Prakash D. Nallathamby

IN VIVO IMAGING OF TRANSPORT AND BIOCOMPATIBILITY OF SINGLE SILVER NANOPARTICLES IN EARLY DEVELOPMENT OF ZEBRAFISH EMBRYOS

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.

Random walk of single gold nanoparticles in zebrafish embryos leading to stochastic toxic effects on embryonic developments

We have synthesized and characterized stable (non-aggregating, non-photobleaching and non-blinking), nearly monodisperse and highly-pure Au nanoparticles, and used them to probe nanoparticle transport and diffusion in 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 the chorionic space of the embryos via their chorionic pore canals and continued their random-walk through chorionic space and into the inner mass of embryos. Diffusion coefficients of single nanoparticles vary dramatically (2.8 x 10(-11) to 1.3 x 10(-8) cm(2) s(-1)) as nanoparticles diffuse through the various parts of embryos, suggesting highly diverse transport barriers and viscosity gradients in the embryos. The amount of Au nanoparticles accumulated in embryos increases with nanoparticle concentration increases. Interestingly, however, their effects on embryonic development are not proportionally related to their concentration. The majority of embryos (74% on average) chronically incubated 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 have developed a new approach to image and characterize individual Au nanoparticles embedded in tissues using histology sample preparation methods and localized surface plasmon resonance spectra of single nanoparticles. We found Au nanoparticles in various parts of normally developed and deformed zebrafish, suggesting that the 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 with (less toxic to) the embryos than the 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 the biocompatibility and toxicity of nanoparticles is highly dependent on their chemical properties, and that the embryos can serve as effective in vivo assays to screen their biocompatibility.

Photostable Single-Molecule Nanoparticle Optical Biosensors for Real-Time Sensing of Single Cytokine Molecules and Their Binding Reactions

We synthesized tiny stable silver nanoparticles (2.6 +/- 1.1 nm) and used its small surface area and functional groups to control single molecule detection (SMD) volumes on single nanoparticles. These new approaches allowed us to develop intrinsic single molecule nanoparticle optical biosensors (SMNOBS) for sensing and imaging of single human cytokine molecules, recombinant human tumor necrosis factor-alpha (TNFalpha), and probing its binding reaction with single monoclonal antibody (MAB) molecules in real-time. We found that SMNOBS retained their biological activity over months and showed exceptionally high photostability. Our study illustrated that smaller nanoparticles exhibited higher dependence of optical properties on surface functional groups, making it a much more sensitive biosensor. Localized surface plasmon resonance spectra (LSPRS) of SMNOBS showed a large red shift of peak wavelength of 29 +/- 11 nm, as single TNFalpha molecules bound with single MAB molecules on single nanoparticles. Utilizing its LSPRS, we quantitatively measured its binding reaction in real time at single molecule (SM) level, showing stochastic binding kinetics of SM reactions with binding equilibrium times ranging from 30 to 120 min. SMNOBS exhibited extraordinarily high sensitivity and selectivity, and a notably wide dynamic range of 0-200 ng/mL (0-11.4 nM). Thus, SMNOBS is well suited for the fundamental study of biological functions of single protein molecules and SM interactions of chemical functional groups with the surface of nanoparticles, as well as development of effective disease diagnosis and therapy.

Design of Stable and Uniform Single Nanoparticle Photonics for In Vivo Dynamics Imaging of Nanoenvironments of Zebrafish Embryonic Fluids

We report here the use of a simple washing approach to reduce the ionic strength of the solution, which increased the thickness of the electric double layer on the surface of silver (Ag) nanoparticles and thereby enhanced their surface zeta-potential. This approach allowed us to prepare optically uniform (75-99%) and purified Ag nanoparticles (11.3 +/- 2.3 nm) that are stable (nonaggregation) in solution for months, permitting them to become robust and widely used single nanoprobes for in vivo optical imaging. These Ag nanoparticles show remarkable photostability and serve as single nanoparticle photonic probes for continuous imaging nanoenvironments of segmentation-stage zebrafish embryos for hours. Unlike other particle tracking experiments, we utilized size-dependent localized surface plasmon resonance spectra (LSPRS) (colors) of single Ag nanoparticles to determine given colored (sized) nanoparticles in situ and used the monodisperse color (size) of nanoparticles to simultaneously measure viscosities and flow patterns of multiple proximal nanoenvironments in segmentation-stage zebrafish embryos in real time. We found new interesting counterclockwise flow patterns with rates ranging from 0.06 to 1.8 microm/s and stunningly high viscosity gradients spanning two orders of magnitude in chorion space of the embryos, with the highest viscosity observed around the center of chorion space and the lower viscosity at the interfacial areas near the surface of both chorion layers and inner mass of the embryos. This study demonstrates the possibility of using individual monodisperse nanophotonics to probe the roles of embryonic fluid dynamics in embryonic development.

