Unravelling the correlation between metal induced aggregation and cellular uptake/subcellular localization of Znsalen: an overlooked rule for design of luminescent metal probes

We demonstrate the importance of speciation of luminescent metal complexes in water on biological behaviours such as cellular uptake and subcellular localization.


General information
All solvents and chemicals for synthesis were purchased from Alfa Aesar and J&K and used as received without further purification, unless otherwise specified. Cellular imaging trackers and endocytosis inhibitors were purchased from Invitrogen (Life Technologies). Mitochondrial Isolation Kit for Cultured Cells is purchased from Cultured Cells BiYunTian company. The 1 H NMR spectroscopic measurements were carried out using a Bruker-400 NMR at 400 MHz with tetramethysilane (TMS) as internal reference. The 13 C NMR spectroscopic measurements were carried out using a 100 MHz NMR (Bruker-400, USA). Electrospray ionization (ESI) mass spectra were performed on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (Bruker, USA), positive-ion mode. FT-IR spectra were taken on a Nicolet iN10 MX Fourier Transform Infrared Spectrometer. The steady-state absorption spectra were obtained with an Agilent 8453 UV-vis spectrophotometer in 1cm path length quartz cells. Single-photon luminescence spectra were recorded using fluorescence lifetime and steady state spectrophotometer (Edinburgh Instrument FLS920). Quantum yields of one photon emission of all the synthesized compounds were measured relative to the fluorescence of Rhodamine B (Ф=0.65) in ethanol, and the two photon absorption cross section of the probes was calculated at each wavelength relative to Rhodamine B as standard. Confocal fluorescent images of living cells were performed using Nikon A1R-si Laser Scanning Confocal Microscope (Japan), equipped with lasers of 405/488/543/638 nm. Morphology images of ZnL 1 and L 1 were taken using Transmission Electron Microscope (JEM-2100, Japan) and Scanning electron microscope (SPI3800/SPA400, Japan). Zeta plus were used to characterize the liposomal particle size and zeta potential. DLS data were recorded using Laser Light Scattering Spectrometer (ALV/DLS/SLS-5022F, ALV/Laser Vertriebsgesellschaft m.b.H, German). Endocytosis inhibition experiments were performed using Flow Cytometry Analyzer (BD LSR Fortessa). Compound 2 3-methoxy-N-methyl-N-(prop-2-ynyl)aniline A reaction mixture of 3-methoxyaniline (2.0 g, 16.0 mmol), 3-bromoprophyne (1.9 g, 16.0 mmol) and K 2 CO 3 (2.2 g, 16.0 mmol) in acetonitrile (50 mL) was refluxed under nitrogen for 12 h. After evaporation, the residue was extracted with CH 2 Cl 2 , washed and dried with anhydrous Na 2 SO 4 . Then the concentrated liquid was further purified by column chromatography to give yellow oil (2.1 g, 81%). 1

Compound L 1
A mixture of compound 6 (220.0 mg, 0.4 mmol) and diaminomaleonitrile (23.0 mg, 0.2 mmol) was dissolved in 10 mL MeOH and refluxed overnight in the presence of a drop of concentrated H 2 SO 4 . Solvent was removed by rotary evaporator, and the product L 2 was recrystallized from DCM/ether as violet solid after filtration (100 mg, 39%). 1

Compound L 2
A mixture of compound 6 (50 mg, 0.088 mmol) and diaminomaleonitrile (5.0 mg, 0.2 mmol) was dissolved in 10 mL MeOH and refluxed for 8 h. Solvent was removed by rotary evaporator, and the product L 2 was recrystallized from methanol and ether as dark red solid after filtration (29 mg, 51%). 1

Quantum yield determination
Quantum yields of one photon emission of L 1, H 2 L 1 , L 2 were measured with Rhodamine B as reference (Ф=0.65). [2] The one photon fluorescence measurements were performed in 1cm quartz cells with 1 μM compound in DMSO on a fluorescence lifetime and steady state spectrophotometer (Edinburgh Instrument FLS920) equipped 450 W Xenon light, slits 2.5 × 2.5. The values of fluorescence quantum yield, Φ (sample), were calculated according to equation as following [

Two photon absorption
The two photon absorption spectra of ZnL 1 and L 1 were determined over a broad spectral region by the typical two photon induced fluorescence method relative to Rhodamine B as standard. The two photon fluorescence data were acquired using a Tsunami femtosecond Ti: Sapphire laser (pulse width ≤100fs, 80 MHz repetition rate, tuning range 720-870 nm Spectra Physics Inc., USA). The two photon fluorescence measurements were performed in a 1cm quartz cell with 40 μM Salen in DMSO and the excitation power density is set to be 100 mW. The quadratic dependence of two photon induced fluorescence intensity on the excitation power was verified (from 34 mW to 127 mW) for excitation wavelength at 840 nm. The two photon absorption cross section of the Salens (δ sample ) was calculated at each wavelength according to equation as following [4]

