Nanoparticle for siRNA delivery and its pancreatic cancer targeting ability |
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Authors: | LI Jia-jia CHEN Yin-ting ZENG Lin-juan LIAN Guo-da CHEN Shao-jie LI Ya-qing HUANG Kai-hong |
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Affiliation: | 1.Department of Gastroenterology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, China;2.Department of Oncology, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, China. |
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Abstract: | AIM: To synthesize a safe, efficient and targeted nanoparticulate carrier for siRNA delivery to pancreatic 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 fragment, was constructed as a cancer-targeting nanocarrier for siRNA delivery. Prussian blue staining and immunofluorescent staining were performed to detect the distribution of scFvCD44v6-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 scFvCD44v6-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 revealed 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)%, respectively. 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. |
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Keywords: | Pancreatic neoplasms Nanoparticles Genetic therapy Targeting |
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