multimodal-analytical-platform-on-multiplexed-surface-plasmon

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Université de Bourgogne Franche-Comté

FEMTO-ST Institute

Blood microparticles (MPs) are small membrane vesicles (50-1000nm), derived from different cell types. They are known to play important roles in various biological processes and also recognized as potential biomarkers of various health disorders. Different methods are currently used for the detection and characterization of MPs, but none of these methods is capable to quantify and qualify total MPs at the same time, hence, there is a need to develop a new approach for simultaneous detection, characterization and quantification of microparticles. Here we show the potential of surface plasmon resonance (SPR) method coupled to atomic force microscopy (AFM) to quantify and qualify platelet-derived microparticles (PMPs), on the whole nano-to micro-meter scale. The different subpopulations of microparticles could be determined via their capture onto the surface using specific ligands. In order to verify the correlation between the capture level and the microparticles concentration in solution, two calibration standards were used: Virus-Like Particles (VLPs) and synthetic beads with a mean diameter of 53nm and 920nm respectively. The AFM analysis of the biochip surface allowed metrological analysis of captured PMPs and revealed that more than 95% of PMPs were smaller than 300nm. Our results suggest that our NanoBioAnalytical platform, combining SPR and AFM, is a suitable method for a sensitive, reproducible, label-free characterization and quantification of MPs over a wide concentration range (≈10 to 10 particles/mL; with a limit of detection (LOD) in the lowest ng/µL range) which matches with their typical concentrations in blood.

Development of a NanoBioAnalytical platform for "on-chip" qualification and quantification of platelet-derived microparticles.

Plasma transfusion induces some transfusion related acute lung injury (TRALI) mediated through neutrophil extracellular traps (NETs). We investigated whether extracellular vesicles (EVs) present in plasma or obtained from resting (N-PEVs) or thrombin activated platelets (T-PEVs) can trigger NETs, and whether 75 nm-nanofiltration, to partially remove EVs, prohibits NETs formation. EVs size and concentration were determined by conventional biophysical approaches and by an original NanoBioAnalytical (NBA) platform based on EV immunocapture biochip, combining Surface Plasmon Resonance Imaging (SPRi) and Atomic Force Microscopy (AFM) exploration. EVs effective diameter was in the 25-1000 nm range, with a majority (≈ 90%) ≤ 100 nm. Both T-PEVs in buffer (but not N-PEVs) and non-nanofiltered plasma containing T-PEVs triggered NETs formation. Nanofiltration depleted large EVs (> 70 nm) and decreased NETs formation. The NBA platform was found to be a suitable tool to investigate the safety of plasma for transfusion.

NanoBioAnalytical characterization of extracellular vesicles in 75-nm nanofiltered human plasma for transfusion: A tool to improve transfusion safety.

Extracellular vesicles (EV) are emergent therapeutic effectors that have reached clinical trial investigation. To translate EV-based therapeutic to clinic, the challenge is to demonstrate quality, safety, and efficacy, as required for any medicinal product. EV research translation into medicinal products is an exciting and challenging perspective. Recent papers, provide important guidance on regulatory aspects of pharmaceutical development, defining EVs for therapeutic applications and critical considerations for the development of potency tests. In addition, the ISEV Task Force on Regulatory Affairs and Clinical Use of EV-based Therapeutics as well as the Exosomes Committee from the ISCT are expected to contribute in an active way to the development of EV-based medicinal products by providing update on the scientific progress in EVs field, information to patients and expert resource network for regulatory bodies. The contribution of our work group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France", created in 2020, can be positioned in complement to all these important initiatives. Based on complementary scientific, technical, and medical expertise, we provide EV-specific recommendations for manufacturing, quality control, analytics, non-clinical development, and clinical trials, according to current European legislation. We especially focus on early phase clinical trials concerning immediate needs in the field. The main contents of the investigational medicinal product dossier, marketing authorization applications, and critical guideline information are outlined for the transition from research to clinical development and ultimate market authorization.

