Optochemical Control of Therapeutic Agents through Photocatalyzed Isomerization

Abstract A Ru(bpy)3Cl2 photocatalyst is applied to the rapid trans to cis isomerization of a range of alkene‐containing pharmacological agents, including combretastatin A‐4 (CA‐4), a clinical candidate in oncology, and resveratrol derivatives, switching their configuration from inactive substances to potent cytotoxic agents. Selective in cellulo activation of the CA‐4 analog Res‐3M is demonstrated, along with its potent cytotoxicity and inhibition of microtubule dynamics.


Cell culture
Stably expressing (under continuous antibiotic selection) EB3-GFP HeLa cells were a kind gift from Prof. Patrick Meraldi (University of Geneva, Switzerland). Cells were grown in DMEM medium (Gibco) containing 10% FCS, 1% pen-strep, 0.3 µg ml −1 puromycin, and 500 µg ml −1 G418 at 37 °C under 5% CO2 in a humidified incubator. Cells were regularly tested for mycoplasma contamination by qPCR using the MycoplasmaCheck test service (Eurofins). Colonies were kept in culture for no more than 15 passages.
Wild type HeLa cells were obtained from the American Type Culture Collection (ATCC) and expanded following their instructions. Cells were grown in DMEM medium (Gibco) containing 10% FCS, 1% pen-strep at 37 °C under 5% CO2 in a humidified incubator. Cells were regularly tested for mycoplasma contamination by qPCR using the MycoplasmaCheck test service (Eurofins). Colonies were kept in culture for no more than 20 passages.

Monitoring of photoisomerization by NMR
Compounds were dissolved to a final concentration of 3.2 mM in the relevant solvent system. Ru(bpy)3Cl2 was added as stock in either DMF-d7 or DMSO-d6 to a final concentration of 32 µM (1 mol %) and an initial NMR was obtained. The sample was then irradiated in the NMR tube for the prescribed time at 455 nm with a collimated LED light 11 cm above the plate. The experiments were protected from external light by performing experiments inside a protected enclosure (see Figure S-1A for experimental setup). After irradiation reaction progression was monitored by proton NMR. . NMR and eppendorf tubes were laid horizontally across the elevated sample stage to maximize irradiation crosssection. A surface flux density of 6.2 mW/cm 2 and 0.82 mW/cm 2 was measured using a PM100USB power meter and S-170c power sensor (Thor labs) for setup A and B respectively. To validate the homogenous irradiation in absence of a collimator, the radiant exposure was measured for 52 seconds in 5 positions of the plate. As shown in

Determination of kinetics of photoisomerization by reverse-phase HPLC
Samples were prepared from a 5 mM stock of trans-CA-4 in DMF or a 3.3 mM stock in DMSO depending on the solvent system tested. Appropriate concentration samples were prepared by dilution and addition of Ru(bpy)3Cl2 from a 25 µM stock in either DMF or water. Samples were irradiated for designated time periods as described for the NMR experiments in Eppendorf tubes, then diluted to a concentration of 100 µM and 20 µL injected onto the HPLC system for analysis. Samples were analyzed over 40-100% B gradient over 2.9 min at 0.68 mL/min, with chromatography monitored at 287 and 329 nm. The area under the curve for trans-CA-4 was determined at 329 nm between 3.0-3.1 min and expressed as a percentage of the initial area at T0 using the formula: Pcis = (Atrans -A) / (Atrans -Acis) where Pcis is the percentage converted to cis, Atrans is the area under the curve for the trans isomer, Acis is the area under the curve for the cis isomer and A is the area under the curve for the sample in question. From this the concentration of the trans isomer remaining was determined. The slope of the relation between the natural log and irradiation time was determined to give the pseudo-first-order rate constant, with this value then divided by the catalyst concentration to give the second-order rate constant of the reaction. All time points were conducted in triplicate and were analyzed independently.

