RICHIESTA DI ASSEGNO DI COLLABORAZIONE ALLA RICERCA

RESEARCH PROJECT

TITLE: Fractionation and characterization of nanovesicles from complex biosamples

During their lifecycle, cells release a plethora of vesicles in the extracellular matrix, both spontaneously and under stimulation. In particular, the vesicles with a diameter between 30 and 100nm (exosomes) are secreted by many cell types following the joining processes between endo/lysosomes and the plasmatic membrane

However, it is yet uncertain the exact biological function of exosomes, which is also cell-line dependent: amongst the studied functions we find intercellular communication, the exchange of membrane matter between cells, immunomodulation over target cells (both as antigens and as carriers of proteins, DNA, RNA and fragments).

The complexity or micro- and nanovesicles, and their versatiliy in the biological environment, make for a challenging research topic on many aspects: characterisation, quantification, chemical composition and biological effect; moreover, the compatibility with biological systems make these vesicles a good candidate for theranostic and therapeutic applications.

One of the main issues regarding micro- and nanovesicle characterisation though is their prior separation from the extracellular matrix; another is the lack of effective and fast methods to define their size distribution. Vesicle isolation is a long process which often causes the loss of a high percentage of the sample; sequencial ultracentrifugation, density gradient centrifugation, and flow cytometry show many disadvantages and drawbacks both in terms of cost and performance.

The use of a flow field-fractionation technique, and in particular the hollow fiber version (HF5), can provide 1. Sorting, 2. Quantificationvia Number Density, 3. Size Distribution e 4. Morphological characterisation.

The most recent projects involving this research group regard the exploitability if HF5 coupled to a multidetection system (UV, FLD, dRI, MALS) during the various steps od micro- and nanovesicles isolation, fractionation and biochemical characterisation.This can be done starting from a complex sample (e.g. cell culture medium), or from refined samples (already pre-centrifuged), developing a second-dimensional separation. Another potential use of this separation platform is the separation/detection of specific exosomal populations through the usage of markers: the current procedures are impractical, given tehe impossibility to wash out the unlinked tag.

The collaborations benefiting this project are inclusive of:

- Collaboration signed between the Chemistry Department and byFlow srl for the development of custom FFF methods and the sharing or research and technology development activities

- Research collaboration between this Department and the Department of biomolecular sciences (Uniurb), to study exosomes and microvesicles derived from muscle and neural cells.

- Collaboration between this Group and the department of Medicina Specialistica, Diagnostica e Sperimentale, to study vesicular subpopulations derived from in vitro cell cultures.

RICHIESTA DI ASSEGNO DI COLLABORAZIONE ALLA RICERCA

WORKPLAN

TITLE: Integration of analytical platforms based on FlFFF and miniaturized-FlFFF for sorting and characterization of subcellular vescicles and particles

The research activity will be focused on the instrumental and methodological development of the separation techniques and their coupling to various apt detectors, to characterize in terms of dimensions and morphology micro and nanovesicles obtained from the ECM, starting both from coplex and pre-centrifuged samples.

These methods will be based on HF5, coupled to UV absoprtion and fluorescence, fotoluminescence, differential refractive index and multi angle laser scattering (UV-FL-RI-MALS).

This platform will provide a further sorting step and particle purification, and can potentially trade in with the longer and less performing procedures as of now required for sample preparation.

Targets

1)Study of the potential application of the HF5-based platform as a post-centrifugation step or instead of centrifugation

2)Development and optimisation of HF5 methods to characterise vesicular subpopulations

3)Development of an in-flow reactor for functional characterisation of nano and microvesicles

References

1) Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates, Tanase, M et al. nature Protocols

2) Hollow-fiber flow field-flow fractionation with multi-angle laser scattering detection for aggregation studies of therapeutic proteins

P Reschiglian, et al.Analytical and bioanalytical chemistry

3) Hollow fiber flow field-flow fractionation and size-exclusion chromatography with multi-angle light scattering detection: A complementary approach in biopharmaceutical industry

V Marassi, et al. Journal of Chromatography A

4) Hollow-fiber flow field-flow fractionation and multi-angle light scattering as a new analytical solution for quality control in pharmaceutical nanotechnology

V Marassi, et al. Microchemical Journal. In press.