Dynamic covalent chemistry in live cells for organelle targeting and enhanced photodynamic action

Organelle-specific targeting enables increasing the therapeutic index of drugs and localizing probes for better visualization of cellular processes. Current targeting strategies require conjugation of a molecule of interest with organelle-targeting ligands. Here, we propose a concept of dynamic covalent targeting of organelles where the molecule is conjugated with its ligand directly inside live cells through a dynamic covalent bond. For this purpose, we prepared a series of organelle-targeting ligands with a hydrazide residue for reacting with dyes and drugs bearing a ketone group. We show that dynamic hydrazone bond can be formed between these hydrazide ligands and a ketone-functionalized Nile Red dye (NRK) in situ in model lipid membranes or nanoemulsion droplets. Fluorescence imaging in live cells reveals that the targeting hydrazide ligands can induce preferential localization of NRK dye and an anti-cancer drug doxorubicin in plasma membranes, mitochondria and lipid droplets. Thus, with help of the dynamic covalent targeting, it becomes possible to direct a given bioactive molecule to any desired organelle inside the cell without its initial functionalization by the targeting ligand. Localizing the same NRK dye in different organelles by the hydrazide ligands is found to affect drastically its photodynamic activity, with the most pronounced phototoxic effects in mitochondria and plasma membranes. The capacity of this approach to tune biological activity of molecules can improve efficacy of drugs and help to understand better their intracellular mechanisms.

Malvern Zetasizer Nano ZSP (Malvern, U.K.). The final DOPC concentration in the LUVs was calculated to be 1 mM.

Preparation of nanoemulsions (NEs)
Nanoemulsions were produced by spontaneous nanoemulsification according to protocols described previously. 1 Labrafac oil (Labrafac WL 1349® from Gattefossé, Saint Priest, France) (50 mg) was mixed with the surfactant Kolliphor ELP® (Sigma-Aldrich) (50 mg) in a sonication bath at 40 °C for 10 min. Then, the mixture was homogenized under magnetic stirring at 40 °C for 10 min up to complete homogenization. Finally, NEs were generated with the addition of ultrapure (Milli-Q) water (230 mg). The hydrodynamic diameter of NEs was 90 nm according to DLS.
Model study of in situ reactions in LUVs/NEs. 1 mL of LUVs (1 mM) in PBS were mixed with 5 μL of 10 mM stock solution (in DMSO) of membrane targeting compound PM-HZ and incubated for 30 min, then the excess of membrane targeting moiety was removed by dialysis using a dialysis membrane with molecular cut off weight of 14000 Da. Afterwards, 5 μL of 3 mM solution of NRK in DMSO were added and further incubated for 1h at r. t. Then, the mixture was extracted by 500 μL distilled water and 500 μL DCM, DCM phase was collected and concentrated in vacuo. The reaction was controlled using TLC by comparing with a control, which was prepared by directly mixing 100 μL of LUVs with 5 μL 3 mM (in DMSO) NRK, followed by the same extraction process. DCM:MeOH=95:5 was used as the eluent. The novel compound, identified by the different polarity in comparing to starting materials, was collected and subjected to mass spectrometry detection. Fluorescence spectra of NRK in LUVs in the presence of the membrane targeting hydrazine PM-HZ were recorded using the following sample preparation: 2 µM NRK in 1 mL in phosphate buffer (pH 7.4) were mixed with 10 µL of the LUVs with PM-HZ, prepared as mentioned above (after the dialysis) and incubated for 30 min. In the two control samples, 2 µM NRK in 1 mL in phosphate buffer were mixed or not with 10 µL of the LUVs (1 mM).
In order to estimate the yield of the hydrazone conjugate of PM-HZ, LD-HZ and Mito-HZ with NRK in LUVs/NEs, the following protocol was used. PM-HZ or Mito-HZ (20 µM) was incubated in 1 mL LUVs (containing 1 mM DOPC) for 20 min at r. t. LD-HZ (20 µM) was incubated in 1 mL NEs (containing 1 mM Labrafac oil) for 20 min at r. t. Then, NRK (10 µM) was added to these solutions and further incubated for 1h at r. t. Finally, the mixture was extracted by 1 mL ethyl acetate twice; the extract was evaporated in vacuum and then purified by TLC (DCM:MeOH=95:5 as an eluent). The formed fractions of NRK-PM-HZ conjugate (with higher polarity, low Rf), NRK-LD-HZ (with lower polarity, high Rf) and NRK-Mito-HZ (with higher polarity, low Rf) and NRK were collected and their absorbance was measured by dissolving in 1 mL MeOH. According to the absorbance of NRK and NRK-PM-HZ, NRK-LD-HZ or NRK-Mito-HZ, the reaction yields of the hydrazone formation were 65 mol%, 63 mol% and 28 mol% for NRK in-situ reaction with PM-HZ, LD-HZ and Mito-HZ, respectively.

