Highly regulated programs for airway epithelial cell proliferation and differentiation during development and repair are often disrupted in disease. These processes have been studied in mouse models; however, it is difficult to isolate and identify epithelial cell-specific responses in vivo. To investigate these processes in vitro, we characterized a model for primary culture of mouse tracheal epithelial cells. Small numbers of cells seeded at low density (7.5 × 104 cells/cm2) rapidly proliferated and became polarized. Subsequently, supplemented media and air-liquid interface conditions resulted in development of highly differentiated epithelia composed of ciliated and nonciliated cells with gene expression characteristic of native airways. Genetically altered or injured mouse tracheal epithelial cells also reflected in vivo patterns of airway epithelial cell gene expression. Passage of cells resulted in continued proliferation but limited differentiation after the first passage, suggesting that transit-amplifying cell populations were present but with independent programs for proliferation and differentiation. This approach provides a high-fidelity in vitro model for evaluation of gene regulation and expression in mouse airway epithelial cells.

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.

Noble metal nanoparticles (NPs) possess size- and shape- dependent optical properties, suggesting the possibility of tuning desired optical properties of ensemble NPs at single NP resolution and underscoring the importance of probing the sizes and shapes of single NPs in situ and in real-time. In this study, we synthesized twelve colloids of Ag NPs. Each colloid contains various sizes and shapes of single NPs, showing rainbow colors with peak-wavelength of absorption spectra from 393 to 738 nm. We correlated the sizes and shapes of single NPs determined by high-resolution transmission electron microscopy (HRTEM) with scattering localized surface plasmon resonance (LSPR) spectra of single NPs characterized by dark-field optical microcopy and spectroscopy (DFOMS). Single spherical (2–39 nm in diameter), rod (2–47 nm in length with aspect ratios of 1.3–1.6), and triangular (4–84 nm in length with thickness of 2–27 nm) NPs show LSPR spectra (λmax) at 476±5 or 533±12, 611±23, and 711±40 nm, respectively. Notably, we observed new cookie-shaped NPs, which exhibit LSPR spectra (λmax) at 725±10 nm with a shoulder peak at 604±5 nm. Linear correlations of sizes of any given shape of single NPs with their LSPR spectra (λmax) enable the creation of nano optical rulers (calibration curves) for identification of the sizes and shapes of single NPs in solution in real time using DFOMS, offering the feasibility of using single NPs as multicolored optical probes for study of dynamics events of interest in solutions and living organisms at nm scale in real time.

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-α (TNFα), 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 TNFα molecules bound with single MAB molecules on single nanoparticles. Utilizing its LSPRS, we quantitatively measured its binding reaction in real time at 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.

At the cellular level, a small number of protein molecules (receptors) can induce significant cellular responses, emphasizing the importance of molecular detection of trace amounts of protein on single living cells. In this study, we designed and synthesized silver nanoparticle biosensors (AgMMUA-IgG) by functionalizing 11.6 +/- 3.5-nm Ag nanoparticles with a mixed monolayer of 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (1:3 mole ratio) and covalently conjugating IgG with MUA on the nanoparticle surface. We found that the nanoparticle biosensors preserve their biological activity and photostability and can be utilized to quantitatively detect individual receptor molecules (T-ZZ), map the distribution of receptors (0.21-0.37 molecule/microm(2)), and measure their binding affinity and kinetics at concentrations below their dissociation constant on single living cells in real time over hours. The dynamic range of detection is 0-50 molecules per cell. We also found that the binding rate (2-27 molecules/min) is highly dependent upon the coverage of receptors on living cells and their ligand concentration. The binding association and dissociation rate constants and affinity constant are k1 = (9.0 +/- 2.6) x 10(3) M(-1) s(-1), k(-1) = (3.0 +/- 0.4) x 10(-4) s(-1), and KB = (4.3 +/- 1.1) x 10(7) M(-1), respectively.