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).