Membrane Targeted Nanoactuators for Cellular Photostimulation

Photostimulation has recently attracted interest for its possible application in the biomedical field. Several approaches have been proposed to perturbate cellular activity using light in a spatiotemporal precise manner. Organic semiconductors (films, nanoparticles) and photochromic molecules (azobenzene and its derivates) have been revealed to be able to perturb the plasma membrane electrical properties under light stimulation. Light absorption by these materials can be converted into an electrical, mechanical, or thermal stimuli leading to a change of membrane electrical properties. In this regard, Ziapin2 an amphiphilic alkyl-substituted 4,4'-diaminoazobene was recently developed. Ziapin2 inserts properly in the cell membrane persisting in a cis configuration. The insertion in the membrane and the consequent formation of Ziapin2 dimers lead to a shrinkage of the membrane and an increase of the membrane capacitance. Light stimulation induces a cis-trans isomerization with a significant perturbation of the cell membrane potentials, able to trigger action potential firing in excitable cells without directly affecting local temperature. Moreover, capacitance increase after Ziapin2 portioning in the membrane induces stable changes in cellular physiology even under dark conditions. We propose to modify Ziapin2 to obtain a push-pull molecule able to overcome such limitations. The new molecule, named PP-2Pyr, reveals be more soluble in water to improve its biocompatibility and clearance. The substitution of the amino group with a nitro group increases the red-shift absorption avoiding the need to use UV light, not suitable for applications in living cells. Here, we report a study of PP-2Pyr biological functions, going through biocompatibility assays and electrophysiological characterization using an immortalized cell line (HEK cells) as cellular model of investigation. In accordance with the theoretical design of the molecule, the absorbance/emission spectra display a significant red-shift in PP-2Pyr absorbance with a peak at 517nm and an emission peak at 600nm. The biological investigation reveals that PP-2Pyr are able to enter properly in cells after few minutes of incubation with no evident cytotoxicity at two different concentrations (5 and 10µM) until 5 days after the exposure. In addition, after molecule internalization cells exposed to an acute light stimulation lasting 30s do not display evidence of a significant phototoxicity. We also demonstrated that in cells loaded with the compound, millisecond pulses of visible light induce a transient and defined depolarization of the membrane potential followed by a delayed and slight hyperpolarization. The amplitude of the membrane depolarization is dependent of both the light power density and the molecule concentration. Despite further studies of the push-pull photophysical features and biological behavior are in progress, data so far available indicate PP-2Pyr as a promising tool for photo-excitation of cells for both in vitro and in vivo applications.