1. Introduction
Breast cancer is the most common
type of cancer, and at the same time, it has the second-highest
mortality rate in women worldwide. Statistically, breast cancer affects
about 100 out of 100,000 women in the United States each year, resulting
in a total of more than 230,000 new patients.[1]Currently, various treatments, such as chemotherapy and hormonal
therapy, are being used to treat breast cancer or prevent a recurrence.
Still, they exhibit drug resistance and can cause non-specific cell
damage.[2,3] Furthermore, chemotherapy’s side
effects include fatigue, diarrhea,
constipation.[4] In addition, hormonal therapy is
limited because it cannot be used in about 30% of
women.[5] Recently, treatment methods that
alleviate systemic toxicity are being studied in chemotherapy and
hormonal therapy. However, there are still reports of side effects such
as nausea, mouth ulcers, and mild cognitive impairment, continuing
concerns about toxicity.[6] For these reasons, it
is necessary to develop a new anticancer drug that is effective without
side effects.
siRNAs are double-stranded synthetic RNAs composed of 21-23 base pairs
and can become a component of the RISC complex, which could cut the
desired mRNA sequence in the cells.[7,8] Due to
specific gene silencing and minimal side effects, siRNAs are currently
being studied to treat various diseases and effectively kill cancer
cells by knocking down the expression of
oncogenes.[9] The FDA has approved three siRNA
drugs of patisiran in 2018, givosiran in 2019, and lumasiran in
2020.[10–12] However, siRNAs are difficult to
deliver into cells due to their strong negative charge and are easily
degraded by nucleases. Therefore, an effective and stable delivery
technique is required for siRNA delivery.
siRNA delivery methods are majorly divided into physical and chemical
methods. First, physical methods included electroporation,
iontophoresis, sonophoresis, magnetofection, gene gun, and laser beam
transduction. They showed high delivery efficiency of nucleic acids.
However, special equipment is
required.[13] Second, chemical methods include
polymer-based, liposome-based, and peptide-mediated carriers. They have
been recognized for their delivery efficiency, but polymer-based
carriers presented clinical difficulties due to their high
toxicity.[14,15] Also, liposome-based carriers
showed low toxicity, instability, drug leakage, and difficult
sterilization due to sensitivity to high
temperature.[16,17]
Peptide-mediated carriers have been studied since cell-penetrating
peptides were first discovered using phage display in
1988.[18,19] Nowadays, cell-penetrating peptides
(CPP) are widely used due to their advantages of excellent penetrating
ability, easy conjugation, imparting additional functions depending on
the amino acid sequence, and relatively low
cytotoxicity.[20] The most frequently used CPPs
include transportan, polyarginine, antennapedia, and TAT. Among them,
polyarginine, an analog of TAT, could penetrate the cellular membrane
most efficiently with relatively low toxicity.[21]Furthermore, because it has a positive charge, it neutralizes the
negative charge of nucleic acids and facilitates intracellular transfer
through hydrogen bonding with the cell
membrane.[22–25] However, polyarginine-based
carriers cannot deliver drugs targeting certain cell types. So, the
non-targeted delivery of cytotoxic drugs can cause various side effects,
i.e., damage to normal tissues. Therefore, a new targeted delivery
method needs to be developed to maximize the therapeutic efficacy and
minimize side effects.
Homing peptides can become a potent ligand for specific delivery due to
the affinity to particular proteins on the cell membrane surface. Among
them, TT1 and SP82 homing peptides were reported as specific to breast
cancer cells. TT1 peptide was screened through the M13 phage display
technique using the peptide library of cyclic nine amino
acids.[26] The TT1 was revealed to bind the p32
receptor on breast cancer cells’ surfaces. Next, protease could cut the
front part of the c-terminus serine of the peptide, leaving a C-end Rule
(or CendR) motif. Then, the cleaved TT1 could be delivered into the
cells through the NRP-1 receptor-mediated endocytosis. And, the SP82
peptide was screened specifically to breast cancer cells through the M13
phage display technique. In addition, the SP82 endocytosis mechanism
still needs to be revealed.[27] Furthermore, these
homing peptides need a conjugation strategy to use for the specific
delivery.
This study developed the nanocomplex-mediated siRNA delivery based on
novel fusion peptides targeting breast cancer cells (Fig. 1). The homing
peptide (SP82 and TT1) was conjugated to oligoarginine
(R11) with a linker (GGGG), respectively (Additional
file 1: Table S1). The oligoarginine with a positive charge could
spontaneously form nanocomplexes (NCs) with siRNAs with electrostatic
attraction. The formed NCs were characterized in size, surface charge,
and stability. The delivery efficiency and specificity of NCs were
quantitatively analyzed using flow cytometry. The cellular
internalization of NCs was confirmed using fluorescence z-stack imaging.
The efficacy of delivered siRNA was confirmed using mRNA gene silencing
and viability test of breast cancer cells. Finally, the biocompatibility
of RS-NC was assessed by histological analysis and cytokine ELISA of
intradermally injected mouse skin.