pretreatment of ROS scavenger could significantly reduce the HClO level.These results confirmed that theAHOH could monitor the fluctuation of HClO levels by dual channel imaging.
Before dual-color monitoring of ROS and mitophagy, we investigated the biocompatibility of AHOH .HeLa cells exhibited above 90% cell viabilities after treatment with AHOH up to 10 μM for 6 h (Figure S13), indicating that AHOH possessed high biocompatibility and low cytotoxicity to living cells since its working concentration was 5 μM.In addition, the photostability of AHOH compared with commercial LTDR and MTDR were evaluated (Figures S14-15).The red channel fluorescence intensity of AHOH stained cells remained over 80% after 40 s irradiation, while that of LTDR showed only 30% to that of the initial intensity.Meanwhile, the green channel fluorescence intensity decrease of AHOH stained cells was lower than that of MTDR incubated cells.The above results confirmed thatAHOH showed better photostability than commercial dyes, suggesting its potential for long term monitoring of mitophagy and oxidative stress.

Monitoring of Mitophagy and Oxidative Stress Induced by Drug Treatment
Next, we evaluated the capability of AHOH for simultaneously monitoring the mitophagy and oxidative stress induced by typical autophagy-inducing drugs (Figure 4).HeLa cells incubated withAHOH exhibited showed poor overlap (PCC, 0.102) between green channel and red channel, indicating low level of lysosomesmitochondria interaction in the untreated group.However, the overlap degree between red channel and green channel increased distinctly when HeLa cell were pretreated with CCCP (PCC, 0.842) and rapamycin (PCC, 0.722), suggesting much higher level of mitochondria engulfment by lysosomes upon mitophagy induction.Meanwhile, compared to the control group, the drug-pretreated groups showed enhanced green channel signals and decreased red channel emission, suggesting higher level of HClO production during mitophagy process.The data demonstrated that AHOH could simultaneously visualize mitophagy and oxidative stress by monitoring the extent of overlap between two channels and fluorescent intensity changes in the two channels.We then investigated whether AHOH is capable of real time monitoring the dynamic interactions between mitochondria and lysosomes and changes in ROS levels under normal and drug-treated conditions (Figure 5).Firstly, HeLa cells were incubated with AHOH for 10 min , the fluorescence images in both channels and the overlap images with PCC values were collected every 40 s.No significant intensity changes in both channels were observed, while the overlap between the two channel images remained at low level.Secondly, HeLa cells were co-incubated with AHOH and autophagy inducer CCCP (20 uM ), the fluorescent images of both channels were collected every 2 min.The fluorescence intensity in the green channel gradually enhanced (7.84-fold at 10 min) and that in the red channel gradually decreased (4.7% left at 10 min).Meanwhile, it could be clearly seen that mitochondria and lysosomes gradually approached and fused over time according to the enlarged merged images, the PCC value of the green channel and red channel gradually increased from 0.221 to 0.812.The above result confirmed that AHOH could be applied for real time tracking of mitophagy dynamics and the oxidative stress changes upon drug treatment.

Dual Channel Imaging in Cells with Mitochondrial Defect
Iron regulatory proteins (IRPs) play an important role in mitochondrial iron homeostasis and function.IRP2 knockout significantly induces up-regulation hypoxia-inducing factor subunits, HIF1α and HIF2α, affecting mitochondrial function due to the activation of cascade reaction chains. [40]A commercial dye (DAPRed) for autophagy was chosen to stain wild type mouse embryonic fibroblasts (WT MEF) and MEF with global IRP2 knockout (IRP2 -/-MEF).Negligible red fluorescence was observed for WT MEF, while strong signal was collected in the IRP2 -/-MEF, indicating mitochondria in IRP2 knockout (KO) MEF were severely damaged and distinct mitophagy occurred (Figure 6a).As depicted in Figure 6b-d, WT MEF cells stained with AHOH showed moderate fluorescence in green channel and strong fluorescence in red channel, concomitant with a small overlap factor (PCC, 0.055 for the whole image and 0.109 for the enlarged image) between green and red channels.However, AHOH stained IRP2 -/-MEF cells showed enhanced green fluorescence (1.51-fold) and reduced red fluorescence (by 50%), with distinct elevated green channel and red channel overlap factor (PCC, 0.755 for the whole image and 0.815 for the enlarged image).These data demonstrated that AHOH was capable of dual-color visualization of increased oxidative stress and mitophagy flux in IRP2 KO MEF.

