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.