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.