Development of a lanthanide-based luminescent nanoparticule for the dynamic quantification of autophagy in vitro and in vivo. – Lumiphage

Self-digestion of the cell, also coined autophagy, is a major biological process involved in the homeostasy of cells and organisms. Its importance is largely recognized in multiple reseach fields of biology such as development, neurodegeneration, cancer, pathogens infection or obesity. As the the role of autophagy in diseases and therapies is firmly recognized; and in consideration of the advent of personnalized therapy, we anticipate that the development of tools that enable to image and quantify non-invasively autophagy in vivo will have an important impact on therapeutic efficiency. Whereas several conventionnal tools have been developped to study the functions of autophagy in vitro, molecules that can be exploited to evaluate dynamically and non-invasively the autophagic activity in vivo are still missing. We propose in this study to develop a nanoparticule to obtain unpreceded dynamic and quantitative informations on the autophagic flux in vitro and in vivo non-invasively.
The autophagic flux is described by the successive steps from the formation of autophagosomes until their fusion with the lysosomes and the degradation of the autophagosomal cargo by lysosomal enzymes at acidic pH. This later step is determinant of the physiological outcome of autophagy but to date no assay can measure it conveniently and dynamically in non-engenieered cells. In this work, we will produce a lanthanide-based luminescent dendrimer targeted to autophagosomes and exploit pH acidification at late stage of autophagy tp quantify autophagic activity. A pH-sensitive system of energy transfert from an organic fluorophore, also called antenna, to lanthanides atomes embedded into the core nanoparticule will be used to measure lanthanide luminescence in the visible range in vitro by fluorescence microscopy, cytometry or in plate readers and in the near-infrared range in vivo with a near-infrared imaging device.
Our expertize in the chemistry of lanthanides, fluorophores and peptides allows the implementation of multiple strategies to produce the four key elements in the synthesis of the probe. Firstly several visible and near-infrared antenna which have been produced, or are still in development in our group will be screened to select optimized transfert to lanthanides. Secondly, we have chosen to develop of a dendrimer-based nanoparticule since as shown by us and others, dendrimers are functionnalizable, they can coordinate lanthanides and they present biocompatible features. Third, the pH sensitivity will be tuned using an imine link which has already been validated in our group in a fret assay between an oganic dye and ytterbium. Finally, the targeting of the nanoparticules towards autophagosomes will be tuned by surface chemistry using recently identified peptides that modulate the autophagy of the nanoparticules.
The photophysical properties of the probe produced will be characterized with state-of-the art equipment by visible and near-infrared spectroscopy, and life-times measurements. Finally, the functionnality of the probe will be tested at first in vitro in cancer cells treated with autophagy inducers such as Class I PI3Kinases inhibitors, autophagic flux dirupting drugs, and in cancer cells knocked-down for ATG7, or Beclin-1. Localization and reporting activity of the probe will be compared to standard procedures currently in use to study of autophagy for validation. Upon completion of these tests in cells, the probe will be tested in mice bearing tumors and treated with chemotherapeutic agents that modulate autophagy. A macroscope equiped with a sensitive camera in the near-infrared range will be used to image mice and to obtain quantitative measurements.
We foresse that the completion of this project will significantly impact the developpement of NIR molecular imaging technics, will provide new tools for experimental reseach, and ultimately open the way towards clinical exploitation of this technology to assist personnalized medicine.