Laser-induced release of neuroactive proteins from polymer microcapsules using one- and two-photon excitation for directed neural cell differentiation

Precise control of neuroactive protein delivery is essential for directing neural cell differentiation and understanding neurodevelopmental processes. Yet existing approaches lack the combination of high spatial resolution, deep tissue penetration, and minimal phototoxicity required for various biological applications. Here, we present a platform for light-induced release of neuroactive proteins from polymer capsules, comparing one-photon and two-photon excitation mechanisms and demonstrating functional neural differentiation. Neuroactive proteins (BDNF and NeuroD1) were loaded in 1.7-micron polyelectrolyte capsules, and then released in neural progenitor cells (NPCs) using two confocal microscopy systems: single-photon (488, 561 nm) and two-photon (795, 982 and 1045 nm). Obtained capsules demonstrated low cytotoxicity and high internalization capacity in NPCs, as well as stable loading with proteins. The loaded capsules were able to release the proteins under the action of single-photon (49 µW for 561 nm) or two-photon (1.5 mW for 1045 nm) excitable lasers at parameters that did not affect cell survival. The microcapsules maintained a high level of fluorescence and detection ability in cells for two weeks after internalization. Additionally, these capsules can be used to activate local differentiation of NPCs. The combination of laser-induced activation, and integrated tracking creates a powerful platform for investigating neural development, regeneration, and cell-cell interactions with single-cell precision.