Intracellular Delivery of Plasmid DNA Using Amphipathic Helical Cell-Penetrating Peptides Containing Dipropylglycine

Abstract

Cell-penetrating peptides (CPPs) serve as potent vehicles for delivering membrane-impermeable compounds, including nucleic acids, into cells. In a previous study, we reported the successful intracellular delivery of small interfering RNAs (siRNAs) with negligible cytotoxicity using a peptide containing an unnatural amino acid (dipropylglycine). In the present study, we employed the same seven peptides as the previous study to evaluate their efficacy in delivering plasmid DNA (pDNA) intracellularly. Although pDNA and siRNA are nucleic acids, they differ in size and biological function, which may influence the optimal peptide sequences for their delivery. Herein, three peptides demonstrated effective pDNA transfection abilities. Notably, only one of the three peptides previously exhibited efficient gene-silencing effect with siRNA. These findings validate our hypothesis and offer insights for the personalized design of CPPs for the delivery of pDNA and siRNA.

Introduction

Cell-penetrating peptides (CPPs) possess the ability to transport membrane-impermeable compounds into intracellular compartments, making them promising candidates for drug delivery systems (DDSs).^1–3) Currently, some CPPs are undergoing clinical trials, indicating their potential in the field.^4,5) Among their various applications, CPPs are being utilized to deliver plasmid DNA (pDNA) for gene therapy. Most CPPs contain multiple cationic amino acids, and they interact with anionic pDNA through electrostatic interactions, resulting in the formation of nano-sized assemblies. These assemblies serve as effective tools for the intracellular delivery of pDNA.

Although numerous CPPs have been identified, the quest for novel CPPs with enhanced cell-penetrating ability and minimal cytotoxicity continues. One of strategies for designing such CPPs involves the incorporation of unnatural amino acids (non-proteinogenic amino acids).^6–8) Introducing unnatural amino acids into peptides can sometimes stabilize their secondary structure and confer resistance against enzymatic degradation. The former property is particularly advantageous for designing amphipathic CPPs capable of adopting an α-helical structure. The latter property ensures prolonged functionality of CPPs without decomposition. We have developed CPPs containing α,α-disubstituted α-amino acids (dAAs), known for their ability to stabilize peptide secondary structure into the α-helix.^9–11) These peptides have shown high resistance to enzymatic degradation and prolonged transfection abilities when utilized for pDNA delivery.^12,13)

In this study, we synthesized seven peptides and evaluated their effectiveness in delivering pDNA into cultured cells (Fig. 1A). Dipropylglycine (Dpg) was employed as a dAA for helical stabilization (Fig. 1B). With the exception of Pep7, these peptides adopted an amphipathic structure upon forming an α-helical structure. Previously, these peptides had been assessed for preferred secondary structures, cell-penetrating abilities, and efficiency in delivering small interfering RNA (siRNA).¹⁴⁾ In that paper, it was observed that Pep2–Pep7 assumed an α-helical structure with varying degrees of helicity. Notably, Pep4 (Fig. 1C) demonstrated the highest cell-penetrating ability and RNA interference efficiency among the peptides evaluated. Although pDNA and siRNA are both nucleic acids, they differ significantly in size and cellular function (pDNA: nucleus; siRNA: cytoplasm). Given this distinction, we aimed to determine if the seven previously designed peptides exhibited similar intracellular delivery efficiency for pDNA as they did for siRNA. Physicochemical properties of peptide/pDNA complexes were evaluated using gel retardation analysis, dynamic light scattering (DLS), and zeta-potential measurements. Transfection efficiencies and cytotoxicities of the peptide/pDNA complexes were then assessed in cultured cells. Not only Pep4 but also Pep2 and Pep3 demonstrated good pDNA transfection abilities, which differed from their performance in siRNA delivery.

