Protein and peptide candidates are screened to apply therapeutic application as a drug. Ensuring that these candidates are delivered and maximized effectiveness is still challenging and a variety of studies are ongoing. Even if predicted as a good candidate, various barriers are encountered in the biological environment because of the drug’s physicochemical properties (Komin et al., 2017). Therefore, drug delivery systems (DDSs) maximizing efficiency without unexpected side effects in appropriate way are important (Su et al., 2015). As DDS vehicles, nano-emulsions, liposomes and nanoparticles (NPs) were developed and applied in skin pharmaceutics and cosmetics (Wu and Guy, 2009). In cancer therapy, nanoparticles can also be modified by considering physicochemical properties such as compositions, sizes, shapes, structures, morphologies, and surface properties. Several DDS have been approved for cancer therapy and are under clinical trials or preclinical evaluations (Sun et al., 2021). Several researchers studied DDS in distinct developmental properties and physiology, accessible stem cell niches. For example, gold NPs were injected to pregnant mice intravenously for placental transport. On the other hand, PAMAM dendritic NPs (5-80 nm) and elastin-like polypeptide NPs could not deliver to the placenta (Menjoge et al., 2011; Rattanapinyopituk et al., 2014; Kuna et al., 2018). Thus, the study of DDS in animal cells is also necessary before application in vivo.

Cell-penetrating peptide (CPP) is defined as a short (~5-30 amino acids) peptide that has cell membrane protein transduction domains or membrane translocating sequence (Milletti, 2012). They are comprised of cationic or amphipathic sequences that can cross the cell membranes (Lindgren et al., 2000). The CPPs can deliver various kinds of cargo into cell cytosols, such as fluorophores, drugs, peptides and nucleic acids (Ye et al., 2017; Allolio et al., 2018; Borrelli et al., 2018). In a previous study, we developed Ara27 CPP, which is a zinc knuckle family protein of Arabidopsis and confirmed internalization in human dermal fibroblast (HDF) and human dental pulp stem cell (hDPSC) at low concentration with short treatment condition. Interestingly, Ara27 exhibited higher cell internalizing ability compared to TAT, conventional cationic CPP (Min et al., 2020). Ara27, as an amphipathic CPP, could be modified and utilized in the biomedical field excluding the risk of toxicity. Therefore, we would like to confirm the non-toxic induced penetrating ability of Ara27 in various cell lines.

The purpose of this study was to screen the cell internalization ability of Ara27 in various cell lines (Table 1). According to the result, Ara27 showed superior penetrating ability in multiple cells, indicating that Ara27 has the potential as a core structure for various DDS applications.

Table 1. Classification of cell lines used in the experiment.

Normal cell lines	Cancer cell lines	Animal cell lines
Human Dermal fibroblast	Non-small-cell lung cancer cell (A549, H460)	Porcine Fetal Fibroblast
Human Keratinocyte (HaCaT)		Bovine Dermal Fibroblast

Culture of multiple cell lines

HDF was obtained from ATCC (ATCC, Manassas, VA). HaCaT was obtained from Cell Lines Service (CLS, Eppelheim, Germany). A549 and H40 were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). BDF and PFF were isolated according to previously studied methods (Park et al., 2014; Kim et al., 2016).

HDF, HaCaT, BDF and PFF cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; WELGENE, Daegu, Korea) with 10% fetal bovine serum and 1% penicillin-streptomycin at 37℃ with 5% CO₂ in the incubator. A549 and H460 cells were cultured in Roswell Park Memorial Institute (RPMI-1640; HyClone, Logan, UT, USA) medium with 10% FBS and 1% P/S at 37℃ with 5% CO₂ in the incubator.

Peptide synthesis and preparation

Ara27 was synthesized by LifeTein LLC (Hillsborough, NJ, USA) and labeled with carboxy-tetramethylrhodamine (TAMRA). Ara27-TAMRA was dissolved in distilled water to prepare a 1 mM stock solution. Detailed information on peptides has been presented in a previous study (Min et al., 2020).

Cell viability assay

Cell Counting Kit-8 (CCK-8; Abbkine, Wuhan, China) based on water-soluble tetrazolium salt (WST-8) was used to measure the cell viability. Cells were seeded on 96-well plates. After 24 h, Ara27 was treated in each well of 96 well plates and incubated at 37℃. After 18 h, the medium was changed with fresh medium containing 10 μL of CCK-8 reagents and incubated at 37℃ for 1 h. Absorbance was measured at 452 nm by a microplate reader (Multiskan^TM GO Microplate Spectrophotometer.; Thermo Scientific^TM, Massachusetts, USA). All analyses were performed using GraphPad Prism 5 software. Data were analyzed using analysis of variance (ANOVA) and were presented as the mean ± standard deviation.

