Nanovesicular Technologies

We are developing a comprehensive catalog of theralytic methodologies for NV isolation, characterization and quality control as well as for their tracking in vitro and in vivo. We are compiling a repertoire of quality control and theralytic technologies as a toolbox for comparative QC of nanovesicles from diverse sources, across kingdoms of life. Building on capabilities established in Salzburg within the EVTT project, the LBI NVPM is developing and consolidating a repertoire of advanced technologies for comprehensive routine characterization of NVs from diverse sources. The suitability for quality control in manufacturing and analytics is continuously evaluated, and technology transfer is performed where applicable. The catalogue includes methods for:

• Physicochemical characterization (particle size, number, zeta potential, stiffness, morphology) based on state-of-the-art technologies such as nanoparticle tracking analysis, zeta potential, cryo-transmission electron microscopy, as well as new developments using infrared, Raman, nuclear magnetic resonance, and circular dichroism (CD) spectroscopy.
• Determination of molecular composition, based on characterization of protein and RNA markers, development of new species-specific and generic detection tools (antibodies, aptamers), bulk characterization of selected markers by RT-qPCR, Western blotting, bead-based flow cytometry, chromatography with in-line probing, (semi-)quantitative omic profiling (proteome, lipidome, transcriptome, metabolome, glycome) using isotope-labeled internal standards.
• Single vesicle analysis: nano-flow cytometry, fluorescence correlation spectroscopy, high-resolution single vesicle fluorescence imaging.
• Assessment of functional integrity, including methods to assess protein functionality and cellular uptake.
• Development of surface chemistries for EV capture, and transfer of relevant assays into a microfluidics format to result in new chip-based prototypes for NV analytics.
• Quantitative assessment of endotoxin levels and development of novel strategies for endotoxin removal in NV therapeutics isolated from non-sterile edible sources.

Methods to track NVs in vivo are still prone to artifacts due to suboptimal labelling strategies and NV characterization methods. Building on know-how, assays, and tools generated in Salzburg within the EVTT program, we are also continuing our development of an extended repertoire of complementary technologies to quantitatively track NVs intracellular and in whole organisms down to the subcellular level:

• Fluorescence and luminescence labelling of extracellular esicles: advanced labelling strategies and EV-tracking in cells, whole organs by epifluorescence imaging, and tissue sections using quantitative histology.
• Strategies beyond fluorescence and luminescence-based tracking: Together with Adjunct PI1 Greetje Vande Velde at KU Leuven we expand vesicle labelling and tracking to methods suitable for clinical monitoring of NVTs including computer tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET)
• Label-free technologies for EV tracking and biodistribution analysis, including single-cell RNA sequencing, isotope-based protein labelling, and spatial transcriptomics. We further explore post-administration detection of EV components in tissue sections using advanced histological approaches such as RNAscope and species-specific antibody labelling.

The translation of nanovesicle (NV) and extracellular vesicle (EV)-based therapeutics into clinical applications requires robust and predictive preclinical models that capture biodistribution, target engagement, biological activity, and safety. Major knowledge gaps remain regarding the influence of EV source and administration routes on tissue distribution, the ability of EVs to cross physiological barriers, their interactions with the immune system, and the relationship between biodistribution and therapeutic efficacy. We are developing complementary preclinical models and functional readouts in in vivo and ex vivo cellular and organoid models:

• Epithelial barrier models: Caco2 epithelial barrier models with and without immune and mucus- producing cells, air liquid interphase respiratory tract, 3D transwell skin models and blood brain barrier models.
• Tracking of the subcellular fate of nanovesicles: Combining high resolution imaging together with advanced subcellular fractionation approaches (e.g. combined with isotope labelling or species specific identification of EV originating components)  and pull-down approaches.
• Functional readouts, measurement of endosomal escape and cargo quantification in cells, serum and tissue.

Open source image quantitation

To enable quantitative single-vesicle analysis and to track NVs down to the single-cell level, robust image quantification is essential. Starting in EVTT, the team has spent several years developing EVAnalyzer, an open access- open-source solution for quantification of images from EV research. The software provides automated, quantitative analysis of single-vesicle imaging data, cellular uptake experiments, and EV quantification in histological sections. Since its initial release in 2022, the tool has gained increasing recognition within the EV community and has been continuously refined and enhanced, culminating in the current stand-alone version, EVAnalyzer 2.0. Today, EVAnalyzer enables EV characterization at the single-vesicle level and supports quantitative analysis across a wide range of biological contexts, including cells, tissues, and serum—facilitating measurements such as tissue and serum pharmacokinetics (PKs). The software integrates classical signal processing techniques with machine learning and AI-based object detection methods for accurate segmentation and particle identification, complemented by an integrated database for efficient data handling and analysis.

Software and documentation are freely available through EVAnalyzer.org.