Kurt H. Jacobson

Gastrointestinal biodurability of engineered nanoparticles: Development of an in vitro assay

The toxicity of engineered nanoparticles is expected to depend in part on their stability in biological systems. To assess the biodurability of engineered nanomaterials in the human digestive system, we adapted an in vitro assay previously used to evaluate the bioaccessibility of metals in contaminated soils. The compositions of the simulated gastric and intestinal fluids, temperature and residence times were designed to closely mimic conditions in the stomach and duodenum of the small intestine. We demonstrated the utility of the assay using CdSecore/ZnSshell quantum dots functionalized with polyethylene glycol (PEG) thiol of two different molecular masses (PEG350 and PEG5000). Under gastric conditions, removal of the PEG ligand diminished the stability of PEG350-quantum dot suspensions, while PEG5000-quantum dots were severely degraded. Inclusion of the glycoprotein mucin, but not the digestive protein pepsin, in simulated gastric fluids provided both PEG350- and PEG5000-coated quantum dots partial protection from transformations induced by gastric conditions.

Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes

Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations and assessment of the potential implications of nanoparticle release into the environment requires understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate the electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the outer leaflet-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed the electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. The association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.

Elongation Kinetics of Polyglutamine Peptide Fibrils: A Quartz Crystal Microbalance with Dissipation Study

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, including Huntingtons disease. Expansion of the polyglutamine (polyQ) domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. Studies of synthetic polyQ peptides have contributed substantially to our understanding of the mechanism of aggregation. In this report, polyQ fibrils were immobilized onto a sensor, and their elongation by polyQ peptides of various length and conformation was examined using quartz crystal microbalance with dissipation monitoring (QCM-D). The rate of elongation increased as the peptide length increased from 8 to 24 glutamines (Q8, Q20, and Q24). Monomer conformation affected elongation rates: insertion of a β-turn template d-Pro-Gly in the center of the peptide increased elongation rates several-fold, while insertion of Pro-Pro dramatically slowed elongation. Dissipation measurements of the QCM-D provided qualitative information about mechanical properties of the elongating fibrils. These data showed clear differences in the characteristics of the elongating aggregates, depending on the specific identity of the associating polyQ peptide. Elongation rates were sensitive to the pH and ionic strength of the buffer. Comparison of QCM-D data with those obtained by optical waveguide lightmode spectroscopy revealed that very little water was associated with the elongation of fibrils by the peptide containing d-Pro-Gly, but a significant amount of water was associated when the fibrils were elongated by Q20. Together, the data indicate that elongation of polyQ fibrils can occur without full consolidation to the fibril structure, resulting in variations to the aggregate structure during elongation.

Attachment of pathogenic prion protein to model oxide surfaces.

Prions are the infectious agents in the class of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies, which affect humans, deer, sheep, and cattle. Prion diseases of deer and sheep can be transmitted via environmental routes, and soil is has been implicated in the transmission of these diseases. Interaction with soil particles is expected to govern the transport, bioavailability and persistence of prions in soil environments. A mechanistic understanding of prion interaction with soil components is critical for understanding the behavior of these proteins in the environment. Here, we report results of a study to investigate the interactions of prions with model oxide surfaces (Al2O3, SiO2) using quartz crystal microbalance with dissipation monitoring and optical waveguide light mode spectroscopy. The efficiency of prion attachment to Al2O3 and SiO2 depended strongly on pH and ionic strength in a manner consistent with electrostatic forces dominating interaction with these oxides. The presence of the N-terminal portion of the protein appeared to promote attachment to Al2O3 under globally electrostatically repulsive conditions. We evaluated the utility of recombinant prion protein as a surrogate for prions in attachment experiments and found that its behavior differed markedly from that of the infectious agent. Our findings suggest that prions would tend to associate with positively charged mineral surfaces in soils (e.g., iron and aluminum oxides).

Inter- and intra-observer repeatability of aortic annulus measurements on screening CT for transcatheter aortic valve replacement (TAVR): Implications for appropriate device sizing

OBJECTIVES To investigate intra- and inter-observer repeatability of aortic annulus CT measurements for transcatheter aortic valve replacement (TAVR) by readers with different levels of experience and evaluate the impact of different multi-reader paradigms to improve prosthesis sizing. METHODS 82 TAVR screening CTAs were evaluated twice by three raters with six (R1 = radiologist), three (R2 = 3D-laboratory technician) or zero (R3 = medical student) years of experience. Results were translated into hypothetical TAVR size recommendations. Intra- and inter-observer repeatability between single readers and three different multi-reader paradigms ([A]: two readers, [B]: three readers, or [C]: two readers + an optional third reader) were evaluated. RESULTS Intra-observer variability did not differ significantly (range: 50.1-67.8mm2). However, we found significant differences in mean inter-observer variance (p = 0.001). Multi-reader paradigms led to significantly increased precision (lower variability) for scenarios [B] and [C] (p = 0.03, p < 0.05). Compared to single readers, all multi-reader strategies clearly lowered the rate of discrepant device size categorization between repeated measurements (22-26% to 5-10%). CONCLUSIONS Aortic annulus CT measurements for TAVR are highly reproducible. Multi-reader strategies provide higher precision than evaluations from single readers with different levels of experience and could effectively be implemented with two readers and an optional third reader (Paradigm C) in a clinical setting.