title: Synthesis of a stabilized 177Lu-siRNA complex and evaluation of its stability and RNAi activity.
abstract: OBJECTIVE
  Serum and intracellular instability limits the therapeutic applications of short interfering RNA (siRNA) as a radiopharmaceutical. Chemical modifications like phosphorothioate (PS) substitution and 2'-O-methoxy (2'-O-Me) modifications could eliminate such limitations. In this study, the effects of PS and 2'-O-Me modifications at the backbone of siRNA on serum stability and RNA interference activity were investigated.
  
  
  METHODS
  Fully PS and 2'-O-Me-modified type 1 insulin-like growth factor receptor (IGF-1R) siRNA was radiolabeled with lutetium-177 ((177)Lu) through p-SCN-Bn-DTPA as a chelator. After purification with Vivaspin and PD-10 columns, the radiolabs were examined for stability in serum by instant thin-layer chromatography and polyacrylamide gel electrophoresis. The level of IGF-1R in response to the modified and labeled IGF-1R siRNA was examined using RT-PCR and ELISA assay in colon cancer cells. The effects of such siRNA on the prevention of proliferation of colon cancer cells and its apoptosis were investigated using MTT assay and Annexin-V/propidium iodide double staining, respectively. Cellular accumulation quantities of the labeled and modified IGF-1R siRNA were determined using a γ-counter by taking advantage of (177)Lu as a γ-emitter.
  
  
  RESULTS
  Both the modified (177)Lu-siRNA complex and the modified nonlabeled siRNA showed significant stability in serum. The levels of IGF-1R mRNA and protein significantly decreased with both the labeled and nonlabeled IGF-1R siRNAs, but no such reduction in IGF-1R was observed with luciferase siRNA (P<0.01). Proliferation decreased significantly and apoptosis increased in the cells treated with modified (177)Lu-IGF-1R siRNA in comparison with either (177)Lu or labeled luciferase siRNA (P<0.001).
  
  
  CONCLUSIONS
  Uniform chemically modified siRNAs can form stable complexes with Lu that pronounce its cytotoxic effect through apoptosis in colon cancer cells.
authors.affiliation: aDepartment of Clinical Biochemistry, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan bDepartment of Radiochemistry, Nuclear Sciences and Technology Research Institute (NSTRI) cDepartment of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Mojtaba
authors.initials: M
authors.lastname: Fathi
authors.forename: Kamal
authors.initials: K
authors.lastname: Yavari
authors.forename: Mohammad
authors.initials: M
authors.lastname: Taghikhani
authors.forename: Mohammad
authors.initials: M
authors.lastname: Ghannadi Maragheh
mesh_terms: D017209:Apoptosis / Q000235:genetics; D001692:Biological Transport; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D002614:Chelating Agents / Q000737:chemistry; D060326:Chemistry Techniques, Synthetic; D055785:Gene Knockdown Techniques; D006801:Humans; D007700:Kinetics; D008187:Lutetium / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D034622:RNA Interference; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011836:Radiation Tolerance; D011868:Radioisotopes / Q000737:chemistry*; D017526:Receptor, IGF Type 1 / Q000172:deficiency / Q000235:genetics
chemical_list: D002614:Chelating Agents; D010710:Phosphates; D034741:RNA, Small Interfering; D011868:Radioisotopes; D008187:Lutetium; C083120:phosphorodithioic acid; D017526:Receptor, IGF Type 1