Nanoparticules fonctionnalisées pour le théranostique du rabdomyosarcome

par Sofia Dominguez-Gil

Projet de thèse en Chimie et Physico-Chimie des Matériaux

Sous la direction de Frédérique Cunin et de Jean-Olivier Durand.

Thèses en préparation à Montpellier , dans le cadre de Sciences Chimiques (Montpellier ; École Doctorale ; 2015-....) , en partenariat avec ICGM - Institut Charles Gerhardt de Montpellier (laboratoire) et de IMNO - Ingéniérie Moléculaire et Nano-Objets (equipe de recherche) depuis le 01-10-2018 .


  • Résumé

    Rhabdomyosarcomas (RMS) are the most common soft tissue sarcomas of childhood. Despite intensified, multimodality treatments, the overall survival for high-risk populations has remained at 5% to 20% over the last decades. Therefore, new innovative selective treatments based on nanomedicine are needed. The gamma-subunit of the AChR has been proposed as a diagnostic marker for this form of cancer because it differentiates between RMS and other tumors, as well as normal tissue. The fetal form of the muscle AChR (fAchR-gamma) has also been suggested as a possible target for RMS [1]. In 2005 a peptide toxin from the venom of the marine cone snail, Conus obscurus that selectively inhibits the mammalian fAChR was isolated [2]. The sequence of this conotoxin peptide is CCGVONAACPOCVCNKTCG, where O is hydroxyproline. This peptide will be synthesized with Dr Oleg Melnyk (Institut Pasteur de Lille) and will be functionalized with an aceto-acetyl group at the N-terminal in order to graft it on the surface of nanoparticles following the method developed by Dr Oleg Melnyk [3]. Here we plan to develop innovative and multifunctional Periodic Mesoporous Organosilica Nanoparticles (PMO) and Porous Silicon Nanoparticles (pSiNP) capable of performing imaging, photodynamic therapy (PDT) and photo-induced siRNA delivery. These nanoparticles will be synthesized and functionalized with the peptide by the PhD candidate. The synthesis of multifunctional PMO NPs will be realized in Dr F. Cunin/J.O. Durand's lab. These PMO NPs will incorporate porphyrins in the wall of the structure letting the porosity free [4]. These PMOs NPs possess biphotonic properties. The large mesopores will allow to load high quantity of molecules of interest such as siRNA [5]. As an example, we recently described PMO loading with gemcitabine with a loading capacity of 50% [4]. In parallel, the synthesis of pSiNP with Dr Frédérique Cunin will be performed. Porous silicon itself presents several advantages: it is luminescent, it absorbs under two photon excitation and it is totally biodegradable in three days. It can also be loaded with porphyrin to reinforce its PDT efficacy [6]. In addition, we will investigate the possibility to load NPs with siRNA. Concerning the choice of siRNA able to block the progression of the disease, we know that RMS tumors are characterized by specific chromosomal translocations that result in the expression of a PAX3-FOXO1 and PAX7-FOXO1 fusion transcription factor [7]. Translation products of these gene fusions lead to increase the expression of a number of oncogenes leading to aggressive phenotype of RMS. Facing the lack of efficiency of therapies against RMS, new concepts are urgently needed. The better understanding of the molecular biology signature of this disease has led to propose new therapeutic approaches. For example, down regulation of PAX3-FOXO1 induced by siRNA led to apoptosis in RMS cells [5]. In this project, we decided to study the effect of siRNA against the PAX3/7-FOXO-1 fusion transcript, loaded in PMO or pSiNP anchored with conotoxin peptide. First, we will characterize the physico-chemical