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Direct cytosolic delivery of siRNA via cell membrane fusion using cholesterol-enriched exosomes

Nature Nanotechnology volume 19, pages 1858–1868 (2024)Cite this article

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Abstract

Efficient cytosolic delivery is a significant hurdle when using short interfering RNA (siRNA) in therapeutic applications. Here we show that cholesterol-rich exosomes are prone to entering cancer cells through membrane fusion, achieving direct cytosolic delivery of siRNA. Molecular dynamics simulations suggest that deformation and increased contact with the target cell membrane facilitate membrane fusion. In vitro we show that cholesterol-enriched milk-derived exosomes (MEs) achieve a significantly higher gene silencing effect of siRNA, inducing superior cancer cell apoptosis compared with the native and cholesterol-depleted MEs, as well as conventional transfection agents. When administered orally or intravenously to mice bearing orthotopic or subcutaneous tumours, the cholesterol-enriched MEs/siRNA exhibit antitumour activity superior to that of lipid nanoparticles. Collectively, by modulating the cholesterol content of exosome membranes to facilitate cell entry via membrane fusion, we provide a promising approach for siRNA-based gene therapy, paving the way for effective, safe and simple gene therapy strategies.

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Fig. 1: The CGMD simulations of effects of cholesterol concentration on the interactions between exosomes and cell membranes.
Fig. 2: Characterization of MEs with varying cholesterol incorporation.
Fig. 3: MEs with varying cholesterol levels enter cells via different internalization pathways.
Fig. 4: SiPLK1-loaded MEs effectively inhibited the growth of HCT116 cells in vitro.
Fig. 5: SiPLK1-loaded MEs effectively to eradicate subcutaneous HCT116 colorectal tumour-bearing mice.
Fig. 6: SiPLK1-loaded MEs effectively to inhibit orthotopic HCT116 colorectal tumour-bearing mice.

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Data availability

All data supporting the findings of this study are included in the article and the Supplementary Information. There are no data from third-party or publicly available datasets. All data generated as part of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We sincerely appreciate the financial support from the National Science Fund of Distinguished Young Scholars (82025032), the National Key Research and Development Program of China (2022YFA1203200), the National Natural Science Foundation of China (82073773), the Young Elite Scientists Sponsorship Program by CAST (2022QNRC001), the Key Research Program of Chinese Academy of Sciences (ZDBS-ZRKJZ-TLC005), the ‘Open Competition to Select the Best Candidates’ Key Technology Program for Nucleic Acid Drugs of NCTIB (NCTIB2022HS01006), the Shanghai Action Plan for Science, Technology and Innovation (23HC1401200) and the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (SIMM0220232001). We thank the staff members of the Integrated Laser Microscopy System, the Electron Microscopy System, the Molecular Imaging System and the Large-scale Protein Preparation System at the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sciences, China, for sample preparation, data collection and analysis. The MD simulations were performed on BSCC-A6 at the Beijing Super Cloud Computing Center. We are grateful to G. Li, Y. Yu, F. Gong and F. Liu for cryo-TEM data collection, cell imaging and AFM data collection, respectively.

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  1. These authors contributed equally: Yan Zhuo, Zhen Luo.

Authors and Affiliations

  1. State Key Laboratory of Drug Research and Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Yan Zhuo, Zhu Zhu, Jie Wang, Xiang Li, Zhuan Zhang, Cong Guo, Bingqi Wang, Di Nie, Yong Gan & Miaorong Yu

  2. School of Pharmacy, Jiangxi Medical College, Nanchang University, Nanchang, China

    Yan Zhuo

  3. Department of Engineering Mechanics, State Key Laboratory of Fluid Power and Mechatronic Systems, Zhejiang University, Hangzhou, China

    Zhen Luo & Guoqing Hu

  4. School of Pharmacy, Henan University, Kaifeng, China

    Zhu Zhu

  5. School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China

    Jie Wang

  6. University of Chinese Academy of Sciences, Beijing, China

    Xiang Li, Cong Guo, Bingqi Wang, Di Nie, Yong Gan & Miaorong Yu

  7. NMPA Key Laboratory or Quality Research and Evaluation of Pharmaceutical Excipients, National Institutes for Food and Drug Control, Beijing, China

    Yong Gan

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Contributions

M.Y., G.H., Y.G., Y.Z. and Z.L. planned and executed the experiments, analysed the data and were involved in discussions of the data. M.Y., G.H., Y.Z., Z.L. and Z. Zhu wrote the paper. Y.Z., Z.L., Z. Zhu, J.W., X.L., Z. Zhang, C.G., B.W. and D.N. performed the experiments. All authors critically reviewed and approved the paper.

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Correspondence to Yong Gan, Guoqing Hu or Miaorong Yu.

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Supplementary information

Supplementary Information

Supplementary Discussion, Methods, Table 1, Figs. 1–22 and References.

Reporting Summary

Supplementary Video 1

Uptake of 5%Chol/MEs in HCT116 cells.

Supplementary Video 2

Uptake of 30%Chol/MEs in HCT116 cells.

Supplementary Video 3

3D distribution of 5%Chol/MEs in HCT116 cells.

Supplementary Video 4

3D distribution of 30%Chol/MEs in HCT116 cells.

Supplementary Data 1

Uncropped and unprocessed scans for Supplementary Fig. 2.

Supplementary Data 2

Uncropped and unprocessed scans for Supplementary Fig. 4.

Source data

Source Data Fig. 1

Statistical source data for Fig. 1a,c,e,f,i.

Source Data Fig. 2

Statistical source data for Fig. 2b,c,f,h–j.

Source Data Fig. 3

Statistical source data for Fig. 3c,e,f.

Source Data Fig. 4

Statistical source data for Fig. 4b–d,f.

Source Data Fig. 4

Unprocessed western blots for Fig. 4c.

Source Data Fig. 5

Statistical source data for Fig. 5b,c,e.

Source Data Fig. 6

Statistical source data for Fig. 6b,d,e.

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Zhuo, Y., Luo, Z., Zhu, Z. et al. Direct cytosolic delivery of siRNA via cell membrane fusion using cholesterol-enriched exosomes. Nat. Nanotechnol. 19, 1858–1868 (2024). https://doi.org/10.1038/s41565-024-01785-0

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