Development and Application of Cationic Nile Blue Probes in Live-Cell Super-Resolution Imaging and Specific Targeting to Mitochondria

Mitochondria are essential organelles involved in various metabolic processes in eukaryotes. The imaging, targeting, and investigation of cell death mechanisms related to mitochondria have garnered significant interest. Small-molecule fluorescent probes have proven to be robust tools for utilizing light to advance the study of mitochondrial biology. In this study, we present the rational design of cationic Nile blue probes carrying a permanent positive charge for these purposes. The cationic Nile blue probes exhibit excellent mitochondrial permeability, unique solvatochromism, and resistance to oxidation. We observed weaker fluorescence in aqueous solutions compared to lipophilic solvents, thereby minimizing background fluorescence in the cytoplasm. Additionally, we achieved photoredox switching of the cationic Nile blue probes under mild conditions. This enabled us to demonstrate their application for the first time in single-molecule localization microscopy of mitochondria, allowing us to observe mitochondrial fission and fusion behaviors. Compared to conventional cyanine fluorophores, this class of dyes demonstrated prolonged resistance to photobleaching, likely due to their antioxidation properties. Furthermore, we extended the application of cationic Nile blue probes to the mitochondria-specific delivery of taxanes, facilitating the study of direct interactions between the drug and organelles. Our approach to triggering cell death without reliance on microtubule binding provides valuable insights into anticancer drug research and drug-resistance mechanisms.


General Methods for Synthesis
All reagents and solvents were used as received from commercial sources (Sigma-Aldrich, Acros Organics, J&K Scientific, Bide Pharm and Energy-Chemical) unless otherwise specified.Anhydrous tetrahydrofuran (THF), diethyl ether (Et 2 O) and dichloromethane (DCM) were collected from a PureSolv MD Solvent Purification System made by Innovative Technology and stored in 4Å molecular sieves.Column chromatography was carried out on silica gel 60 (particle size of 0.040-0.063mm, from various commercial sources) and eluted with solvents specified.Preparative HPLC separations were performed with Waters HPLC system equipped with photodiode array detector using XBridge Prep C18 10 μm OBD column (10 μm, 300 Å, 30 × 250 mm) at a flow rate of 15 mL/min.Mobile phases of HPLC used are as follows, Solvent A: acetonitrile; Solvent B: 0.1% TFA (v/v) in water.Nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance Fourier Transform Spectrometers at room temperature.NMR peaks were reported in δ ppm, after calibration using appropriate NMR solvent peaks for 1 H and 13 C NMR, or the tetramethylsilane (TMS) peak for 1 H NMR. Highresolution mass spectra were acquired using a Thermo Scientific Electron Finnigan Gas Chromatography High Resolution MS System (for EI), and Bruker Impact II/Maxis II UHR-TOF LC-MS Systems (for ESI).  Figure S2. 1 H NMR spectrum of S1, which was isolated from the reaction shown in Scheme S1, while tetraethyl Nile blue S2 could not be isolated due to its low yield.
m/z: 346.30m/z: 374.44 In our pursuit of Nile blue derivatives as mitochondrial targeting probes, we initially focused on preparation of N, N'-tetraethyl Nile blue (compound S2, Scheme S1).However, only triethyl Nile blue S1 was isolated as predominant product, whose structure was unambiguously confirmed by LC/MS and NMR spectrum (Figures S1-S2).According to the reaction outcome, a possible pathway for the condensation and formation of S1 blue was proposed (Scheme S1).In the first step, nucleophilic addition of amino naphthalene to nitroso group occurred under the catalysis of acetic acid.Simultaneously, C−N single bond in amino naphthalene was converted to double bond (C=N) and hybridization of this nitrogen was converted from sp 3 to sp 2 , by which the two ethyl groups on this nitrogen were placed on the same plane with naphthalene ring.In addition, the bond length was greatly shortened during transformation from C−N to C=N.These two factors resulted in the formation of an unstable iminium cation (showing in intermediate S0) and this instability mainly arose from steric hindrance experienced between one of the ethyl groups on iminium and the naphthalene ring (highlighted in red in S0).Therefore, it made this ethyl group easily attacked by the solvent acetic acid at elevated temperatures.A lower temperature (~100 o C) was unable to drive the reaction to happen, probably due to the instability of S0 (high activation energy for this step).
Since steric hindrance played a deterministic role in the reaction outcome, we next replaced the two ethyl groups on the amino naphthalene with two methyl groups considering the relatively small space occupied (Scheme S2).As could be predicted from its LC/MS profile (Figure S3), the steric hindrance was greatly alleviated after carrying out such replacement.Therefore, dimethyl Nile blue S4, instead of monomethyl one S3, was generated as the major product (Scheme S2).However, we envisioned that the former product could still suffer from nucleophilic attack in live cells due to the existence of large amounts of thiol and aminecontaining species, which complicates its application.

