A Cryptand-Type Aluminum Tris ( salophen ) Complex : Synthesis , Characterization , and Cell Imaging Application

Metal salen/salophen complexes have been used as fluorescent probes for cell imaging with various metal centers. Herein we synthesized cryptand-type aluminum salophen complexes LAl3 and the corresponding mononuclear compound LAl. X-ray crystal diffraction verifies the cryptand-type structure of LAl3 with C3h symmetry. Both LAl3 and LAl show moderate green fluorescence with quantum yields of 0.17 and 0.05, respectively. The hydrophilic and cationic nature of these aluminum salophen complexes renders them enhanced cellular uptake. Both complexes are internalized into cells by energy-dependent pathways and they distribute in lysosomal organelles.

However, the trinuclear Zn salophen complexes suffer from little intracellular fluorescence signal, probably due to low cellular uptake arising from the hydrophobicity and neutral form and thus could not be directly used as cell imaging probes.To enhance the cellular uptake, we envisioned to use the main group element Al (3+) cation for (1) form the cation complexes; (2) closed shell structure similar to Zn 2+ ; and (3) Lewis acid reactivity.These features render Al salen/salophen complexes better cell imaging probes than Zn complexes [33,34].Previously, we demonstrated that monomeric [AlSalen] + Cl − displayed higher sensitivity and stronger intracellular fluorescence in monitoring intracellular microvisicosity than Zn analogue.In this work, we synthesized the trinuclear aluminum salophen complex LAl 3 and the corresponding mononuclear complex LAl and performed live cell imaging experiments to reveal the ability of LAl 3 as a fluorescent probe, which highlights the importance of choice of metal ion in design of metal probes.

Synthesis and Characterization
Mononuclear salophen ligand and Al salophen complexes were synthesized according to the reported methods [35][36][37][38][39][40].Salicylaldehyde and o-phenylenediamine were refluxed in ethanol for 16 h to give the orange salophen ligand.The reaction of salophen ligand with aluminum chloride in acetonitrile afforded the target mononuclear complex LAl.More importantly, we synthesized the trinuclear aluminum salophen complex LAl 3 to study the potential synergistic effect (Figure 1).Trinuclear cryptand-type salophen ligand was prepared according to the procedure reported by T. Nabeshima [41], and LAl 3 was obtained by stirring ligand and aluminum chloride in refluxing acetonitrile for 24 h.The axial chloride was replaced by fluoride to get LAl 3 -F, which was used for crystal growth.
Inorganics 2018, 6, x FOR PEER REVIEW 2 of 11 monitoring intracellular microvisicosity than Zn analogue.In this work, we synthesized the trinuclear aluminum salophen complex LAl3 and the corresponding mononuclear complex LAl and performed live cell imaging experiments to reveal the ability of LAl3 as a fluorescent probe, which highlights the importance of choice of metal ion in design of metal probes.