Fig. 1. (A) Sequences of Peptides Described in the Current Study; (B) Structure of Dipropylglycine (Dpg, X); (C) Schematic Illustration of the α-Helical Structure of Pep4 as Viewed along the Helical Axis

Results and Discussion

Physicochemical Properties of Peptide/pDNA Complexes

We synthesized seven peptides, Pep1–Pep7, as described previously.¹⁴⁾ An N-terminal tyrosine (Tyr) residue was incorporated into each peptide to determine the peptide concentration. We combined a peptide solution with a pDNA solution to generate peptide/pDNA complexes at different mixing ratios. The N/P ratio was defined as the molar ratio of the amino or guanidino groups in the peptides to the phosphate groups in the pDNA. The formation of peptide/pDNA complexes was verified using gel retardation analysis conducted at N/P ratios of 2, 4, and 8 (Fig. 2), although smear bands were observed in Pep1, Pep3, and Pep4 at N/P = 2, suggesting incomplete complex formation.

Fig. 2. Gel Retardation Analysis of Peptide/pDNA Complexes Prepared at N/P Ratios of 2, 4, and 8

DLS measurements were utilized to determine the size and polydispersity index (PDI) of the peptide/pDNA complexes (Table 1). Pep1 and Pep5–Pep7 exhibited cumulant diameters in the range of several thousand nanometers, with the exception of Pep5 and Pep7 at N/P = 8. In contrast, Pep2–4 afforded smaller complex sizes compared with the other peptides, suggesting that these complexes may be suitable for intracellular pDNA delivery. The zeta-potentials of complexes are also important for their intracellular internalization of nano-sized DDS. Pep1 (non-helical peptide), Pep5 (short peptide), Pep6 (different type of amphipathic peptide), and Pep7 (non-amphipathic peptide) exhibited a tendency towards negative and/or small positive zeta-potentials compared with Pep2–4 (Table 1). In particular, in Pep4, peptide/pDNA complexes prepared at N/P ratios of 4 and 8 exhibited zeta-potentials exceeding +30 mV. Pep4 is the longest peptide and contains the highest number of cationic amino acids (lysine (Lys)) among all peptides. Therefore, its binding affinity to pDNA may be the highest. Taking into account the physicochemical properties determined by DLS and zeta-potential measurements, it can be concluded that Pep2–4/pDNA complexes are well-suited for nano-sized DDS targeting cultured cells.

Table 1. Sizes, PDI, and Zeta-Potentials of the Peptide/pDNA Complexes Prepared at N/P Ratios of 2, 4, and 8

Peptide	N/P ratio	Size (nm)	PDI (µ/Γ2)	Zeta-potential (mV)
Pep1	2	5439 ± 431	0.308 ± 0.088	−22.4 ± 1.0
	4	5197 ± 318	0.353 ± 0.064	−14.0 ± 1.2
	8	5742 ± 1674	0.510 ± 0.046	−8.1 ± 1.1
Pep2	2	602 ± 9	0.480 ± 0.013	+22.6 ± 0.5
	4	178 ± 3	0.215 ± 0.030	+27.4 ± 0.5
	8	161 ± 1	0.199 ± 0.008	+26.5 ± 0.6
Pep3	2	1603 ± 139	0.533 ± 0.058	+25.9 ± 0.3
	4	149 ± 1	0.180 ± 0.001	+29.8 ± 1.9
	8	168 ± 1	0.204 ± 0.021	+26.6 ± 1.8
Pep4	2	307 ± 2	0.230 ± 0.013	+15.2 ± 0.5
	4	209 ± 4	0.186 ± 0.006	+33.9 ± 2.1
	8	207 ± 1	0.204 ± 0.021	+37.5 ± 2.0
Pep5	2	5331 ± 674	0.245 ± 0.110	−12.3 ± 0.6
	4	3359 ± 256	0.548 ± 0.012	+8.7 ± 2.1
	8	395 ± 4	0.405 ± 0.012	+4.1 ± 0.2
Pep6	2	4561 ± 354	0.279 ± 0.051	−1.1 ± 0.5
	4	4214 ± 369	0.291 ± 0.076	+9.3 ± 0.4
	8	5916 ± 446	0.557 ± 0.029	+15.4 ± 0.5
Pep7	2	3977 ± 162	0.284 ± 0.006	+8.3 ± 0.1
	4	3841 ± 143	0.281 ± 0.104	+15.3 ± 0.5
	8	1175 ± 3	0.440 ± 0.069	+20.0 ± 0.9

