| Objective:The RNAi-based therapy represents a promising prospect to correct the aber-rant expression of disease-related proteins and virus infection. The key factor of RNAi is looking for a delivery system that has high transfection efficiency, high loading capacity, non-toxic, no immunogenicity and easy to commercial production. Nonviral vectors have been investigated intensely in siRNA delivery due to their controllable synthesis, less immunogenicity and protect siRNA from RNAase. Cationic polysaccharide is one of the most applied vectors due to its biocompatibility and easy to modify. The absorption of serum protein by cationic polysaccharide reduced the RNAi efficiency. To avoid the problems of serum protein, the serum-free medium is currently required, but the cationic polysaccharide usually brings the cytotoxicity in the serum-free medium, and for in vivo application, the serum is inevitable. Mechanisms under the phenomena cited above re-main elusive. We previously reported that the serum concentration affected the escape from lysosome of Ps/siRNA, and then affected the RNAi efficiency. But it is also unclear that the relationship between Ps and serum protein.The chronic myeloid leukemia (CML) is a clonal haemopoietic stem cell disorder characterized by a reciprocal translocation between the long arms of chromosomes 9 (ch9) and 22 (ch22), generating the fusion gene BCR-ABL. The main treatmsent of CML in-clude TKIs and allografting. Durg resistance and morbidity are the shortage of currently therapy methods. RNAi has been also useded as an important tool to verify the role of leukemia-related genes, along with its widely application in tumor therapy. However, it’s a big challenge to transfect CML cells. So far, except for electroporation and viral vectors, little conventional polymeric carriers are available to meet the demand. However, the elec-troporation is not suitable to apply in vivo, and viral vectors may bring risks of im-mune toxicity and cause host genetic mutation. Therefore, new siRNA carriers require intensive exploition for CML cells.Pullulan-spermine (Ps) is a typical cationic polysaccharide. Ps has been widely used as drug and gene delivery system due to the characteristic of biocompatibility, non-toxic and no immunogenicity. In this work, two projects were conducted by using Ps as the siRNA carrier:(1) The influence of the mass ratio of serum to Ps onthe Ps/siRNA com-plex particle size and RNAi efficiency. (2) Investigation and comparison of RNAi effi-ciency in Chronic Myeloid Leukemia cells by using Ps, and in RAW264.7 and Jurkat cells.Methods:To detect the absorption of Ps to bovine serum albumin (BSA) and the stabil-ity of Ps/siRNA in BSA solution, the isothermal titration calorimetry (ITC) and gel elec-trophoresis were used in this study. The regulation of Pr/Ps to the particle size of Ps/siRNA was detected by using dynamic light scattering (DLS). Flow cytometry was used to test the effect of Pr/Ps on the ability to enter cells in MDA-MB-231 and the RNAi efficiency of Ps/siRNA in MDA-MB-231-EGFP.The RNAi efficiency induced by Ps/siRNA or Lipo 3000/siRNA to K562, KU812, MEG-01, RAW and Jurkat cells under serum free and 10% FBS conditions was verified by RT-PCR. The DLS was used to detect the Ps/siRNA particle size under different FBS concentration. Flow cytometry and confocol microscopy was used to test the cell positive rate of Ps/siRNA in K562. The BCR-ABL RNAi efficiency in CML cells under different FBS concentration modulated by Ps/siRNA was tested by RT-PCR and western blot methods. K562, RAW and Jurkat cells were transfected by Ps/PGL4.51 and the expres-sion of Luc was measured by Microplate multimode Reader.Results:The quality ratio of the combination between Ps and siRNA was about 0.75:1. Ps could absorb BSA.The stability of Ps/siRNA was not be affected by the absorption. With the increase of Pr/Ps, the particle size of Ps/siRNA decreased; both the ability to enter cells and the efficiency of RNAi were reduced gradually. When Ps and serum con-centration was ensured, with the increase of the amount of siRNA, the RNA interference efficiency increased, but if the dosage of siRNA reached 1 μg/mL, the RNA interference efficiency was not increased anymore. CQ can improve the efficiency of RNA interfer-ence. Compared with the method that CQ and RNA interference complex incubated the cell as the same time, the RNA interference efficiency was higher by using CQ post-processing method.In the conditions of N/P 2.5, the activity of K562 cells was higher than 80%, no matter the serum existing. The positive rate of Ps/siRNA in three kinds of CML cells was over 95%, but in both RAW and Jurkat cells was about 85%. In the interference of β-actin gene of the 5 kinds of cells,the interference efficiency was higher by using Ps than Lipo 3000 on CML cells in the Opti-MEM medium, but the interference efficiency was higher by using Lipo 3000 than Ps on the RAW and Jurkat cells in the Opti-MEM me-dium. It was have no RNA interference efficiency by using both Lipo 3000 and Ps under the condition of the 10% serum concentrations. About the interference of BCR-ABL gene, the interference efficiency was higher by using Ps than Lipo 3000 on K562 cells in the two culture mediums. There was no difference between the two carriers on KU812 cells in the two culture mediums. On MEG-01 cells, the interference efficiency was higher by using Ps than Lipo 3000 in the Opti-MEM medium, but it was higher by using Lipo 3000 than Ps in the 10% serum medium. Ps/siRNA particle size and RNAi efficiency on K562 and the drug-resistantce in K562 were not affected by the serum concentration under N/P 2.5, but the efficiency was decreased with the increase of serum concentration in HELA cells. The expression of Ps/PGL 4.51 was the highest in K562 cells.Conclutions:The mass ratio of serum to cationic polysaccharide plays an important role in regulating the particle size and the ability to enter cells of the RNAi complex, which affect the efficiency of RNAi. Ps can be used as gene vector in CML cells especially K562 cells with low cytotoxicity. We provided a new approach for treatment of CML. |
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