M.Mazzaa,b, C. Alliota,c, C. Sinquind, P.E. Reillerce, S. Colliec-Jouaultd, and S. Huclier-Markaia,b*
a GIP ARRONAX, 1 rue Arronax, F-44817 Nantes Cedex 3, France.
b SUBATECH, 4 rue Alfred Kastler, BP 20722, 44307 Nantes Cedex 3, France
c INSERM U892- 8 quai Moncousu, F-44007 Nantes Cedex 1, France.
d IFREMER, Institut français de recherche pour l’exploitation de la mer, rue de l’Ile d’Yeu, BP 21105 F-44311 Nantes Cedex 3, France.
e Den – Service d’Etudes Analytiques et de Réactivité des Surfaces (SEARS), CEA, Université Paris-Saclay, F-91191, Gif sur Yvette, France.
An oversulphated exopolysaccharide (OS-EPS) derivative, produced by a deep-sea hydrothermal bacterium named Alteromonas infernus, has shown to inhibit the establishment of lung metastasis in patients with osteosarcoma. These EPSs have anticoagulant properties that could reduce the thrombotic complication affecting some kind of tumors. Heparin is currently used in therapy for that purpose, but with the risk of prion, that is not the case of polysaccharides. Thus, this new class of molecules could pave the way to new type of applications in oncology and the idea of this work is to investigate the coupling of these EPS with a theranostic radionuclide pair, such as 44Sc/47Sc.
A very important part of this work is dedicated to an extensive characterization of these very complexe molecules to be further used for medical purposes. EPSs have been characterized in terms of molecular weight and dispersity using High Performance Size Exclusion Chromatography (HPSEC) coupled to Multi Angle Light Scattering (MALS) as well as Asymmetrical Flow Field-Flow Fractionation (A4F) coupled to MALS. A4F-MALS showed that EPSs are monodisperse polysaccharides with a polydispersity index (IP) of 1.4, suitable for therapeutic uses.
To have an idea on the conformation of these polymers in solution, the intrinsic viscosity for EPS and heparin as reference, has been measured at different ionic strength of solvent revealing that polymer conformation could become more folded increasing the added salt. The conformation may lay an important role then on the biodistribution of these molecules in vivo.
Finally, the stability constants of Sc-EPS complexes must be determined. We have based our approach on two techniques: potentiometric titrations and time-resolved laser fluorescence spectroscopy (TRLFS). TRLFS requires a fluorescent probe, such as Eu3+ which has also a coordination chemistry close to Sc3+. Moreover, TRLFS allows to measure stability constants only using traces of EPS, avoiding wastes and chemical degradation compared to titrations. Potentiometric titrations are not suitable for such complex systems, but this method was employed on purpose for the determination of the pKa of heparin (3,65) and the number of heparin dimers (33). Work is still on-going for Sc/Eu-EPS systems.
The corresponding half-life T =(1.8 ±0.5(stat) ± 0.1 (syst))x10^22 y is longest ever measured directly.