Nanoparticle for siRNA delivery and its pancreatic cancer targeting abili-ty / 中国病理生理杂志

ABSTRACT

AIM:To synthesize a safe , efficient and targeted nanoparticulate carrier for siRNA delivery to pan-creatic cancer cells .METHODS: Iron oxide nanocrystal with carboxylic acid group-polyethyleneimine ( IONP-PEI ) was synthesized and investigated as a nonviral carrier of siRNA to the pancreatic cells .The size, surface and charge using zeta potential were characterized .The perfect charge ratio between amino groups of IONP-PEI and phosphate groups of siRNA ( N/P) was determined by the transfection efficiency detection , gel retardation assay and MTS assay .An antibody-directed nonviral vector , scFvCD44v6-IONP-PEI nanoparticle attaching to the cancer-associated CD44v6 single-chain variable frag-ment, was constructed as a cancer-targeting nanocarrier for siRNA delivery .Prussian blue staining and immunofluorescent staining were performed to detect the distribution of scFv CD44v6-IONP-PEI/siRNA complexes in the cells .The transfection efficiency , fluorescence intensity and the expression of KRAS at mRNA and protein levels in the cells transfected by IONP -PEI/siRNA and scFv CD44v6-IONP-PEI/siRNA were detected by flow cytometry , fluorescence microscopy , real-time PCR and Western blotting, respectively.RESULTS:The mass ratio of IONP to PEI was 0.75.The suitable ratio of N/P was 20. The averaged size and surface zeta potential of IONP-PEI/siRNA in deionized water were (51.3 ±2.2)nm (diameter) and (21.73 ±8.07)mV, respectively.Red fluorescence was seen in both targeting and nontargeting groups , which clearly re-vealed the intracellular distribution of siRNA and delivery agents .Transfection efficiencies in targeting and nontargeting groups were (89.75 ±1.81)%and (59.87 ±4.52)%, respectively.Down-regulation of the KRAS mRNA in Panc-1 cells transfected with siKRAS by scFvCD44v6-IONP-PEI and IONP-PEI was up to (34.02 ±6.15)%and (51.09 ±6.70)%, re-spectively .The protein level of KRAS was lower in targeting group than that in nontargeting group .CONCLUSION:scFvCD44v6-IONP-PEI is a safe and efficient nanoparticulate carrier for gene delivery .It is more effective to transfer siRNA into the cells and mediate gene silencing effect in vitro than the nontargeting group .