4. Discussion
This research has developed
an NC-mediated targeted siRNA delivery method using novel RS and RT
fusion peptides (Fig. 1). First, RS and RT fusion peptides were composed
of polyarginine peptide interacting siRNAs, a linker, and the SP82 and
TT1 peptides targeting breast cancer cells in series, respectively.
Then, RS and RT peptides electrostatically attracted siRNAs and formed
self-assembled NCs (Fig. 2). Especially, RS-NCs penetrated breast cancer
cells selectively and highly efficiently (Fig. 3&4). In addition,
RS-NC-delivered siRNAs effectively inhibited breast cancer cells (Fig.
6). These results implied the synergistic effect of NC-forming and
membrane-penetrating abilities of poly-arginine and selectively binding
to breast cancer cells of SP82 peptide. Moreover, improved penetration
could be explained by the combinatorial effect of proper size, evenness,
weakly positive surface charge, high stability, and high
biocompatibility of the RS-NC (Fig. 3).
The different cell-penetrating efficiencies between RS- and RT-NCs could
be caused by the inherent property of SP82 and TT1 peptides and the
difference of formed NCs. First, the SP82 peptide had more
hydrophobicity than the TT1 peptide. The RS peptide had phenylalanine,
glycine, alanine, isoleucine, and three leucines. On the other hand, the
RT peptide consists of glycine and three alanines. Therefore, the
hydrophobicity of RS peptides could form NC more compactly after charge
neutralization. It was also reported that peptides’ hydrophobicity could
increase cell penetration efficiencies.[22]Moreover, the predicted alpha-helical structure of RS peptide and
hydrophobicity could help to interact with membrane and escape from
endosome (Figs. S5 and S6).[23]
Furthermore, the hydrophobicity of RS peptides also affected the size
and evenness of NCs when an electrical charge was neutralized. For
example, the size and PdI of RS-NC increased and decreased as the N/P
ratio increased. On the other hand, the size and PdI of RT-NCs decreased
as the N/P ratio increased. This result indicated that electrical
attraction is major interaction when the N/P ratio is low. However, as
the N/P ratio increases, hydrophobic interaction between peptides or
siRNAs becomes more significant when an electrical charge is
neutralized. Therefore, when the N/P ratio increased over 5, the RS
peptide’s hydrophobic interaction could help from more compact NCs,
favorable for cell penetration.[29]
Second, hydrogen bonding of RS peptides could affect the NC formation
and cell penetration. For example, in Fig. 2, the RS peptide formed more
stable and compact NCs than the RT peptide. These compact and stable NCs
could increase cell penetration efficiencies. First, RS peptide had more
hydrogen bonding groups, including threonine (hydroxyl), glutamine
(amino), tyrosine (hydroxyl), glutamic acid (carboxylate), than RT
peptide’s serine (hydroxyl) and threonine (hydroxyl). Therefore, RS
peptides could form more hydrogen bonding with siRNAs, and hydrogen
bonds could stabilize NCs. Second, hydroxyl, amino, carboxylic, and
guanidinium groups of peptides were known to form hydrogen bonds with
phosphate of nucleic acid or cellular
membrane.[24,30–32] Therefore, hydrogen bonding
of RS peptides could increase interactions with membrane and
cell-penetrating efficiencies. Also, hydrogen bonding and hydrophobicity
of RS peptides increase the stability, improving cellular penetration
efficiency. NC stability test indicated RS-NCs are more stable than
R11- or RT-NCs (Fig. S2, Table S3). The extended
stability of RS-NCs also contributes to cell penetration.
The endocytosis pathways of RS-NC were revealed as lipid raft-mediated,
clathrin-mediated endocytosis, micropinocytosis, and caveolin-mediated
endocytosis in high order (Fig. 5). Generally, endocytosis pathways had
a size range for effective working such as lipid raft-mediated
endocytosis for particles less than 150 nm, clathrin-mediated
endocytosis for particles less than 200 nm, micropinocytosis for
particles over 1,000 nm, and caveolin-mediated endocytosis for particles
less than 90 nm.[33,34] Thus, because the size
distribution of RS-NCs ranged from 60 nm to 1,000 nm (Fig. S3), RS-NCs
could be uptaken through four endocytosis pathways. Also, smaller NCs
could become dissociated from big NCs. Moreover, the specificity of
RS-NCs could be explained by the interaction between SP82 and specific
surface receptors on breast cancer cells.[26,27]
Biosafety of NC was proved by maintaining the cell viability of RS
peptide and that intradermally injected RS-NC did not induce tissue
damage or inflammatory cell infiltration. Also, intradermally injected
RS-NC did not raise TNF-alpha and IL-5 cytokine levels, causing
inflammation. As a result, the RS-NC showed proper biocompatibility.