Estimation of IC50 for CA-4 and ReS-3-M
As comets number is a well-known indicator of tubulin polymerization state, we decided to measure the IC50 of isomerized Res-3M and CA-4 using comets number as a descriptor [2] . To do so, HeLa cells expressing EB3-GFP [3] were seeded at a concentration of 100000 cells ml −1 in a 96-wells black/clear bottom plate one day before the experiment. The in vitro isomerization was carried out by mixing 10 µL of a 100 mM Res-3M solution in DMSO and 10 µL of a 1 mM Ru(bpy)3Cl2 to a final concentration of 50 mM and 1% mol. of catalyst, or 10 µL of a 20 mM CA-4 solution in DMSO and 10 µL of a 0.2 mM Ru(bpy)3Cl2 to a final concentration of 10 mM and 1% mol. of catalyst. Then, the sample was irradiated in a microcentrifuge tube for 30 minutes at 455 nm with a collimated LED light 11 cm above the tube (flux density 6.2 mW/cm 2 -radiant exposure 11.16 J/cm 2 ). While the drugs were isomerizing, cells were treated with a 2 µg/mL solution of Hoechst 33342 (Chemodex) for 30 minutes. Then, cells were washed 3 times with warm media and treated with dilution series of the drugs for 30 minutes. Finally, cells were imaged using a Molecular DevicesTM ImageXpress Micro (IXM) XL automated microscope equipped with a 60x dry objective. A total of 16 tiles and 3 z stacks were imaged using DAPI and FITC settings. Cells were kept at 37 °C and with 5% CO2 for the whole duration of the experiment. The isomerization was carried out using the setup shown in figure S-1A.

Comet's analysis protocol
Microscopy images were analyzed using the MetaXpress® software. For nuclear segmentation, DAPI was used with the following settings: minimum width = 8 µm, maximum width = 25 µm, intensity above background = 1000. FITC channel was used to segment the whole cell body using the following settings: minimum width = 8 µm, maximum width = 50 µm, intensity above background = 100. Cells touching the frame's border were excluded from the analysis. To reduce the background and facilitate the segmentation of EB3 comets, a top hat modification was applied. This modification was only used to ease the segmentation but not for the intensity quantification of the comets. To remove artifacts like protein aggregates or condensates, the comets were segmented and filtered by area. In particular, all the objects with an intensity above 80 and a width between 0.3 µm and 3 µm were identified as a comet. All objects with a minimum width below 0.2 µm were removed from the mask. This threshold has been used to analyze images taken with a Leica SP8 dive falcon used during MP-Laser isomerization. For the pictures taken using IXM-C microscope the threshold is way higher due to the higher GFP signal.

Cell toxicity using in vitro isomerized trans-Res-3M
To assess whether isomerized Res-3M can induce cell death HeLa cells expressing EB3-GFP were seeded at a concentration of 100'000 cells ml −1 in a 96-wells black/clear bottom plate one day before the experiment. The in vitro isomerization was carried out by mixing 10 µL of a 100 mM Res-3M solution in DMSO and 10 µL of a 1 mM Ru(bpy)3Cl2 to a final concentration of 50 mM and 1% mol. of catalyst. Then, the sample was irradiated in a microcentrifuge tube for 30 minutes at 455 nm with a collimated 455 nm LED light 11 cm above the tube (flux density 6.2 mW/cm 2 -radiant exposure 11.16 J/cm 2 ). Finally, cells were treated with 50, 5, 0.5, and 0.05 µM solution of the isomerized compound, same concentrations of trans-Res-3M, 0.5 µM of Ru(bpy)3Cl2, or 0.1 % of DMSO. After 8 or 20 hours of incubation, the supernatant was gently decanted to minimize cells loss, and cells were fixed in 4 % PFA for 30 minutes. Before imaging, the cells were stained for 30 minutes with a 2 µg/mL solution of Hoechst 33342 (Chemodex). Cells were imaged using a Molecular DevicesTM ImageXpress Micro (IXM) XL automated microscope with DAPI settings. Excitation = 100 ms. Objective 20×. The number of healthy cells was quantified by using a nuclei count protocol where the particle mask size was width = 10 μm; height = 25 μm; fluorescence cutoff threshold = 1000. Each well was analyzed by the acquisition of a matrix of images covering the well (16 tiles). The isomerization was carried out using the setup shown in figure S-1A.