Lop P determination
Equal amounts of n-octanol and water were thoroughly mixed by an oscillator for 24 h. The mixture was then left to separate for another 24 h to finally yield water and octanol phase, each saturated with the other. Each complex was dissolved in water (C o ) and water saturated with octanol to form a 20 μM solution. Then the latter was mixed with equal amounts of octanol (saturated with water) and shaken again as described above. After separation, the final concentrations of compounds in water corresponded to C w . The final concentration in 1-octanol corresponded to C oil. [5] ( . S3)  oil water C P Equation C

The Mitochondria Isolation
Mitochondrial permeation capabilities more exact using freshly isolated mitochondria. To prepare mitochondrial fractions, HeLa cells were harvested and the pellet processed using a Mitochondrial Isolation Kit for Cultured Cells (BiYunTian company). All isolation operations were completed under sterile conditions at 4 °C.

GUV preparation
GUVs were prepared by gentle hydration method. [6] Lipids dissolved in chloroform were mixed at designated ratios in a glass flask with a final mass of 1 mg. The chloroform was evaporated under reduced pressure distillation followed by storage overnight. The dried lipids film was stored at -20 o C prior to use. To hydrate the lipid, 1 mL buffer PBS 8.0 buffer was added to the film for about an hour after prehydration. The liposome size and zeta-potential were measured using Zeta Plus. Measurements were performed by dispersing liposomes at a concentration of 1 mg mL -1 in buffer pH 8.0. The temperature was set at 25 o C for all measurements. ZnL 1 and L 1 solution were added into the well prepared GUVs solution, and the mixture was stirred softly for half an hour. To get confocal images, the vesicles were filled with sucrose and were in a glucose environment at osmolarity 200 mOsm g -1 . The effect of gravity deforming the vesicle is visible from the snapshot.

Colocalization assay
HeLa cells were placed onto 0.1 mM poly-D-lysine coated glasses in complete media and the cells were incubated for 24 h. A stock solution of Salens in chromatographic grade, anhydrous DMSO was prepared as 2 mM. The solution was diluted to a final concentration of 2 μM by complete growth medium. Stock solutions of Lyso Tracker Green DND-26, MitoTracker Green FM were prepared as 1 mM, and the stock solution was diluted to the working concentrations in complete medium (Lyso Tracker: 75 nM, Mito Tracker: 100 nM). Transfection with EHD1-EGFP, and FYVE-EGFP plasmids: Hela cells were grown to about 80% confluency and then reseeded in 24-well plates; cells were transfected with 0.8 μg plasmids, using LipfectamineTM 2000 according to manufacturer's instruction. After incubation of 2 μM Salens for different period of time, cells were washed with PBS buffer twice before confocal experiments. Images were taken under conditions as follows: 60× immersion lens with a resolution of 1024×1024 and a speed of 0.5 frame per second, 543 nm excitation wavelength and 552 to 617 nm detector slit, 100% laser power for dye, and 80% laser power for LysoTracker (ex: 488 nm, em: 505-560 nm), MitoTracker (ex: 488 nm, em: 505-560 nm), FYVE-EGFP (ex: 488 nm, em: 505-560 nm), EHD1-EGFP (ex: 488 nm, em: 505-560 nm). Differential interference contrast (DIC) and fluorescent images were processed and analyzed using ImageJ. The Pearson's Coefficient was calculated by ImageJ.

