## 3 Results and Discussion
### In-Cell Expression of mCherry mRNA Encapsulated in Artificial Viral Capsid  
Uptake of mCherry mRNA-encapsulated capsids into human hepatoma HepG2 cells were analyzed by CLSM to evaluate mCherry mRNA expression inside cells. mCherry mRNA and a strong transfection reagent (TransIT-mRNA) were incubated with the cells for 48 h at 37 \({}^{\circ}\)C as a positive control; significant red fluorescence of mCherry was observed (Figure 7(Aa)). Unfortunately,  
Figure 5: (**A**) Size-distributions obtained from DLS and (**B**) TEM images for co-assembly peptides (dT\({}_{20}\)-SS-\(\beta\)-annulus:\(\beta\)-annulus = 1:9) and mCherry mRNA in PBS (pH 7.4) obtained after incubation at 25 \({}^{\circ}\)C.  
Figure 6: (**A**) Illustration of digestion of mRNA by RNase A: (**a**) mRNA alone, (**b**) mRNA-encapsulated artificial viral capsid, and (**c**) Mixture of mRNA and capsid modified with dT20 at the exterior. (**B**) Gel electrophoresis assay of digestions mRNA by RNase A at 37 \({}^{\circ}\)C for 10 min (mRNA alone and \(\beta\)-annulus-dT\({}_{20}\)) and 10–60 min (dT\({}_{20}\)-SS-\(\beta\)-annulus:\(\beta\)-annulus = 1:9 co-assembly). The gel was stained with GelRed.  
minimal expression of encapsulated mCherry mRNA was observed as similar to the naked mCherry mRNA(Figure 7A(b,c)). We employed a histidine 16-mer (His16), a cell-penetrating peptide uncharged under physiological conditions [36; 37] to enhance cell permeability of capsids. We constructed artificial viral capsids modified with His16 on the exterior and using the 1:9 co-assembly of dT\({}_{20}\)-SS-\(\beta\)-annulus peptide and \(\beta\)-annulus peptide modified with His16 at the C-terminal (\(\beta\)-annulus-His16). Significant red fluorescence of mCherry was observed in cells incubated with His16-modified capsids (Figure 7(Ad)). The expression level of mCherry was quantified by fluorescence intensity in the cell areas of the CLSM image using the software ImageJ. The expression level using His16-modified capsids was 26.5 times the expression observed with unmodified capsids (Figure 7B). This indicates that the intracellular expression of mCherry was significantly enhanced by the modification of His16 on the surface of the artificial viral capsid. We have demonstrated in our previous work that the reductive cleavage of the disulfide bond of the artificial viral capsid by dithiothreitol (DTT) caused the controlled-release of ssDNA with the destruction of the capsid [35]. mCherry mRNA was likely released from capsids in the reducing environment in cells.  
Figure 7: **(A)** Confocal laser scanning microscopy (CLSM) images of HepG2 Cells transfected with mCherry mRNA encapsulated in artificial viral capsids: **(a)** positive control (2 \(\upmu\)M TransIT-mRNA with 1 \(\upmu\)M mCherry mRNA), **(b)** 1 \(\upmu\)M Naked mCherry mRNA, **(c)** mRNA-encapsulated artificial viral capsid (50 \(\upmu\)M dT\({}_{20}\)-SS-\(\beta\)-annulus, 450 \(\upmu\)M \(\beta\)-annulus, and 1 \(\upmu\)M mCherry mRNA), and **(d)** mRNA-encapsulated histidine 16-mer (His16)-artificial viral capsid (50 \(\upmu\)M dT\({}_{20}\)-SS-\(\beta\)-annulus, 450 \(\upmu\)M \(\beta\)-annulus-His16, and 1 \(\upmu\)M mCherry mRNA). **(B)** Relative fluorescence intensity of mCherry from HepG2 Cells.