Efficient cytosolic delivery of luminescent lanthanide bioprobes in live cells for two-photon microscopy

Lanthanide(iii) (Ln3+) complexes have desirable photophysical properties for optical bioimaging. However, despite their advantages over organic dyes, their use for microscopy imaging is limited by the high-energy UV excitation they require and their poor ability to cross the cell membrane and reach the cytosol. Here we describe a novel family of lanthanide-based luminescent probes, termed dTAT[Ln·L], based on (i) a DOTA-like chelator with a picolinate moiety, (ii) a two-photon absorbing antenna to shift the excitation to the near infrared and (ii) a dimeric TAT cell-penetrating peptide for cytosolic delivery. Several Tb3+ and Eu3+ probes were prepared and characterized. Two-photon microscopy of live cells was attempted using a commercial microscope with the three probes showing the highest quantum yields (>0.15). A diffuse Ln3+ emission was detected in most cells, which is characteristic of cytosolic delivery of the Ln3+ complex. The cytotoxicity of these three probes was evaluated and the IC50 ranged from 7 μM to >50 μM. The addition of a single positive or negative charge to the antenna of the most cytotoxic compound was sufficient to lower significantly or suppress its toxicity under the conditions used for two-photon microscopy. Therefore, the design reported here provides excellent lanthanide-based probes for two-photon microscopy of living cells.

The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure.The crude product was dissolved in EtOH (20 mL).NaOH 6 M (4 mL) was added dropwise and the reaction mixture was stirred for 10 min at room temperature.The solution was neutralized by dropwise addition of HCl 6 M (ca. 4 mL) and a precipitate was formed.
The suspension was centrifugated and the supernatant was removed under reduced and the residue was solubilized in AcOEt.The solution was washed with water, dried over Na2SO4 and the solvent was removed under reduced pressure.The product obtained was purified by HPLC to give a white powder (259 mg, 49 % calculated based of the formula L-Ar-NH(BocbAla)(tBu)3×4TFA).HPLC (anal.):tR = 10.7 min (method B);  1, 52.5, 50.6, 38.3, 37.9, 28.7, 28.4, 20.8The solution was stirred for 10 min at 0 °C; then DCC (524 mg, 2.5 mmol) was added and a precipitate was formed.The solution was stirred at 0 °C to room temperature for 4 h.The reaction mixture was diluted in DCM and filtered.The organic phase was washed using a saturated NaHCO3 then water.The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure.The crude product was purified by flash chromatography (silica gel, AcOEt/cyclohexane 30:70) to give a colourless oil (732 mg, 75 %).7, 170.1, 142.0, 139.1, 138.2, 120.9, 118.8, 94.1, 81.4, 34.0, 32.5, 30.8, 28 6, 170.1, 146.3, 140.2, 137.0, 120.4, 115.6, 83.3, 81.2, 32.6, 30.9, 30.8, 28.1, 24.9, 22.3 was diluted using AcOEt and filtrated over Celite.The solution was extracted with saturated NaHCO3 then water.The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure.The crude product was dissolved in EtOH (20 mL).NaOH 6 M (4 mL) was added dropwise and the reaction mixture was stirred for 10 min at room temperature.The solution was neutralized by dropwise addition of HCl 6 M (ca. 4 mL) and a precipitate was formed.
The suspension was centrifugated and the supernatant was removed under reduced and the residue was solubilized in AcOEt.The solution was washed with water, dried over Na2SO4 and the solvent was removed under reduced pressure.

CK-TAT-RA resin:
The peptide was synthesized on Rink-PEG-PS resin (NovaPEG Rink Amide, 0.25 mmol, 0.4 mmol/g).Automated peptide elongation was performed as described above using Fmoc-Lys(Alloc)-OH to introduce the last lysine and Boc-Cys(Trt)-OH to introduce the N-terminal cysteine.Removal of the Alloc protecting group was performed as described above for AcK-TAT-RA resin.

