Membrane Environment Enables Ultrafast Isomerization of Amphiphilic Azobenzene

Abstract

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.

Keywords: amphiphilic; azobenzene; cell membranes; cell stimulation; ultrafast isomerization.

Figure 1

a) Computed UV–vis electronic transition and molecular structures for trans and cis ZIAPIN2 at the DFT and TDDFT level of theory (see Supporting Information for details). b) UV–vIS and c) PL spectra of ZIAPIN2 in DMSO, SDS 100 mm, and water (25 µm). PL spectra were normalized to both lamp intensity and ground state absorption, to obtain a relative PL quantum yield among the three solutions. d) Time‐lapse bright‐field and fluorescence confocal images of ZIAPIN2 (in DMSO 25 µm) loading in HEK293 cells (scale bar = 50 µm).

Figure 2

UV–vis absorption spectra of ZIAPIN2 in a) DMSO, c) SDS (100 mm), and e) water taken under illumination with a blue LED (470 nm). The inset in Figure 2c highlights the 10% decay in the trans absorption of ZIAPIN2 in SDS. Isomerization kinetics of ZIAPIN2 in DMSO taken at b) 490 nm, d) SDS (100 mm), and f) water as a function of illumination time.

Figure 3

a) TA spectra in DMSO (top panel), SDS (100 mm, central panel) and water (bottom panel). The system was excited at 470 nm (π–π*) and probed with a white light super continuum. The three solutions (25 µm) were flowed by means of a peristaltic pump, to permit relaxation from the cis state. b) Global analysis of the experimental TA spectra into two fitting components. The first and second components observed in DMSO and SDS can be associated with the trans and cis isomer spectra, respectively. In H₂O, this latter component can be linked to the excimer formation. c) Population evolution (normalized) of the two SVD components in DMSO, SDS, and H₂O. d) TA experimental dynamics of ZIAPIN2 in DMSO, SDS 100 mm, and H₂O (25 µm) excited at 470 nm and probed in the photobleaching region (500–520 nm) and e) in the 530–580 nm region. Solid lines represent the best‐fit of the experimental data. f) Sketch of the photodynamics in DMSO, SDS, and water as suggested by the TA data.

Figure 4

a–d) Box plots of the parameters investigated for voltage membrane modulation analysis as a function of illumination wavelength. Hyperpolarization (a,c) and depolarization (b,d) changes of HEK293 cells or exposed to DMSO/ZIAPIN2, subjected to 20 or 200 ms light stimulation (n = 14, 18, 20 for CTRL, DMSO, and ZIAPIN2, respectively). Hyperpolarization and depolarization were measured as the minimum and maximum voltage, respectively, reached within 350 ms from the light‐onset. e,f) Action spectrum versus wavelength of the |ΔV| of hyperpolarization (e) and depolarization (f) in HEK293 treated with 25 µm ZIAPIN2, for both short (20 ms) and long (200 ms) light stimuli. We also report the absorption spectrum of ZIAPIN2 in SDS for comparison (dotted grey line). All experiments were carried out at 24 ± 1 °C. **p < 0.01; ***p < 0.001; ****p < 0.0001, Kruskal–Wallis test.