Real-time in vivo imaging of size-dependent transport and toxicity of gold nanoparticles in zebrafish embryos using single nanoparticle plasmonic spectroscopy

Noble metal nanoparticles (NPs) show distinctive plasmonic optical properties and superior photostability, enabling them to serve as photostable multicoloured optical molecular probes and sensors for real-time in vivo imaging. To effectively study biological functions in vivo, it is essential that the NP probes are biocompatible and can be delivered into living organisms non-invasively. In this study, we have synthesized, purified and characterized stable (non-aggregated) gold (Au) NPs (86.2 +/- 10.8 nm). We have developed dark-field single NP plasmonic microscopy and spectroscopy to study their transport into early developing zebrafish embryos (cleavage stage) and their effects on embryonic development in real-time at single NP resolution. We found that single Au NPs (75-97 nm) passively diffused into the embryos via their chorionic pore canals, and stayed inside the embryos throughout their entire development (120 h). The majority of embryos (96 +/- 3%) that were chronically incubated with the Au NPs (0-20 pM) for 120 h developed to normal zebrafish, while an insignificant percentage of embryos developed to deformed zebrafish (1 +/- 1)% or dead (3 +/- 3)%. Interestingly, we did not observe dose-dependent effects of the Au NPs (0-20 pM) on embryonic development. By comparing with our previous studies of smaller Au NPs (11.6 +/- 0.9 nm) and similar-sized Ag NPs (95.4 +/- 16.0 nm), we found that the larger Au NPs are more biocompatible than the smaller Au NPs, while the similar-sized Ag NPs are much more toxic than Au NPs. This study offers in vivo assays and single NP microscopy and spectroscopy to characterize the biocompatibility and toxicity of single NPs, and new insights into the rational design of more biocompatible plasmonic NP imaging probes.