In Vivo Quantitative Study of Sized-Dependent Transport and Toxicity of Single Silver Nanoparticles Using Zebrafish Embryos

Nanomaterials possess distinctive physicochemical properties (e.g., small sizes and high surface area-to-volume ratios) and promise a wide variety of applications, ranging from the design of high quality consumer products to effective disease diagnosis and therapy. These properties can lead to toxic effects, potentially hindering advances in nanotechnology. In this study, we have synthesized and characterized purified and stable (nonaggregation) silver nanoparticles (Ag NPs, 41.6 ± 9.1 nm in average diameter) and utilized early developing (cleavage-stage) zebrafish embryos (critical aquatic and eco- species) as in vivo model organisms to probe the 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 into 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 into 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), we found 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).

Study of cytotoxic and therapeutic effects of stable and purified silver nanoparticles on tumor cells

We have synthesized and purified silver nanoparticles (Ag NPs) (11.3+/-2.3 nm) that are stable (non-aggregated) in cell culture medium and inside single living cells. We have developed new imaging methods to characterize sizes and number of single NPs in the medium and in single living cells in real-time and determine their stability (non-aggregation) in the medium and in single living cells at single NP resolution. These new approaches allow us to study toxic and therapeutic effects of single Ag NPs on tumor cells (L929, mouse fibroblast cells) with determined sizes and concentrations (doses) of NPs over time at single NP and single cell resolution. We found that Ag NPs inhibited the growth and division of tumor cells and their nuclei, in a dose and time dependent manner, showing significant inhibitory effects and abnormal cells with giant undivided nuclei or multiple nuclei beyond 12 h incubation. The results show that Ag NPs inhibited the segregation of chromosomes, but not their replication. Intracellular Ag NPs were well distributed in the cell population, and located in the nuclei and cytoplasm with higher numbers in the cytoplasm. This study demonstrates the possibility of using Ag NPs to inhibit the growth and division of tumor cells and using their cytotoxicity for potential therapeutic treatments. This study offers a new method to count the number of single NPs in the medium for characterization of their concentration and stability at single NP resolution over time.

Study of Charge-Dependent Transport and Toxicity of Peptide-Functionalized Silver Nanoparticles Using Zebrafish Embryos and Single Nanoparticle Plasmonic Spectroscopy

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 diameter) 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.

Design and characterization of optical nanorulers of single nanoparticles using optical microscopy and spectroscopy

Current conventional imaging methods cannot determine sizes of single nanoparticles (NPs) in solution and living organisms at the nanometre scale, which limits the applications of NPs. In this study, we developed new imaging calibration approaches to characterize the sizes of single Ag NPs in solution at nanometre resolution by measuring their size-dependent scattering localized-surface-plasmon-resonance (LSPR) spectra and scattering intensity using dark-field optical microscopy and spectroscopy (DFOMS). We synthesized nearly spherical shape Ag NPs, ranging from 2 to 110 nm in diameter, and characterized the sizes of single NPs using high-resolution transmission electron microscopy, and the LSPR spectra and scattering intensity of single NPs using DFOMS. We constructed calibration curves of the peak wavelength (lambda(max)) of LSPR spectra or scattering intensity of single NPs versus their sizes. These calibration curves allow us to determine the sizes of single NPs at 1 nm resolution by measuring the LSPR spectra or scattering intensity of single NPs using DFOMS. These new approaches enable us to create optical nanorulers (calibration curves) of single Ag NPs for simultaneously imaging and measuring sizes of multiple single NPs in solution in real time at nanometre resolution using optical microscopy. One can now use these new imaging calibration approaches to study and characterize single NPs in solution and living organisms in real time for a wide variety of applications.

Study of the Multidrug Membrane Transporter of Single Living Pseudomonas aeruginosa Cells Using Size-Dependent Plasmonic Nanoparticle Optical Probes

Multidrug membrane transporters (efflux pumps) in both prokaryotes and eukaryotes are responsible for impossible treatments of a wide variety of diseases, including infections and cancer, underscoring the importance of better understanding of their structures and functions for the design of effective therapies. In this study, we designed and synthesized two silver nanoparticles (Ag NPs) with average diameters of 13.1 +/- 2.5 nm (8.1-38.6 nm) and 91.0 +/- 9.3 nm (56-120 nm) and used the size-dependent plasmonic spectra of single NPs to probe the size-dependent transport kinetics of MexAB-OprM (multidrug transporter) in Pseudomonas aeruginosa in real time at nanometer resolution. We found that the level of accumulation of intracellular NPs in wild-type (WT) cells was higher than in nalB1 (overexpression of MexAB-OprM) but lower than in DeltaABM (deletion of MexAB-OprM). In the presence of proton ionophores (CCCP, inhibitor of proton motive force), we found that intracellular NPs in nalB1 were nearly doubled. These results suggest that MexAB-OprM is responsible for the extrusion of NPs out of cells and NPs (orders of magnitude larger than conventional antibiotics) are the substrates of the transporter, which indicates that the substrates may trigger the assembly of the efflux pump optimized for the extrusion of the encountered substrates. We found that the smaller NPs stayed inside the cells longer than larger NPs, suggesting the size-dependent efflux kinetics of the cells. This study shows that multisized NPs can be used to mimic various sizes of antibiotics for probing the size-dependent efflux kinetics of multidrug membrane transporters in single living cells.

Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 ± 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c << 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target specific pathways in development, and potentially enable target specific study and therapy for early embryonic development.