siRNA转染试剂的全面对比研究

doi:10.5246/jcps.2024.01.003

中国药学(英文版) ›› 2024, Vol. 33 ›› Issue (1): 26-34.DOI: 10.5246/jcps.2024.01.003

siRNA转染试剂的全面对比研究

佟淑文¹^,³^,⁴, 齐宪荣¹^,²^,^*()

^1. 北京大学药学院药剂学系, 北京 100191
^2. 北京大学药学院分子药剂学与新释药系统北京市重点实验室, 北京 100191
^3. 北京大学前沿交叉学科研究院, 北京 100871
^4. 北京多纳医药科技有限公司, 北京 102206

收稿日期:2023-10-23 修回日期:2023-11-15 接受日期:2023-12-09 出版日期:2024-01-31 发布日期:2024-01-31
通讯作者: 齐宪荣

A comprehensive comparative analysis of transfection reagents for siRNA delivery

Shuwen Tong¹^,³^,⁴, Xianrong Qi¹^,²^,^*()

¹ Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
² Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery System, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
³ Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
⁴ D-Nano Therapeutics Inc. Beijing 102206, China

Received:2023-10-23 Revised:2023-11-15 Accepted:2023-12-09 Online:2024-01-31 Published:2024-01-31
Contact: Xianrong Qi
Supported by:
"Open Competition to Select the Best Candidates" Key Technology Program for Nucleic Acid Drugs of NCTIB (Grant No. NCTIB2022HS01001), Beijing Natural Science Foundation (Grant No. L202044).

摘要/Abstract

摘要：

siRNA已经成为生物研究的常用工具, 也是目前最具潜力的下一代基因治疗药物。将siRNA成功转染进入细胞, 仍然是限制siRNA广泛应用的关键技术, 对于难转染细胞尤其如此。siRNA转染效率是摄取、细胞利用率及细胞毒性的综合作用结果, 更高的转染效率通常需要高摄取、高细胞利用率和低细胞毒性。本研究旨在比较不同的转染试剂递送siRNA的摄取、siRNA敲低效率和毒性情况。新开发的CALNP RNAi转染试剂与传统转染试剂的摄取行为不同, 转染效率显著高于传统转染试剂, 同时毒性最低。CALNP RNAi转染试剂为难转染细胞的RNA干扰带来了新的选择。

关键词: 转染试剂, siRNA, RNA干扰, 难转染细胞

Abstract:

siRNAs have emerged as essential tools in biological research and hold promise as next-generation therapeutics for gene therapy. However, achieving successful and safe siRNA transfection remains a challenge, especially for cells that are notoriously difficult to transfect. The efficiency of siRNA transfection is influenced by factors such as uptake, cellular availability, and cytotoxicity. Generally, higher transfection efficiency is associated with increased uptake, improved cellular availability, and reduced cytotoxicity. This study aimed to compare the performance of different transfection reagents in delivering siRNAs, focusing on their uptake, knockdown efficacy, and toxicity. All reagents measured in here were good uptaken in HepG2 and HEK293T cells, but low uptake in RAW264.7 cells. Among them, the newly developed CALNP RNAi transfection reagent exhibited distinct uptake profiles when compared to other transfection reagents across various tested cell lines. CALNP RNAi consistently demonstrated superior transfection efficiency and minimal cell toxicity in all the tested cell lines. Furthermore, CALNP RNAi held promise for enabling RNA interference in "hard-to-transfect" cells. This research contributed to our understanding of effective siRNA delivery methods and highlighted the potential of CALNP RNAi transfection reagent as a valuable tool for gene silencing, especially in challenging cellular contexts.

Key words: Transfection reagent, siRNA, RNA interfering, "Hard-to-transfect" cells

Supporting:

佟淑文, 齐宪荣. siRNA转染试剂的全面对比研究[J]. 中国药学(英文版), 2024, 33(1): 26-34.

Shuwen Tong, Xianrong Qi. A comprehensive comparative analysis of transfection reagents for siRNA delivery[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(1): 26-34.

图/表 11

Figure 1. The percentage of Cy3-positive cells and MFI at 4 h after incubation with different Cy3-siRNA-transfection mixtures. (A) HepG2; (B) HEK-293T; (C) RAW264.7; 1) 10 nM Cy3-siRNA; 2) 2 nM Cy3-siRNA; 3) Cy3+ cells % and MFI values.

Figure 2. The percentage of Cy3-positive cells and MFI at 24 h after incubation with different Cy3-siRNA-transfection mixtures. (A) HepG2; (B) HEK-293T; (C) RAW264.7; 1) 10 nM Cy3-siRNA; 2) 2 nM Cy3-siRNA; 3) Cy3+ cells % and MFI values.

Figure 3. GAPDH mRNA level of HepG2 cells transfected with different transfection reagents (3 × 104 cells/well; hGAPDH-siRNA 10 and 2 nM; n = 3; compared with the same concentration of siRNA-CALNP RNAi, **P < 0.01, ***P < 0.001).

Figure 4. GAPDH mRNA level of HepG2 cells transfected with different transfection reagents (1 × 105 cells/well; hGAPDH-siRNA 10 and 2 nM; n = 3; compared with the same concentration of siRNA-CALNP RNAi, **P < 0.01, ***P < 0.001).

Figure 5. GAPDH mRNA level of HEK293T cells transfected with different transfection reagents (3 × 104 cells/well; hGAPDH-siRNA 10 and 2 nM; n = 3; compared with the same concentration of siRNA-CALNP RNAi, **P < 0.01, ***P < 0.001).

Figure 6. GAPDH mRNA level of HEK293T cells transfected with different transfection reagents (1 × 105 cells/well; hGAPDH-siRNA 10 and 2 nM; n = 3; compared with the same concentration of siRNA-CALNP RNAi, **P < 0.01, ***P < 0.001).

Figure 7. GAPDH mRNA level of RAW264.7 cells transfected with different transfection reagents (3 × 104 cells/well; mGAPDH-siRNA 10 and 2 nM; n = 3; compared with the same concentration of siRNA-CALNP RNAi, **P < 0.01, ***P < 0.001).

Figure 8. GAPDH mRNA level of RAW264.7 cells transfected with different transfection reagents (1 × 105 cells/well; mGAPDH-siRNA 10 and 2 nM; n = 3; compared with the same concentration of siRNA-CALNP RNAi, ***P < 0.001).

Figure 9. Cell viability of HepG2 cells transfected with different transfection reagents (1 × 105 cells/well; corresponding siRNA concentration 50 and 10 nM; n = 3; compared with the PBS group, *P < 0.05, **P < 0.01).

Figure 10. Cell viability of HEK293T cells transfected with different transfection reagents (1 × 105 cells/well; corresponding siRNA concentration 50 and 10 nM; n = 3; compared with the PBS group, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 11. Cell viability of RAW264.7 cells transfected with different transfection reagents (1 × 105 cells/well; corresponding siRNA concentration 50 and 10 nM; n = 3; compared with the PBS group, *P < 0.05, **P < 0.01).

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siRNA转染试剂的全面对比研究

A comprehensive comparative analysis of transfection reagents for siRNA delivery

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