In cells cytotoxicity
HeLa cells were seeded at a concentration of 20'000 cells ml −1 in a 96-wells black/clear bottom plate one day before the experiment. The next day, the cells were treated with trans-Res-3M (5 µM), Ru(bpy)3Cl2 (0.5 µM), or a combination of both. After 20 minutes, the plate was placed at 37 °C in an aluminum-coated box on a shaker and irradiated for 60 minutes (flux density 0.82 mW/cm 2 -radiant exposure 3.3 J/cm 2 ). Once irradiated the cells were incubated with the compounds for 24 hours. Before imaging, Hoechst 33342 (Chemodex) was added to the cells to a final concentration of 2 µg/mL for 1 hour. No washing was performed to avoid any loss of dead or dying cells. After that, cells were imaged using a Molecular DevicesTM ImageXpress Micro (IXM) XL automated microscope with DAPI settings. Excitation = 100 ms. Objective 20×. The temperature was maintained constantly at 37 °C and CO2 was kept at 5%. The number of healthy cells was quantified by using a nuclei count protocol where the particle mask size was width = 10 μm; height = 25 μm; fluorescence cutoff threshold = 1000. Each well was analyzed by the acquisition of a matrix of images covering the well (6 tiles). Nuclei were counted using the MetaXpress® software and the statistical analysis was performed using GraphPad. The statistical significance was determined using Student's t-test where **** is equal to pvalue ≤ 0.0001, *** is equal to pvalue < 0.001, ** is equal to pvalue < 0.01, * is equal to pvalue < 0.05 and ns is equal to pvalue > 0.05 ( Figure S-31). The isomerization was carried out using the setup shown in figure S-1B. Since the plate was irradiated without a collimator, we needed to be sure that no significant difference in radiant exposure could be measured by changing the position of the sensor inside the box. To this end, we recorded a brief light exposure by placing an S-170c power sensor connected to a PM100USB power meter in different positions for 52 seconds (Fig. S-1B). The maximal positional variation in radiant exposure between the various positions was measured to be < 2%. Therefore, we concluded that our setup was suitable for in cells isomerization. . Each dot is representative of a technical replicate. Nuclei were detected and a mask was applied to discriminate between healthy and dying cells using the MetaXpress® software. pvalues ≥ 0.05 denoted as ns (no significance), pvalues < 0.001 denoted as ***, pvalues < 0.01 denoted as **.

Apoptosis induction and quantification using in vitro isomerized trans-Res-3M
To assess whether treatment with isomerized trans-Res-3M was able to induce apoptosis, cells were seeded at a concentration of 160'000 cells ml −1 in a µ-Slide 8 Well chamber. After one day, solutions containing Ru(bpy)3Cl2 0.5 µM, trans-Res-3M 50 mM, DMSO 0.1% or a combination of Ru(bpy)3Cl2 (0.5 µM) and trans-Res-3M (50 mM) were either irradiated or not using setup S-1A for 30 minutes(flux density 6.2 mW/cm 2 -radiant exposure 11.16 J/cm 2 ). After the isomerization, the cells were treated with one of the aforementioned solutions and 3 µM of CellEvent™ Caspase-3/7 Green Detection Reagent (Thermo -C10740). Finally, after 24 hours, cells were imaged using a Nikon BioStation widefield microscope (objective 10x -irradiation light 488 nm). A total of four tiles per condition were taken ( Figure S-32). The isomerization was carried out using the setup shown in figure S-1A. . Bright green spots are indicative of apoptotic cells with a fluorogenic substrate to measure caspase 3/7 acticity. Images for GFP channel were acquired as follows: excitation = 488 nm -emission = 500 to 550 nm. Panels "trans-Res-3M + Ru(bpy)3Cl2" and Res-3M + Ru(bpy)3Cl2 -irradiated" also appear in the main text ( Figure 4E).