Synthesis and Characterization
Mononuclear salophen ligand and Al salophen complexes were synthesized according to the reported methods [35][36][37][38][39][40].Salicylaldehyde and o-phenylenediamine were refluxed in ethanol for 16 h to give the orange salophen ligand.The reaction of salophen ligand with aluminum chloride in acetonitrile afforded the target mononuclear complex LAl.More importantly, we synthesized the trinuclear aluminum salophen complex LAl3 to study the potential synergistic effect (Figure 1).Trinuclear cryptand-type salophen ligand was prepared according to the procedure reported by T. Nabeshima [41], and LAl3 was obtained by stirring ligand and aluminum chloride in refluxing acetonitrile for 24 h.The axial chloride was replaced by fluoride to get LAl3-F, which was used for crystal growth.To further confirm the structure of LAl3, we grew a single crystal (CCDC 1814298) suitable for X-ray structure analysis via the diffusion of N-hexane to a methanol/toluene mixed solution of LAl3-F.The detailed parameters are listed in Table S1.As shown in Figure 2, each Al 3+ shows octahedral coordination geometry, with a fluorinion and a methanol molecule as the axial ligand.Al 3+ only 0.109 above the N2O2 plane and the angles between each two N2O2 planes are near 60°, indicating the C3 symmetry of the molecule.Given that there is a symmetry plane perpendicular to the C3 axial, the LAl3 molecule belongs to C3h point group.Each salophen moiety shows great distortion, with the angle between two phenyl planes being near 58.57°, larger than that in the crystal of mononuclear LAl reported by Darensbourg et al. [35].The octahedral coordination geometry as well as the distortion of salophen moieties inhibit the aggregation of the molecules.The three compounds were characterized by 1 H NMR, ESI-MS, and IR spectroscopies. 1 H-NMR spectra in DMSO-d6 show good resolution of the proton signals.Aromatic and imine protons in LAl 3 display nearly same chemical shifts with the mononuclear LAl, revealing the use of cryptand-like tri-salophen ligand has little effect to the conjugated system in the salophen moieties.The disappearance of an aromatic peak at δ = 7.68 and the emergence of a single peak at δ = 8.27 corresponding to the methenyl protons are strong proofs supporting the cryptand-type structure of LAl 3 .IR spectra show characteristic C=N vibration (1625, 1620 cm −1 ) of metal salophen complexes.The ESI mass spectra of the mononuclear and trinuclear Al salophen complexes show main peaks corresponding to m/z peak at 341.0866 and 1191.1954,respectively.The accurate exact mass and the successful assignment of 1 H NMR signals ensure that the cryptand structure of LAl 3 is right.
To further confirm the structure of LAl 3 , we grew a single crystal (CCDC 1814298) suitable for X-ray structure analysis via the diffusion of N-hexane to a methanol/toluene mixed solution of LAl 3 -F.The detailed parameters are listed in Table S1.As shown in Figure 2, each Al 3+ shows octahedral coordination geometry, with a fluorinion and a methanol molecule as the axial ligand.Al 3+ only 0.109 Å above the N 2 O 2 plane and the angles between each two N 2 O 2 planes are near 60 • , indicating the C 3 symmetry of the molecule.Given that there is a symmetry plane perpendicular to the C 3 axial, the LAl 3 molecule belongs to C 3h point group.Each salophen moiety shows great distortion, with the angle between two phenyl planes being near 58.57• , larger than that in the crystal of mononuclear LAl reported by Darensbourg et al. [35].The octahedral coordination geometry as well as the distortion of salophen moieties inhibit the aggregation of the molecules.

UV-Vis and Fluorescent Spectra
The absorption spectra of two Al salophen complexes in DMSO are shown in the Figure 3.The two main absorption bands at near 300 and 400 nm can be assigned to salophen-centered π-π* transitions.Different from the previously reported Zn salen complexes [17,21,31], no intramolecular charge-transfer transition bands between 500-600 nm is observed for there is no "D-π-A" structure in the salophen ligands.The absorption peaks of LAl3 red-shifts ca.20 nm compared to LAl.Besides, the extinction coefficient of LAl3 is obviously larger than LAl because of the increasing number of the salophen chromophores in one molecule.(Table 1  Emission spectra were recorded to investigate the excited state properties of two Al salophen complexes.(Figure 3) LAl and LAl3 show similar single emission bands with emission maxima at 500 and 523 nm, respectively.The large Stokes shift (ca.115 nm) of LAl3 is consistent with the previous report of cryptand-type Zn salophen complexes.Fluorescence lifetime of the two complexes were monitored at their emission maxima, and fluorescence quantum yields (±10%) were determined as 0.054 and 0.17 for LAl and LAl3 in DMSO, using quinine sulfate (Φf = 0.54 in 0.1 N H2SO4) as standard.The longer lifetime and higher fluorescence quantum yield of LAl3 can be attributed to its high structure rigidity.The fluorescence quantum yields of Al salophen complexes were only slightly larger than corresponding Zn complexes reported by us previously, indicating that similar closedshell metal centers show little effect to the fluorescence quantum yield of salophen complexes.The thermal ellipsoids are scaled to the 50% probability level.Carbon atoms are depicted in grey, oxygen in red, nitrogen in blue, fluorine in green, and aluminum in pink.

