title: Effects of silencing the RET/PTC1 oncogene in papillary thyroid carcinoma by siRNA-squalene nanoparticles with and without fusogenic companion GALA-cholesterol.
abstract: BACKGROUND
  RET/PTC1 is the most prevalent type of gene rearrangement found in papillary thyroid carcinoma (PTC). Previously, we introduced a new noncationic nanosystem for targeted RET/PTC1 silencing by efficient delivery of small interfering RNA (siRNA) using the "squalenoylation" approach. With the aim of improving these results further, we designed new squalenoyl nanostructures consisting of the fusogenic peptide GALA-cholesterol (GALA-Chol) and squalene (SQ) nanoparticles (NPs) of siRNA RET/PTC1.
  
  
  METHODS
  The siRNA RET/PTC1-SQ bioconjugate was synthesized. The corresponding NPs were prepared with or without GALA-Chol by nanoprecipitation and then characterized for their size and zeta potential. The effects of NPs on BHP 10-3 SCmice and TPC-1 cell viability (MTT assay), gene and protein silencing (reverse transcription-quantitative polymerase chain reaction [rt-qPCR], Western blot), and cellular uptake (fluorescent microscopy) were studied. In vivo gene silencing efficiency of siRNA RET/PTC1-SQ NPs was assessed by administration in nude mice via either intratumoral (i.t.) or intravenous (i.v.) routes. Tumor growth was followed for 19 days. Tumors were then collected, and RET/PTC1 gene and protein inhibitions were assessed by RT-qPCR and Western blot.
  
  
  RESULTS
  The combination of siRNA RET/PTC1-SQ bioconjugate and GALA-Chol leads to stable NPs of ∼200 nm diameter. In vitro, the results revealed that combining GALA-Chol with siRNA RET/PTC1-SQ NPs decreased cell viability, enhanced cellular internalization, and induced gene silencing efficiency in both human PTC (BHP 10-3 SCmice and TPC-1) cell lines. On the contrary, in vivo, the siRNA RET/PTC1-SQ GALA-Chol NPs were not found to be efficient either in gene silencing or in tumor growth inhibition, compared to siRNA RET/PTC1-SQ NPs both via i.t. and i.v. routes (p<0.001).
  
  
  CONCLUSIONS
  Conversely to siRNA RET/PTC1-SQ NPs, the siRNA RET/PTC1-SQ GALA-Chol NPs are efficient in vitro but not in vivo. Finally, NPs of siRNA RET/PTC1-SQ were found to be efficient silencers of the RET/PTC1 fusion oncogene in in vivo applications even at a concentration lower than used in a previously published study.
authors.affiliation: 1 Laboratory of Vectorology and Anti-Cancer Therapies (UMR 8203 CNRS), Gustave Roussy , Villejuif, France .
authors.forename: Hafiz Muhammad
authors.initials: HM
authors.lastname: Ali
authors.forename: Andrei
authors.initials: A
authors.lastname: Maksimenko
authors.forename: Giorgia
authors.initials: G
authors.lastname: Urbinati
authors.forename: Hubert
authors.initials: H
authors.lastname: Chapuis
authors.forename: Mouna
authors.initials: M
authors.lastname: Raouane
authors.forename: Didier
authors.initials: D
authors.lastname: Desmaële
authors.forename: Hayashi
authors.initials: H
authors.lastname: Yasuhiro
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Harashima
authors.forename: Patrick
authors.initials: P
authors.lastname: Couvreur
authors.forename: Liliane
authors.initials: L
authors.lastname: Massaad-Massade
mesh_terms: D000818:Animals; D002277:Carcinoma / Q000188:drug therapy*; D002291:Carcinoma, Papillary / Q000188:drug therapy*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000008:administration & dosage / Q000031:analogs & derivatives*; D015321:Gene Rearrangement; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D053758:Nanoparticles / Q000008:administration & dosage*; D015514:Oncogene Proteins, Fusion / Q000187:drug effects; D010455:Peptides / Q000008:administration & dosage*; D051096:Proto-Oncogene Proteins c-ret / Q000187:drug effects* / Q000235:genetics; D034741:RNA, Small Interfering / Q000008:administration & dosage*; D013185:Squalene / Q000008:administration & dosage*; D000077273:Thyroid Cancer, Papillary; D013964:Thyroid Neoplasms / Q000188:drug therapy*
chemical_list: D015514:Oncogene Proteins, Fusion; D010455:Peptides; D034741:RNA, Small Interfering; C066951:GALA peptide; D013185:Squalene; D002784:Cholesterol; D051096:Proto-Oncogene Proteins c-ret