Transfection Efficiencies of Peptide/pDNA Complexes

The pDNA transfection efficiency of each peptide was assessed against human hepatoma Huh-7 cells using a Firefly luciferase (fLuc) pDNA. Peptide/pDNA complexes were prepared at N/P ratios of 2, 4, and 8, followed by quantification of their transfection efficiencies compared to a control using the commercially available transfection reagent jetPEI after 48-h incubation (Fig. 3A). The N/P ratio of 8 utilized for jetPEI falls within the recommended range of N/P ratios. Pep2–4 exhibited higher transfection efficiencies than other peptides and jetPEI. With an increase in N/P ratios, the transfection efficiencies of Pep2 and Pep3 decreased, whereas those of Pep4 increased. The cell viabilities of Huh-7 cells treated with peptide/pDNA and jetPEI/pDNA complexes were evaluated after 24-h incubation (Fig. 3B). The cytotoxicity varied significantly among peptides, with the cytotoxicity ranking as follows: Pep5 > Pep2, Pep3, Pep7 > Pep4, Pep6 > Pep1. This ranking was generally consistent with previous results of peptide/siRNA complexes.¹⁴⁾ Considering the results of fLuc pDNA transfection, Pep2 and Pep3/pDNA complexes with a low N/P ratio, as well as Pep4/pDNA complexes with a high N/P ratio had good transfection ability alongside relatively low cytotoxicity. These complexes exhibited small sizes and high positive values of zeta-potentials (Table 1), making them suitable nano-sized DDS for intracellular internalization. Additionally, it was reported that the cell-penetrating abilities of peptides were highest for Pep4, followed by Pep1–Pep3 and Pep6.¹⁴⁾ The synergic effect of the physicochemical properties of peptide/pDNA complexes and the cell-penetrating abilities of peptides appeared to contribute to their pDNA transfection ability.

Fig. 3. Transfection Efficiencies and Cytotoxicities of Peptide/pDNA Complexes against Huh-7 Cells

(A) fLuc pDNA introduction using peptides (N/P ratios of 2, 4, and 8) and commercially available reagent jetPEI (N/P ratio of 8) after 48-h incubation. (B) Cell viabilities of Huh-7 cells treated with peptide/pDNA complexes (N/P ratios of 2, 4, and 8) and jetPEI/pDNA complex (N/P ratio of 8) after 48-h incubation. Error bars represent the standard deviation, n = 5.

Subsequently, the time-dependent transfection efficiencies of peptide/pDNA complexes were assessed against Huh-7 cells using Gaussia luciferase (gLuc) pDNA (Fig. 4). Pep2–Pep4 were selected due to their demonstrated high transfection abilities (Fig. 3A). Pep2 and Pep3 at a low N/P ratio, as well as Pep4 at a high N/P ratio, demonstrated relatively high transfection efficiencies. Pep2 and Pep3/pDNA complexes prepared at an N/P ratio of 8 exhibited negligible transfection efficiency across all incubation times due to their high cytotoxicity (Fig. 3B). The transfection efficiency of Pep4/pDNA complexes at an N/P ratio of 2 was moderate. The time-dependent transfection efficiencies were also assessed against human cervical carcinoma HeLa cells using the same peptide/pDNA complexes (Supplementary Fig. S1). The results obtained showed a similar trend compared to those presented in Fig. 4. Comparing the transfection efficiencies of fLuc pDNA and gLuc pDNA after incubation for 48 h sometimes yielded different results (Figs. 3A, 4). The luciferase assay was conducted using cell lysate for fLuc pDNA introduction, but cell culture medium was used for gLuc pDNA introduction, which might lead to varying results. In Fig. 4, jetPEI was also evaluated and exhibited slightly decreased gene expression post 24-h incubation time, possibly due to its cytotoxicity. All peptide/pDNA complexes exhibited increased or plateau transfection efficiencies after incubation for 72 h. Specifically, the transfection efficiency of the Pep4/pDNA complexes at an N/P ratio of 8 after incubation for 72 h was significantly higher than that after 48 h. Peptides containing dAAs were reported to exhibit resistance against enzymatic degradation.^9,11,15) Therefore, it is likely that pDNA in the Pep4/pDNA complexes was gradually released from the complexes within the cells.

