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.

The authors thank Dr Olga Guslyakova (Skolkovo Institute of Science and Technology) for helping with obtaining AuNRs; Dr Mikhail Berestovoy (Federal Center for Brain and Neurotechnologies) and Dina Bass (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences) for helping with cell culturing. This work was supported by the Russian Science Foundation (project No. 25-63-00019). Development of the multiphoton imaging approaches was partially supported by Russian Science Foundation (grant 25-12-00211) and the Development Program of MSU and the National Project “Science and Universities”.

Cell culture works, including cytotoxicity studies and flow cytometry, were supported by Russian Science Foundation (grant no. 23-75-30023). I.S. personally acknowledges Brain Program of the IDEAS Research Center. Center for Bio- and Medical Technologies, Skolkovo Institute of Science and Technology, Moscow, Russia Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency, Moscow, Russia Veronika Usatova, Olga Astakhova, Vladimir Baklaushev & Vsevolod Belousov Life Improvement by Future Technologies (LIFT) Center, Moscow, Russia Andrei Fedotov, Aleksandr Lanin, Vsevolod Belousov & Gleb Sukhorukov Physics Department, Lomonosov Moscow State University, Moscow, Russia Institute of Neuroscience and Neurotechnology, Pirogov Russian National Research Medical University, Moscow, Russia Department of Metabolism and Redox Biology, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia Correspondence to Ivan Smirnov or Gleb Sukhorukov.

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