Cell experiments
HeLa cells (ATCC® CCL-2) were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco-Invitrogen), supplemented with penicillin (100 U/mL), 1% glycan (100 U/mL) and 10 % FBS at 37 °C in a humidified atmosphere containing 5% CO2. HeLa cells (ATCC) were then seeded on 36 mm ibidi coverslips at 30,000 cell per well in this DMEM medium with overnight. The DMEM medium was firstly removed and the cells were washed with PBS for three times. Then, medium was changed to Opti-MEM containing or not (in case of control) 100 μM of targeting hydrazides: LD-HZ for lipid droplets, PM-HZ for plasma membrane or Mito-HZ for mitochondria. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 30 min. Then, after washing the cells gently once with PBS, the medium was changed to Opti-MEM containing 1 μM NRK (except 2.5 μM for mitochondria) and further incubated for 30 min at room temperature followed by fluorescence imaging. In the case of protocol without washing out the targeting molecule, the cells were seeded into labtek (8 well chambered coverslips) at 4000 cells per well in this DMEM medium and left overnight. The DMEM medium was then removed and the cells were washed with PBS for three times. The Opti-MEM medium (200 µL) containing certain concentration (1, 5, 20, 100 µM) of HZ-PM was added to HeLa cells. The cells were incubated for 10 min at r.t. followed by addition of NRK (1 µM) and further incubation for 30 min before imaging. For all co-localization experiments, the cells were seeded into LabTek (8 well chambered coverslips) at 4000 cells per well in the DMEM medium and left overnight. The DMEM medium was then removed and the cells were washed with PBS for three times. Cells were preincubated with Opti-MEM medium (200 µL) containing or not (in cases of control) 20 µM corresponding targeting moieties of PM-HZ, LD-HZ or Mito-HZ for 10 min at 37 °C followed by addition of NRK or doxorubicin at 1 µM concentration and further incubated for 30 min. In case of co-localization in plasma membranes, F2N12SM (400 nM) was added after the cells were treated with Mito-HZ and NRK/doxorubicin and incubated for 15 min before imaging. For LDs labeling, LDs marker SMCy5.5 (1 μM) was added to the cells, incubated for 3h at 37 °C before adding targeting hydrazine LD-HZ and NRK/doxorubicin. For mitochondria labeling, MitoTracker Deep Red FM 644/665 (200 nM) was added after the cells were treated with Mito-HZ and NRK/doxorubicin and further incubated for 5 min at 37 °C. Finally, the corresponding medium was changed to Opti-MEM followed by fluorescence imaging. In cases of nucleus staining, Hoechst 33342 (10 µg/mL) in Opti-MEM was used to stain cell nucleus over 1 h and washed with PBS once before all the processes. For reversibility analysis of reaction of NRK with PM-HZ at plasma membrane interface, the cells in LabTek were pre-incubated with Opti-MEM medium (200 µL) containing or not (in cases of control) 20 µM PM-HZ for 10 min at 37 °C followed by addition of 1 µM NRK and further incubated for 30 min at 37 °C. For experiments with different pH (short incubation time), the cells were washed with PBS three times and the medium was changed to Opti-MEM (pH 7.4) or to phosphate-acetate buffer (pH 5.8). For long incubation in Opti-MEM (pH 7.4), after washing with PBS three times, cell medium was changed to Opti-MEM medium (200 µL) and the cells were incubated for 1 h at 37 °C. For each experimental condition, just before imaging the cells were incubated for 5 min at 37 °C with reference plasma membrane probe F2N12SM (400 nM). 3 Fluorescence imaging of NRK was done with the excitation wavelength at 488 nm, and fluorescence emission was collected at two different spectral channels: 550-600 nm and 600-670 nm. In the colocalization measurements with LDs marker SMCy5.5, for NRK channel, excitation wavelength was set at 488 nm and emission wavelength was collected in the range of 550-600 nm. For doxorubicin channel, the excitation wavelength was at 488 nm and fluorescence emission was collected in range of 520-650 nm (images of for membrane and mitochondria targeting were collected over average of 20 frames for this channel due to low brightness of doxorubicin). For the SMCy5.5 and MitoTracker Deep Red FM 644/665 channel, the excitation wavelength was set at 635 nm and the emission wavelength was collected in the range of 650-800 nm. Colocalization analysis was conducted in two ways. The first way was by calculating Pearson's and Mander's correlation coefficients. This was conducted by using an ImageJ plugin named "Just Another Colocalisation Plugin (JACoP)". A certain threshold for both Green and Red channels was applied to select the ROI area for the calculation of Pearson's and Mander's correlation coefficients. The second way was to calculate the ratio of the mean fluorescence intensity from organelle areas versus the whole cells. The organelle areas were selected by using fluorescence image of corresponding reference markers, F2N12SM for plasma membrane, SMCy5.5 for lipid droplet or Mito Tracker Deep Red FM for mitochondria. Cell selection and calculation of the mean fluorescence intensity were done using ImageJ.

