Understanding the Adsorption of Peptides and Proteins onto PEGylated Gold Nanoparticles.

Polyethylene glycol (PEG) surface conjugations are widely employed to render passivating properties to nanoparticles in biological applications. The benefits of surface passivation by PEG are reduced protein adsorption, diminished non-specific interactions, and improvement in pharmacokinetics. However, the limitations of PEG passivation remain an active area of research, and recent examples from the literature demonstrate how PEG passivation can fail. Here, we study the adsorption amount of biomolecules to PEGylated gold nanoparticles (AuNPs), focusing on how different protein properties influence binding. The AuNPs are PEGylated with three different sizes of conjugated PEG chains, and we examine interactions with proteins of different sizes, charges, and surface cysteine content. The experiments are carried out in vitro at physiologically relevant timescales to obtain the adsorption amounts and rates of each biomolecule on AuNP-PEGs of varying compositions. Our findings are relevant in understanding how protein size and the surface cysteine content affect binding, and our work reveals that cysteine residues can dramatically increase adsorption rates on PEGylated AuNPs. Moreover, shorter chain PEG molecules passivate the AuNP surface more effectively against all protein types.

Using NMR Spectroscopy To Measure Protein Binding Capacity on Gold Nanoparticles.

A simple one-dimensional 1H NMR experiment that quantifies protein bound to gold nanoparticles has been developed for upper-division biochemistry and physical chemistry students. This laboratory experiment teaches the basics of NMR techniques, which is a highly effective tool in protein studies and supports students to understand the concepts of NMR spectroscopy and nanoparticle-protein interactions. Understanding the interactions of gold nanoparticles (AuNPs) with biological macromolecules is becoming increasingly important as interest in the clinical use of nanoparticles has been on the rise. Applications in drug delivery, biosensing, diagnostics, and enhanced imaging are all tangible possibilities with a better understanding of AuNP-protein interactions. The ability to use AuNPs as biosensors for drug delivery methods in cellular uptake is dependent on the amount of protein that is able to bind to the surface of the nanoparticle. This laboratory experiment solidifies concepts such as quantitative NMR spectroscopy while reinforcing precision laboratory titrations. Students learn how 1H proton NMR spectra can be used to measure free protein in solution and protein bound to AuNPs. A simple formula is used to determine the binding capacity of the nanoparticle. This analysis helps students to understand the impact of nanoparticle-protein interactions, and it allows them to conceptualize macromolecular binding using NMR spectroscopy.

A single-vector intersectional AAV strategy for interrogating cellular diversity and brain function.

As discovery of cellular diversity in the brain accelerates, so does the need for tools that target cells based on multiple features. Here we developed Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), an adeno-associated virus-based, single-construct, intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches to deliver molecular cargo to specific neuronal subtypes. ConVERGD offers benefits over existing intersectional expression platforms, such as expanded intersectional targeting with up to five recombinase-based features, accommodation of larger and more complex payloads and a vector that is easy to modify for rapid toolkit expansion. In the present report we employed ConVERGD to characterize an unexplored subpopulation of norepinephrine (NE)-producing neurons within the rodent locus coeruleus that co-express the endogenous opioid gene prodynorphin (Pdyn). These studies showcase ConVERGD as a versatile tool for targeting diverse cell types and reveal Pdyn-expressing NE+ locus coeruleus neurons as a small neuronal subpopulation capable of driving anxiogenic behavioral responses in rodents.

A Novel Single Vector Intersectional AAV Strategy for Interrogating Cellular Diversity and Brain Function.

As the discovery of cellular diversity in the brain accelerates, so does the need for functional tools that target cells based on multiple features, such as gene expression and projection target. By selectively driving recombinase expression in a feature-specific manner, one can utilize intersectional strategies to conditionally promote payload expression only where multiple features overlap. We developed Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), a single-construct intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches. ConVERGD offers benefits over existing platforms, such as expanded intersectionality, the ability to accommodate larger and more complex payloads, and a vector design that is easily modified to better facilitate rapid toolkit expansion. To demonstrate its utility for interrogating neural circuitry, we employed ConVERGD to target an unexplored subpopulation of norepinephrine (NE)-producing neurons within the rodent locus coeruleus (LC) identified via single-cell transcriptomic profiling to co-express the stress-related endogenous opioid gene prodynorphin (Pdyn). These studies showcase ConVERGD as a versatile tool for targeting diverse cell types and reveal Pdyn-expressing NE+ LC neurons as a small neuronal subpopulation capable of driving anxiogenic behavioral responses in rodents.