Physicochemical characterization of mRNA-encapsulated NPs and RNA loading efficiency study

Physicochemical characterization of mRNA-encapsulated NPs and RNA loading efficiency study

The hydrodynamic diameter and morphological structure of mRNA-encapsulated NPs were determined by DLS (Brookhaven Instruments Corporation) and TEM [Tecnai G2 Spirit BioTWIN (FEI Company)], respectively. 

1. For TEM, a small drop of NPs (about 10 µl) is pipetted onto a TEM grid and simply allowed to dry at room temperature. The grid were then stained with 1% uranyl acetate for 1 min and washed with pure water. Finally the grid observed in a TEM once the medium is evaporated. 
2. To measure the hydrodynamic diameter of NPs, the NPs samples were first passed through syringe filters (0.45 μm, Millipore) and then detected by DLS (Brookhaven Instruments Corporation).

To check the long-term in vitro stability of mRNA-encapsulated NPs, the NPs were incubated in PBS and cell culture medium solution at 37°C, and their hydrodynamic diameters were measured at various points (0, 2, 4, 8, 12, 24, and 48 hours). 

To measure the mRNA complexation ability of cationic G0-C14 and its loading efficiency, naked EGFP mRNA and EGFP mRNA complexed with different doses of G0-C14 (ratio of weight from 3.2 to 25) are prepared as follows,

1), Dilute 1 µg EGFP mRNA in RNase free water (20 µl). 

2), Add G0-C14 (in varying weight ratios from 3.2 to 25 to mRNA) to diluted EGFP mRNA and incubated for 20 min.

3), Add loading dye (Invitrogen) to G0-C14/mRNA mixture.

4), The volumes of samples were run into an E-gel 2% (w/v) agarose (Invitrogen). Electrophoresis was performed at 70 V for 30 min.

5), The gel was imaged under ultraviolet (UV) light and the bands were analyzed using ImageJ software. 

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