Cell culture
HeLa cells or COS-7 cells were cultured as sub-confluent monolayers on 100 mm cell culture dishes in complete growth medium, i.e.Dulbecco's Modified Eagle Medium (DMEM) supplemented with heat inactivated Fetal Bovine Serum (FBS, 10%, v/v) and Penicillin-Streptomycin (PS, 1%, v/v), in a humidified incubator (70 -95 %) at 37 o C with CO 2 (5%).Cells grown to sub-confluence were enzymatically dissociated from the surface with 1 mL of a solution of trypsin (0.05%)/EDTA and washed with 2 mL of fresh medium.The cells were spun down (1000 rpm × 3 min) for counting.U-2 OS cells were cultured under the same conditions used for HeLa culturing despite that McCoy's 5A Modified Medium supplemented with heat inactivated FBS (10%, v/v) and PS (1%, v/v) was used as complete growth medium.

Confocal imaging of live cells
The cells were plated in 35 mm glass bottom confocal dishes (Mat-Tek) at density of 1 × 10 5 cell/mL in 2-mL seeding volume 1 day prior to the imaging, or at density of 5×10 4 cell/mL in 2-mL seeding volume 2 days prior to the imaging.These conditions produced a monolayer at sub-confluence for the experiments.After incubation overnight, the medium was discarded and replaced with Hanks' Balanced Salt Solution (HBSS) containing probes of specified concentrations for different time.Before imaging, cells were washed with HBBS for 3 times and placed in DMEM (phenol red free).Fluorescence microscopy was performed with Zeiss LMS 780 or 880 confocal microscope with a 64× or 40×/1.3oil-immersion objective lens.A stage-top incubator was used to maintain an imaging environment at 37 o C with 5% CO 2 .Excitation wavelength: 633 nm for CNB, CNB-Cl, CNB-PTX and MTDR; 561 nm for MTR and NR-PTX; 488 nm for Cyt c monoclonal antibody-Alexa Fluor 488 conjugates, LTG and MTG; 405 nm for Hoechst.

Mapping the distribution of cytochrome c in probes treated cells
The cells were plated in 35 mm glass bottom confocal dishes (Mat-Tek) at density of 5×10 4 cell/mL in 2-mL seeding volume two days prior to the imaging.After overnight incubation, the medium was changed with fresh medium containing various concentrations of probes as specified in the figure descriptions containing 0.1% DMSO.After incubation for 24 hours at 37 °C in a 5% CO 2 incubator, the medium was discarded, and cells were washed with PBS twice.The cells were fixed with 4% PFA in PBS at room temperature for 15 min, washed with PBS twice, blocked with 3% BSA at room temperature for 30 min, washed with PBS twice successively.The cells were than incubated with cyt c monoclonal antibody (6H2.B4)-Alexa fluor 488 conjugates (Thermo Fisher) at a dilution of 5 µg/mL in blocking buffer for 1 hour.The cells were washed with PBS three times before mounted onto confocal microscope.Fluorescence imaging was performed with Zeiss LMS 780 or 880 confocal microscope with a 64× or 40×/1.3oil-immersion objective lens.

Cell viability assay
The effects of different probes on cell viability were analysed using CellTiter-Glo® (Promega) Luminescent cell viability assay.Cells were seeded at density of 5000 cells/well into a 96-well microplate (black plate, clear flat bottom with lid) and incubated in DMEM containing 10% FBS and 1% PS at 37 °C in a 5% CO 2 incubator overnight.The medium was then replaced with fresh medium containing various concentrations of probes 1% DMF.After incubation for 24 h, cells were equilibrated at room temperature for approximately 30 min before loaded with 50 μL of Cell-Titer Glo reagent.The microplates were subjected to gentle shaking for cell lysis at room temperature (approximately 10 min).The microplate was mounted onto a DTX multimode plate reader (Molecular Devices) for luminescence detection (550 nm), by using cellular ATP contents as a measure of cell viability.