Pharmacodynamic Evaluation of Drugs Regulating Mitochondria Damage
We speculated that AHOH could serve as a potent imaging agent to build a fluorescence-based screening platform for evaluating drugs regulating mitochondria dysfunction.WT MEF cells and IRP2 -/-MEF cells were treated with PX-478 and PT-2385, specific inhibitors of HIF1α and HIF2α, respectively. [41]PX-478 can inhibit HIF1α expression while PT-2385 disrupts the heterodimerization of HIF2α with HIF1β and restore normal mitochondrial function.In WT MEF cells, the fluorescence intensities in red and green channel and the related PCC values between the two channels remained basically unchanged after treatment with PT-2385 and PX-478 (Figure 7a, c).While in IRP2 -/-MEF cells, the overlap between mitochondria and lysosomes decreased after treatment with PT-2385 and PX-478 (PCCs 0.380, and 0.595 v.s.PCC 0.617), concomitant with the decreased fluorescence in green channel and enhanced signal in red channel (Figure 7b,  d).Meanwhile, when IRP2 -/-MEF were treated with PX-478 and PT-2385 combination, negligible overlap between red and green channels (PCC 0.098) as well as more distinct decrease in green channel and increase in red channel were observed.These data suggested that PX-478 or PT-2385 single drug treatment could reduce mitochondria oxidative stress and mitophagy flux in IRP2 -/-MEF to some extent, PT-2385 showed better regulation performance.In addition, the combination treatment of both PX-478 and PT-2385 showed much better efficacy and almost completely restored normal mitochondria function.Collectively, the above results revealed that AHOH could function as a reliable fluorescence-based platform for evaluating drugs regulating mitochondria dysfunction.

Conclusion
We rationally constructed a merocyanine dye called AHOH , which showed dual-color/dual-target feature and could selectively response to HClO in a ratiometric manner.AHOH aggregates selectively locate in lysosome via endocytosis and show red emission, while its monomer preferentially enters mitochondria through electrostatic interaction and shows green emission due to oxidation by mitochondrial HClO.By quantifying overlap factors (PCC value) and the fluorescence intensity changes in the two channels, we could simultaneously monitor both mitochondria-lysosomes interactions and oxidative stress induced by drugs and genetic knockout through dual channel super-resolution fluorescent imaging.Moreover, AHOH was also applied in an imaging platform for evaluating drugs that regulate mitochondrial diseases.This study provided a powerful tool for visualization of mitochondria-lysosome interactions and redox status during mitophagy, which is of great importance for understanding the physiological functions of mitophagy and regulation of mitochondria dysfunction.

Acknowledgements
This study was financial supported from the National Natural Science Foundation of China (22122701, 22293050, 22293051, 91953201, 92153303, 22377050), the Excellent Research Program of Nanjing University (ZYJH004), the Natural Science Foundation of Jiangsu Province ( BK20232020, BK20230977).Financial support from the National Postdoctoral Program for Innovative Talents (BX2021123), the China Postdoctoral Science Foundation (2021M691505), the Jiangsu Postdoctoral Research Funding Program (2021K125B) and Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (23KJB150020) were also gratefully acknowledged.

Conflict of Interests
The authors declare no conflict of interests.

Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.
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Scheme 1 .
Scheme 1.(a) Synthesis route of probe AHOH ; (b) proposed mechanism of AHOH response to HClO and its ability to image mitochondria and lysosomes in dual emission colors.

Figure 3 .
Figure 3. Imaging of intracellular HClO.(a) Confocal fluorescence images in detection exogenous and endogenous HClO in HeLa cells, right column: enlarged view of selected merged regions shown in left column; (b), (c) relative fluorescence intensity of the corresponding green channel and red channel by different treatment.Scale bars in original images: 5 μm; Scale bars in enlarged images: 1 μm.

Figure 4 .
Figure 4. Monitoring the mitophagy induced by CCCP and rapamycin.(a) Fluorescence images of HeLa cells for tracking mitochondria-lysosomes fusion in mitophagy induced by CCCP and rapamycin.Top, living HeLa cells were incubated with AHOH (5 μM) for 10 min and then directly imaged; middle and bottom, HeLa cells were pretreated with CCCP (20 μM) or rapamycin (0.5 μM) for 30 min and then stained with 5 μM AHOH for another 10 min; (b) PCC values in (a); (c) Normalized average fluorescence intensities in green channel and red channel.Scale bars in original images: 5 μm; Scale bars in enlarged images: 1 μm.

Figure 5 .
Figure 5. Real-time imaging of dynamic interactions between mitochondria and lysosomes upon exogenous stimulations in HeLa cells,AHOH (5 μM) was added to the culture plates of two groups and incubated for 10 min.(a) Merged fluorescence images of green and red channels with AHOH at various time points (0-200 s) after washing with HBSS buffer solution; (b) Merged fluorescence images of green and red channels of AHOH loaded cells co-stained with CCCP (20 μM) at virous time points (0-10 min); lower panel: enlarged view of selected regions shown in upper panel; (c) (d) Mean fluorescence intensity changes in green and red channels at different time in (a) and (b).Scale bars in original images: 5 μm; Scale bars in enlarged images: 1 μm.

Figure 6 .
Figure 6.Dual channel monitoring of oxidative stress and mitophagy flux in MEF with mitochondrial dysfunction.(a)Imaging of WT MEF cells and IRP2 -/-MEF cells stained with DAPRed, the relative fluorescence intensities are shown in the right panel; (b) fluorescence images in WT MEF cells and IRP2 -/-MEF cells, all cells were incubated with AHOH (5 μM) at 37 for 10 min; (c) PCC values in green channel and red channel in WT MEF cells and IRP2 -/-MEF cells.(d) analysis of the average fluorescence intensity in green channel and red channel in WT MEF cells and IRP2 -/-MEF cells.Scale bars in original images: 5 μm; Scale bars in enlarged images: 1 μm.