Full
Outline
To construct an engineered milk-derived exosome (mExos) vector for efficient messenger RNA (mRNA) delivery to the respiratory tract by systematically screening cationic modification materials.
mExos were isolated via ultracentrifugation, followed by electroporation-mediated loading of firefly luciferase (Fluc) or enhanced green fluorescent protein (EGFP) mRNA. Chitosan (CS), polyethyleneimine (PEI), poly(beta-amino ester) (PBAE), cationic lipid (DOTAP), and protamine were used to modify mRNA-loaded mExos. Dynamic light scattering, nanoparticle tracking analysis, flow cytometry and luciferase reporter assay were employed to evaluate particle size, zeta potential, colloidal stability, cellular uptake, transfection efficiency, and cytotoxicity. Fifty female BALB/c mice (6 to 8 weeks, about 20 g) received intranasal administration of dye-labeled or Fluc-mRNA-loaded formulations. In vivo distribution and pulmonary transfection kinetics were monitored by IVIS imaging, while histopathology and serum biochemistry of major organs were analyzed at 24 h post-administration.
Cationic-modified mExos were successfully constructed. CS-modified mExos (CS-mExos) demonstrated surface charge reversal while maintaining optimal size and stability (PDI unchanged within 48 h). In vitro, CS-mExos enhanced cellular uptake in 16HBE and A549 cells by 2.56-fold (P<0.001) and 4.56-fold (P<0.001), respectively, and increased Fluc-mRNA transfection efficiency by 28.9-fold (P<0.001) and 26.5-fold (P<0.001) versus unmodified mExos, with >95% cell viability. In vivo imaging revealed CS-mExos specifically accumulated in the lungs with sustained retention >72 h, mediating 9.5-fold higher peak Fluc-mRNA expression at 6 h post-administration. Histopathology and serum biochemistry confirmed no significant organ damage or abnormal hepatic/renal indices.
Systematic screening identified chitosan as the optimal cationic material for engineering milk exosomes. The developed CS-mExos vector enables efficient targeted mRNA delivery to the respiratory tract with enhanced pulmonary transfection and favorable biosafety.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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