Res-3M isomerization on cells using an LED light source
EB3-GFP HeLa cells were seeded at a concentration of 75'000 cells ml −1 in a 3.5 cm glass-bottom dish with a 10 mm microwell (Mattek). After one day cells were treated with either 1% DMSO, 5 µM Res-3M, 0.05 µM Ru(bpy)3Cl2 or a combination of drug and catalyst. After 15 minutes, the cells were irradiated for 180 seconds with a collimated 455 nm LED light 11 cm above the plate in a thermal oven set at 37 °C (flux density 6.2 mW/cm 2 -radiant exposure 11.16 J/cm 2 ). The cells were irradiated for no longer than 5 minutes and then left to equilibrate in a humidified incubator for at least 15 minutes before imaging. The experiments were protected from external light by performing the experiments inside a protected enclosure. Finally, the EB3-GFP comets were imaged using a Leica SP8 microscope equipped with a 488 nm laser and a 63x oil objective, taking stacks of the whole cell ( Figure 5). Shown data are a representative selection of a biological triplicate. The isomerization was carried out using the setup shown in figure S-1A.

Res-3M isomerization on cells using a multiphoton laser
HeLa cells stably expressing EB3-GFP were seeded at a concentration of 75'000 cells ml −1 in a 3.5 cm glass-bottom dish with a 10 mm microwell (Mattek). After one day cells were incubated with 10 µM Ru(bpy)3Cl2 for 3 hours. The spernatant containing the catalyst that was not taken up cells was then removed, and cells were extensively washed with warm media before the addition of 5 µM Res-3M. To trigger the isomerization, the dishes were irradiated using a multiphoton laser (InSight X3 -Spectra-Physics) at 916 nm and real-time images were acquired on the green channel. Cells were maintained at 37 °C for the full duration of the experiment ( Figure

Ru(bpy)3Cl2-induced singlet oxygen generation
As Ruthenium is a well know photosynthesizer used in photodynamic therapy, [4] we asked ourselves whether singlet oxygen production might be responsible for the observed phenotypes. To address this question, we leveraged a recently developed singlet oxygen sensor called BioTracker Si-DMA Singlet Oxygen Live Cell Dye (Sigma -SCT063 -Figure S-34) [5] . HeLa cells were seeded at a concentration of 75'000 cells ml −1 in a 3.5 cm glass-bottom dish with a 10 mm microwell (Mattek). After one day, Ru(bpy)3Cl2 was added to a final concentration of 10 µM (0.1% DMSO). The catalyst was incubated for 24 hours and then washed three times with warm DPBS. A solution of Si-DMA (500 nM in Fluoro Brite media -Gibco) was added to the cells for 1 hour. Finally, cells were washed two times with warm PBS before imaging. Ruthenium was irradiated using a multi-photon laser at 916 nm (laser power 1% equal to 4 mW -measured with a PM100USB -Thorlabs) for 60 seconds. At the same time, the Si-DMA probe was irradiated with a 640 nm white light laser. For the whole duration of the experiment fluorescence between 665 and 675 nm was detected. As a positive control, HeLa cells were incubated with 5-aminolevulinic acid (5-ALA -Acros) for 4 hours to increase the endogenous production of protoporphyrin IX (PpIX). Differently, the positive control was irradiated with an Argon laser at 405 nm, where PpIX shows a maximum of absorption, for 60 seconds. Just before imaging, trans-Res-3M was added to a final concentration of 5 µM for 15 minutes. The detection of Si-DMA over time was carried out identically to the Ru(bpy)3Cl2 treated cells. For the quantification, cytoplasmatic regions of interest were defined at T0, and fluorescence was quantified within these regions throughout the temporal scan ( Figure S-34, S35-A, S35-B). Each experiment was carried out as a biological replicate, statistical analysis was carried out via a Student's t-test using the program GraphPad.