Biology subjects

Hook, F.

Publications and source records attributed to Hook, F..

4 recordsLinked to original sources

bioRxiv2026

SIZE DETERMINATION AND MULTIPLEXED FLUORESCENCE-BASED PHENOTYPING OF SINGLE CELL-DERIVED MEMBRANE VESICLES USING A NANOFLUIDIC DEVICE

Extracellular vesicles (EVs) are cell-secreted biological nanoparticles that play a crucial role in intercellular communication and are gaining increasing attention as diagnostic biomarkers, therapeutic agents, and drug delivery vehicles. Consequently, the development of robust and sensitive methods for their characterization is essential. Herein we present the use of a microscope-mounted nanofluidic device for direct size determination and multi-parametric (3-color) fluorescence-based phenotyping of single biological nanoparticles that are in the size range of 20-200 nm in a method we denote Nano-SMF (SMF; size and multiplexed fluorescence). We demonstrate that it is possible to accurately determine the size of nanoparticles by analyzing their one-dimensional Brownian motion during directional flow through nanochannels, achieving size distributions for monodisperse nanoparticle solutions that are on par with TEM analysis, and size discrimination of nanoparticle mixtures that is significantly improved compared to conventional nanoparticle tracking analysis (NTA). Furter, we demonstrate that the method can be applied to analyze EVs directly in minute volumes of cell supernatant, avoiding pre-isolation or concentration steps. The method was applied to phenotype CD63- and CD81-positive EVs from a human embryonic kidney cell model, demonstrating that vesicle sub-populations defined by these two tetraspanin biomarkers differ significantly in size.

biophysics↗

bioRxiv2024

Time Resolved Inspection of Ionizable-Lipid Facilitated Lipid Nanoparticle Disintegration and Cargo Release at an Endosomal Membrane Mimic

This study investigates pH-triggered fusion dynamics of lipid nanoparticles (LNPs) with an endosomal membrane mimic, addressing mechanistic aspects of a crucial yet elusive process for effective mRNA delivery. Utilizing time-resolved total internal reflection fluorescence (TIRF) imaging, we observed a delayed onset of LNP fusion upon pH drop, lasting seconds to minutes depending on pH and LNP size. Once initiated, LNP fusion and cargo escape occurred rapidly within tens to hundreds of milliseconds. While LNP disintegration is observed to lead to release of a significant portion of mRNA into the acidic environment, some mRNA molecules remained mobile on the endosomal membrane mimic due to deprotonation-resistant complex salt formation. Comparison of the fusion efficiency of two LNP formulations correlated with protein translation in human primary cell transfection data, emphasizing the importance of biophysical investigations in understanding ionizable-lipid-containing LNP-assisted mRNA delivery mechanisms and providing insights for optimizing mRNA-LNP design for enhanced endosomal escape.

biophysics↗

bioRxiv2021

Single-molecule Dynamic In-Solution Inhibition Assay: A Method for Full Kinetic Profiling of Drug Candidate Binding to GPCRs in Native Membranes

Kinetic profiling of drug-target interactions using surface-based label-free technologies is well established for water-soluble pharmaceutical targets but is difficult to execute for membrane proteins in general and G-protein-coupled receptors (GPCRs) in particular. That is because surface immobilization of GPCRs tends to alter their configuration and function, leading to low target coverage and non-specific binding. We here describe a novel assay for kinetic profiling of drug binding to the GPCR human beta 2 adrenergic receptor ({beta}2AR). The assay involves temporally-resolved imaging of the binding of individual {beta}2AR-containing cell membrane-derived liposomes to a surface-immobilized ligand in the presence of screened drugs. This approach allowed to determine association and dissociation constants of {beta}2AR and suspended alprenolol (antagonist) and fenoterol (agonist). The set-up combines a 384 well-plate sensor chip with automated liquid handling and the assay takes minutes to complete, making it well adapted for drug screening campaigns.

biophysics↗

bioRxiv2021

Time evolution of PEG-shedding and serum protein coronation determines the cell uptake kinetics and delivery of lipid nanoparticle formulated mRNA

Development of efficient lipid nanoparticle (LNP) vectors remains a major challenge towards broad clinical translation of RNA therapeutics. New lipids will be required, but also better understanding LNP interactions with the biological environment. Herein, we model protein corona formation on PEG-ylated DLin-MC3-DMA LNPs and identify time-dependent maturation steps that critically unlock their cellular uptake and mRNA delivery. Uptake requires active serum proteins and precedes after a significant ([~]2 hours) lag-time, which we show can be eliminated by pre-incubating LNPs for 3-4 hours in serum-containing media. This indicates an important role of protein corona maturation for the pharmacokinetic effects of these LNPs. We show, using single-nanoparticle imaging, NMR diffusometry, SANS, and proteomics, that the LNPs, upon serum exposure, undergo rapid PEG-shedding ([~]30 minutes), followed by a slower rearrangement of the adsorbed protein layer. The PEG-shedding coincides in time with high surface abundance of Apolipoprotein A-II, whereas the LNPs preferentially bind Apolipoprotein E when their maximum uptake-competent state is reached. Finally, we show that pre-incubation of the LNPs enables rapid uptake and allows pulse-chase video-microscopy colocalization experiments with sufficiently short pulse durations to gain improved mechanistic understanding of how intracellular trafficking events determine delivery efficacy, emphasizing early endosomes as important delivery-mediating compartments.

biophysics↗