Real-time study of the transport and biocompatibility of nanomaterials in early embryonic development at single-nanoparticle resolution can offer new knowledge about the delivery and effects of nanomaterials in vivo and provide new insights into molecular transport mechanisms in developing embryos. In this study, we directly characterized the transport of single silver nanoparticles into an in vivo model system (zebrafish embryos) and investigated their effects on early embryonic development at single-nanoparticle resolution in real time. We designed highly purified and stable (not aggregated and no photodecomposition) nanoparticles and developed single-nanoparticle optics and in vivo assays to enable the study. We found that single Ag nanoparticles (5-46 nm) are transported into and out of embryos through chorion pore canals (CPCs) and exhibit Brownian diffusion (not active transport), with the diffusion coefficient inside the chorionic space (3 x 10(-9) cm(2)/s) approximately 26 times lower than that in egg water (7.7 x 10(-8) cm(2)/s). In contrast, nanoparticles were trapped inside CPCs and the inner mass of the embryos, showing restricted diffusion. Individual Ag nanoparticles were observed inside embryos at each developmental stage and in normally developed, deformed, and dead zebrafish, showing that the biocompatibility and toxicity of Ag nanoparticles and types of abnormalities observed in zebrafish are highly dependent on the dose of Ag nanoparticles, with a critical concentration of 0.19 nM. Rates of passive diffusion and accumulation of nanoparticles in embryos are likely responsible for the dose-dependent abnormalities. Unlike other chemicals, single nanoparticles can be directly imaged inside developing embryos at nanometer spatial resolution, offering new opportunities to unravel the related pathways that lead to the abnormalities.

We have synthesized and characterized stable (non-aggregation, non-photobleaching and nonblinking), nearly monodisperse and highly-purified Au nanoparticles, and used them to probe transport of cleavage-stage zebrafish embryos and to study their effects on embryonic development in real time. We found that single Au nanoparticles (11.6 ± 0.9 nm in diameter) passively diffused into chorionic space of the embryos via their chorionic-pore-canals and continued their random-walk through chorionic space and into inner mass of embryos. Diffusion coefficients of single nanoparticles vary dramatically (2.8×10 -11 to 1.3×10 -8 cm 2 /s) as nanoparticles diffuse through various parts of embryos, suggesting highly diverse transport barriers and viscosity gradients of embryos. The amount of Au nanoparticles accumulated in embryos increase with its concentration. Interestingly, their effects on embryonic development are not proportionally related to the concentration. Majority of embryos (74% on average) incubated chronically with 0.025-1.2 nM Au nanoparticles for 120 h developed to normal zebrafish, with some (24%) being dead and few (2%) deformed. We developed a new approach to image and characterize individual Au nanoparticles embedded in tissues using histology sample preparation methods and LSRP spectra of single nanoparticles. We found that Au nanoparticles in various parts of normally developed and deformed zebrafish, suggesting that random-walk of nanoparticles in embryos during their development might have led to stochastic effects on embryonic development. These results show that Au nanoparticles are much more biocompatible (less toxic) to the embryos than Ag nanoparticles that we reported previously, suggesting that they are better suited as biocompatible probes for imaging embryos in vivo. The results provide powerful evidences that biocompatibility and toxicity of nanoparticles highly depend on their chemical properties, and the embryos can serve as effective in-vivo assays to screen their biocompatibility.

Nanomaterials possess distinctive physicochemical properties (e.g., small sizes, high surface area-to-volume ratios) and promise a wide variety of applications, ranging from design of high quality consumer products to effective disease diagnosis and therapy. These properties can lead to toxic effects, potentially hindering advance in nanotechnology. In this study, we have synthesized and characterized purified and stable (non-aggregation) silver nanoparticles (Ag NPs, 41.6±9.1 nm in average diameters), and utilized early-developing (cleavage-stage) zebrafish embryos (critical aquatic and eco- species) as in vivo model organisms to probe diffusion and toxicity of Ag NPs. We found that single Ag NPs (30–72 nm diameters) passively diffused into the embryos through chorionic pores via random Brownian motion and stayed inside the embryos throughout their entire development (120 hours-post-fertilization, hpf). Dose and size dependent toxic effects of the NPs on embryonic development were observed, showing the possibility of tuning biocompatibility and toxicity of the NPs. At lower concentrations of the NPs (≤ 0.02 nM), 75–91% of embryos developed to normal zebrafish. At the higher concentrations of NPs (≥ 0.20 nM), 100% of embryos became dead. At the concentrations in between (0.02–0.2 nM), embryos developed to various deformed zebrafish. Number and sizes of individual Ag NPs embedded in tissues of normal and deformed zebrafish at 120 hpf were quantitatively analyzed, showing deformed zebrafish with higher number of larger NPs than normal zebrafish, and size-dependent nanotoxicity. By comparing with our previous studies of smaller Ag NPs (11.6±3.5 nm), the results further demonstrate striking size-dependent nanotoxicity that, at the same molar concentration, the larger Ag NPs (41.6±9.1 nm) are more toxic than the smaller Ag NPs (11.6±3.5 nm).

