Single nanoparticle spectroscopy for real-time in vivo quantitative analysis of transport and toxicity of single nanoparticles in single embryos

Abstract

Nanomaterials exhibit distinctive physicochemical properties and promise a wide range of applications from nanotechnology to nanomedicine, which raise serious concerns about their potential environmental impacts on ecosystems. Unlike any conventional chemicals, nanomaterials are highly heterogeneous, and their properties can alter over time. These unique characteristics underscore the importance of study of their properties and effects on living organisms in real time at single nanoparticle (NP) resolution. Here we report the development of single-NP plasmonic microscopy and spectroscopy (dark-field optical microscopy and spectroscopy, DFOMS) and ultrasensitive in vivo assay (cleavage-stage zebrafish embryos, critical aquatic species) to study transport and toxicity of single silver nanoparticles (Ag NPs, 95.4 ± 16.0 nm) on embryonic developments. We synthesized and characterized purified and stable (non-aggregation) Ag NPs, determined their sizes and doses (number), and their transport mechanisms and effects on embryonic development in vivo in real time at single-NP resolution. We found that single Ag NPs passively entered the embryos through their chorionic pores via random Brownian diffusion and stayed inside the embryos throughout their entire development (120 h), suggesting that the embryos can bio-concentrate trace NPs from their environment. Our studies show that higher doses and larger sizes of Ag NPs cause higher toxic effects on embryonic development, demonstrating that the embryos can serve as ultrasensitive in vivo assays to screen biocompatibility and toxicity of the NPs and monitor their potential release into aquatic ecosystems.

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

“…We calculated their molar concentrations by dividing moles of single Au NPs by the solution volume, and characterized rsfs.royalsocietypublishing.org Interface Focus 3: 20120098 them as we described previously [11,16,18,19,35]. Briefly, we calculated the weight of Au (W Au ) generated by the complete reduction of ).…”

“…Unlike any other imaging tools, such as transmission electron microscopy (TEM) or scanning probe microscopy, DFOMS offers high temporal resolution and throughput to trace, quantify and characterize multiple individual NPs in vivo in real-time simultaneously [11,13,16,18,19]. We have recently developed photostable optical nanoscopy (PHOTON) and single molecule nanoparticle optical biosensors, which enable DFOMS to image single molecules and single NPs at millisecond (ms) temporal and nanometre spatial resolutions [17,22].…”

“…A pair of zebrafish can produce massive amounts (200-300) of embryos overnight at a very low cost. These superior characteristics enable zebrafish embryos to serve as in vivo high-throughput assays to study the biocompatibility and toxicity of nanomaterials [11,16,18,19].…”

“…In our previous studies, we used cleavage-stage zebrafish embryos to study the transport, biocompatibility and toxicity of purified Ag NPs with three distinctive sizes (11.6 + 3.5, 41.6 + 9.1 and 95.4 + 16.0 nm) and the smaller Au NPs (11.6 + 0.9 nm) [11,16,18,19]. In this study, we use zebrafish embryos to characterize the transport, biocompatibility and toxicity of the larger Au NPs (86.2 + 10.8 nm), aiming to determine the dependence of the biocompatibility and toxicity of the NPs upon their sizes and chemical compositions for rational design of biocompatible NP probes.…”

“…To overcome the limitations of current nanotoxicity studies, we have developed ultrasensitive in vivo assays (cleavage-stage zebrafish embryos) to characterize the biocompatibility and toxicity of nanomaterials [11,16,18,19]. To depict the dependence of NP toxicity on their physicochemical properties, and to rationally design biocompatible NP imaging probes, we have synthesized a mini-library of Au and Ag NPs with various sizes and surface functional groups, purified them, determined their stability, and developed DFOMS to characterize them throughout their incubation with living organisms in situ in real-time at single NP resolution [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21].…”