A systematic study on the use of multifunctional nanodiamonds for neuritogenesis and super-resolution imaging

Regeneration of defective neurons in central nervous system is a highlighted issue for neurodegenerative disease treatment. Various tissue engineering approaches have focused on neuritogenesis to achieve the regeneration of damaged neuronal cells because damaged neurons often fail to achieve spontaneous restoration of neonatal neurites. Meanwhile, owing to the demand for a better diagnosis, studies of super-resolution imaging techniques in fluorescence microscopy have triggered the technological development to surpass the classical resolution dictated by the optical diffraction limit for precise observations of neuronal behaviors. Herein, the multifunctional nanodiamonds (NDs) as neuritogenesis promoters and super-resolution imaging probes were studied.

To investigate the neuritogenesis-inducing capability of NDs, ND-containing growing medium and differentiation medium were added to the HT-22 hippocampal neuronal cells and incubated for 10 d. In vitro and ex vivo images were visualized through custom-built two-photon microscopy using NDs as imaging probes and the direct stochastic optical reconstruction microscopy (dSTORM) process was performed for the super-resolution reconstruction owing to the photoblinking properties of NDs. Moreover, ex vivo imaging of the mouse brain was performed 24 h after the intravenous injection of NDs.

NDs were endocytosed by the cells and promoted spontaneous neuritogenesis without any differentiation factors, where NDs exhibited no significant toxicity with their outstanding biocompatibility. The images of ND-endocytosed cells were reconstructed into super-resolution images through dSTORM, thereby addressing the problem of image distortion due to nano-sized particles, including size expansion and the challenge in distinguishing the nearby located particles. Furthermore, the ex vivo images of NDs in mouse brain confirmed that NDs could penetrate the blood-brain barrier (BBB) and retain their photoblinking property for dSTORM application.

It was demonstrated that the NDs are capable of dSTORM super-resolution imaging, neuritogenic facilitation, and BBB penetration, suggesting their remarkable potential in biological applications.