Membrane Environment Enables Ultrafast Isomerization of Amphiphilic Azobenzene.

The non-covalent affinity of photoresponsive molecules to biotargets represents an attractive tool for achieving effective cell photo-stimulation. Here, an amphiphilic azobenzene that preferentially dwells within the plasma membrane is studied. In particular, its isomerization dynamics in different media is investigated. It is found that in molecular aggregates formed in water, the isomerization reaction is hindered, while radiative deactivation is favored. However, once protected by a lipid shell, the photochromic molecule reacquires its ultrafast photoisomerization capacity. This behavior is explained considering collective excited states that may form in aggregates, locking the conformational dynamics and redistributing the oscillator strength. By applying the pump probe technique in different media, an isomerization time in the order of 10 ps is identified and the deactivation in the aggregate in water is also characterized. Finally, it is demonstrated that the reversible modulation of membrane potential of HEK293 cells via illumination with visible light can be indeed related to the recovered trans→cis photoreaction in lipid membrane. These data fully account for the recently reported experiments in neurons, showing that the amphiphilic azobenzenes, once partitioned in the cell membrane, are effective light actuators for the modification of the electrical state of the membrane.

temperature and the solvent is removed under reduced pressure. The resulting red powder is washed with DCM and Et 2 O, the combined organic layers are collected, and the solvent is evaporated under reduced pressure, to give 540 mg of the desired product 1 as an orange powder in 51% yield.
Steady-state UV-Vis and PL measurements. The solutions were prepared by suspending the proper amount of ZIAPIN2 in DMSO, SDS above its critical micellar concentration (100 mM) and H 2 O to obtain a concentration of 25 µM. For the UV-Vis absorption measurements, we employed both a halogen a deuterium 1 Azo-Br2 ZIAPIN 2 lamp as transmission probe. The transmitted light was focused to a fibre-fed spectrometer (Ocean Optics Maya Pro 2000). Isomerisation was triggered by means of a LED lamp centred at 470 nm at a power of 100 mW, which was placed on top of a quartz cuvette (1 mm path) containing the ZIAPIN2 solutions. The sample was illuminated uniformly. The micro-PL on HEK293 cells was acquired by exciting with a CW diode laser (excitation energy of 10 mW mm -2 ). The emission was collected with a 50x objective (Zeiss), filtered to remove the excitation line and sent to the camera (Hamamatsu, acquisition time 100 ms).
Scanning electron microscopy. We used Tescan MIRA3 microscope. The measurements were performed at a voltage of 5 kV and backscattered electrons were detected. ZIAPIN2 solutions were drop-cast on top of silicon substrates and let to dry. The sample were covered with carbon paste to improve conductivity.
Confocal microscopy. Live HEK cells were loaded with CellMask TM Deep Red plasma membrane stain (1 μl/ml; Thermo Fischer) for the evaluation of the localization of ZIAPIN2 at the plasma membrane. Cells were cultured as described in the electrophysiology section (see below). Briefly, HEK cells were exposed to ZIAPIN2 in DMSO (25 μM) for 7 min; the molecule was then washed away, and cells were treated with CellMask TM Deep Red, following the manufacturer instructions. Coverslips with cells were washed with fresh medium and mounted on a confocal laser scanning microscope (CLSM) Leica SP8 (Leica Microsystem) for live-cell z-series stack acquisition of consecutive confocal sections (40x 1.4 NA objective).
For time lapse experiments, HEK cells were loaded with 25 μM ZIAPIN2 in DMSO and imaged in 2Dconfocal mode every 30 seconds for 10 min; bright-field images were acquired to maintain the focus and visualize the localization of ZIAPIN2 overtime.
Ultrafast time-resolved spectroscopy. For the femtosecond TA measurements, we used a Ti:Sapphire laser with a repetition rate of 1 kHz and a pulse width 100-150 fs. We excited the solutions with a wavelength of 470 nm that was generated by means of an optical parameter amplifier and probed with a white light beam generated by a CaF 2 plate. The excitation energy was of ≈ 20 nJ and the beam spot size of ≈ 200 µm in diameter. We had to circulate the solution containing the sample because ZIAPIN2 recovery time to the trans ground state far exceeds the time interval between pulses (set by the laser repetition rate). Circulation assures that a "fresh" non-excited molecule is measured by each pulse. The solutions were circulated by means of a peristaltic pump (200 rpm). The light source for excitation was provided by a LED system (Lumencor Spectra X) fibre-coupled to the fluorescence port of the microscope; the illuminated spot on the sample has an area of 0.23 mm 2 . Cyan emitting LED was used as light source, characterized by maximum emission wavelength at 470 nm and at a photoexcitation density of 50 mW/mm 2 , as measured at the output of the microscope objective (Pobj). We also irradiated at different wavelengths to record action spectra. For this, we employed a Lumencor Spectra light engine® generation-III (8 LED light sources). Data are represented as mean ± MSE. All experiments were carried out at 24 ± 1 °C. **p<0.01; ***p<0.001; ****p<0.0001, Kruskal-Wallis test.
Cytotoxicity. The Alamar Blue® is a cytotoxicity/proliferation assay that is based in the emission properties of Resazurin. [1] In particular, Resazurin is reduced by cell respiration when added to the cell culture. The reduced form of the molecule (Resorufin) has a higher emission yield at 590 nm compared to the oxidized Resazurin, allowing to study cells proliferation by monitoring the emission intensity at 590 nm from a fixed volume of Resazurin. In such procedure, Alamar Blue is firstly added to DMEM without phenol red (Alamar Blue® volume = 10% of the whole volume of cellular buffer). A fixed volume of this solution (500 µl) is then added to the cellular buffer and let to react with cells (3 hours). Finally, three aliquots (100 µl) are taken from each samples for emission measurements (PL wavelength 590 nm). Each emission point is acquired at 24, 48, 72 and 96 hours after ZIAPIN2 incubation. Control measurements were taken on Alamar Blue treated HEK cells without ZIAPIN2. Figure S1. a, PL spectra of ZIAPIN as a function of added water volume. To establish a quantitative relationship among the relative quantum yields, sample volumes were kept constant and the obtained PL spectra were normalised to lamp intensity and ground state absorption. b, Stokes shift and integrated PL area (normalised to absorbance) as a function of water volume.    For the measurements in cuvette, we employed a fluorimeter equipped with a blue LED (470 nm), and took the PL signal at the peak maxima (resolution 20 ms). For the PL measurements in HEK cells, we used a micro-PL set-up, equipped with an objective (40x) and a CW laser (450 nm). In this case, we had to increase the acquisition time to 100 ms to obtain a good signal-to-noise ratio.