Valentina Sesti1,2,Matteo Moschetta2,Arianna Magni2,Paola Moretti1,2,Giuseppe Maria Paternò1,2,Matteo Tommasini1,Guglielmo Lanzani2,1,Chiara Bertarelli1,2
Politecnico di Milano1,Istituto Italiano di Tecnologia2
Valentina Sesti1,2,Matteo Moschetta2,Arianna Magni2,Paola Moretti1,2,Giuseppe Maria Paternò1,2,Matteo Tommasini1,Guglielmo Lanzani2,1,Chiara Bertarelli1,2
Politecnico di Milano1,Istituto Italiano di Tecnologia2
Cationic membrane-targeted azobenzenes have been recently proposed as light-induced nanoactuators for neuronal photostimulation.<sup>1,2</sup> In previous works, we developed an azobenzene as a promising nanoscale tool for cell photostimulation, namely Ziapin2. Our approach resides in non-specific binding, and Ziapin2 has an amphiphilic character allowing it to partition spontaneously within the cell membrane. The cationic terminal groups interact with the phospholipid polar heads of the membrane while the hydrophobic part of the molecule stabilizes inside the lipidic region. We demonstrated that this azobenzene is an effective light-driven intramembrane actuator, able to modulate neuronal firing both in vitro and in vivo.<sup>1</sup><br/>These previous results open to the next generation of light-driven photoswitches aimed at enhancing the performances and improving the partition stability. In this work, new amphiphilic push-pull azobenzenes (ppABs) were designed and synthesized, which feature a strong electron donor group in the <i>para</i>-position on one phenyl ring and a strong electron acceptor group in the <i>para</i>-position on the opposite phenyl ring. This push-pull substitution of the phenyl rings delocalizes electrons of the azobenzene and shifts the absorption wavelengths towards red, which is a valuable feature for biological application, allowing for a better light penetration in tissues. Interestingly, light-activated electrophysiology of cells loaded with ppABs shows a depolarization of the membrane followed by a small hyperpolarization, and this is owed to the significant variation of the dipole moment.<sup>3</sup> Within this series of homologous ppABs, we also identified the key role of the cationic heads in the partitioning and potential modulation of the cell membrane.<br/><br/><br/><sup>1</sup> DiFrancesco, M.L., Lodola, F., Colombo, E. et al. “Neuronal firing modulation by a membrane-targeted photoswitch.” Nat. Nanotechnol. 15, 296–306 (2020)<br/><sup>2</sup> Vurro, V., Bondelli, G., Sesti, V., Lodola, F., Paternò, G.M., Lanzani, G. and Bertarelli, C. “Molecular Design of Amphiphilic Plasma Membrane-Targeted Azobenzenes for Nongenetic Optical Stimulation.” Front. Mater. 7:631567 (2021)<br/><sup>3</sup> Bandara, H. M. Dhammika., Burdette, Shawn C. “Photoisomerization in different classes of azobenzene.” Chem. Soc. Rev., 41, 1809-1825 (2012)