Carbon nanotubes have been reported to cause pulmonary fibrosis in mice after inhalation exposure. When inhaled, multiwall carbon nanotubes (MWCNTs) activate macrophage inflammasomes and interleukin (IL)-1β release, key cellular components of the innate immune response. Macrophages are the first line of defense that engulf and remove inhaled MWCNTs from the lungs. Macrophages are also a source of secreted osteopontin (OPN), which promotes tissue matrix remodeling and fibrosis. These responses may be triggered by the unique aspect ratio, aggregation or surface chemistry of MWCNTs. In previous studies, we explored atomic layer deposition (ALD) as a means to modify the surface functionality of MWCNTs and studied how the surface coating affected the toxic response of THP-1 cells, a widely used human monocyte/macrophage cell line, and primary peripheral blood monocytes (PBMCs) obtained from normal human donors. Compared to uncoated MWCNTs, we found that nanotubes with Al₂O₃ nanocoatings showed enhanced IL-1β secretion and decreased OPN production in THP-1 cells and PBMCs, indicating that the coating enhances the innate immune response and decreases pro-fibrotic activity.

In this study we examined the effect of ALD ZnO coatings on the fibrogenic response in human lung fibroblast (HLFs) using mRNA expression and secretion of transforming growth factor (TGF)-b 1 and CXCL10, mediators that promote and deter fibrosis respectively. We find that the ALD ZnO layer thickness can be controlled down to ~5nm, and the thickness scaled directly with the number of ALD cycles, as observed by TEM. Thicker coatings inhibited MWCNT aggregation, and sonication allowed us to induce fiber fragmentation. In this way the ALD coating allowed us to independently adjust surface termination, fiber aggregation, and fiber aspect ratio, providing us a unique tool to examine how each of these factors influences cellular response. Initial results show that the ZnO coating significantly increased TGF-β1 mRNA expression and stimulated a larger pro-fibrogenic response in HLFs compared to uncoated MWCNTs. Control experiments using ZnO nanoparticles also showed potent induction of TGF-β1 mRNA in HLFs. Also, the response tends to correlate with extent of dispersion, and is nearly independent of MWCNT aspect ratio. These experiments show that nanoscale surface functionalization of nanoscale materials may help us gain better understanding of the mechanisms associated with toxicology of nanomaterials, and expand knowledge of biological response at nano/bio interfaces.

Ultrasound (US) is a common medical imaging modality due to its flexibility, low-cost and therapeutic potential. 500 nm silica nanoshells were synthesized as a contrast agent to improve US imaging signal for better diagnostic performance. Iron (III) was included into the silica network to enhance the biodegradability of the silica nanoshells. Previously, ferric iron was shown to facilitate silica nanoshell biodegradation due to its strong binding affinity with serum transferrin proteins. The removal of iron from the silica network by serum proteins fragments the nanoshells enabling effective biodegradation for in vivo applications. The silica nanoshells are filled with perfluorocarbon (PFC) vapor which expands and shatters the nanoshells during US irradiation. A mechanically weaker silica nanoshell increases US signal at lower power. A range of alkoxysilanes with selected R-groups such as long chain hydrocarbons, fluorinated carbon chains, fluorinated phenyl groups and vinyl groups were employed along with tetramethyl orthosilicate and iron (III) ethoxide in a modified sol-gel synthesis to create structural defects that alter the mechanical properties of the nanoshells. Monodispersed 500 nm polystyrene beads were used as a soft template during the reaction. The silica nanoparticles were calcined at 550 C to remove the polystyrene core and form hollow nanoshells. SEM and TEM showed that 500 nm silica nanoshells with different microstructures were synthesized incorporating alkoxysilanes with different R-groups. Formulations with higher concentrations of alkoxysilanes with large R-groups such as long chain hydrocarbons resulted in stronger in vitro contrast enhanced ultrasound (CEUS) signals due to the increase of structural voids that resulted in weaker shell strength. CEUS experiments demonstrated that mechanically weaker silica nanoshells exhibited longer signal life time and required a lower mechanical index (MI) for imaging. The high intensity focused ultrasound (HIFU) properties of the modified silica nanoshells were tested for potential therapeutic applications. Mechanically weaker silica nanoshells were shown in vitro to require a lower HIFU power to fracture which is consistent with safer HIFU therapy. By synthesizing strength tunable silica nanoshells as US contrast agents, it is possible to improve diagnostic US imaging performance in order to detect smaller tissue structures or early stage tumors. Additionally, mechanically weaker silica nanoshells may also increase the efficiency of HIFU enabling HIFU at lower US power and/or higher speed.