F 1P spectroscopy
Determination of molar extinction coefficients: A solution of mTAT [L] or CTAT [L] peptide in HEPES buffer (10 mM, pH 7.5) was titrated by a solution of TbCl3 or EuCl3 of known concentration with absorption monitoring, as previously described. [8]The concentration of the peptide was determined from the endpoint of the titration, allowing determination of extinction coefficient using Beer Lambert law.The titration of mTAT[L-Me Ar-OMe] by Tb 3+ is given in Fig. S4 as typical example.Luminescence decay: Ln 3+ luminescence decays were measured for each compound in aerated and de-oxygenated PBS, prepared in H2O, in aerated PBS prepared in D2O and lifetimes, tLn, were determined by mono-exponential fit (or biexponential fit when required).They are indicated in Table S1 as well as hydration number q determined using Parker's equations: q Tb = 5.0 × (1/τ(H2O) − 1/τ(D2O) − 0.06) and q Eu =1.2 × (1/τ(H2O) − 1/τ(D2O) − 0.325)) for Tb 3+ and Eu 3+ ,respectively, with τ in ms. [9]Examples of decay curves and their fits are given in Fig. S6.0.0 a Error on t and q values are estimated ± 0.03 ms and ± 0.2, respectively.b Since back energy transfer to the triplet state contributes significantly to Tb 3+ 5 D4 state decay, estimation of q by Parker's equation is not reliable and thus, q was not calculated.
Quantum yields measurements: Quantum yields were determined using a Fluorolog FL3-22 spectrophotometer by a relative method with quinine sulphate in 0.5 M H2SO4 as a reference compound (F = 0.545) [10,11] using solutions of various concentrations having absorption below 0.1 at the excitation wavelength.The excitation wavelength was the same for the sample compound (S) and the reference.To determine the quantum yields of the sample compound, the following equation was used: where A is the absorbance at the excitation wavelength, I the integrated emission intensity and n the refractive index.Estimated experimental error for the quantum yield determination is ~10 %.OMe] in PBS (red: conjugate; black: quinine sulfate).The slopes that correspond to I/A in Equation ( 1) were used to determine the quantum yields.

Determination of energy of S1 and T1 excited states:
The energy of the S1 state was determined using the cut-off of the absorption band.For the energy of T1 excited state, a solution of the Gd-loaded peptide in PBS/glycerol 9:1 (v/v) was prepared and frozen at 77 K.A time-gated (delay = 100 µs) emission spectrum was recorded to monitor the antenna phosphorescence emission.The energy of the T1 state was estimated using the wavelength at half-maximum on the onset of the phosphorescence spectrum.

2P spectroscopy
Determination of 2P cross-sections s2P: Two-photon excitation spectra and two-photon cross-sections were obtained by two-photon excited fluorescence measurements of diluted PBS solutions of the compounds (ca. 10 µM, the exact concentration was determined from absorption spectrum) using a femtosecond Ti:sapphire laser (Coherent Chameleon Ultra II, 80 MHz, 140 fs) in the range 690-990 nm.The excitation beam (2.6 mm diameter) was focused with a 75 mm focal length lens to the sample.The up-converted fluorescence was collected at right-angle using a 30 mm focal length doublet lens.After filtering the scattered excitation beam by low-pass filters, the florescence was coupled to a fiber optic spectrometer (Avantes Hero).The sample was contained in a 1´1 cm quartz cell and continuously stirred with a magnetic stirrer to avoid thermal effects and photodegradation.After verifying that the emission intensity exhibited quadratic power dependence for each sample (Fig. S9), the incident power was adjusted to 35 mW to characterize the two-photon absorption spectra.Calibration of the 2P absorption spectra was performed at each excitation wavelength by comparison with that of fluorescein (10 µM, pH 11) as reference compound. [12]2P absorption spectra are provided in Fig. S10.

2P microscopy
General: HeLa, MRC5 and HEK293 cell lines were purchased from the ATCC and agreed by French ethic boards (single pulsed excitation with the 7.56 μs temporal resolution). [13] Image analysis:    NHAc] in HeLa cells (1 h incubation at 10 µM), recorded by TSLIM [13] with the LSM710 microscope (APD; lex = 720 nm for A and C, 700 nm for B).Experimental data were fitted to bi-exponential (A and B) or mono-exponential (C) decays yielding t values indicated on the graphs and in the text.