Phototoxicity study
HeLa cells (ATCC® CCL-2) were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco-Invitrogen), supplemented with penicillin (100 U/mL), 1% glycan (100 U/mL) and 10 % FBS at 37 °C in a humidified atmosphere containing 5% CO2. HeLa cells (ATCC) were then seeded on 36 mm coverslips at 30,000 cell per well in this DMEM medium with overnight. The DMEM medium were first removed and washed with PBS for three times before changing the medium to Opti-MEM (as control) or Opti-MEM containing PM-Hz, Mito-HZ or LD-HZ (100 μM), then the cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 30 min. After that the cell were washed with PBS and the medium was changed to Opti-MEM containing 1 μM of NRK, followed by an incubation for 30 min at r. t. To understand PDT effect when NRK is targeted to different cell organelles, we used CellTox™ Green, a nucleus maker for monitoring cell death induced by membrane disruption. After targeting NRK to cell organelles with corresponding ligands, the dishes with cells were washed once with PBS and subjected to illumination with excitation of 550 nm (50% LED source power) continuously over 1 min followed by changing the medium to 1 mL CellTox™ Green assay containing 12 µg/mL Hoechst nucleus staining agent and incubated for 30 min. The transmission and fluorescence (CellTox™ Green channel) images were taken before and after illumination using 20x air objective Nikon CFI Plan Apo, NA = 0.75 (for data in Figure 3) or 10x air objective Nikon Plan Fluor NA = 0.3 (for data in Figure S5); excitation: 470 nm, emission filter: 531/40 nm for CellTox™ Green channel and excitation: 405 nm, emission filter: 475/50 nm for Hoechst 33342 channel. To observe the cell morphology change induced by phototoxicity, cell images (both bright field and epi-fluorescence channel (4% power, 300 ms integration period) with excitation of 550 nm and filter of 600/50 nm) were firstly taken before the application of high source power. Then video was prepared by taking images of the bright field transmission channel and epi-fluorescence channel (source power: 30% for lipid droplets, plasma membrane and their controls; 50 % for mitochondria and its control) and short integration period (30 ms) continuously for 30 slices over 10 min. After the video recording, images were taken in dic channel (300 ms) and epi channel (4% power, 300 ms integration period). This cellular imaging studies were done using epi-fluorescence mode with a Nikon Ti-E inverted microscope, equipped with CFI Plan Apo ×60 oil (numerical aperture = 1.4) objective, and a Hamamatsu Orca Flash 4 sCMOS camera.
Compound 1a was prepared as described previously. 4 Compound 2a. 400 mg of compound 1a were dissolved in 7 mL of dry CH3CN together with 589 mg (3.5 equiv., 794 µL) of DIPEA. After that, a solution of 196 mg (1.5 eq.) of succinic anhydride 3 mL of dry CH3CN was added dropwise and the resulting mixture was stirred for 12h (control by TLC) under Ar atmosphere. After the reaction the solvent was evaporated in vacuo and the crude product was purified by column chromatography (SiO2, DCM:MeOH:HCOOH 80:20:2).Yield: 550 mg as a colourless solid (DIPEA salt is present, its signals are not presented Compound 3a. 200 mg of compound 2a were dissolved in 3 mL of dry DMF together with 238 mg (1.5 eq.) of HATU, 28 mg (0.5 eq.) of HOBt and 215 mg (4 eq., 291 µL) of DIPEA under Ar atmosphere. After 5 minutes, a solution of 83 mg (1.5 eq.) of tert-Butyl carbazate in 1.5 mL of dry DMF was added and the mixture was stirred for 24h (control by TLC) under Ar atmosphere. After the reaction the solvent was evaporated in vacuo and the crude product was purified by column chromatography (SiO2, DCM:MeOH 80:20, redone twice).Yield: 165 mg as a colourless solid (DIPEA salt is present, its signals are not presented C-NMR spectrum of 3a in MeOH-D4.

