Ultrasonic neural regulation over two-dimensional graphene analog biomaterials: Enhanced PC12 cell differentiation under diverse ultrasond excitation

Two-dimensional (2D) biomaterials, with unique planar topology and quantum effect, have been widely recognized as a versatile nanoplatform for bioimaging, drug delivery and tissue engineering. However, during the complex application of nerve repair, in which inflammatory microenvironment control is imperative, the gentle manipulation and trigger of 2D biomaterials with inclusion and diversity is still challenging. Herein, inspired by the emerging clinical progress of ultrasound neuromodulation, we systematically studied ultrasound-excited 2D graphene analogues (graphene, graphene oxide, reduced graphene oxide (rGO) and carbon nitride) to explore their feasibility, accessibility, and adjustability for ultrasound-induced nerve repair in vitro. Quantitative observation of cell differentiation morphology demonstrates that PC12 cells added with rGO show the best compatibility and differentiation performance under the general ultrasound mode (0.5 w/cm(2), 2 min/day) compared with graphene, graphene oxide and carbon nitride. Furthermore, the general condition can be improved by using a higher intensity of 0.7 w/cm(2), but it cannot go up further. Later, ultrasonic frequency and duty cycle conditions were investigated to demonstrate the unique and remarkable inclusion and diversity of ultrasound over conventional electrical and surgical means. The pulse waveform with power of 1 MHz and duty cycle of 50 % may be even better, while the 3 MHz and 100 % duty cycle may not work. Overall, various graphene analog materials can be regarded as biosafe and accessible in both fundamental research and clinical ultrasound therapy, even for radiologists without material backgrounds. The enormous potential of diverse and personalized 2D biomaterials-based therapies can be expected to provide a new mode of ultrasound neuromodulation.