4. Discussion
Novel fusion peptides were designed using SPACE and oligo-arginine and evaluated for siRNA delivery with a self-assembled complex. The newly-designed fusion peptides successfully formed a self-assembled complex stably with siRNAs via electrostatic interaction. All fusion peptide complex enhanced siRNA penetration similar to or better than that of commercialized Lipofectamine™ 2000. Among fusion peptides, the S-R15 complex induced the highest knockdown of GAPDH mRNA expression comparable to that of commercialized Lipofectamine™ 2000. This mRNA knockdown by S-R15 could be affected by uniform small size and stability of complex because the stability of complex increased as arginine residues increase. Co-localization and cellular internalization of siRNA/S-R15 complexes were verified peripherally around the nucleus. The primary penetration mechanism of the S-R15 complex was identified as lipid raft-mediated endocytosis. Also, each fusion peptide was biocompatible to human dermal fibroblast cells under a concentration of 200 μg/mL.
First, R11 and fusion peptides formed a self-assembled and condensed complex with siRNAs based on complete retardation of the siRNA band. In contrast, SPACE and TAT did not form a condensed complex with siRNA based on almost no retardation of the siRNA band. These results coincided with the result using oligo arginine (Kim et al., 2010) and indicated that a positively charged region with high-density was crucial to forming a complex with siRNA. Using amino acid analysis via the peptide 2.0 web (www.peptide2.com), percentages of positive basic residues were R11 of 100%, SPACE peptide of 9.1% among 11 amino acids, TAT peptide of 88.9% basic residue among nine amino acids. As a result, successive positive charge and longer length of R11 was supposed to interact with negatively charged siRNAs sufficient to form a stable complex. For fusion peptides, percentages of positive basic residues were S-R7 of 38.1%, S-R11 of 48%, and S-R15 of 55.2%. The successive and longer positive charge of the peptide could increase the electrostatic interaction with the negative charge of siRNA and result in charge neutralization. Moreover, the long length of fusion peptides was favorable to form and maintain condensed complexes with siRNA via van der Waals or hydrophobic interactions.
Interestingly, the S-R15 peptide formed the smallest and uniform self-assembled complex with weakly positive zeta potential based on the light scattering analysis. Other fusion peptide complexes had small and medium-size, respectively, with acceptable PdI and weak positive zeta potential. Weakly positive zeta potential of three fusion peptide complexes could mean that positively charged fusion peptides covered the surface of the complex with siRNA. Furthermore, slightly positive zeta potential and uniform size could become favorable to cellular internalization of the complex (Kim et al., 2003). Also, the R11 complex had a medium size, acceptable PdI, and negative zeta potential. Its negative zeta potential could mean that R11 peptide did not completely shield the negative charge of siRNA on the complex surface. In contrast, the SPACE peptide appeared to fail to form a condensed complex based on the large size, high polydispersity index, and highly negative zeta potential. Consequently, the strategy of fusion peptides enabled to form self-assembled complexes of the proper size, PdI, and weakly positive zeta potential, which could be expected to enhance the cellular internalization.
S-R15 formed the most stable complex, which might show increased stability of cellular uptake. The stability of the S-R15 complex could be affected by charge neutralization and cohesive strength caused by the electrostatic interaction between peptide and siRNA (Tai and Gao, 2017). That is, long and successive positive charge residues of peptides could shield negative charge of siRNA effectively as well as interact with other siRNAs. Therefore, due to long positive charged residues, S-R15 could neutralize the negative charge of siRNA and interact with other siRNAs most effectively. Besides, SPACE part of S-R11 enhanced complex stability compared to that of R11 peptide. This enhanced stability might be affected by hydrophobic and van der Waals interactions.
Co-localized particles and dissociated spread of siRNA/S-R15 complexes were distributed peripherally around the nucleus. Co-localization of the complex was identified via white fluorescence merged between siRNA of magenta fluorescence and S-R15 of green fluorescence. The co-localized siRNA/S-R15 complexes were distributed peripherally around the nucleus in particle-like forms within the cytoplasm. Particle-like forms might represent endosomes containing complexes. This co-localization supported the complex formation. On the other hand, the spread of magenta fluorescence might indicate siRNAs dissociated from complexes after the endosomal escape. These results coincided with those of previous reports (Chiu et al., 2004; Kim et al., 2010; Wang et al., 2014; Wang et al., 2007).
GAPDH mRNA knockdown by the S-R15 complex showed the highest reduction among the three fusion peptides. This result could be affected by the synergistic effect of cellular uptake efficiency and complex stability. That is, high cellular uptake efficiency and higher stability of the S-R15 complex could enable prolonged cellular uptake compared to those of other complexes. Resultantly, prolonged cellular uptake could enhance the knockdown of the GAPDH mRNA expression. As a result, the increase of arginine length enhanced complex stability. Enhance complex stability could affect increased efficiencies of cellular uptake and mRNA knockdown.
The endocytosis pathway of S-R15 peptide agrees with not the micropinocytosis of SPACE peptide (Hsu and Mitragotri, 2011) but the clathrin-mediated endocytosis of arginine-rich peptides (Schmidt et al., 2010). This result indicated that S-R15 peptide could dominantly bind a specific cell surface receptor resulting in the clustering formed by the assembly of clathrin (Schmidt et al., 2010). On the other hand, the endocytosis pathway of the siRNA/S-R15 complex was different from that of S-R15 peptide only. The siRNA/S-R15 complex associated with the cell membrane and then became trapped in lipid raft. The possible reasons could be the large size of the S-R15 complex and weak binding to a specific cell surface receptor caused by strong interactions between oligo-arginine and siRNA. This result coincides with the endocytosis pathway of arginine-rich peptide fusion proteins or large cargo (Jones et al., 2005).