properties of these multifunctionalized NPs and then, their biological action on RMS will be investigated. The first step of the experimental studies will be the establishment of the in vitro “proof of concept”. Jochen Roessler (University Medical Center Freiburg) will provide us with the cell lines of RMS through a network of specialists in the field, in France and Germany. We will verify the recognition and the sub-cellular localization of fluorescent PMO-conotoxin and pSiNP-conotoxin in RMS cells by using confocal microscopy on living cells and fluorescent markers of different organites. These cell lines will also be used to determine the efficiency for siRNA delivery of multifunctional PMO and pSiNP for photo-induced siRNA delivery. In fact, when the optimal incubation time for a quantifiable lysosomal accumulation of NPs will be determined, we will irradiate the cells with two-photon excitation in order to stimulate the lysosomal escape and allow the transfer of NP and its genetically material to the cytoplasm. This strategy will be first verified on MCF-7 cells transfected with a genetic construction inducing a constitutive expression of luciferase (MCF-7 Luc) already available in the lab. In this case, NPs will be loaded with siRNA against Luciferase to extinguish the luminescence of these cells. Then NPs loaded with siRNA PAX3/7-FOXO-1 will be tested, in the same conditions, on RMS cells to validate their use. Finally, preliminary in vivo experiments will be conducted on RMS Zebrafish model. Langenau et al. discovered that a transgene in which expression of human activated KRAS (kRASG12D) was driven by a rag2 promoter rapidly induced tumors in zebrafish which are fully consistent with a human RMS [8]. Zebrafish embryos were injected at the one-cell stage of development with a human kRASG12D-containing transgene (rag2-kRASG12D) and develop externally visible tumors at 10 days post fecondation. We will inject zebrafish with multifunctional NPs at this stage. The measurement of NPs accumulation in tumor will be possible thanks to the relative transparency of the fish and overall the luminescence of NPs used under a near infra-red excitation. In addition, the effects of NPs within the tumor will be assessed by the size measurement and by the extent of the knock-down of PAX3/7-FOXO-1 at mRNA and protein level. The life time will also be a criteria for the biocompatibility and the success of the treatment. References [1] Gattenloehner S, et al. The fetal form of the acetylcholine receptor distinguishes rhabdomyosarcomas from other childhood tumors. Am J Pathol. 1998, 152: 437-44. [2] Teichert RW, et al. A uniquely selective inhibitor of the mammalian fetal neuromuscular nicotinic acetylcholine receptor. J Neurosci. 2005, 25: 732-6. [3] Boll, E. et al., Access to Large Cyclic Peptides by a One-Pot Two-Peptide Segment Ligation/Cyclization Process, Org. Lett. 2015, 17, 130-133. [4] Mauriello-Jimenez, C. et al. Nouvelles nanoparticules d'organosilice mésoporeuses, leur méthode de préparation et leurs utilisations. FR Patent, N°16 50396 (2015). [5] Improved gene transfer with histidine-functionalized mesoporous silica nanoparticles Brevet D., et Al. Int. J. Pharm. 2014, 471, 197–205 [6] Secret E, et al. Two-photon excitation of porphyrin-functionalized porous silicon nanoparticles for photodynamic therapy. Adv Mater. 2014, 26: 7643-8. [7] Kubo T, et al. Prognostic value of PAX3/7-FOXO1 fusion status in alveolar rhabdomyosarcoma: Systematic review and meta-analysis. Crit Rev Oncol Hematol. 2015, 96: 46-53. [8] Langenau DM, et al. Effects of RAS on the genesis of embryonal rhabdomyosarcoma. Genes Dev. 2007, 21: 1382-95.

