Complementary Nanoparticle Characterization by Resistive-Pulse Sensing, Electron Microscopy, and Charge Detection Mass Spectrometry.

Nanotechnology has provided novel modalities for the delivery of therapeutic and diagnostic agents. In particular, nanoparticles (NPs) can be engineered at a low cost for drug loading and delivery. For example, silica NPs have proven useful as a controlled release platform for anti-inflammatory drugs. Despite the wide-ranging potential applications for NPs, robust characterization across all size ranges remains elusive. Electron microscopy (EM) is the conventional tool for measuring NP diameters. However, imitations in throughput and the inability to provide comprehensive information on physical properties, such as mass and density, without underlying assumptions, hinder a complete analysis. In addition, assessing sample heterogeneity, aggregation, or coalescence in solution by traditional EM analysis is not possible. Resistive-pulse sensing (RPS) provides a high throughput, solution-phase method for characterizing particle heterogeneity based on volume. Complementing these methods, charge detection mass spectrometry (CD-MS), a single particle technique, provides accurate mass information for heterogeneous samples including NPs. By combining EM, RPS and CD-MS, accurate volume, mass, and densities were obtained for silica NPs of various sizes. The results show that the density for 20 nm silica NPs is close to the density of fused silica (2.2 g/cm3). Larger silica NPs were found to have densities that were either smaller or larger, while also falling outside the range of densities usually found for silica colloids and NPs (1.9-2.3 g/cm3). Lower densities are attributed to pores (i.e., porous particles). For one sample, the mass distribution showed two components attributed to two populations of particles in the sample with different densities. The synergistic combination of EM, RPS, and CD-MS measurements outlined here for NP samples, allows much more extensive information to be obtained than from any of the techniques alone.

Integrated In-Plane Nanofluidic Devices for Resistive-Pulse Sensing.

Single-particle (or digital) measurements enhance sensitivity (10- to 100-fold improvement) and uncover heterogeneity within a population (one event in 100 to 10,000). Many biological systems are significantly influenced by rare or infrequent events, and determining what species is present, in what quantity, and the role of that species is critically important to unraveling many questions. To develop these measurement systems, resistive-pulse sensing is used as a label-free, single-particle detection technique and can be combined with a range of functional elements, e.g., mixers, reactors, filters, separators, and pores. Virtually, any two-dimensional layout of the micro- and nanofluidic conduits can be envisioned, designed, and fabricated in the plane of the device. Multiple nanopores in series lead to higher-precision measurements of particle size, shape, and charge, and reactions coupled directly with the particle-size measurements improve temporal response. Moreover, other detection techniques, e.g., fluorescence, are highly compatible with the in-plane format. These integrated in-plane nanofluidic devices expand the toolbox of what is possible with single-particle measurements.

Characterization of Extracellular Vesicles by Resistive-Pulse Sensing on In-Plane Multipore Nanofluidic Devices.

Extracellular vesicles (EVs) are cell-derived, naturally produced, membrane-bound nanoscale particles that are linked to cell-cell communication and the propagation of diseases. Here, we report the design and testing of in-plane nanofluidic devices for resistive-pulse measurements of EVs derived from bovine milk and human breast cancer cells. The devices were fabricated in plane with three nanopores in series to determine the particle volume and diameter, two pore-to-pore regions to measure the electrophoretic mobility and zeta potential, and an in-line filter to prevent cellular debris and aggregates from entering the nanopore region. Devices were tested with and without the channels coated with a short-chain PEG silane to minimize electroosmotic flow and permit an accurate measurement of the electrophoretic mobility and zeta potential of the EVs. To enhance throughput of EVs, vacuum was applied to the waste reservoir to increase particle frequencies up to 1000 min-1. The nanopores had cross-sections 200 nm wide and 200 nm deep and easily resolved EV diameters from 60 to 160 nm. EVs from bovine milk and human breast cancer cells had similar particle size distributions, but their zeta potentials differed by 2-fold, -8 ± 1 and -4 ± 1 mV, respectively.