UV-Vis and Fluorescent Spectra
The absorption spectra of two Al salophen complexes in DMSO are shown in the Figure 3.The two main absorption bands at near 300 and 400 nm can be assigned to salophen-centered π-π* transitions.Different from the previously reported Zn salen complexes [17,21,31], no intramolecular charge-transfer transition bands between 500-600 nm is observed for there is no "D-π-A" structure in the salophen ligands.The absorption peaks of LAl 3 red-shifts ca.20 nm compared to LAl.Besides, the extinction coefficient of LAl 3 is obviously larger than LAl because of the increasing number of the salophen chromophores in one molecule.(Table 1)

UV-Vis and Fluorescent Spectra
The absorption spectra of two Al salophen complexes in DMSO are shown in the Figure 3.The two main absorption bands at near 300 and 400 nm can be assigned to salophen-centered π-π* transitions.Different from the previously reported Zn salen complexes [17,21,31], no intramolecular charge-transfer transition bands between 500-600 nm is observed for there is no "D-π-A" structure in the salophen ligands.The absorption peaks of LAl3 red-shifts ca.20 nm compared to LAl.Besides, the extinction coefficient of LAl3 is obviously larger than LAl because of the increasing number of the salophen chromophores in one molecule.(Table 1) Emission spectra were recorded to investigate the excited state properties of two Al salophen complexes.(Figure 3) LAl and LAl3 show similar single emission bands with emission maxima at 500 and 523 nm, respectively.The large Stokes shift (ca.115 nm) of LAl3 is consistent with the previous report of cryptand-type Zn salophen complexes.Fluorescence lifetime of the two complexes were monitored at their emission maxima, and fluorescence quantum yields (±10%) were determined as 0.054 and 0.17 for LAl and LAl3 in DMSO, using quinine sulfate (Φf = 0.54 in 0.1 N H2SO4) as standard.The longer lifetime and higher fluorescence quantum yield of LAl3 can be attributed to its high structure rigidity.The fluorescence quantum yields of Al salophen complexes were only slightly larger than corresponding Zn complexes reported by us previously, indicating that similar closedshell metal centers show little effect to the fluorescence quantum yield of salophen complexes.Emission spectra were recorded to investigate the excited state properties of two Al salophen complexes.(Figure 3) LAl and LAl 3 show similar single emission bands with emission maxima at 500 and 523 nm, respectively.The large Stokes shift (ca.115 nm) of LAl 3 is consistent with the previous report of cryptand-type Zn salophen complexes.Fluorescence lifetime of the two complexes were monitored at their emission maxima, and fluorescence quantum yields (±10%) were determined as 0.054 and 0.17 for LAl and LAl 3 in DMSO, using quinine sulfate (Φ f = 0.54 in 0.1 N H 2 SO 4 ) as standard.The longer lifetime and higher fluorescence quantum yield of LAl 3 can be attributed to its high structure rigidity.The fluorescence quantum yields of Al salophen complexes were only slightly larger than corresponding Zn complexes reported by us previously, indicating that similar closed-shell metal centers show little effect to the fluorescence quantum yield of salophen complexes.2.90 0.17 1 All data was determined in DMSO. 2 Wavelength of absorption peaks and the corresponding extinction coefficients. 3Wavelength of fluorescence emission peaks. 4Fluorescence quantum yield with the quinine sulfate (0.1 N H 2 SO 4 , Φ = 0.54) as standard, the uncertainty is ±10%.