Fig. 4. Time-Dependent Transfection Efficiencies Peptide/pDNA Complexes against Huh-7 Cells

gLuc pDNA introduction using Pep2–Pep4 (N/P ratios of 2, 4, and 8) and commercially available reagent jetPEI (N/P ratio of 8). Error bars represent the standard deviation, n = 5.

Cellular Uptake of Peptide/pDNA Complexes

To elucidate the cellular uptake amounts of pDNA delivered by peptides, we conducted flow cytometric analysis on Huh-7 cells using Cy5-labeled pDNA (Cy5-pDNA). For this analysis, peptide/Cy5-pDNA complexes prepared with Pep2 at an N/P ratio of 2, Pep3 at an N/P ratio of 2, and Pep4 at N/P ratios of 2, 4, and 8 were utilized (Fig. 5), because these complexes demonstrated relatively good transfection efficiency without severe cytotoxicity (Figs. 3, 4). Overall, Huh-7 cells treated with Pep4/Cy5-pDNA complexes exhibited higher fluorescence intensity, whereas those treated with Pep3/Cy5-pDNA complexes displayed lower intensity. Among Pep4/Cy5-pDNA complexes, we observed no definitive correlation between the cellular uptake amount of Cy5-pDNA and transfection efficiency. For instance, complexes at an N/P ratio of 8, which exhibited the highest transfection ability, did not necessarily demonstrate the highest cellular uptake amounts of pDNA. These results suggested that the cellular uptake amounts of pDNA may not be critical for achieving high transfection efficiencies with Pep4 at high N/P ratios.

Fig. 5. Cellular Uptake of Cy5-pDNA Delivered by Pep2 (N/P Ratio of 2), Pep3 (N/P Ratio of 2), and Pep4 (N/P Ratios of 2, 4, and 8)

Error bars represent the standard deviation, n = 3. * p < 0.05 and ** p < 0.01.

To gain insights into the intracellular distribution of pDNA after uptake, we conducted confocal laser scanning microscopy (CLSM) observations of Cy5-pDNA (green) delivered by Pep2 (N/P ratio of 2), Pep3 (N/P ratio of 2), and Pep4 (N/P ratios of 2, 4, and 8) with staining for late endosomes/lysosomes (red) and nuclei (blue) using LysoTracker Red and Hoechst 33342, respectively (Fig. 6A). The observed green signals were lower for Pep3 and higher for Pep4, which was consistent with the results shown in Fig. 5. The colocalization ratios of Cy5-pDNA with late endosomes/lysosomes were quantified (Fig. 6B). Pep4/Cy5-pDNA complexes exhibited similar values regardless of N/P ratios, and these values were significantly lower than those of Pep2 and Pep3/Cy5-pDNA complexes. These results indicated that Cy5-pDNA internalized into cells by Pep4 was distributed in similar intracellular compartments regardless of N/P ratios and may escape from late endosomes/lysosomes into the cytosol, compared with that by Pep2 and Pep3. Pep2, Pep3, and Pep4 are amphipathic helical peptides composed of cationic and hydrophobic amino acids. It is worth noting that such amphipathic helical peptides sometimes exhibited antimicrobial activity and membrane destabilization activity.^16,17) Pep4, being longer than Pep2 and Pep3, was expected to have stronger membrane destabilization activity. This may potentially lead to better endosomal escaping ability of Pep4/Cy5-pDNA complexes. In general, nano-sized DDS internalizes into cells via the endocytosis pathway and is subsequently transported to late endosomes/lysosomes. In the case of pDNA delivery, pDNA is susceptible to degradation by nucleases in lysosomes. Therefore, it needs to migrate into the cytoplasm for successful transfection. In the current study, a significant difference in colocalization ratios was observed between Pep2/Pep3 and Pep4, with no significant difference observed among different N/P ratios of Pep4. Endosomal escape of pDNA appeared to be essential for efficient transfection to some extent, but not necessarily essential for Pep2, Pep3, and Pep4 in this study.