Immunofluorescence staining

Cells were seeded on gelatin-coated coverslips to an appropriate density. After 24 h of stabilization, the cells were treated with Ara27-TAMRA for 1 and 18 h. After treatment, immunofluorescence staining was conducted. Briefly, the cells on coverslips were three times with heparin-containing PBS and fixed with 4% paraformaldehyde for 10 min. And then, the coverslips were rinsed thoroughly with PBS and mounted on slides using a mounting solution containing Hoechst 33342. All slides were analyzed by confocal microscopy (LSM800, Zeiss, Munich, Germany).

Cell viability of Ara27 in HDF and HaCaT

To confirm the toxicity of Ara27, cell viability was analyzed on HDF and HaCaT. Concentrations of Ara27 were started at 0.1-1 μM for 18 h treatment. The viability of HDF and HaCaT treated with Ara27 was maintained (n = 5). There were no significant changes in HDF (Fig. 1A) and HaCaT (Fig. 1B) cell viability fold changed from the non-treatment group. These results indicated that the 1 μM for 18 h treatment of Ara27 did not affect the cell viability of normal human cells.

Figure 1.Cytotoxic effect of Ara27 at various concentrations on HDF and HaCaT. Cell viability was measured using the CCK8 assay. (A) Cell viabilities of HDF after treatment of Ara27 from 0.1 to 1 μM for 18 h. (B) Cell viabilities of HaCaT after treatment of Ara27 from 0.1 to 1 μM for 18 h. The experiments were repeated at least three times independently and the results were shown as mean ± SD. ns = not significant.

Internalization of Ara27 in HDF and HaCaT

To evaluate the penetrating effect of Ara27 as a CPP, TAMRA labeled Ara27 was treated HDF and HaCaT. The concentration of Ara27 was 1 μM, which is already confirmed, and treatment times were 1 and 18 h. The cells without CPP treatment were used as negative controls. Ara27-TAMRA was internalized in HDF cytosol and the intensity of TAMRA fluorescence was remarkably increased at 18 h compared to 1 h treatment (Fig. 2A). Also in HaCaT, Ara27 was located in a cell at 1 h and the intensity was increased at 18 h treatment (Fig. 2B). These indicate that Ara27 permeabilized and localized in the normal cell lines and time-dependent increased internalization was happened.

Figure 2.Cellular internalization of Ara27-TAMRA in normal cell lines. (A) Cellular uptake in HDF with 1 μM of Ara27-TAMRA for 1 and 18 h. (B) Cellular uptake of Ara27 in HaCaT with 1 μM of Ara27-TAMRA for 1 and 18 h. The fluorescence signal was analyzed by confocal microscopy. Scale bar = 20 μm.

Internalization of Ara27 in A549 and H460

To investigate whether Ara27 can work as a CPP in cancer cell lines, Ara27-TAMRA was treated with A549 (Fig. 3A) and H460 (Fig. 3B). The concentration and time were the same as those treated in HDF. The fluorescence was detected both in 1 and 18 h treatments. And, the dots of CPPs were more compacted and located throughout the cytoplasm as time passed. The result shows that Ara27 also can internalize in cancer cell lines and the dose of internalized CPP was increased through the time.

Figure 3.Cellular internalization of Ara27-TAMRA in cancer cell lines. (A) Cellular uptake in A549 with 1 μM of Ara27-TAMRA for 1 and 18 h. (B) Cellular uptake of Ara27 in H460 with 1 μM of Ara27-TAMRA for 1 and 18 h. The fluorescence signal was analyzed by confocal microscopy. Scale bar = 20 μm.

Internalization of Ara27 in BDF and PFF

Ara27, as a CPP, was already confirmed the permeability in normal cells and cancer cells (Fig. 1 and 2) without cell cytotoxicity at 1 μM until 18 h of treatment. We also conducted the same experiments on bovine and porcine fibroblasts (BDF; Fig. 4A and PFF; Fig. 4B). Surprisingly, Ara27-TAMRA was internalized both in BDF and PFF and the time-dependent increased permeation was also found in the animal cell lines. This indicates that Ara27 can be used in bovine and porcine cells as CPP.