Lipophilicity
Molecular lipophilicity is an important parameter affecting cellular uptake pathway and subcellular localization.To evaluate lipophilicity of the two complexes, we measured the N-octanol-water partition coefficient (log P, Table 2) according to Leo's method [42].Compared to LAl, trinuclear LAl 3 showed larger log P.This increase of the hydrophobicity might be due to the cage-like structure of the cryptand-type ligand, exposing the hydrophobic groups and protecting the hydrophilic metal centers inside the cage.Log P of Al complexes are significantly smaller compared to corresponding Zn complexes (log P > 1.0), indicating Al salophen complexes are much more hydrophilic, which might be due to the cationic nature of the Al salophen complexes.

Cell Imaging
In order to demonstrate different cellular behaviors of two Al salophen complexes, the cytotoxicity of the two compounds was evaluated by CCK-8 assay after 24 h incubation of the complexes with HeLa cells firstly (Figure 4).The result showed that in a concentration of 5 µM, the high cell viability up to 95% indicated low cytotoxicity.We chose this concentration for the subsequent imaging experiments.2.90 0.17 1 All data was determined in DMSO. 2 Wavelength of absorption peaks and the corresponding extinction coefficients. 3Wavelength of fluorescence emission peaks. 4Fluorescence quantum yield with the quinine sulfate (0.1 N H2SO4, Φ = 0.54) as standard, the uncertainty is ±10%.

Lipophilicity
Molecular lipophilicity is an important parameter affecting cellular uptake pathway and subcellular localization.To evaluate lipophilicity of the two complexes, we measured the N-octanolwater partition coefficient (log P, Table 2) according to Leo's method [42].Compared to LAl, trinuclear LAl3 showed larger log P.This increase of the hydrophobicity might be due to the cagelike structure of the cryptand-type ligand, exposing the hydrophobic groups and protecting the hydrophilic metal centers inside the cage.Log P of Al complexes are significantly smaller compared to corresponding Zn complexes (log P > 1.0), indicating Al salophen complexes are much more hydrophilic, which might be due to the cationic nature of the Al salophen complexes.