Fig. 6. CLSM Observations of Cy5-pDNA Delivered by Pep2 (N/P Ratio of 2), Pep3 (N/P Ratio of 2), and Pep4 (N/P Ratios of 2, 4, and 8)

(A) Intracellular distribution of Cy5-pDNA (green) was observed after staining late endosomes/lysosomes (red) and nuclei (blue) with LysoTracker Red and Hoechst 33342, respectively. White bars represent 20 µm. (B) Quantification of Cy5-pDNA colocalized with late endosomes/lysosomes. Error bars represent the standard deviation, n = 16. ** p < 0.01.

Flow cytometric analysis and CLSM observation elucidated certain biological functions of each peptide/pDNA complex, yet these did not fully account for their respective transfection abilities. For encapsulated pDNA in the nano-sized DDS to function effectively, it must be shielded from enzymatic degradation, successfully penetrate the nuclear membrane, and be liberated from the complexes. In addition to these considerations, various other factors could impact the transfection capability of peptide/pDNA complexes. Further research is required to elucidate the mechanisms behind efficient transfection.

Conclusion

In this study, we used seven peptides, designated Pep1–Pep7, and assessed their capabilities of pDNA transfection. The results obtained here indicated that Pep2 and Pep3 exhibit effective transfection efficiency at a low N/P ratio, while Pep4 demonstrates effective transfection efficiency at a high N/P ratio when applied to cultured cells. We previously reported intracellular siRNA delivery using the same seven peptides,¹⁴⁾ wherein only Pep4 exhibited efficient RNA interference. Pep4 demonstrated the ability to intracellularly deliver both pDNA and siRNA, whereas Pep2 and Pep3 were capable of delivering only pDNA. In addition, Pep2 and Pep3 exhibited almost the same transfection ability of pDNA as Pep4 at a low N/P ratio, although Pep2 and Pep3 were shorter than Pep4. In this study, intracellular behaviors of pDNA delivered by Pep2/Pep3 and Pep4 exhibited variations, yet their transfection efficiencies did not differ significantly. The sequence and length of peptides play a crucial role in influencing the physicochemical properties of complexes with both pDNA and siRNA, as well as their biological activities. Our findings, encompassing this study and our previous research, will be valuable for informing the design of CPPs tailored for the delivery of nucleic acids.

Experimental

Materials

The peptides were synthesized as described previously.¹⁴⁾ Briefly, solid-phase synthesis was employed using Fmoc chemistry on a standard commercially available Rink amide resin and Fmoc amino acids. The synthesized peptides were purified using reverse-phase HPLC (RP-HPLC) and characterized by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS). A pDNA encoding fLuc under the control of the CAG promoter was obtained from the RIKEN Gene Bank (Tsukuba, Japan). A pDNA encoding Gaussia luciferase (gLuc) was purchased from New England BioLabs (Ipswich, CA, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM) was a product of Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). Hoechst 33342 and Cell Counting Kit-8 were purchased from Dojindo Laboratories (Kumamoto, Japan). A luciferase assay kit was obtained from Promega (Madison, WI, U.S.A.). LysoTracker Red was purchased from Molecular Probes (Eugene, OR, U.S.A.). pDNA was labeled with Cy5 (Cy5-pDNA) using the Label IT^® Tracker™ intracellular Nucleic Acid Localization Kit obtained from Mirus Bio Co. (Madison, WI, U.S.A.).

Preparation of Peptide/pDNA Complexes

Each peptide and pDNA were dissolved separately in 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (pH 7.3). Peptide solutions of various concentrations were added to the pDNA solution to form peptide/pDNA complexes with different compositions in HEPES buffer. Complex solutions were stored at room temperature for 15 min prior to use. The N/P ratio was defined as the molar ratio of the amino or guanidino groups in peptides to the phosphate groups in pDNA.

Agarose Gel Electrophoresis

Gel electrophoresis was performed at 100 V for 45 min using 1% agarose gel. pDNA in the gel was stained with SYBR Gold and detected using a GelDoc Go Gel Imaging System (BIO-RAD Laboratories, Inc., Hercules, CA, U.S.A.).

