Biology subjects

Alexandre, L.

Publications and source records attributed to Alexandre, L..

3 recordsLinked to original sources

bioRxiv2024

In situ investigation of extracellular vesicles in viscous formulations: interplay of nanoparticle transport and nanorheology through interferometric light microscopy analysis

While extracellular vesicles (EVs) demonstrate growing potential as innovative therapeutics in diverse medical context (cancer, regenerative medicine, etc.) or as naturally circulant diagnostic / prognostic probes, their physical properties (size, transport, etc.) remains a critical concern. Here, we introduce a pipeline that relies on interferometric light microscopy (ILM) for measuring not only nanoparticle concentration and size distribution but also for analyzing the interactions of these nanoparticles with their environment. The analysis of interference patterns allows for the physical characterization of (bio)nanoparticles not only in aqueous solutions but also in challenging media with relatively high viscosity, particularly pertinent for characterizing gel-based EV-delivery systems. Through exploration of the instruments functionality and the use of calibrated NPs of various known sizes, we successfully obtained information about the local viscosity characteristics of a complex fluid embedding EVs. We present a proof-of-concept for characterizing EVs suspended in unconventional media and their interactions with their surroundings. Leveraging the outcomes of this investigation, we not only highlight the advantages of using ILM for characterizing EVs in complex fluid, particularly pertinent for the development of optimized biological carriers for targeted drug delivery and therapeutic applications, but we also validate a new method for measuring viscosity at the nanoscale.

biophysics↗

bioRxiv2024

Magnetic Microtweezers for High-Throughput Bioseparation in Sub-Nanoliter Droplets

Multiomics studies at single-cell level require small volume manipulation, high throughput analysis and multiplexed detection, characteristics that droplet microfluidics can tackle. However, the initial step of molecules bioseparation remains challenging. Here, we describe a unique magnetic device to trap and extract magnetic particles in sub-nanoliter droplets, for compartmentalisation of detection steps. Relying on electrodeposition of NiFe structures and microfluidic manipulation, this technology has allowed the purification of genetic material at single-cell scale and was able to reach an extraction rate of 72% for a sample of purified oligonucleotides.

bioengineering↗

bioRxiv2023

Effect of sample preprocessing and extraction methods on the physical and molecular profiles of extracellular vesicles

Extracellular vesicles (EVs) are nanometric lipid vesicles that shuttle cargo between cells. Their analysis could shed light on health and disease conditions, but EVs must first be preserved, extracted and often pre-concentrated. Here we firstly compare plasma preservation agents, and secondly, using both plasma and cell supernatant, four EV-extraction methods including (i) ultracen-trifugation (UC), (ii) size exclusion chromatography (SEC), (iii) centrifugal filtration (LoDF), and (iv) accousto-sorting (AcS). We benchmarked them by characterizing integrity, size-distribution, concentration, purity and the expression profiles for nine proteins of EVs, as well as overall throughput, time-to-result and cost. We found that the difference between EDTA and citrate anticoagulants vary with the extraction method. In our hands, ultracentrifugation produced a high yield of EVs with low contamination; SEC is low-cost, fast, and easy to implement, but the purity of EVs is lower; LoDF and AcS are both compatible with process automation, small volume requirement, and rapid processing times. When using plasma, the LoDF was susceptible to clogging and sample contamination, while the AcS featured high purity but a lower yield of extraction. Analysis of protein profiles suggest that extraction methods extract different sub-population of EVs. Our study highlights the strength and weakness of sample preprocessing methods, and the variability in concentration, purity, and EV expression profiles of the extracted EVs. Pre-analytical parameters such as collection or pre-processing protocols must be considered as part of the entire process in order to address EV diversity and their use as clinically actionable indicators.

bioengineering↗