Cell Imaging
In order to demonstrate different cellular behaviors of two Al salophen complexes, the cytotoxicity of the two compounds was evaluated by CCK-8 assay after 24 h incubation of the complexes with HeLa cells firstly (Figure 4).The result showed that in a concentration of 5 μM, the high cell viability up to 95% indicated low cytotoxicity.We chose this concentration for the subsequent imaging experiments.Intracellular fluorescence of the two complexes in HeLa cells was studied by laser scanning confocal microscopy.As shown in Figure 5, incubation with 5 µM LAl or LAl 3 for 12 h led to green punctuate luminescence recorded in a perinuclear pattern.The corresponding Zn complexes showed almost invisible intracellular fluorescence under the same incubation condition, reflecting the relatively higher cellular uptake levels of Al salophens than that of Zn salophens.We ascribed this difference to the positive central charge of Al salophens, which allows Al salophens to traverse the negatively charged cellular membrane more easily than Zn salophens, driven by the membrane potential.
Intracellular fluorescence of the two complexes in HeLa cells was studied by laser scanning confocal microscopy.As shown in Figure 5, incubation with 5 μM LAl or LAl3 for 12 h led to green punctuate luminescence recorded in a perinuclear pattern.The corresponding Zn complexes showed almost invisible intracellular fluorescence under the same incubation condition, reflecting the relatively higher cellular uptake levels of Al salophens than that of Zn salophens.We ascribed this difference to the positive central charge of Al salophens, which allows Al salophens to traverse the negatively charged cellular membrane more easily than Zn salophens, driven by the membrane potential.Then we examined the subcellular localization of LAl and LAl3 by co-incubating the complex with a commercial lysosome tracker LysoTracker Red.As shown in Figure 5, the perinuclear punctuate green fluorescence of LAl and LAl3 overlapped well with the red fluorescence of the lysosome tracker, with Pearson's co-localization coefficients being ca.0.85 and 0.96, respectively.This result indicated that Al salophen complexes were mainly distributed in lysosomal organelles following internalization.
To determine whether the cellular uptake mechanisms of LAl and LAl3 is energy dependent or independent, we studied the effects of low temperature and ATP depletion in HeLa cells.Incubating cells under low temperature (4 °C) or in the presence of metabolic inhibitors blocks cellular uptake processes requiring energy supply.2-deoxy-D-glucose and sodium azide, known as inhibitors of oxidative phosphorylation and glycolytic pathway respectively, having been widely used in cellular uptake mechanism studies [43][44][45].As shown in Figure 6, no obvious intracellular luminescence of LAl or LAl3 displayed after incubation under 4 °C, revealing that their uptake was greatly inhibited.When cells were pre-incubated with 10 mM sodium azide or 6 mM 2-deoxy-D-glucose to deplete the cellular ATP level, a smaller but still significant inhibition on the uptake of Al complexes was observed.The above temperature and ATP dependence of the cell uptake suggests that internalization of Al salophen complexes is energy-dependent.Little difference between LAl and LAl3 is observed.Then we examined the subcellular localization of LAl and LAl 3 by co-incubating the complex with a commercial lysosome tracker LysoTracker Red.As shown in Figure 5, the perinuclear punctuate green fluorescence of LAl and LAl 3 overlapped well with the red fluorescence of the lysosome tracker, with Pearson's co-localization coefficients being ca.0.85 and 0.96, respectively.This result indicated that Al salophen complexes were mainly distributed in lysosomal organelles following internalization.
To determine whether the cellular uptake mechanisms of LAl and LAl 3 is energy dependent or independent, we studied the effects of low temperature and ATP depletion in HeLa cells.Incubating cells under low temperature (4 • C) or in the presence of metabolic inhibitors blocks cellular uptake processes requiring energy supply.2-deoxy-D-glucose and sodium azide, known as inhibitors of oxidative phosphorylation and glycolytic pathway respectively, having been widely used in cellular uptake mechanism studies [43][44][45].As shown in Figure 6, no obvious intracellular luminescence of LAl or LAl 3 displayed after incubation under 4 • C, revealing that their uptake was greatly inhibited.When cells were pre-incubated with 10 mM sodium azide or 6 mM 2-deoxy-D-glucose to deplete the cellular ATP level, a smaller but still significant inhibition on the uptake of Al complexes was observed.The above temperature and ATP dependence of the cell uptake suggests that internalization of Al salophen complexes is energy-dependent.Little difference between LAl and LAl 3 is observed.

General Experimental Information
All solvents and chemicals were purchased from Alfa Aesar (Haverhill, MA, USA) and J&K (Beijing, China) and used without further purification, unless specifically mentioned.Cellular imaging trackers were purchased from Invitrogen (Life Technologies, Carlsbad, CA, USA).The 1 H NMR spectroscopic measurements were carried out using a Bruker-400 NMR spectrometer (Bruker, Billerica, MA, USA), at 400 MHz.Tetramethysilane (TMS) is used as the internal reference.The 19 F NMR spectroscopic measurements were carried out using a Varian-400 NMR (Varian, Palo Alto, CA, USA).Electrospray ionization (ESI) mass spectra were performed on a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR, Bruker, Billerica, MA, USA).FTIR spectra were taken on a Nicolet iN10 MX Fourier transform infrared spectrometer (ThermoFisher, Carlsbad, CA, USA).Elemental analysis is performed on an Elementar Vario EL CUBE (Elementar, Langenselbold, Germany).The steady-state absorption spectra were attained on an Agilent 8453 UV-vis spectrophotometer (Agilent, Santa Clara, CA, USA) in 1 cm path length quartz cells.Single-photon luminescence spectra were recorded using fluorescence lifetime and steady state spectrophotometer (Edinburgh Instrument FLS920, Livingston, UK).Quantum yields of one photon emission of all the synthesized compounds were measured relative to the fluorescence of quinine sulfate in 0.1 N H2SO4.Confocal fluorescent images of living cells were performed using Nikon A1R-si laser scanning confocal microscope (Nikon, Tokyo, Japan), equipped with the laser of 405 nm.