Dynamic Light Scattering (DLS) Measurements

The sizes of the peptide/pDNA complexes were evaluated by DLS using Nano ZS (ZEN3600, Malvern Instruments, Ltd., U.K.). A He-Ne ion laser (633 nm) was used as the incident beam. Light scattering data were obtained at a detection angle of 173° and a temperature of 25 °C, and were subsequently analyzed using the cumulant method to determine the hydrodynamic diameters and polydispersity index (PDI) (µ/Γ²) of the complexes. Results were presented as the mean and standard deviation derived from three measurements.

Zeta-Potential Measurements

The zeta-potentials of the peptide/pDNA complexes were evaluated using the laser-Doppler electrophoresis method with Nano ZS with a He-Ne ion laser (633 nm). Zeta-potential measurements were performed at 25 °C, utilizing a scattering angle of 173° for the measurements. Results were presented as the mean and standard deviation derived from three measurements.

fLuc pDNA Transfection

Human hepatoma Huh-7 cells were seeded onto 96-well culture plates (5000 cells/well) and incubated overnight in 100 µL of DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS). The culture medium was then replaced with fresh medium, and peptide/fLuc pDNA complex solutions at various N/P ratios (N/P 2, 4, and 8) were added to each well. The amount of pDNA was adjusted to 0.25 µg per well. After incubation for 48 h, fLuc gene expression was evaluated based on photoluminescence intensity using a luciferase assay kit and the Lumat3 LB9508 luminometer (Berthold Technologies, Bad Wildbad, Germany). Results were presented as the mean and standard deviation derived from five measurements.

gLuc pDNA Transfection

Huh-7 cells or human cervical carcinoma HeLa cells were seeded onto 96-well culture plates (5000 cells/well) and incubated overnight in 100 µL of DMEM containing 10% FBS and 1% PS. The culture medium was then replaced with fresh medium, and peptide/gLuc pDNA complex solutions were added to each well, with the amount of pDNA adjusted to 0.25 µg per well. After each time incubation (3, 7, 24, 48, and 72 h), 5 µL of supernatant of the culture medium was used to evaluate gLuc gene expression. Results were presented as the mean and standard deviation derived from five measurements.

Cell Viability

Huh-7 cells were seeded onto 96-well culture plates (5000 cells/well) and incubated overnight in 100 µL of DMEM containing 10% FBS and 1% PS. The culture medium was then exchanged with fresh medium, and peptide/pDNA complex solutions were added to be at 0.25 µg pDNA per well. After 24-h incubation, cell viabilities were evaluated using a Cell Counting Kit-8 in accordance with the manufacturer’s protocol.

Cellular Uptake of pDNA

Huh-7 cells were seeded onto 24-well culture plates (50000 cells/well) and incubated overnight in 500 µL of DMEM containing 10% FBS and 1% PS. The culture medium was then replaced with fresh medium, and peptide/Cy5-pDNA complex solutions were added to each well, with the amount of pDNA adjusted to 1 µg per well. After 24-h incubation, the medium was removed, and the cells were washed with phosphate-buffered saline (PBS). Subsequently, they were trypsinized, collected, and analyzed using flow cytometry Guava^® Muse™ cell analyzer (BM Equipment Co., Ltd., Tokyo, Japan). The results are presented as a mean and standard deviation derived from three samples. p values were calculated using Student’s t-test.

CLSM Observation

Huh-7 cells were seeded onto 8-well glass plates (20000 cells/well) and incubated overnight in 200 µL of DMEM containing 10% FBS and 1% PS. After exchanging the medium, peptide/Cy5-pDNA complexes containing 0.5 µg of pDNA were added to each well, and the cells were then incubated for 24 h. The medium was subsequently removed, and the cells were washed 3 times with PBS before being fixed with 10% formalin. The intracellular uptake of Cy5-pDNA was visualized using an LSM900 Airyscan (Carl Zeiss, Oberkochen, Germany) after staining the nuclei with Hoechst 33342 and the late endosomes/lysosomes with LysoTracker Red. Observations were performed using a 63× objective lens. The results are presented as a mean and standard deviation derived from 16 samples. p-Values were calculated using Student’s t-test.

Acknowledgments

This work was financially supported in part by the Japan Science and Technology Agency COI-NEXT (Grant Number: JPMJPF2114 to MO) and the Hoansha Foundation (to M.O.). We thank S. Ibuki for technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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