TFF3 interacts with LINGO2 to regulate EGFR activation for protection against colitis and gastrointestinal helminths.

Intestinal epithelial cells (IEC) have important functions in nutrient absorption, barrier integrity, regeneration, pathogen-sensing, and mucus secretion. Goblet cells are a specialized cell type of IEC that secrete Trefoil factor 3 (TFF3) to regulate mucus viscosity and wound healing, but whether TFF3-responsiveness requires a receptor is unclear. Here, we show that leucine rich repeat receptor and nogo-interacting protein 2 (LINGO2) is essential for TFF3-mediated functions. LINGO2 immunoprecipitates with TFF3, co-localizes with TFF3 on the cell membrane of IEC, and allows TFF3 to block apoptosis. We further show that TFF3-LINGO2 interactions disrupt EGFR-LINGO2 complexes resulting in enhanced EGFR signaling. Excessive basal EGFR activation in Lingo2 deficient mice increases disease severity during colitis and augments immunity against helminth infection. Conversely, TFF3 deficiency reduces helminth immunity. Thus, TFF3-LINGO2 interactions de-repress inhibitory LINGO2-EGFR complexes, allowing TFF3 to drive wound healing and immunity.

Yeast cell wall supplementation alters the metabolic responses of crossbred heifers to an endotoxin challenge.

This study examined the effect of feeding yeast cell wall (YCW) products on the metabolic responses of newly-received feedlot cattle to an endotoxin challenge. Heifers were separated into treatment groups receiving either a Control diet, YCW-A or YCW-C, and were fed for 52 d. Heifers were weighed on d 0, 14, 36, 38 and 52. On d 37 heifers were challenged i.v. with LPS [0.5 µg/kg body weight (BW)] and blood samples were collected relative to LPS challenge. Heifer BW increased from d 0 to 36 and from d 38 to 52, but was not affected by treatment. Post-LPS, glucose concentrations increased and were less in YCW-A than Control and YCW-C heifers. Pre-LPS, insulin concentrations were greater in YCW-A and YCW-C than Control heifers. Post-LPS, insulin concentrations increased with YCW-C having greater insulin than Control heifers. Pre-LPS, NEFA concentrations tended to be less in YCW-C than Control heifers. Post-LPS non-esterified fatty acids (NEFA) concentrations were less in YCW-C than Control and YCW-A heifers. Post-LPS, blood urea nitrogen (BUN) concentrations were greater in YCW-A than Control and YCW-C. These data indicate, based on NEFA and BUN data, that certain YCW products can enhance energy metabolism during an immune challenge without causing lipolysis or muscle catabolism.

Yeast cell wall supplementation alters aspects of the physiological and acute phase responses of crossbred heifers to an endotoxin challenge.

A study was conducted to determine the effect of feeding yeast cell wall (YCW) products on the physiological and acute phase responses of crossbred, newly-received feedlot heifers to an endotoxin challenge. Heifers (n = 24; 219 ± 2.4 kg) were separated into treatment groups receiving either a control diet (n = 8), YCW-A (2.5 g/heifer/d; n = 8) or YCW-C (2.5 g/heifer/d; n = 8) and were fed for 52 d. On d 37 heifers were challenged i.v. with LPS (0.5 µg/kg body mass) and blood samples were collected from -2 h to 8 h and again at 24 h relative to LPS challenge. There was an increase in vaginal temperature in all heifers post-LPS, with YCW-C maintaining a lower vaginal temperature post-LPS than control and YCW-A heifers. Sickness behavior scores increased post-LPS in all heifers, but were not affected by treatment. Cortisol concentrations were greatest in control heifers post-LPS compared with YCW-A or YCW-C heifers. Concentrations of IFN-γ and TNF-α increased post-LPS, but were not affected by treatment. Serum IL-6 concentrations increased post-LPS and were greater in control heifers than YCW-A and YCW-C heifers. These data indicate that YCW supplementation can decrease the physiological and acute phase responses of newly-received heifers following an endotoxin challenge.