Synthesis and Characterization
LAl was synthesized according to the reported methods.First, we synthesized the mononuclear salophen ligand.Salicylaldehyde (10 mmol, 10.4 mL) and o-phenylenediamine (5 mmol, 540.7 mg) were refluxing in 20 mL ethanol for 16 h, after cooling to the room temperature, the mixture was filtered to achieve the ligand (1.3 g, yield 85%).The ligand (158.2 mg, 0.5 mmol) and aluminum chloride (68 mg, 0.51 mmol) were refluxing in 20 mL acetonitrile solution for 16 h, then filtered and the precipitate was wash by CH3CN to afford the aimed product (147 mg, yield: 78%). 1

General Experimental Information
All solvents and chemicals were purchased from Alfa Aesar (Haverhill, MA, USA) and J&K (Beijing, China) and used without further purification, unless specifically mentioned.Cellular imaging trackers were purchased from Invitrogen (Life Technologies, Carlsbad, CA, USA).The 1 H NMR spectroscopic measurements were carried out using a Bruker-400 NMR spectrometer (Bruker, Billerica, MA, USA), at 400 MHz.Tetramethysilane (TMS) is used as the internal reference.The 19 F NMR spectroscopic measurements were carried out using a Varian-400 NMR (Varian, Palo Alto, CA, USA).Electrospray ionization (ESI) mass spectra were performed on a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR, Bruker, Billerica, MA, USA).FTIR spectra were taken on a Nicolet iN10 MX Fourier transform infrared spectrometer (ThermoFisher, Carlsbad, CA, USA).Elemental analysis is performed on an Elementar Vario EL CUBE (Elementar, Langenselbold, Germany).The steady-state absorption spectra were attained on an Agilent 8453 UV-vis spectrophotometer (Agilent, Santa Clara, CA, USA) in 1 cm path length quartz cells.Single-photon luminescence spectra were recorded using fluorescence lifetime and steady state spectrophotometer (Edinburgh Instrument FLS920, Livingston, UK).Quantum yields of one photon emission of all the synthesized compounds were measured relative to the fluorescence of quinine sulfate in 0.1 N H 2 SO 4 .Confocal fluorescent images of living cells were performed using Nikon A1R-si laser scanning confocal microscope (Nikon, Tokyo, Japan), equipped with the laser of 405 nm.

Photophysical Properties
Quantum yields of emission of synthesized two compounds were measured with quinine sulfate (dissolved in 0.1 N H 2 SO 4 ) as reference.The fluorescence measurements were performed in 1 cm 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 the equation Equal volume (200 mL) of N-octanol and water were thoroughly mixed by an oscillator and separated after 24 h.Two compounds (1 mg each) were then dissolved in 40 mL of the separated N-octanol and the solution was allowed to equilibrate for further 24 h.The extinction coefficient was then calculated and 40 mL of water (previously separated from the mixture) was added.The new octanol-water system was allowed to equilibrate for additional 24 h.After separating, both fractions were analyzed by UV-vis spectra.The log P values were calculated by log P = log C octanol C water where C octanol and C water refer to the concentration of two compounds in the N-octanol and water, respectively.