Compound 4a (PM-HZ).
To the solution of 3a (165 mg, 0.31 mmol) in DCM (5 mL), TFA (5 mL) and 1 drop of MiliQ water were added and stirred at room temperature for 2 h. After confirming the reaction completely proceeded with TLC, the solvents were evaporated under reduced pressure, and then the residue was dissolved in MeOH and evaporated 3 times to remove TFA and give desired product 4a as a colorless solid (153 mg) (DIPEA salt is present, its signals are not presented Compound 1b. 500 mg of (3-carboxypropyl)triphenylphosphonium bromide were dissolved in 8 mL of dry DMF together with 465 mg (1.05 eq.) of HATU and 450 mg (3 eq., 610 µL) of DIPEA. After 5 minutes, a solution of 161 mg (1.05 eq.) of tert-Butyl carbazate in 2 mL of dry DMF was added and the mixture was stirred for 24h (control by TLC) under Ar atmosphere. After the reaction the solvent was evaporated in vacuo, then the solid residue was dissolved in MeOH and precipitated with Et2O, filtered and washed with Et2O (redone twice). After that, the solid residue was dissolved in DCM, washed with water (x2) and brine, dried over Na2SO4 and the solvent was evaporated in vacuo. The crude product was purified by gradient column chromatography (SiO2, DCM:MeOH 90:10 to 80:20

Fig. S12.
Phototoxicity study based on cell morphology changes due to excitation of NRK targeted to lipid droplets, plasma membrane and mitochondria by the corresponding targeting agents. Bright-field images were taken every 1 min after excitation at 550 nm for 300 ms using high illumination power (30% for lipid droplets and plasma membrane and its control; 50 % for mitochondria and its control), images were selected for display at indicated periods of 1 min, 7 min. Red arrows show example of cells undergoing the morphological change.