Evaluation of cellular uptake of cationic Nile blue derivatives
The HeLa cells were seeded in 5 cm of cultural dishes at density of 2 × 10 5 cell/mL in 4-mL seeding volume and allowed to grow at 37 o C. 24 hours later, the medium (DMEM) was replaced with fresh medium containing various concentrations of taxane derivatives or cationic Nile blue containing 0.1% DMSO.After incubation for 6 hours, the medium was discarded, and the cells were washed with PBS twice and trypsinized.After centrifugation, the cell pellet was resuspended in PBS and recentrifuged.The pellet was then extracted with 1 ml of 95% ethanol.After sonication for 20 min and further centrifugation, the concentration of probes in supernatant was analyzed by reading fluorescence intensity at 680 nm with excitation at 645 nm.

Absorption and Fluorescence analysis
Fluorescent dyes were dissolved in DMF or DMSO to make a 10 mM stock solution, which was diluted to the required concentration of testing solution for measurement in a 1 cm × 1 cm quartz cuvette.UV-Visible absorption spectra of sample solutions in spectral grade solvents were measured using a Shimadzu UV-3150 spectrometer or an L6S Split Beam UV-VIS Spectrophotometer (INESA Analytical Instrument Co., Ltd.).Fluorescence measurements were carried out at room temperature on a Hitachi F-7000 fluorescence spectrophotometer or an FS5 Spectrofluorometer (Edinburgh Instruments Ltd.).

Fluorescence quantum yield (Q.Y.) measuring
Fluorescence quantum yields were determined at a temperature of 25 °C employing a relative method, which utilized Nile blue A (Φ F = 0.27 in EtOH) 3 as a standard.The relative fluorescence quantum yield was ascertained using the subsequent equation: where A is the absorbance (below 0.1 A.U.), F is the area under the emission curve, n is the refractive index of the solvents (at 25 °C) used in the measurements, and the subscripts s and x represent standard and unknown, respectively.The refractive index values used in these measurements were 1.36 for ethanol and 1.33 for PBS.

Sample preparation and data analysis of single-molecule localization microscopy
For fixed cells: U-2 OS cells were seeded in a 35 mm culture dish containing an 18 mm, round glass coverslip (Marienfeld) at density of 1 × 10 5 cell/mL in 2-mL seeding volume one day prior to the imaging.After overnight incubation at 37 °C in a 5% CO 2 incubator, the medium was discarded, and the cells were incubated with 200 nM of probes in HBSS for 1 hour.Cells were then washed with PBS twice and fixed with 4% PFA at room temperature for 15 min.The cells were then washed with PBS 3 times and further treated with Triton TM X-100 (0.1% in PBS) for 3 min at room temperature.The resulting fixed permeabilized cells were washed 3 times with PBS before further treatment.The coverslip was lifted from culture dish and placed in imaging chamber.The cells were immersed with 380 µl of imaging buffer (containing 1mM ascorbic acid, 1 mM methyl viologen and 50 mM Tris-HCl 8.0 in MilliQ H 2 O) and covered with an 18 mm × 18 mm square coverslip.The imaging chamber was then mounted onto the microscope for both wide-field and super-resolution imaging.
For live cells: U-2 OS cells, COS-7 cells or HeLa cells were seeded in a 35 mm culture dish containing an 18 mm, round glass coverslip (Marienfeld) at density of 1 × 10 5 cell/mL in 2-mL seeding volume one day prior to the imaging.After overnight incubation at 37 °C in a 5% CO 2 incubator, the medium was discarded, and the cells were incubated with 100~250 nM of probes in HBSS for 1 h.The cells were then washed with DMEM twice.The coverslip was lifted from culture dish and placed in imaging chamber.The cells were immersed with 380 µL of DMEM (phenol red free) and covered with an 18 mm × 18 mm square coverslip.The imaging chamber was then mounted onto the microscope for both wide-field and super-resolution imaging.SMLM data were pre-processed with the HAWK plugin 4 using five levels to remove potential artifacts, followed by single-emitter fitting with ThunderSTORM plugin. 5Fluorescent spots that were too dim or too bright were discarded.

2 .
Scheme S1.Attempt to prepare tetraethyl Nile blue and proposed mechanism for the formation of of N, N'-tetraethyl Nile blue.Briefly, diethylamino naphthalene and nitroso compound were heated to 130 o C in acetic acid under air for 4 hours.Afterwards, an aliquot of reaction mixture was subjected to LC/MS analysis.Major products were isolated by column chromatography on silica gel.

Figure S1 .
Figure S1.LC/MS analysis of the reaction components in Scheme S1.It shows that the major product (retention time of 5.23 min) has a m/z of 346.30, corresponding to triethyl Nile blue S1.
Scheme S2.Attempts to prepare dimethyl Nile blue S4.Briefly, dimethylamino naphthalene and nitroso compound were heated to 130 o C in acetic acid under air for 2 hours.Afterwards, an aliquot of the reaction mixture was subjected to LC/MS analysis.