Cytotoxicity
MTT proliferation assay: Inhibition of cell proliferation by dTAT[Ln•L] conjugates was measured by a MTT assay.HeLa cells were seeded into 96-well plates (3×10 3 cells per well) in 100 μL of culture medium (RPMI with 10% of fetal calf serum).After 24 h, cells were washed with PBS three times, then treated with compound dissolved at various concentrations in RPMI medium (without phenol red and without serum) during 2 h.A control with culture medium only was prepared also.Following incubation of the cells with the samples, the solution was discarded, the cells were washed twice with PBS and fresh culture medium was added to the wells.After 24 h at 37 °C, plates were centrifugated for 5 min at 400 g.The medium was then discarded and replaced with fresh culture medium containing MTT (0.5 mg/mL, Euromedex, Mundolsheim, France).After 3 h at 37 °C, 100 µL of solubilizing solution (10% Triton-X100, 0.1 M HCl, in isopropanol) were added in each well.Plates were incubated at room temperature under shaking until solubilization of water insoluble purple formazan crystals.Absorbance was then measured on an ELISA reader (Tecan, Männedorf, Switzerland) at a test wavelength of 570 nm and a reference wavelength of 650 nm.Absorbance obtained by cells in control medium was rated as 100 % cell survival.Each data point is the average of three independent experiments.Data were then fitted using GraphPad Prism to determine IC50 values.
Fiji/ImageJ was used to analyze images and particularly for linear unmixing to deconvolute autofluorescence and Ln 3+ emission maps.They were performed using the Stowers ImageJ plugins developed by Jay Unruh at the Stowers Institute for Medical Research.Pure 2P-excited autofluorescence and Ln 3+ emission spectra used for linear unmixing were acquired with the LSM710 microscope from control HeLa cells (Fig. S9) or droplets of mTAT[Ln•L] conjugates (100 µM in PBS).They are displayed in Fig. S10.

Fig. S9
Fig. S9 2PM imaging (lex = 720 nm) of control living HeLa cells (not incubated with dTAT[Ln•L]).Left panel shows the differential interference contrast (DIC) image, middle panel shows the luminescence image, and right panel shows the merge.Scale bar corresponds to 10 µm.

Fig. S17
Fig. S17 2PM imaging (lex = 720 nm) of living HeLa cells incubated 1 h with dTAT[Tb•L-Me Ar-NHSuc(-)] (10 µM) in RPMI medium.(A) Left panel: DIC image; Middle panel: luminescence (detected with APD) image; Right panel: merge.Scale bars correspond to 10 µm.(B) Left panel: 2P-excited emission spectra (detection with PMT) of cells outlined in red, green and blue in panel A; Middle and right panels: autofluorescence and Tb 3+ emission maps obtained by linear unmixing of 2P-excited spectral images recorded with the PMT.
HeLa cells were seeded at 3´10 4 cells/well onto 8-chamber Labtek-I coverglass system.After 24 h, cells were washed three times with PBS.Cells were incubated with dTAT [Ln•L] (10 µM) in RPMI medium (without phenol red and without serum) or PBS at 37°C for 1 h.Then, cells were washed three times with PBS before adding RPMI medium supplemented with fetal bovine serum (200 µL) for observation under microscope.Confocal microscopy: Confocal 2P experiments were performed by using an LSM-DuoScan-Confocor3 NLO microscope (Carl Zeiss) composed of a LSM710 confocal module and an inverted motorized stand (AxioObserver) equipped with an on-stage cell incubator.Excitation was provided by a Ti:Sapphire femtosecond laser (Chameleon, Ultra II, Coherent) featuring chirp precompensation.The C-apochromat 40×/1.2water-immersion objective was used throughout experiments.The pinhole was open during 2P acquisition in descanned detection mode.The spectral PMT detector (Quasar) or avalanche photodiodes were used to register the emission signal in each pixel of the confocal image.Temporal Sampling Lifetime Imaging Microscopy (TSLIM) was used to record luminescence lifetime decay in cells