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Marie-Thérèse Aloy,1,* Jacqueline Sidi Boumedine,2,* Agathe Deville,2,3 David Kryza,2 Arnaud Gauthier,1,4 Delphine Brichart-Vernos,1,5 Grégoire Ollier,1 Veronica La Padula,5 François Lux,5 Olivier Tillement,5 Claire Rodriguez-Lafrasse,1,4,* Marc Janier2,3,* 1Laboratory of Cellular and Molecular Radiobiology, UMR CNRS5822/IP2I, Lyon-Sud Medical School, Univ Lyon, Lyon 1 University, Oullins, France; 2CNRS, LAGEPP, UMR5007, IMTHERNAT, Lyon 1 University, Hospital Edouard Herriot, Lyon, France; 3Department of Nuclear Medicine, Groupement Hospitalier Est, Hospices Civils de Lyon, Lyon, France; 4Department of Biochemistry and Molecular Biology, Groupement Hospitalier Sud, Hospices Civils de Lyon, Pierre-Bénite, France; 5Light Matter Institut UMR CNRS 5306, Lyon 1 University, Villeurbanne, France*These authors contributed equally to this workCorrespondence: Marc Janier, Department of Nuclear Medicine, Groupement Hospitalier Est, Hospices Civils de Lyon, Lyon, 69634, France, Tel +33472356999, Fax +33472357345, Email marc.janier@univ-lyon1.frPurpose: Chondrosarcomas (CHSs), which represent 20% of primary bone tumors in adults, are mostly resistant to radio- and chemotherapy. It is therefore essential that new therapeutic approaches, targeted to the tumour, be developed to improve the prognosis of patients. The effectiveness, as a radiosensitizing agent, of gadolinium oxide nanoparticles (GdoNP, AGuIX®) nanoparticles in CHS was evaluated in vitro, in spheroid CHS models allowing to reproduce cell-cell extracellular matrix interactions, and, in vivo, in a nude mouse model with heterotopic tumour xenograft.Methods: Spheroids from SW1353 and HEMC-SS cells were characterized by confocal microscopy with or without GdoNP treatment. Real-time microscopy enabled quantification of cell viability, cell migration and invasion. In vivo, the efficacy of the association of GdoNP combined with a single (4Gy) or fractionated (4x1Gy) irradiation was evaluated in HEMC-SS tumor-bearing mice by monitoring tumor growth, mouse survival and gene expression profile.Results: The expression of proteoglycans in the extra-cellular matrix (ECM) of spheroids demonstrated the relevance of the 3-D model. The combination of GdoNP with single or fractionated irradiation increased the lethal effects of irradiation on 2-D- and 3-D-cultured cells. In vivo, a single or a fractionated dose of 4 Gy associated with IT or IV injection of GdoNP decreased tumor growth significantly. Only IT injection increased mice survival. Unexpectedly, the radiosensitizing effect of GdoNP was associated, in vitro, with a significant decrease in invasion-migration capacities and, in vivo, with the decreased expression of PTX3, a protein involved in the epithelial-to-mesenchymal transition process, suggesting a potential impact of GdoNP on metastasis formation.Conclusion: These results provide the first proof of concept of the radiosensitizing effect of GdoNP in CHSs and opened the way for a multicentre, randomized Phase 2 trial evaluating the association of GdoNP with radiotherapy for the therapeutic management of patients with symptomatic inoperable musculoskeletal tumor lesions.Keywords: chondrosarcoma, AGuIX® nanoparticle, radiosensitization, PTX3

Mickaël Gries, 1 Noémie Thomas, 1 Joël Daouk, 1 Paul Rocchi, 2 Laurence Choulier, 3 Justine Jubréaux, 1 Julien Pierson, 1 Aurélie Reinhard, 1 Valérie Jouan-Hureaux, 1 Alicia Chateau, 1 Samir Acherar, 4 Céline Frochot, 5 François Lux, 2– 6 Olivier Tillement, 2 Muriel Barberi-Heyob 1 1Université de Lorraine, Centre National de la Recherche Scientifique (CNRS), Research Center for Automatic Control of Nancy (CRAN), Nancy, France; 2Université de Lyon, CNRS, Institut Lumière Matière, Lyon, France; 3Université de Strasbourg, CNRS, Biotechnologie et Signalisation Cellulaire, Illkirch, France; 4Université de Lorraine, CNRS, Laboratoire de Chimie-Physique Macromoléculaire, Nancy, France; 5Université de Lorraine, CNRS, Laboratoire Réactions et Génie des Procédés, Nancy, France; 6Institut Universitaire de France, Paris, FranceCorrespondence: Muriel Barberi-Heyob Email muriel.barberi@univ-lorraine.frBackground: Local recurrences of glioblastoma (GBM) after heavy standard treatments remain frequent and lead to a poor prognostic. Major challenges are the infiltrative part of the tumor tissue which is the ultimate cause of recurrence. The therapeutic arsenal faces the difficulty of eradicating this infiltrating part of