Nanovesicular technologies

In program line 3, we are developing a comprehensive catalog of theralytic methodologies for NV characterization and quality control, their tracking in vivo, and for biomarker discovery. 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 catalog 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, and atomic force 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 [Reference: Corso et al., JEV 2019], high-resolution single vesicle fluorescence imaging [Reference: Schürz M et al., JEV 2022].
• Assessment of functional integrity, including methods to assess protein folding, biochemical activity assays in situ, assays for in situ receptor-ligand interactions
• 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.

Methods to track NVs in vivo are still prone to artifacts due to suboptimal labeling 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 in whole organisms down to the subcellular level. We initially focus on technologies for preclinical studies in rodents but gradually move to larger animals (Adjunct PI2) and eventually non-invasive and well-tolerated methods suitable for clinical monitoring of the NVTx. We further investigate potential biomarkers for monitoring and predicting the therapeutic benefit of NV precision medicines side by side with their development in programs 1 and 2. With the overall aim of the LBI NVPM to increase patients’ quality of life, we focus on minimally invasive strategies using easily accessible body fluids such as urine, saliva, and blood/serum.