In situgeneration and biological effect evaluation of intracellular nanobubbles

Nanobubbles (NBs) exhibit unique physicochemical properties with significant promise for biomedical applications. Current strategies rely on ex situ preparation of NBs-containing media followed by introducing them into organisms, which imposes limitations including special storage requirements, reduced NB concentration, and limited in vivo targeting. In principle, in situ NBs generation offers a more efficient approach to harnessing their functional properties. Here, we investigated whether NBs could be generated in situ within cells and evaluated preliminarily their biological effects. Intracellular O₂-NBs in a cell model were generated via a compression-decompression method and directly visualized by synchrotron-based scanning transmission X-ray microscopy (STXM) in a cryogenic mode. The effects of NBs on cell viability, proliferation, and overall cellular oxidative activity were explored, which indicates that the composition and the gas-liquid interface of NBs can affect cell metabolism. These findings provide a unique strategy for modulating the cellular microenvironment to achieve biological effects and potential therapeutic outcomes through NB generation by purely physical means.