Development of extracellular vesicle-based medicinal products: A position paper of the group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France".

Extracellular vesicles (EVs) originate from eukaryotic and prokaryotic cells and play a crucial role in intercellular communications. They are found in the environment of cells and tissues, and contribute to the complexity of different biological media, in particular biofluids. Due to their high diversity of cell origin, size range, concentration and composition, EVs offer some of the most important challenges in (pre-)analytical fields. To tackle these challenges, many works deal with the development and implementation of a wide variety of approaches, technologies and methodologies to enrich, isolate, quantify and characterize EVs and their subsets. Nevertheless, other components such as lipoproteins or viruses in complex samples, can interfere with EVs qualification, and make difficult, even today, to standardize biochemical and physical approaches for this purpose. The present chapter presents EVs and the mostly used technics for their isolation and characterization. Performances of methods in terms of resolution, discrimination, throughput and also ability to be or not applied in clinics, are also discussed.

[Extracellular vesicles: Definition, isolation and characterization].

For the last 30 years, molecular surveys have shown that human norovirus (HuNoV), predominantly the GII.4 genotype, is one of the main causative agents of gastroenteritis. However, epidemiological surveys have revealed the worldwide emergence of GII.17 HuNoVs. Genetic analysis confirmed that GII.17 strains are distributed into three variants (i.e., Kawasaki 308, Kawasaki 323, and CS-E1). Here, virus-like particles (VLPs) were baculovirus-expressed from these variants to study putative interactions with HBGA. Qualitative analysis of the HBGA binding profile of each variant showed that the most recent and predominant GII.17 variant, Kawasaki 308, possesses a larger binding spectrum. The retrospective study of GII.17 strains documented before the emergence of the dominant Kawasaki 308 variant showed that the emergence of a new GII.17 variant could be related to an increased binding capacity toward HBGA. The use of duodenal histological sections confirmed that recognition of enterocytes involved HBGA for the three GII.17 variants. Finally, we observed that the relative affinity of recent GII.17 VLPs for HBGA remains lower than that of the GII.4-2012 variant. These observations suggest a model whereby a combination of virological factors, such as polymerase fidelity and increased affinity for HBGA, and immunological factors was responsible for the incomplete and non-persistent replacement of GII.4 by new GII.17 variants.

Epidemiological Impact of GII.17 Human Noroviruses Associated With Attachment to Enterocytes.

Breast cancer is one of the leading causes of cancer-related death among females worldwide. A major challenge is to develop innovative therapy in order to treat breast cancer subtypes resistant to current treatment. In the present study, we examined the effects of two Troglitazone derivatives Δ2-TGZ and AB186. Previous studies showed that both compounds induce apoptosis, nevertheless AB186 was a more potent agent. The kinetic of cellular events was investigated by real-time cell analysis system (RTCA) in MCF-7 (hormone dependent) and MDA-MB-231 (triple negative) breast cancer (TNBC) cells, followed by cell morphology analysis by immuno-localization. Both compounds induced a rapid modification of both impedance-based signals and cellular morphology. This process was associated with an inhibition of cell migration measured by wound healing and transwell assays in TNBC MDA-MB-231 and Hs578T cells. In order to identify cytoplasmic targets of AB186, we performed surface plasmon resonance (SPR) and pull-down analyses. Subsequently, 6 cytoskeleton components were identified as potential targets. We further validated α-tubulin as one of the direct targets of AB186. In conclusion, our results suggested that AB186 could be promising to develop novel therapeutic strategies to treat aggressive forms of breast cancer such as TNBC.

Wilfrid Boireau

FEMTO-ST Institute,
CNRS UMR-6174

Université de Bourgogne Franche-Comté

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Wilfrid Boireau

FEMTO-ST Institute,CNRS UMR-6174

Université de Bourgogne Franche-Comté

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

FEMTO-ST Institute,
CNRS UMR-6174