the tumor tissue while increasing the targeting of tumor and endogenous stromal cells such as angiogenic endothelial cells. In this aim, neuropilin-1 (NRP-1), a transmembrane receptor mainly overexpressed by endothelial cells of the tumor vascular system and associated with malignancy, proliferation and migration of GBM, highlighted to be a relevant molecular target to promote the anti-vascular effect of photodynamic therapy (VTP).Methods: The multiscale selectivity was investigated for KDKPPR peptide moiety targeting NRP-1 and a porphyrin molecule as photosensitizer (PS), both grafted onto original AGuIX design nanoparticle. AGuIX nanoparticle, currently in Phase II clinical trials for the treatment of brain metastases with radiotherapy, allows to achieve a real-time magnetic resonance imaging (MRI) and an accumulation in the tumor area by EPR (enhanced permeability and retention) effect. Using surface-plasmon resonance (SPR), we evaluated the affinities of KDKPPR and scramble free peptides, and also peptides-conjugated AGuIX nanoparticles to recombinant rat and human NRP-1 proteins. For in vivo selectivity, we used a cranial window model and parametric maps obtained from T2*-weighted perfusion MRI analysis.Results: The photophysical characteristics of the PS and KDKPPR molecular affinity for recombinant human NRP-1 proteins were maintained after the functionalization of AGuIX nanoparticle with a dissociation constant of 4.7 μM determined by SPR assays. Cranial window model and parametric maps, both revealed a prolonged retention in the vascular system of human xenotransplanted GBM. Thanks to the fluorescence of porphyrin by non-invasive imaging and the concentration of gadolinium evaluated after extraction of organs, we checked the absence of nanoparticle in the brains of tumor-free animals and highlighted elimination by renal excretion and hepatic metabolism.Conclusion: Post-VTP follow-ups demonstrated promising tumor responses with a prolonged delay in tumor growth accompanied by a decrease in tumor metabolism.Keywords: vascular photodynamic therapy, AGuIX nanoparticle, NRP-1, peptide, brain tumors, glioblastoma, radiation therapies

Yoann Montigaud,1 Jérémie Pourchez,1 Lara Leclerc,1 Olivier Tillement,2 Anthony Clotagatide,3,4 Clémence Bal,5 Noël Pinaud,5 Nobuyasu Ichinose,6 Bei Zhang,7 Sophie Perinel,3,4 François Lux,2,8 Yannick Crémillieux,5 Nathalie Prevot3,4 1Mines Saint-Etienne, Univ Lyon, Univ Jean Monnet, INSERM, U 1059 Sainbiose, Centre CIS, Saint-Etienne, France; 2Institut Lumière Matière, Université de Lyon, Villeurbanne, France; 3INSERM U 1059 Sainbiose, Université Jean Monnet, Saint-Etienne, France; 4CHU Saint-Etienne, Saint-Etienne, France; 5IBIO, Bordeaux, France; 6Canon Medical Systems Corporation, Otawara, Japan; 7Canon Medical Systems Europe, Zoetermeer, Netherlands; 8Institut Universitaire de France (IUF), Paris, FranceCorrespondence: Jérémie PourchezMines Saint-Etienne, 158 Cours Fauriel, CS 62362, Saint-Etienne, Cedex 2 42023, FranceTel +33 4 77 42 01 80Email pourchez@emse.frPurpose: This study aims at determining lung distribution of gadolinium-based polysiloxane nanoparticles, AGuIX® (small rigid platform – SRP), as a potential theranostic approach by the pulmonary route.Methods: First, the aerodynamic size distribution and the aerosol output rate were thoroughly characterized. Then, a multimodal approach using magnetic resonance (MR) and gamma-camera (GC) imaging allows to assess the deposition of the aerosolised nanoparticles in the respiratory tract using isolated ventilated porcine lungs.Results: The SRP has proven to be radiolabelled by radioisotope with a good yield. Crude SRP or radiolabelled ones showed the same aerodynamic size distribution and output as a conventional molecular tracer, as sodium fluoride. With MR and GC imaging approaches, the nebulised dose represented about 50% of the initial dose of nanoparticles placed in the nebuliser. Results expressed as proportions of the deposited aerosol showed approximately a regional aerosol deposition of 50% of the deposited dose in the lungs and 50% in the upper airways. Each technique assessed a homogeneous pattern of deposited nanoparticles in Lungs. MR observed a strong signal enhancement with the SRP, similar to the one obtained with a commonly used MRI contrast agent, gadoterate meglumine.Conclusion: As a known theranostic approach by intravenous administration, SRP appeared to be easily aerosolised with a conventional nebuliser. The present work proves that pulmonary administration of SRP is feasible in a human-like model and allows multimodal imaging with MR and GC imaging. This work presents the proof of concept of SRP nebulisation and aims to generate preclinical data for the potential clinical transfer of SRP for pulmonary delivery.Keywords: aerosol, pulmonary delivery, ex vivo model, MRI, SPECT/CT, theranostic