Figure 4.Cellular internalization of Ara27-TAMRA in animal cell lines. (A) Cellular uptake in BDF with 1 μM of Ara27-TAMRA for 1 and 18 h. (B) Cellular uptake of Ara27 in PFF with 1 μM of Ara27-TAMRA for 1 and 18 h. The fluorescence signal was analyzed by confocal microscopy. Scale bar = 20 μm.

Nowadays, a fundamental consideration of DDS is how efficiently delivered to its target (Zununi Vahed et al., 2019). Although highly developed DDS, unexpected hurdles can exist and cause insufficient cellular uptake for effects. CPPs, as an emerging DDS platform, are possible to deliver cargos intracellular with less toxic side effects (Soler et al., 2014). TAT, the first reported CPP, has been widely used and modified to enhance cell penetration (Frankel et al., 1988). However, most of CPPs require high concentrations of treatment (>5 μM) for cell permeation and internalization (Said Hassane et al., 2010). In a previous study, an amphipathic CPP, Ara27 had high cell-penetrating efficiency without cell toxicity at low peptide concentrations (0.2-2 μM) for a short time in HDF and hDPSC compared to TAT-PTD (Min et al., 2020). In addition to the permeability of Ara27 in HDF and hDPSC, we would like to apply Ara27 in various cell lines to broaden the scope of research.

In the present study, we evaluated the cell permeability of Ara27 in multiple cell lines such as normal cell lines, cancer cell lines, and animal cell lines. First, concentration screening was performed to set a concentration that did not affect cell viability (Fig. 1). We confirmed that 1 μM of Ara27 did not induce cell cytotoxicity until 18 h. Then, the cellular internalization of TAMRA labeled Ara27 was analyzed in various cell lines. In human-derived normal cell lines (HDF and HaCaT), Ara27 was located intracellular membrane (Fig. 2). Ara27 also penetrated cancer cells of A549 and H460 (Fig. 3). Even in the animal cell lines (BDF and PFF), Ara27 was localized in the cytosol (Fig. 4). In addition, the intensity of TAMRA fluorescence and the number of dots was remarkably increased in 18 h compared to 1 h (Fig. 2, 3, 3 and 4). These results indicate that Ara27, a promising CPP, can be applied to normal, cancer, and animal cell lines in various biomedical fields without cellular toxicity.

In the transdermal and skin researches, CPPs have been used to promote cell proliferation for skin regeneration and wounds healing by increasing the translocation of regenerative cargo into cells by conjugation of the growth factor (Nakayama et al., 2014; Horn et al., 2018). In our previous study, Ara27-conjugated human fibroblast growth factor 2 penetrated HDF and re-released from the cells after 1 h treatment and an HDF condition medium increased the proliferation of HDF (Lee et al., 2020). CPP can also deliver nucleic acid such as DNA, mRNA and siRNA (Meade et al., 2007; Srimanee et al., 2018; Yokoo et al., 2021; Mahjoubin-Tehran et al., 2022) and can mediate anti-cancer effects (Klimpel et al., 2019). CPP can promote neural stem cell differentiation (Ma et al., 2018) and CPP-conjugated with reprogramming factors increased the rate of induced pluripotent stem cell formation (Lim et al., 2014). CPP-conjugated morpholino oligos has been used in vivo broadly (Moulton et al., 2013). Crotamine, one of the CPPs, was used in vitro fertilized bovine embryos and affected several gene expressions (Campelo et al., 2016).

Therefore, Ara-27, as a core CPP structure, could be combined with functional proteins, peptides, nucleic acids, or non-permeable drugs and be overcame the hurdles of the currently existing CPPs in various cell lines in vitro and in vivo.

This study shows that Ara27 is internalized in normal, cancer, and animal cell lines without cell toxicity in time-dependent manner. The result indicates Ara27 can be used as a core CPP structure in DDS applications.

None.

Conceptualization, M.K., S.P.; methodology, M.K., S.P.; investigation, M.K.; data curation, M.K., S.P.; writing—original draft preparation, M.K.; writing—review and editing, S.P., S.R.; supervision J.S., S.R.

This work was supported by a grant from the National Research Foundation of Korea (NRF-2020R1F1A1070433) and by the Technology Development Program (S2910763) funded by the Ministry of SMEs and Startups (Korea).

Not applicable.

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No potential conflict of interest relevant to this article was reported.