Live Cell Imaging
All cells were incubated in complete medium (Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin) at 37 • C in atmosphere containing 5% CO 2 .For imaging, HeLa cells were grown in poly-D-lysine-coated dishes and incubated in 2 mL of complete medium for 24 h.Cells were washed with PBS, and stocked dyes (1 mM in DMSO) were added to obtain a final concentration of 5 µM.The treated cells were incubated for 12 h at 37 • C. A few minutes prior to confocal imaging cells were washed twice with PBS.A confocal laser scanning microscope (A1R-si, Nikon, Tokyo, Japan) was used to obtain images.Cells were imaged via the fluorescence mode with a 60× immersion lens with the following parameters: laser power 100%, pinhole 4.0 a.u., excitation wavelength 405 nm, detector slit 500-530 nm, resolution 1024 × 1024, and a scan speed 0.5 frames per second.
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 salophen complexes in chromatographic grade, anhydrous DMSO was prepared as 1 mM.The solution was diluted to a final concentration of 5 µM by complete growth medium and coincubated with HeLa cells for 12 h.Stock solutions of LysoTracker Red was prepared as 1 mM, and the stock solution was diluted to the working concentrations in complete medium (1 µM).After incubating for half an hour, cells were washed with PBS buffer twice before confocal experiments.Differential interference contrast (DIC) and fluorescent images were processed and analyzed using Image J (version 1.8.0).
For the cellular uptake experiment, the cells were incubated with LAl or LAl 3 at 37 • C for 2 h, while cells at 4 • C were treated with precooled complete growth medium containing 5 µM LAl or LAl 3 , respectively.For the ATP depletion groups, HeLa cells were preincubated with 10 mM sodium azide or 6 mM 2-deoxy-D-glucose for 1 h, then treated with 10 mM sodium azide or 6 mM 2-deoxy-D-glucose complete growth medium containing 5 µM LAl or LAl 3 for 2 h.Hela cells were seeded in flat-bottomed 96-well plates, 10 4 cells per well, with 200 µL complete culture media in the dark for 24 h.After being washed with PBS three times (200 µL × 3), the cells were incubated with 5 µM concentrations of the studied salophens for another 24 h in the dark while untreated-cells and wells containing no cells are set as the controls.HeLa cells were then washed with PBS three times (200 µL × 3). 10 µL Cell Counting Kit-8 (CCK-8) solution and 90 µL PBS were added per well.After 2 h, the absorbance at 450 nm was read by 96-well plate reader.The viability of Hela cells was calculated by CV stands for the viability of cells, A s , A c , and A b stand for the absorbance of cells containing 2-Glu, cell control (0 µM 2-Glu), and blank control (wells containing neither cells nor 2-Glu).

Conclusions
In summary, we reported the synthesis and characterization of the cryptand-type Al salophen complex LAl 3 .The cryptand-type structure was confirmed by the X-ray crystal diffraction.LAl 3 showed green fluorescence emission similar to the mononuclear LAl, and can be internalized into HeLa cells by energy-dependent pathways.It could be used as lysosome localized fluorescent probes for live cell imaging, which was superior to their Zn analogues.

Figure 1 .
Figure 1.Chemical structures of the Al salophen complexes.

Figure 1 .
Figure 1.Chemical structures of the Al salophen complexes.

Figure 2 .
Figure 2.An ORTEP diagram of the single crystal X-ray structure of LAl3: (a) top view and (b) side view.The thermal ellipsoids are scaled to the 50% probability level.Carbon atoms are depicted in grey, oxygen in red, nitrogen in blue, fluorine in green, and aluminum in pink. )

Figure 3 .
Figure 3. UV-Vis and fluorescence spectra of the Al salophen complexes.(a) Normalized UV-Vis absorbance spectra in DMSO at 298 K of LAl and LAl3; (b) Normalized emission spectra in DMSO at 298 K of LAl and LAl3 (λex = 396 nm).

Figure 2 .
Figure 2.An ORTEP diagram of the single crystal X-ray structure of LAl 3 : (a) top view and (b) side view.The thermal ellipsoids are scaled to the 50% probability level.Carbon atoms are depicted in grey, oxygen in red, nitrogen in blue, fluorine in green, and aluminum in pink.

Figure 2 .
Figure 2.An ORTEP diagram of the single crystal X-ray structure of LAl3: (a) top view and (b) side view.The thermal ellipsoids are scaled to the 50% probability level.Carbon atoms are depicted in grey, oxygen in red, nitrogen in blue, fluorine in green, and aluminum in pink.

