DS0304 -

Coordination Complexes to Explore the Recognition of Phospholipids and to Develop a Membrane-active Probe – COCERP

Tracking extracellular microvesicles in biological fluids

Development of a robust and low-cost test for the identification of membrane microvesicles (MVs) bio-markers of cardiovascular risk. The method is based on the selective recognition of phosphatidylserine (PS) present on the surface of the MVs by coordination complexes. The enumeration and complete characterization of MVs for early diagnosis will allow for more efficient management of patients.

Probing phospholipids and activated plasma membranes: towards a high-sensitive diagnosis tool

In response to different stimuli (infectious, inflammation), cells are activated, resulting in cell membrane remodeling and the release of microvesicles (MV). In biological fluids, these MVs, exposing phosphatidylserine on their surface, are considered as biomarkers of cell activation studied in many pathologies (diabetes, hypertension, thrombosis, sepsis). There is therefore a real interest in studying them for early detection, management and therapeutic monitoring. To date, the lack of techniques to separate, preserve, analyze and characterize MVs are barriers to understanding their functions. The objective of this project is the development of a new method for capturing microvesicles by bimetallic coordination complexes immobilized on a surface thanks to their ability to coordinate with phosphatidylserine. After studying the interactions of metal complexes with phospholipids in the form of monomers and model vesicles, tests on microvesicles from standardized biological samples were carried out for application in clinical cohorts.

The multidisciplinary approach involves:
1-Syntheses and characterizations of new zinc and copper complexes capable of selectively recognizing phosphatidylserine exposed on the surface of microvesicles
2-The implementation of complexes for interaction studies (in solution and on supports) with phospholipids arranged in structures of increasing complexity (monomers, model vesicles, microvesicles from biological samples).
3-The grafting of the complexes on different supports (multi-well supports, functionalized microbeads)
4-In parallel the culture of mesenchymal stromal cells from different healthy donors
5-The isolation of stromal mesenchymal stem cell microvesicles and their characterization as a reference
6-Standardized measurement of microvesicles in patient plasmas using reference microvesicles in functional tests.

1)Comparison of the selectivity of new zinc or copper coordination complexes for phosphatidylserine (PS)
2) Proof of concept for the detection of microvesicles (MVs) thanks to the introduction of a fluorescent probe
3) Qualification of mesenchymal stromal cell banks and MVs (phenotype and functionality, size)
4)Selection of PS sensor immobilization support in microplate
5) Validation of the ultrasensitive assay of MVs in pancreatic islet transplant recipients as early markers of graft loss and cell activation sequence

We have demonstrated that fluorescence can be a detection method to be considered in the context of the detection of microvesicles. This is why one of the perspectives of this work would be to integrate other fluorescent groups to amplify the signals.
• The characterization studies by interferometry of biolayers initiated with the project have made it possible to demonstrate the relevance of this still recent technique for the characterization of extracellular vesicles. Systematic tests should still be carried out to validate its application.
• This project opens up new perspectives in the study of MVs in clinical use by so-called routine laboratories which are accustomed to carrying out ELISA (Enzyme-Linked ImmunoSorbent Assay) type tests.

The progress of the project makes it possible to qualify the microvesicles of mesenchymal stromal cells as a reference for the detection of enzymatic activities, or antigenic markers of MVs in several pathologies. The method is therefore versatile and its principle has already been the subject of a joint CNRS-University of Grenoble patent. Articles on the methodology and its clinical applications have already been published in international journals (9) and others (3) are to be submitted/in preparation.


The COCERP project aims to the synthesis and study of new binuclear Zn(II) and Cu(II) complexes capable of preferentially recognize anionic phospholipids such as phosphatidylserine (PS) on microvesicles (MVs). The mechanisms of recognition / selectivity will be studied for different phospholipids in increasingly complex structures (monomers, liposome vesicle and cellular microvesicle models). The parameters (groups involved in the binding, mode of binding, geometry of the coordination, distance between the metallic ions, affinity constants …) governing interactions and selectivity will be determined at this stage. They will be pivotal for the design of new types of assembly and a second generation of sensors with improved selectivity for MVs and the detection of low MV concentrations in biological fluids. MVs are shed by the plasma membrane of cells (vascular cells, blood ..) that expose PS to the outer leaflet in response to different stimuli (in contrast to healthy cells). Flow cytometry, despite its limitations, has shown the diagnosis value of MVs in various vascular or degenerative diseases with high morbidity. Therefore, quantification and phenotypic and functional characterization of MVs are a major challenge for public health. To resolve the flaws and weaknesses of existing methods (cytometry, prothrombinase assay, annexin A5 labelling) we propose to develop an assay, whereby MVs are captured via our coordination complexes immobilized onto a solid surface. This type of compounds will avoid the disadvantages encountered with assays using biological molecules (interference from other coincidental ligands constitutive of biological fluids leading to proteolytic antibody degradation, calcium sensitivity,...). For validation purposes, the assay will be conducted on MVs from isolated cells and from physiological and pathological plasmas and the outcome will be compared with the existing methods. This is a necessary prerequisite for its development and is an important part of our project. COCERP creates opportunities for the development of a robust test and a low cost identification of MVs as sensors of cardiovascular risk. The socio-economic consequences of coronary heart disease or stroke augur of a very significant demand for simple and fast methodology, for the routine enumeration and complete characterization of microvesicles, an early diagnosis and a better monitoring of the patients.



















Project coordination

Catherine Belle (Département de Chimie Moléculaire)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LBP - CNRS_UNISTRA Laboratoire de Biophotonique et Pharmacologie_ CNRS_UNISTRA
UPD - UMRS 1140 Université Paris 5 Descartes - Innovations thérapeutiques en hémostase
DCM-CNRS ALPES Département de Chimie Moléculaire

Help of the ANR 450,427 euros
Beginning and duration of the scientific project: September 2016 - 48 Months

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