Eloïse Thomas,1 Ludovic Colombeau,2 Mickaël Gries,3,4 Thibaut Peterlini,3,4 Clélia Mathieu,1 Noémie Thomas,3,4 Cédric Boura,3,4 Céline Frochot,2 Régis Vanderesse,5 François Lux,1 Muriel Barberi-Heyob,3,4 Olivier Tillement1 1Université Lyon, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique (CNRS), Institut Lumière Matière, Lyon, 2Laboratoire Réactions et Génie des Procédés, Université de Lorraine-CNRS, Nancy, 3Université de Lorraine, Research Center for Automatic Control of Nancy (CRAN), 4CNRS, CRAN, Vandœuvre-lès-Nancy, 5Laboratoire de Chimie Physique Macromoléculaire, Université de Lorraine-CNRS, Nancy, France Abstract: Despite combined treatments, glioblastoma outcome remains poor with frequent local recurrences, indicating that a more efficient and local therapy is needed. In this way, vascular-targeted photodynamic therapy (VTP) could help tumor eradication by destroying its neovessels. In this study, we designed a polysiloxane-based nanoparticle (NP) combining a magnetic resonance imaging (MRI) contrast agent, a photosensitizer (PS) and a new ligand peptide motif (KDKPPR) targeting neuropilin-1 (NRP-1), a receptor overexpressed by angiogenic endothelial cells of the tumor vasculature. This structure achieves the detection of the tumor tissue and its proliferating part by MRI analysis, followed by its treatment by VTP. The photophysical properties of the PS and the peptide affinity for NRP-1 recombinant protein were preserved after the functionalization of NPs. Cellular uptake of NPs by human umbilical vein endothelial cells (HUVEC) was increased twice compared to NPs without the KDKPPR peptide moiety or conjugated with a scramble peptide. NPs induced no cytotoxicity without light exposure but conferred a photocytotoxic effect to cells after photodynamic therapy (PDT). The in vivo selectivity, evaluated using a skinfold chamber model in mice, confirms that the functionalized NPs with KDKPPR peptide moiety were localized in the tumor vessel wall. Keywords: nanoparticles, PDT, vascular targeting strategy, brain tumor, NRP-1, peptide ligand, MRI

Thomas Schlathölter,1 Pierre Eustache,2 Erika Porcel,2 Daniela Salado,2 Lenka Stefancikova,2 Olivier Tillement,3 Francois Lux,3 Pierre Mowat,3 Aleksandra K Biegun,4 Marc-Jan van Goethem,4 Hynd Remita,5 Sandrine Lacombe2 1Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands; 2Institut des Sciences Moléculaires d’Orsay (ISMO), Univ. Paris Sud, CNRS, Université Paris Saclay, Orsay Cedex, 3Institut Lumière Matière, Villeurbanne Cedex, France; 4Kernfysisch Versneller Instituut – Center for Advanced Radiation Technology (KVI-CART), University of Groningen, Groningen, the Netherlands; 5Laboratoire de Chimie Physique, Universite Paris-Sud, Orsay Cedex, France Abstract: The use of nanoparticles to enhance the effect of radiation-based cancer treatments is a growing field of study and recently, even nanoparticle-induced improvement of proton therapy performance has been investigated. Aiming at a clinical implementation of this approach, it is essential to characterize the mechanisms underlying the synergistic effects of nanoparticles combined with proton irradiation. In this study, we investigated the effect of platinum- and gadolinium-based nanoparticles on the nanoscale damage induced by a proton beam of therapeutically relevant energy (150 MeV) using plasmid DNA molecular probe. Two conditions of irradiation (0.44 and 3.6 keV/µm) were considered to mimic the beam properties at the entrance and at the end of the proton track. We demonstrate that the two metal-containing nanoparticles amplify, in particular, the induction of nanosize damages (>2 nm) which are most lethal for cells. More importantly, this effect is even more pronounced at the end of the proton track. This work gives a new insight into the underlying mechanisms on the nanoscale and indicates that the addition of metal-based nanoparticles is a promising strategy not only to increase the cell killing action of fast protons, but also to improve tumor targeting. Keywords: gadolinium-based nanoparticles, platinum nanoparticles, nanosensitization, theranostics