Development of an Organ-Directed Exosome-Based siRNA-Carrier Derived from Autologous Serum for Lung Metastases and Testing in the B16/BL6 Spontaneous Lung Metastasis Model
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Purification and Identification of Exosomes
2.3. Preparation of siRNA-Loaded Exosomes
2.4. Cell Culture
2.5. Measuring GPC3 Levels in B16/BL6 Cells Treated with siRNA-Loaded Exosomes Derived from Serum
2.6. Cell Viability Assay
2.7. Mice
2.8. The B16/BL6 Spontaneous Lung Metastasis Mouse Model
2.9. Distribution of Exosomes and siRNA Delivered by Exosomes in the B16/BL6 Spontaneous Lung Metastasis Mouse Model
2.10. Measuring the Anti-Metastatic Effects of siRNA-Loaded Exosomes in the B16/BL6 Spontaneous Lung Metastasis Mouse Model
2.10.1. Evaluation of Exosome Collection Time to Use for siRNA Carrier
2.10.2. Effect of Survival Rate and Anti-Metastasis Effects of siRNA Loaded Exosome Compared with Dacarbazine or Anti PD-1 Antibody
2.11. Western Blot Analysis
2.12. Statistical Analysis
3. Results
3.1. Size Distribution and Detection of Exosome Markers in Exosomes Isolated from Serum
3.2. Measuring Cell Permeability by Flow Cytometry
3.3. GPC3 Expression in B16/BL6 Cells Treated with siRNA-Loaded Exosomes
3.4. Cell Viability Assay
3.5. Protein Levels in Exosomes
3.6. Anti-Metastatic Effects of siRNA-Loaded Exosomes in the B16/BL6 Spontaneous Lung Metastasis Mouse Model
3.7. Localization of ICG-Labeled Exosomes in the B16/BL6 Spontaneous Lung Metastasis Mouse Model
3.8. Localization of Alexa 647-Labeled siRNA-Loaded Exosomes in the B16/BL6 Spontaneous Lung Metastasis Mouse Model
3.9. Survival Rate and Anti-Metastatic Effects of siRNA-Loaded Exosomes in the B16/BL6 Spontaneous Lung Metastasis Mouse Model Compared with Dacarbazine and Anti-PD-1 Antibody
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Hazekawa, M.; Nishinakagawa, T.; Hosokawa, M.; Ishibashi, D. Development of an Organ-Directed Exosome-Based siRNA-Carrier Derived from Autologous Serum for Lung Metastases and Testing in the B16/BL6 Spontaneous Lung Metastasis Model. Pharmaceutics 2022, 14, 815. https://doi.org/10.3390/pharmaceutics14040815
Hazekawa M, Nishinakagawa T, Hosokawa M, Ishibashi D. Development of an Organ-Directed Exosome-Based siRNA-Carrier Derived from Autologous Serum for Lung Metastases and Testing in the B16/BL6 Spontaneous Lung Metastasis Model. Pharmaceutics. 2022; 14(4):815. https://doi.org/10.3390/pharmaceutics14040815
Chicago/Turabian StyleHazekawa, Mai, Takuya Nishinakagawa, Masato Hosokawa, and Daisuke Ishibashi. 2022. "Development of an Organ-Directed Exosome-Based siRNA-Carrier Derived from Autologous Serum for Lung Metastases and Testing in the B16/BL6 Spontaneous Lung Metastasis Model" Pharmaceutics 14, no. 4: 815. https://doi.org/10.3390/pharmaceutics14040815