Meade, T. J. | Search bioRxiv

bioRxiv2023

HaloTag display enables quantitative single-particle characterization and functionalization of engineered extracellular vesicles

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells, and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity, and other properties. Measuring how incorporation varies across a population of EVs is important for characterizing such materials and understanding their function, yet it remains challenging to quantitatively characterize the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterization platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labeling to antibody-labeling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labeling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterization tool which complements and expands existing methods.

bioengineering↗

bioRxiv2020

Cobalt(III) Schiff Base complexes stabilize non-fibrillar amyloid-β aggregates with reduced toxicity

The aggregation of A{beta} is believed to be foundational to the pathogenesis of Alzheimers disease (AD). In vitro aggregation kinetics have been shown to correlate with rates of disease progression in both AD patients and animal models, thus proving to be a useful metric for testing A{beta}-targeted therapeutics. Here we present evidence of Cobalt(III) Schiff base complex (Co(III)-sb) modulation of A{beta} aggregation kinetics by a variety of complementary techniques. These include Thioflavin T (ThT) fluorescence, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). Our data was fitted to kinetic rate laws using a mathematical model developed by Knowles et al. in order to extract mechanistic information about the effect of Co(III)-sb on aggregation kinetics. Our analysis revealed that Co(III)-sb significantly decreases the kinetic parameter k+, and significantly increases the polymerization rate kn, suggesting that Co(III)-sb causes A{beta} to rapidly form stable oligomeric species that are unable to elongate into mature fibrils. This result was corroborated by TEM and AFM of A{beta} aggregates in vitro. We also demonstrate that A{beta} aggregate mixtures produced in the presence of Co(III)-sb exhibit decreased cytotoxicity compared to untreated samples. Statement of SignificanceAmyloid-{beta} is thought to be a key mediator in the pathology of Alzheimers disease, yet its precise mechanisms of toxicity are poorly understood. The interaction of A{beta} with endogenous metal ions via its N terminal Histidine residues has been shown to alter the peptides aggregation and toxicity. As such, metal-based complexes have been developed both as therapeutic agents as well as tools for investigating the role of metal binding in the pathogenesis of AD. This work expands on our previous studies developing Cobalt(III) Schiff base complexes as amyloid inhibitors. Here we demonstrate effective inhibition of aggregation by various complementary modalities. Additionally we show that Co(III)-sb reduces the toxicity of A{beta} aggregates to cells in culture.

neuroscience↗