  • Titre traduit

    Theranostic of rhabdomyosarcoma with functionalized nanoparticles


  • Résumé

    Rhabdomyosarcomas (RMS) are the most common soft tissue sarcomas of childhood. Despite intensified, multimodality treatments, the overall survival for high-risk populations has remained at 5% to 20% over the last decades. Therefore, new innovative selective treatments based on nanomedicine are needed. The gamma-subunit of the AChR has been proposed as a diagnostic marker for this form of cancer because it differentiates between RMS and other tumors, as well as normal tissue. The fetal form of the muscle AChR (fAchR-gamma) has also been suggested as a possible target for RMS [1]. In 2005 a peptide toxin from the venom of the marine cone snail, Conus obscurus that selectively inhibits the mammalian fAChR was isolated [2]. The sequence of this conotoxin peptide is CCGVONAACPOCVCNKTCG, where O is hydroxyproline. This peptide will be synthesized with Dr Oleg Melnyk (Institut Pasteur de Lille) and will be functionalized with an aceto-acetyl group at the N-terminal in order to graft it on the surface of nanoparticles following the method developed by Dr Oleg Melnyk [3]. Here we plan to develop innovative and multifunctional Periodic Mesoporous Organosilica Nanoparticles (PMO) and Porous Silicon Nanoparticles (pSiNP) capable of performing imaging, photodynamic therapy (PDT) and photo-induced siRNA delivery. These nanoparticles will be synthesized and functionalized with the peptide by the PhD candidate. The synthesis of multifunctional PMO NPs will be realized in Dr F. Cunin/J.O. Durand's lab. These PMO NPs will incorporate porphyrins in the wall of the structure letting the porosity free [4]. These PMOs NPs possess biphotonic properties. The large mesopores will allow to load high quantity of molecules of interest such as siRNA [5]. As an example, we recently described PMO loading with gemcitabine with a loading capacity of 50% [4]. In parallel, the synthesis of pSiNP with Dr Frédérique Cunin will be performed. Porous silicon itself presents several advantages: it is luminescent, it absorbs under two photon excitation and it is totally biodegradable in three days. It can also be loaded with porphyrin to reinforce its PDT efficacy [6]. In addition, we will investigate the possibility to load NPs with siRNA. Concerning the choice of siRNA able to block the progression of the disease, we know that RMS tumors are characterized by specific chromosomal translocations that result in the expression of a PAX3-FOXO1 and PAX7-FOXO1 fusion transcription factor [7]. Translation products of these gene fusions lead to increase the expression of a number of oncogenes leading to aggressive phenotype of RMS. Facing the lack of efficiency of therapies against RMS, new concepts are urgently needed. The better understanding of the molecular biology signature of this disease has led to propose new therapeutic approaches. For example, down regulation of PAX3-FOXO1 induced by siRNA led to apoptosis in RMS cells [5]. In this project, we decided to study the effect of siRNA against the PAX3/7-FOXO-1 fusion transcript, loaded in PMO or pSiNP anchored with conotoxin peptide. First, we will characterize the physico-chemical properties of these multifunctionalized NPs and then, their biological action on RMS will be investigated. The first step of the experimental studies will be the establishment of the in vitro “proof of concept”. Jochen Roessler (University Medical Center Freiburg) will provide us with the cell lines of RMS through a network of specialists in the field, in France and Germany. We will verify the recognition and the sub-cellular localization of fluorescent PMO-conotoxin and pSiNP-conotoxin in RMS cells by using confocal microscopy on living cells and fluorescent markers of different organites. These cell lines will also be used to determine the efficiency for siRNA delivery of multifunctional PMO and pSiNP for photo-induced siRNA delivery. In fact, when the optimal incubation time for a quantifiable lysosomal accumulation of NPs will be determined, we will irradiate the cells with two-photon excitation in order to stimulate the lysosomal escape and allow the transfer of NP and its genetically material to the cytoplasm. This strategy will be first verified on MCF-7 cells transfected with a genetic construction inducing a constitutive expression of luciferase (MCF-7 Luc) already available in the lab. In this case, NPs will be loaded with siRNA against Luciferase to extinguish the luminescence of these cells. Then NPs loaded with siRNA PAX3/7-FOXO-1 will be tested, in the same conditions, on RMS cells to validate their use. Finally, preliminary in vivo experiments will be conducted on RMS Zebrafish model. Langenau et al. discovered that a transgene in which expression of human activated KRAS (kRASG12D) was driven by a rag2 promoter rapidly induced tumors in zebrafish which are fully consistent with a human RMS [8]. Zebrafish embryos were injected at the one-cell stage of development with a human kRASG12D-containing transgene (rag2-kRASG12D) and develop externally visible tumors at 10 days post fecondation. We will inject zebrafish with multifunctional NPs at this stage. The measurement of NPs accumulation in tumor will be possible thanks to the relative transparency of the fish and overall the luminescence of NPs used under a near infra-red excitation. In addition, the effects of NPs within the tumor will be assessed by the size measurement and by the extent of the knock-down of PAX3/7-FOXO-1 at mRNA and protein level. The life time will also be a criteria for the biocompatibility and the success of the treatment. References [1] Gattenloehner S, et al. The fetal form of the acetylcholine receptor distinguishes rhabdomyosarcomas from other childhood tumors. Am J Pathol. 1998, 152: 437-44. [2] Teichert RW, et al. A uniquely selective inhibitor of the mammalian fetal neuromuscular nicotinic acetylcholine receptor. J Neurosci. 2005, 25: 732-6. [3] Boll, E. et al., Access to Large Cyclic Peptides by a One-Pot Two-Peptide Segment Ligation/Cyclization Process, Org. Lett. 2015, 17, 130-133. [4] Mauriello-Jimenez, C. et al. Nouvelles nanoparticules d'organosilice mésoporeuses, leur méthode de préparation et leurs utilisations. FR Patent, N°16 50396 (2015). [5] Improved gene transfer with histidine-functionalized mesoporous silica nanoparticles Brevet D., et Al. Int. J. Pharm. 2014, 471, 197–205 [6] Secret E, et al. Two-photon excitation of porphyrin-functionalized porous silicon nanoparticles for photodynamic therapy. Adv Mater. 2014, 26: 7643-8. [7] Kubo T, et al. Prognostic value of PAX3/7-FOXO1 fusion status in alveolar rhabdomyosarcoma: Systematic review and meta-analysis. Crit Rev Oncol Hematol. 2015, 96: 46-53. [8] Langenau DM, et al. Effects of RAS on the genesis of embryonal rhabdomyosarcoma. Genes Dev. 2007, 21: 1382-95.