Cell uptake mechanism assay
The cellular uptake of luminescent metal complexes is primarily examined using two complementary methods, flow cytometry and confocal microscopy. In the temperature effect assay, cells were placed at 4 o C for 15 minutes, and then incubated with ZnL 1 in absence or presence of pyridine for another hour at 4 o C. For endocytosis mechanism investigation, various endocytosis inhibitors including chlorpromazine (inhibitor of clathrin-mediated endocytosis), genistein (inhibitor of caveolae-mediated endocytosis), cytochalasin D (inhibitor of macropinocytosis) were applied to cells for 30 minutes. Then medium containing both inhibitors and complex was used for incubation for another hour. For flow cytometry, cells are detached from culture either before or after incubation with the metal complex to produce a cell suspension. Untreated cells are used for as a control for autofluorescence. The cells are inspected individually as they pass single file through the laser beam of 561 nm and the instrument records their light scatter and luminescence from 570 to 610 nm. The distribution of luminescence for the cell population is depicted as a histogram of the number of cells versus luminescence intensity.
In the membrane potential effect assay, HeLa cells were detached from culture and washed three times with either HBSS (containing 5.8 mM K + ) or high K + -HBSS (containing 170 mM K + ). Some of the cells in HBSS were pretreated with 10 µM nigericin for 30 min at 37°C. The cells were incubated with 2µM ZnL 1 for 1h at 37 °C in one of the following solutions: HBSS, HBSS with nigericin (to hyperpolarize the cells), or high K + -HBSS (to depolarize the cells). After incubation, the cells were rinsed, and the extent of uptake was analyzed by confocal imaging and dealt with ImageJ. Images were taken under conditions as follows: 60× immersion lens with a resolution of 1024×1024 and a speed of 0.5 frame per second, 543 nm excitation wavelength and 552 to 617 nm detector slit, 100% laser power for dye. Differential interference contrast (DIC) and fluorescent images were processed and analyzed using ImageJ.
In the Organic Cation Transporter effect assay, HeLa cells were detached from culture and washed three times with HBSS (containing 5.8 mM K + ), and then pretreated for 20 min with either 1 mM cation transport inhibitor (Tetrabutylammonium bromide). The cells were then incubated with 2µM ZnL 1 for 1h at 37 °C in one of the following solutions: HBSS, HBSS with 1 mM cation transport inhibitor (Tetrabutylammonium bromide). After incubation, the cells were rinsed, and the extent of uptake was analyzed by confocal imaging and dealt with ImageJ. Images were taken under conditions as follows: 60× immersion lens with a resolution of 1024×1024 and a speed of 0.5 frame per second, 543 nm excitation wavelength and 552 to 617 nm detector slit, 100% laser power for dye. Differential interference contrast (DIC) and fluorescent images were processed and analyzed using ImageJ.

Estimation of aggregation constants of ZnL 1
The aggregation constant for ZnL 1 was calculated by pyridine titration at 298K. The basic method were according to Kleij`s work [7] . 0-350 000 equiv. pyridine were added to a water solution of 20 μM ZnL 1 . The reaction and correlative equilibrium constants are shown as equation (1): (ZnSalen) n + nPy n ZnSalen(Py) K 1 =K n py /K agg K agg =K n-1 n/n+1 , K agg and K n/n+1 represent the overall aggregation constant and stepwise aggregation constant, as shown in equation (2) and (3): (ZnSalen) n n ZnSalen (2) K 2 = K agg (ZnSalen) n (3) The equilibrium constant K 1 was determined by fitting for A 590 nm and equiv. of pyridine.
For each A 590 nm obtained from the titration, there was relationship as A = ε 1 c 1 + ε 2 c 2 (4) nc 1 + c 2 = c total (5) A is the absorbance at 590 nm; n is the aggregation number of ZnL 1 aggregates; ε 1 and ε 2 are the molar extinction coefficient of ZnL 1 aggregates and its pyridine complex in H 2 O, which are obtained from the limit value of fitting curve; c 1 , and c 2 represent the concentration of ZnL 1 aggregates, ZnL 1 -pyridine complex, respectively; and c total is the total concentration of ZnSalen species, which is 20 μM in this case. Combining equation (4) and (5), c 1 and c 2 could be represented as And the equilibrium constant of equation (1) could be written as c py is the concentration of pyridine, which depends on the equiv. of pyridine q; A 1 and A 2 are the limit absorbance of fitting curve, representing A 590 nm of totally aggregated and dissociated states, respectively.
To estimate K agg and K n/n+1 , the coordination constant of pyridine was measured first. 0-40 equiv. pyridine were added to the 20 μM ZnL 1 dichloromethane solution. As shown in equation (7), K py was calculated by the curves of A 587 nm and equiv. of pyridine.

ZnSalen + Py
ZnSalen(Py) K 4 =K py = (9) c 4 c 3 c py c 3 and c 4 are the concentration of ZnL 1 and its pyridine complex in dichloromethane solution, which can also be represented as

c total A-A 4 A 3 -A 4 (10) c 3 = c total A 3 -A A 3 -A 4 (11)
A is the absorbance at 587 nm for each titration; A 3 and A 4 are the absorbance of ZnL 1 without pyridine coordination and totally corrdination, obtained from inital spectra and the limit value from data fitting, respectively. Thus, K agg and K n/n+1 could be determined by K 1 , K py , and n, according to equation (1).

DOSY experiments and estimation of molecular mass of ZnL 1
The DOSY spectroscopic were recorded using a Bruker-500 NMR. The calculation of molecular mass was according to Bella's report [8] . The molecular mass of two species is followed by m B = m A (D A /D B ) 2 (12) Different amount of D 2 O were added to 2 mM ZnL 1 dissolved in DMSO-d 6 solution. The 1 H signal of HDO was used as the reference, with molecular mass of 19 Da. Diffusion coefficient of ZnL 1 was averaged by diffusion coefficients of all peaks on salen. Thus, the molecular mass of ZnL 1 species in these condition could be estimated using equation 12. Quantum yields were measured using Rhodamine B in ethanol as reference.                 14(m, 4H). The 1 H NMR spectrum was shown in Figure S18. 13   The 1 H NMR spectrum was shown in Figure S19. 13