Figure 3 .
Figure 3. UV-Vis and fluorescence spectra of the Al salophen complexes.(a) Normalized UV-Vis absorbance spectra in DMSO at 298 K of LAl and LAl3; (b) Normalized emission spectra in DMSO at 298 K of LAl and LAl3 (λex = 396 nm).

Figure 3 .
Figure 3. UV-Vis and fluorescence spectra of the Al salophen complexes.(a) Normalized UV-Vis absorbance spectra in DMSO at 298 K of LAl and LAl 3 ; (b) Normalized emission spectra in DMSO at 298 K of LAl and LAl 3 (λ ex = 396 nm).

Figure 4 .
Figure 4. Cytotoxicity of the two indicated complexes toward HeLa cells using CCK-8 assay.HeLa cells were incubated with 5 μM dye for 24 h in complete media (Mean ± SD).

Figure 4 .
Figure 4. Cytotoxicity of the two indicated complexes toward HeLa cells using CCK-8 assay.HeLa cells were incubated with 5 µM dye for 24 h in complete media (Mean ± SD).

Figure 5 .
Figure 5. Co-localization studies of the two Al salophen complexes and LysoTracker Red: (a) images of the two compounds indicated; (b) images of LysoTracker Red; (c) merged images of (a,b); (d) Differential interference contrast (DIC).5 μM Al salophens were incubated with HeLa cells for 12 h and then 50 nm LysoTracker Red was incubated with HeLa cells for 20 min.Scale bar: 10 μm.

Figure 5 .
Figure 5. Co-localization studies of the two Al salophen complexes and LysoTracker Red: (a) images of the two compounds indicated; (b) images of LysoTracker Red; (c) merged images of (a,b); (d) Differential interference contrast (DIC).5 µM Al salophens were incubated with HeLa cells for 12 h and then 50 nm LysoTracker Red was incubated with HeLa cells for 20 min.Scale bar: 10 µm.

Figure 6 .
Figure 6.Cellular uptake of LAl and LAl3 inhibited at 4 °C and by depletion of cellular ATP in HeLa cells.(a) Imaging of LAl and LAl3 which were incubated for 30 min at 37 °C or at 4 °C or after depletion of the cellular ATP pool by 10 mM NaN3 or 6 mM 2-deoxy-D-glucose.(1) Images of the two compounds at the same experimental conditions.(2) Merged images of (1) and Differential interference contrast (DIC).(b) Uptake is expressed as the median of cell fluorescence.Mean ± SD are indicated.Values significantly (p < 0.005) (n = 30) different from control (internalization at 37 °C) are marked with asterisk.For depletion of cellular ATP pool, cells were preincubated for 1 h with 10 mM sodium azide and 6 mM 2-deoxy-D-glucose.a.u., arbitrary unit.

Figure 6 .
Figure 6.Cellular uptake of LAl and LAl 3 inhibited at 4 • C and by depletion of cellular ATP in HeLa cells.(a) Imaging of LAl and LAl 3 which were incubated for 30 min at 37 • C or at 4 • C or after depletion of the cellular ATP pool by 10 mM NaN or 6 mM 2-deoxy-D-glucose.(1) Images of the two compounds at the same experimental conditions.(2) Merged images of (1) and Differential interference contrast (DIC).(b) Uptake is expressed as the median of cell fluorescence.Mean ± SD are indicated.Values significantly (p < 0.005) (n = 30) different from control (internalization at 37 • C) are marked with asterisk.For depletion of cellular ATP pool, cells were preincubated for 1 h with 10 mM sodium azide and 6 mM 2-deoxy-D-glucose.a.u., arbitrary unit.

Φ 3 . 4 .
ref : The values of fluorescence quantum yield of the reference.I: integrated emission intensity.OD: optical density at the excitation wavelength.d: the refractive index of solvents.d DMSO = 1.478, d H2O = 1.333.Determination of the Octanol-Water Partition Coefficients (Log P)