The wide-spread use of silver nanoparticles in consumer products raises questions of environmental impact and toxicity. Because both silver particles, and silver ions formed by particle dissolution, may impact biological systems, it is important to understand the characteristics of silver nanoparticles as they are made and their stability and dissolution in the medium relevant to environmental and toxicological studies. Silver nanoparticles produced by different synthesis routes can have significantly varying physical and chemical characteristics. In this talk we will summarize the characterization and dissolution stability of three types of silver nanoparticles (20 nm particles synthesized with and without gold core (~7 nm) and 110 nm particles with gold core) in cell culture media with serum proteins: FBS10%/RPMI, the culture media used at Pacific Northwest National Laboratory for in-vitro toxicity studies. These nanoparticles were synthesized and prepared for biological study in aqueous solution. They were examined in situ using dynamic light scattering, zeta potential measurements and optical adsorption and ex situ with x-ray photoelectron spectroscopy and transmission electron microscopy. For the dissolution studies, concentrations of particles examined were varied from 1 µg/ml to 50 µg/ml, consistent with the range of concentrations typically used during in-vitro studies. Silver particles with gold cores had smaller crystallite size and higher apparent solubility than three different batches of pure ~ 20 nm silver particles. A simple dissolution model was found to describe the time variation of particle size and amount of dissolved silver for particle loadings above 9 µg/ml. The effective solubility product obtained from fitting the data was higher for the 20 nm particles with the gold core in comparison to the pure silver or 110 nm particles. The dissolution of silver nanoparticles was also found to be enhanced by presence of serum proteins contained in fetal bovine serum (FBS). In addition, the protocol of dispersion in cell culture medium was found to influence particle agglomeration and the rate of dissolution. In these measurements focusing on a 24 hour time point, we found that the structure of the silver nanoparticles can have a significant impact on the concentration of dissolved silver in media and thus the dosimetry to which cells would be exposed during in vitro studies.

This work has been supported by the NIEHS under Center grant U19 ES019544. Portions of this work were performed using EMSL, a national scientific user facility sponsored by the US Department of Energy, Biological and Environmental Research and located at PNNL.

Diabetic, pressure, venous and arterial ulcers are a large social, economic and healthcare burden. These chronic non-healing wounds show delayed and incomplete healing processes exposing patients to high risk of infection. The design of wound dressings that combine the necessary morphological and physical requirements for wound healing with the value-added capability to address optimal cell responses and impair bacterial proliferation represents a major challenge in chronic wound care. Polymeric nanofibrous meshes fabricated through the electrospinning process are promising candidates as wound dressings due to their high surface area, micro-porosity and non-woven structure. In this study, the parameters of the electrospinning process (such as spinning rate and electric field intensity) were optimized to fabricate nanofibrous membrane in Polystyrene (M.W. 250.000). The morphological properties of the electrospun meshes were analysed by bright microscopy, three-dimensional optical profiler, Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Electrospun materials have been used as scaffolds for tissue engineering for a number of years, but there is surprisingly little literature on the interactions of fibres with bacteria. In order to understand microbial infiltration and control in wound dressings, a number of microbiological assays (MTT, MTS and live/dead) were completed using E. Coli, P. Aeruginosa, S. Aureus in an effort to understand how the morphological and structural properties of the electrospun meshes influence bacterial attachment, proliferation and growth.

We have investigated the field-extension of tethered DNA in nanoslits with slit heights ranging from 30 to 130 nm, and performed an analysis from an approximated modified worm-like chain (mWLC) field-extension relation. DNA molecules attached to microspheres were anchored at a micro-nanofluidic interface and the molecules electrophoretically extended. We demonstrated that both the DNA segmental correlation and equilibrium lengths increased as the slit height decreased. Furthermore, for extremely confined DNA where h≤30 nm, we observed reptation of the DNAs’ contours within the nanoslit, a phenomena that may be induced by inhomogeneous surface charge distributions. This nano-confined system may have implications for single-molecule sensors on detecting and analyzing genetic, epigenetic markers, and related nanobiotechnological applications.

DNA performs a carefully choreographed ballet during the cell cycle. The organization is driven by the specific binding of proteins to form tertiary DNA-protein-DNA complexes. The search process that precedes the formation must overcome the challenge of very low effective mobility of genomic-sized DNA pieces in the dense cellular environment.

In this paper we will discuss a group of nanofluidic device that force two DNA molecules to either slide past each other in parallel, or cross over each other at a steep angle. Nanochannel cross-sections are 100x100 nm², and are hundreds of microns long. Because DNA is elongated through confinement, loop with a length down to 2 kb can be directly observed in real time. Channels are made of fused silica, enabling single-molecule observation of both DNA and proteins. Because the effective concentration of DNA inside channels exceeds 1 mg/ml with the channel at the point of DNA-DNA contact, protein-mediated capture cross-sections are very high.

We will present analyses of different DNA-binding proteins that demonstrate that we can distinguish dense and sparse binding modes and the compensation of electrostatic DNA-DNA repulsion through protein binding. We further report the detection of long-lived tertiary complexes acting as a lock for looped DNA configurations, and the presence of very short-lived transient links. We further demonstrate a pathway for loop formation that is enhanced in nanochannel devices, and that may be important in a cellular context. By using precision hydrodynamic flows, we are able to measure free energies of the search process.