Figure S3 .CNB
Figure S3.LC/MS analysis of reaction components in Scheme S2.It shows that the major product has a m/z of 346, corresponding to the desired major product S4 in Scheme S2.
Microscope setup for single-molecule localization microscopy SMLM experiments were performed on a custom-built microscope (Nano BioImaging SRiS 2.0) based on a Nikon eclipse Ti-E inverted microscope.A single activation/imaging photodiode laser (647 nm, 500 mW) was focused to the back focal plane of the objective (CFI Apochromat TIRF 100× Oil N.A. 1.49).The laser intensity was controlled directly by a Rhodea 2.0 software.A dichroic beam splitter (T760LPXR-UF2) and a bandpass filter (FF01-692/40-25) separated the fluorescence emission from the excitation light.The fluorescence was recorded with an electron-multiplying CCD camera (Andor iXon Ultra 897).10000 -20000 frames were recorded.During data acquisition, a Perfect Focus System was used to maintain a constant focal plane.
Scheme S5.Commercially available fluorescent probes used in this study.

Figure S5 .Figure S6 .Figure S7 .
Figure S5.(A) Application of CNB in live cell imaging of HeLa cells.Yellow signals in the merged images arising from the co-localization fluorescence of MTR and CNB confirm mitochondrial accumulation of CNB.(B) Fluorescence line plots of the merged image in (A).(C) Correlation plot of CNB and MTR.

Figure S9 .
Figure S9.Comparison of background signal of CNB-Cl and MTG in U-2 OS cells.(A) U-2 OS cells were incubated with mitotracker green (MTG, 100 nM) and CNB-Cl (100 nM) for 1 hour.A set of images taken from cells so that the distance from the objective (z-axis) is different for each image but the image area along the x-and y-axes remains the same.(B) Concentrations are 500 nM for both dyes.Green colour is fluorescence of MTG.Red color is fluorescence of CNB-Cl.Yellow signals arise from the co-localization fluorescence of MTG and CNB-Cl.Scale bars, 20 μm.

Figure S10 .
Figure S10.Evaluation of performance of CNB-Cl in no-wash imaging of live cells.HeLa cells were incubated with solution of CNB-Cl or MTDR in DMEM for 1 hour and directly viewed under confocal laser scanning microscopy.(A) 50 nM of CNB-Cl.(B) 250 nM of CNB-Cl.(C) 500 nM of CNB-Cl.(D) 500 nM of MTDR.Characteristic morphology of mitochondria was observed in (A) (B) and (C) with no or very weak background signals while diffusion of MTDR into cytosol and nucleus were clearly observed (D).Scale bars, 10 μm.

Figure S11 .
Figure S11.Evaluation of performance of CNB in no-wash imaging of live cells.Characteristic morphology of mitochondria was observed with no background signals at broad range of concentrations.Scale bars, 10 μm.

Figure S12 .
Figure S12.Mitochondrial damage effects on HeLa cells of MTDR and CNB-Cl at 100 nM after treatment for 24 hours.Scale bars, 20 μm.

Figure S13 .
Figure S13.(A) A confocal image of typical mitochondria in U-2 OS cells stained by CNB-Cl.(B).Enlargement of the region boxed in figure (A).(C) Transverse profile of single mitochondrion along the green dotted line in figure (B).It indicates the FWHM of single mitochondrion in confocal image is around 400 nm.Scale bars: (A) 10 μm, (B) 2 μm.

Figure S14 .
Figure S14.(A) Histogram of photon numbers per dye molecule CNB-Cl per imaging frame.(B) Histogram of localization uncertainty per dye molecule per imaging frame.Corresponding to Figure 2D.

Figure S15 .
Figure S15.(A) A typical SMLM image of mitochondria in living U-2 OS cells stained by CNB-Cl.(B) Histogram of photon numbers per dye molecule per imaging frame.(C) Histogram of localization uncertainty per dye molecule per imaging frame.Scale bar, 4 μm.

Figure S16 .
Figure S16.Mitochondria dynamics in live Hela cells were revealed by CNB-Cl uncer SMLM condition.Each image was reconstructed with 2000 consecutive frames under rate of 100 frame/sec.

Figure S17 .
Figure S17.Subcellular localization of CNB-PTX in live cells.HeLa cells were incubated with solution of Mitotracker green (MTG, 100 nM) and CNB-PTX (2 μM).Yellow signals in the merged images arising from the co-localization fluorescence of MTG and CNB-PTX confirm mitochondrial accumulation of CNB-PTX.