Currently, molecular mechanisms of multidrug ABC (ATP-binding cassette) membrane transporters remain elusive. In this study, we synthesized and characterized purified spherically shaped silver nanoparticles (Ag NPs) (11.8 ± 2.6 nm in diameter), which were stable (non-aggregation) in PBS buffer and inside single living cells. We used the size-dependent localized surface plasmon resonance (LSPR) spectra of single Ag NPs to determine their sizes and to probe the size-dependent transport kinetics of the ABC (BmrA, BmrA-EGFP) transporters in single living cells (Bacillus subtilis) in real time at nanometer resolution using dark-field optical microscopy and spectroscopy (DFOMS). The results shows that the smaller NPs stayed longer inside the cells than larger NPs, suggesting size-dependent efflux kinetics of the membrane transporter. Notably, accumulation and efflux kinetics of intracellular NPs for single living cells depended upon the cellular expression level of BmrA, NP concentrations, and a pump inhibitor (25 µM, orthovanadate), suggesting that NPs are substrates of BmrA transporters and that passive diffusion driven by concentration gradients is the primary mechanism by which the NPs enter the cells. The accumulation and efflux kinetics of intracellular NPs for given cells are similar to those observed using a substrate (Hoechst dye) of BmrA, demonstrating that NPs are suitable probes for study of multidrug membrane transporters of single living cells in real-time. Unlike fluorescent probes, single Ag NPs exhibit size-dependent LSPR spectra and superior photostability, enabling them to probe the size-dependent efflux kinetics of membrane transporters of single living cells in real-time for better understanding of multidrug resistance.

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.

Nanomaterials possess unusually high surface area-to-volume ratios, and surface-determined physicochemical properties. It is essential to understand their surface-dependent toxicity in order to rationally design biocompatible nanomaterials for a wide variety of applications. In this study, we have functionalized the surfaces of silver nanoparticles (Ag NPs, 11.7 ± 2.7 nm in diameters) with three biocompatible peptides (CALNNK, CALNNS, CALNNE) to prepare positively (Ag-CALNNK NPs+ζ), negatively (Ag-CALNNS NPs−2ζ), and more negatively charged NPs (Ag-CALNNE NPs−4ζ), respectively. Each peptide differs in a single amino acid at its C-terminus, which minimizes the effects of peptide sequences and serves as a model molecule to create positive, neutral and negative charges on the surface of the NPs at pH 4–10. We have studied their charge-dependent transport into early-developing (cleavage-stage) zebrafish embryos and their effects on embryonic development using dark-field optical microscopy and spectroscopy (DFOMS). We found that all three Ag-peptide NPs passively diffused into the embryos via their chorionic pore canals, and stayed inside the embryos throughout their entire development (120 h), showing charge-independent diffusion modes and charge-dependent diffusion coefficients. Notably, the NPs create charge-dependent toxic effects on embryonic development, showing that the Ag-CALNNK NPs+ζ (positively charged) are the most biocompatible while the Ag-CALNNE NPs–4ζ (more negatively charged) are the most toxic. By comparing with our previous studies of the same sized citrated Ag and Au NPs, the Ag-peptide NPs are much more biocompatible than the citrated Ag NPs, and nearly as biocompatible as the Au NPs, showing the dependence of nanotoxicity upon the surface charges, surface functional groups and chemical compositions of the NPs. This study also demonstrates powerful applications of single NP plasmonic spectroscopy for quantitative analysis of single NPs in vivo and in tissues, and reveals the possibility of rational design of biocompatible NPs.

Noble metal nanoparticles (NPs) show distinctive plasmonic optical properties and superior photostability, enabling them to serve as photostable multi-coloured 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.

Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions.