Plasmid DNA Delivery Using Cell-Penetrating Peptide Foldamers

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/abseba

Plasmid DNA Delivery Using Cell-Penetrating Peptide Foldamers Composed of Arg−Arg−Aib Repeating Sequences Makoto Oba,*,† Yurika Ito,† Tomohiro Umeno,† Takuma Kato,†,‡ and Masakazu Tanaka† †

Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan Osaka University of Pharmaceutical Sciences, 40-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

‡

ACS Biomater. Sci. Eng. Downloaded from pubs.acs.org by UNIV OF OTAGO on 04/22/19. For personal use only.

S Supporting Information *

ABSTRACT: Arginine (Arg)-rich cell-penetrating peptides (CPPs) are promising tools for plasmid DNA (pDNA) delivery. αAminoisobutyric acid (Aib) is known to stabilize peptide helical secondary structures and has been used as a building block for foldamers. In the current study, we prepared Aib CPP foldamers, (Arg−Arg−Aib)n (n = 1−6) and examined their pDNA transfection abilities. Transfection efficiencies of peptide/pDNA complexes are dependent on peptide chain length, with longer peptides (n ≥ 4) exhibiting better transfection abilities than an Arg nonapeptide. Furthermore, Aib CPP foldamers achieve prolonged transfection abilities compared with commercially available transfection reagents, which is probably because of the high resistance of the peptides to enzymatic degradation, thereby protecting the encapsulated pDNA for a long period. The obtained results demonstrated promising features of Aib CPP foldamers as pDNA delivery carriers. KEYWORDS: cell-penetrating peptide, plasmid DNA delivery, peptide foldamer, helical structure, α-aminoisobutyric acid

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INTRODUCTION α-Aminoisobutyric acid (Aib) is the simplest α,α-disubstituted α-amino acid (dAA) with an additional alkyl substituent at the α-position of the α-amino acid instead of a hydrogen atom. Aib has been discovered in a number of natural antimicrobial peptides as building blocks1−3 and is well-known to stabilize peptide helical secondary structures. 4−6 Owing to its usefulness, a variety of types of protected Aib are commercially available. Aib has been widely used as a helix inducer for the design and synthesis of functional helical peptide foldamers, such as antimicrobial peptides,7−9 bioactive peptides,10,11 and peptide catalysts.12,13 Cell-penetrating peptides (CPPs), which can deliver membrane-impermeable compounds into living cells, are also one of the targets of Aib-containing peptide foldamers.14,15 Cationic CPPs are promising tools for the delivery of biomacromolecules including proteins, DNA, and RNA.16,17 Electrostatic interactions between cationic CPPs and anionic biomacromolecules result in the formation of nanoparticles, whose sizes and zeta potentials are suitable for their intracellular internalization via endocytosis. We have reported CPP foldamers composed of arginine (Arg) and dAAs18−20 and used some of them as plasmid DNA (pDNA) delivery tools.21,22 CPP foldamers containing dAAs adopted a stable helical structure and acquired resistance to protease digestion, © XXXX American Chemical Society

resulting in enhanced and prolonged cell-penetrating abilities.18−20 Furthermore, pDNA was efficiently delivered by fully cationic CPP foldamers containing cyclic dAAs with basic functional groups in the side chain.21,22 The transfection efficiency using CPP foldamers was better than that with commercially available transfection reagents in the conditions with longer incubation times, which was probably due to the CPP foldamers’ high tolerance to protease digestion. However, it takes several steps to synthesize dAAs, as used in these studies, for 9-fluorenylmethoxycarbonyl (Fmoc)-based solidphase peptide synthesis. Readily available dAAs such as Fmoc− Aib are better for the preparation of CPP foldamers with high transfection efficiency and negligible toxicity. We have developed Aib CPP foldamers composed of Aib and Arg and showed their good cell-penetrating ability.23 pDNA delivery experiments were also performed using Aib CPP foldamers.24 However, the transfection efficiency of (Arg−Arg−Aib)3 was much lower than that of Arg nonapeptide (R9 peptide) and almost the same level as that of Special Issue: Biomaterials Science and Engineering in Japan Received: November 20, 2018 Accepted: April 11, 2019

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DOI: 10.1021/acsbiomaterials.8b01451 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

5C18-AR-II, 4.6 mm ID × 250 mm) and MALDI-TOF MS. RP-HPLC was performed utilizing a JASCO-PU-2089 Plus (JASCO Corporation, Tokyo, Japan) with a JASCO-2075-Plus as a UV detector. Solvent A was 0.05% TFA in H2O, and solvent B was 0.05% TFA in acetonitrile. The purification procedure required gradient conditions (from 5 to 50% solvent B over 15 min) with a flow rate of 8 mL/min and detection at 220 nm. The purities of the final compounds were further confirmed using similar RP-HPLC conditions (from 5 to 95% solvent B over 30 min) with a flow rate of 1 mL/min. Synthetic yields of each peptide were as follows: peptide 1 (13 mg, 24%); peptide 2 (10 mg, 11%); peptide 3 (22 mg, 19%); peptide 4 (5.7 mg, 3.9%); peptide 5 (12 mg, 6.8%); peptide 6 (13 mg, 6.0%). Circular Dichroism (CD) Spectra Measurement. CD spectra were recorded using a JASCO J-725N spectropolarimeter with a 1.0 mm path length cell. Data are expressed in terms of [θ]R, in other words, residue molar ellipticity (deg·cm2·dmol−1). 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (10 mM; pH 7.3) and trifluoroethanol (TFE) were used as solvents. Cellular Uptake. Huh-7 or HeLa cells were seeded on 24-well culture plates (50 000 cells/well) and incubated in 400 μL of DMEM containing 10% fetal bovine serum (FBS). The medium was then replaced with fresh medium, and each peptide solution was added to each well at a concentration of 1 μM. After incubation for 2 h, the medium was removed, and cells were washed with ice-cold phosphatebuffered saline (PBS) with heparin (20 units/mL) and trypsinized. After the addition of medium containing 10% FBS, fluorescence intensity was detected using an On-chip Flow instrument (On-chip Biotechnologies Co., Ltd., Tokyo, Japan). The results are presented as a mean and standard deviation of three samples. Preparation of Peptide/pDNA Complex. Each peptide and pDNA were dissolved separately in HEPES buffer (10 mM; pH 7.3). A 2-fold excess volume of the peptide solutions at various concentrations was added to the pDNA solution to form peptide/ pDNA complexes with different compositions. The final pDNA concentration was adjusted to 33.3 μg/mL, and complex solutions were stored at room temperature for 15 min prior to use. The N/P ratio is defined as the molar ratio of the guanidino groups in the peptide to the phosphate groups in pDNA. Fluorescence Measurement. The fluorescence intensities of peptide/pDNA complex solutions prepared at various N/P ratios were measured using a spectrofluorometer (515 nm; ND-3300, NanoDrop, Wilmington, DE). Results were presented as the mean and standard deviation of three measurements. Dynamic Light Scattering (DLS) Measurement. The size and size distribution of the peptide/pDNA complexes were evaluated by DLS using Nano ZS (ZEN3600, Malvern Instruments, Ltd., UK) and a He−Ne ion laser (633 nm) as the incident beam. DLS measurements were performed at a temperature of 25 °C and a detection angle of 173°. Data were subsequently analyzed by the cumulant method to obtain the hydrodynamic diameters (nm) and polydispersity indices (PDI) (μ/Γ2). Results were presented as the mean and standard deviation of three measurements. Zeta-Potential Measurement. The zeta potential of the peptide/pDNA complexes was evaluated using the laser Doppler electrophoresis method with a Nano ZS equipped with a He−Ne ion laser (633 nm). Zeta-potential measurements were performed at a temperature of 25 °C and a scattering angle of 173°. Results were presented as the mean and standard deviation of three measurements. Transfection. Huh-7 or HeLa cells were seeded on 24-well culture plates (10 000 cells/well) and incubated overnight in 400 μL of DMEM containing 10% FBS. The medium was replaced with fresh medium, and the peptide/pDNA complexes prepared at various N/P ratios (N/P ratios = 2, 4, 8, and 16), and commercially available transfection reagent jetPEI/pDNA complexes, prepared in accordance with the manufacturer’s protocol, were applied to each well. The amount of pDNA was adjusted to 1 μg per well. After a 24 h incubation period, the medium was replaced with fresh medium, followed by further incubations (postincubation) for the indicated times (24 h in Figure 5). Luciferase expression was evaluated based on photoluminescence intensity using the luciferase assay kit and a

naked pDNA. The (Arg−Arg−Aib)3 peptide could not form appropriate complexes with pDNA. In order to improve transfection efficiency, Aib CPP foldamers should be redesigned. For this purpose, we prepared peptides composed of Arg−Arg−Aib repeating sequences with different peptide chain lengths, (Arg−Arg−Aib)n (n = 1−6), in the current study (Figure 1). Through evaluation of the physicochemical

Figure 1. Structure of Aib CPP foldamers in the current study.

properties and transfection abilities of these peptide/pDNA complexes, we identified an appropriate peptide chain length of (Arg−Arg−Aib)n Aib foldamers for pDNA delivery.

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MATERIALS AND METHODS

General. Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometer (MS) spectra were taken on an Ultraflex (Bruker Daltonics, Fermont, CA). Fmoc-protected amino acids were obtained from Watanabe Chemical Industries, Ltd. (Hiroshima, Japan). All commercial materials were used without further purification. Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Sigma-Aldrich Co. (St. Louis, MO). jetPEI was a product of Polyplus-transfection (Illkirch, France). A micro BCA protein assay reagent kit was purchased from Pierce (Rockford, IL). The luciferase assay kit and passive lysis 5× buffer were obtained from Promega (Madison, WI). The plasmid pCAcc+Luc, coding for firefly luciferase under the control of the CAG promoter, was provided by the RIKEN Gene Bank (Tsukuba, Japan) and amplified and purified using GenScript (Tokyo, Japan). Hoechst 33342 and Cell Counting Kit-8 were purchased from Dojindo Laboratories (Kumamoto, Japan). LysoTracker Red was purchased from Thermo Fisher Scientific (Rockford, IL). pDNA was labeled with Cy5 using a Label IT Tracker Intracellular Nucleic Acid Localization Kit, which was obtained from Mirus Bio Co. (Madison, WI). Peptide Synthesis. Peptides 1−6 were synthesized on a solid support by Fmoc-based solid-phase methods using Rink Amide resin (Peptide Institute, Inc., Osaka, Japan) and Fmoc-protected amino acids at a scale of 50 μmol, as reported previously.25 Resin (100 mg, loading: 0.5 mmol/g) was soaked in N,N-dimethylformamide (DMF) for 1 h. After the DMF had been removed, 20% piperidine in DMF was added to the resin for deprotection. After Fmoc-protected amino acid or 5(6)-carboxyfluorescein (CF) (3 equiv) was removed and washed out of piperidine, coupling reagents (O-(7-azabenzotriazol-1yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate/1-hydroxy-7azabenzotriazole) (3 equiv) and N,N-diisopropylethylamine (6 equiv) dissolved in DMF were added for the coupling reaction. The resin was then suspended in a cleavage cocktail (trifluoroacetic acid (TFA): 1.9 mL; H2O: 50 μL; triisopropylsilane: 50 μL) at room temperature for 90 min. The TFA solution was evaporated to a small volume, and cold diethyl ether was then added to the solution to precipitate the peptides. The dried crude peptides were dissolved in acetonitrile and/or H2O and purified by reverse-phase highperformance liquid chromatography (RP-HPLC) using a COSMOSIL Packed Column 5C18-AR-II (20 mm ID × 250 mm) (Nacalai, Kyoto, Japan). Lyophilization afforded yellow crystals, which were characterized using an analytical RP-HPLC (COSMOSIL Packed Column B


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Figure 2. CD spectra of peptides 1−6 and R9 peptide in (a) TFE/buffer = 1:1 and (b) TFE/buffer = 9:1. Peptide concentration: 25 μM.

Figure 3. Cellular uptake of peptides 1−6 and R9 peptide by Huh-7 cells (a) and HeLa cells (b). Incubation time: 2 h. Peptide concentration: 1 μM. Error bars represent the standard deviation, n = 3. luminometer (Gene Light GL-210A, Microtec, Co., Ltd., Chiba, Japan). The amount of protein in each well was concomitantly assessed using a Micro BCA protein assay kit. Results were presented as the mean and standard deviation of four samples. Cell Viability. Huh-7 or HeLa cells were seeded on 96-well culture plates (2500 cells/well) and incubated in 100 μL of DMEM containing 10% FBS. The medium was replaced with fresh medium, and the peptide/pDNA complexes (N/P ratios = 8 and 16) and jetPEI/pDNA complexes were added to each well to be adjusted to 0.25 μg of pDNA per well. Cell Counting Kit-8 was used after a 24 h incubation in accordance with the manufacturer’s protocol. Cell viability was evaluated based on the absorbance of formazan from each well, where 100% cell viability was calculated from the wells without complexes. Results were presented as the mean and standard deviation of four samples. Confocal Laser Scanning Microscope (CLSM) Observation. HeLa cells were seeded onto eight-well chambered cover glasses (20 000 cells/well) and incubated overnight in 200 μL of DMEM containing 10% FBS. The medium was then replaced with fresh

medium, and peptide/Cy5-labeled pDNA (Cy5−pDNA) complexes at an N/P ratio = 8 and jetPEI/Cy5−pDNA complexes were applied to each well at 0.5 μg of pDNA/well. After a 24 h of incubation, the medium was removed, and cells were washed three times with icecold PBS supplemented with heparin (20 units/mL). Intracellular distributions were observed by CLSM after staining late endosomes/ lysosomes with LysoTracker Red and nuclei with Hoechst 33342, respectively. CLSM observations were performed using an LSM 710 (Carl Zeiss, Oberkochen, Germany) with a Plan-Apochromat 63×/ 1.4 objective (Carl Zeiss) at an excitation wavelength of 405 nm (UV laser) for Hoechst 33342, 488 nm (Ar laser) for peptides, 543 nm (He−Ne laser) for LysoTracker Red, and 633 nm (He−Ne laser) for Cy5−pDNA. The rate of colocalization of Cy5−pDNA with LysoTracker Red was quantified as follows

colocalization ratio = Cy5−pDNA pixelscolocalization − Lyso /Cy5−pDNA pixels total C


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ACS Biomaterials Science & Engineering where Cy5−pDNA pixelscolocalization‑Lyso represents the number of Cy5−pDNA pixels colocalizing with LysoTracker Red in the cell, and Cy5−pDNA pixelstotal represents the total number of pixels in the cell. The rate of un-colocalization of Cy5−pDNA with peptides was quantified as follows

Table 1. Sizes and PDI of Peptide/pDNA Complex Solutions peptide

N/P ratio

size (nm)

1

2 4 8 16 2 4 8 16 2 4 8 16 2 4 8 16 2 4 8 16 2 4 8 16 2 4 8 16

282 ± 24 236 ± 21 62.9 ± 0.3 1113 ± 37 61.6 ± 0.8 431 ± 10 1679 ± 105 253 ± 5 687 ± 32 817 ± 65 248 ± 49 136 ± 2 689 ± 139 147 ± 3 140 ± 4 109 ± 2 183 ± 5 135 ± 3 101 ± 3 119 ± 2 128 ± 7 104 ± 1 100 ± 1 124 ± 2 182 ± 7 122 ± 4 102 ± 4 119 ± 1

un‐colocalization ratio = 1 − (Cy5−pDNA pixelscolocalization − peptide /Cy5−pDNA pixels total) 2

where Cy5−pDNA pixelscolocalization‑peptide represents the number of Cy5−pDNA pixels colocalizing with peptides in the cell.

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RESULTS Characterization of the Synthesized Peptides. Peptides 1−6 (Figure 1), which were labeled with CF through the glycine linker to monitor their cell-penetrating abilities, were synthesized using an Fmoc-based solid-phase method. Synthesized peptides were purified by RP-HPLC and characterized by MALDI-TOF MS (Figure S1). The R9 peptide, which was a control for a typical CPP, was also synthesized as reported previously.20,21 The CD spectra of peptides 1−6 and R9 peptide (peptide concentration: 25 μM) were measured in a mixture of TFE and HEPES buffer (10 mM, pH 7.3) (TFE/buffer = 1:1 and 9:1) to obtain information on their secondary structures (Figure 2). A positive maximum at 192 nm and negative maxima at 208 and 222 nm are diagnostic of a right-handed (P) helical structure.26−28 R9 peptide exhibited the typical CD spectrum of a random structure. The CD spectra of peptides except for peptide 1 showed negative maxima at 204−208 nm and 222− 225 nm, indicating that the dominant structure of peptides 2− 6 was a right-handed (P) helical structure. Introduction of Aib into peptides definitely led to the formation of a helical structure even in short peptides (hexapeptide: peptide 2). The intensity of CD spectra was enhanced with an increase in the peptide chain length, suggesting the enhancement of helicity. Cellular Uptake. The uptake of peptides 1−6 and R9 peptide by Huh-7 cells (Figure 3a) and HeLa cells (Figure 3b) was evaluated with a 2 h incubation and a peptide concentration of 1 μM. The cellular uptake of Aib-containing peptides increased with an increase in peptide chain length and reached similar level as the R9 peptide at n = 3 (peptide 3). Peptides 5 (n = 5) and 6 (n = 6) showed much higher cellpenetrating abilities than the R9 peptide. Fluorescence Measurement of Peptide/pDNA Complexes. The formation of complexes between peptides and pDNA was confirmed by fluorescence measurements of peptide/pDNA solutions at various N/P ratios (Figure S2). It was expected that fluorescence quenching occurred with the formation of assembled structures through self-quenching.29,30 The fluorescence intensity of peptide 1/pDNA increased linearly with an increase in the N/P ratios, and that of peptide 2/pDNA increased slightly up to an N/P ratio = 2 and then elevated drastically. These results were different from those of peptides 3−6/pDNA, in which fluorescence intensities remained the same, and then, marked increases in the fluorescence intensities were observed over an N/P ratio = 1.5. The association behavior of the peptide with pDNA was different according to the peptide chain length, and shorter peptides, such as peptides 1 and 2, were less capable of associating with pDNA than longer peptides. Size and Zeta Potentials of Peptide/pDNA Complexes. The mean sizes (Table 1) and zeta potentials (Figure 4) of peptide/pDNA complexes were also different between

3

4

5

6

R9

PDI (μ/Γ2) 0.45 0.51 0.19 0.23 0.19 0.06 0.25 0.12 0.16 0.18 0.14 0.07 0.22 0.16 0.32 0.18 0.10 0.05 0.17 0.18 0.07 0.10 0.18 0.19 0.21 0.12 0.15 0.18

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.12 0.00 0.02 0.01 0.05 0.03 0.01 0.05 0.06 0.04 0.01 0.02 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.01 0.02 0.02 0.00 0.01 0.02

Figure 4. Zeta potentials of peptide/pDNA complex solutions. Error bars represent the standard deviation, n = 3.

shorter peptides and longer peptides. Zeta potentials of peptide 1/pDNA complexes remained the same, ∼−35 mV at all N/P ratios, and those of peptides 2 and 3/pDNA complexes increased with N/P ratios but were still negative values even at an N/P ratio = 8. On the other hand, peptides 4−6/pDNA complexes showed positive zeta potentials even at an N/P ratio = 2 and reached zeta potentials of over +10 mV (peptide 4 at an N/P ratio = 8; peptide 5 at an N/P ratio = 4; peptide 6 at an N/P ratio = 2). The sizes of peptide/pDNA complexes D


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Figure 5. Transfection efficiencies of peptide/pDNA complexes at various N/P ratios with Huh-7 cells (a) and HeLa cells (b). Error bars represent the standard deviation, n = 4.

Figure 6. Transfection efficiencies of peptide/pDNA complexes (N/P ratio = 8) and jetPEI/pDNA complexes with Huh-7 cells (a) and HeLa cells (b) at various postincubation times. Error bars represent the standard deviation, n = 4.

showing zeta potentials of over +10 mV were less than 150 nm with narrow PDI (except for peptide 4 at an N/P ratio = 8), and these values were similar to R9 peptide/pDNA complexes and suitable for intracellular pDNA delivery. Transfection. The transfection efficiency of pDNA encoding luciferase complexed with peptides 1−6 and R9 peptide at N/P ratios = 2, 4, 8, and 16 was evaluated using Huh-7 cells (Figure 5a) and HeLa cells (Figure 5b) using luciferase assay. Transfection experiments in Figure 5 were performed with a 24 h incubation of cells with complexes followed by a 24 h postincubation without complexes. Peptides 1−3/pDNA complexes showed almost no transfection at all N/P ratios against both cell types. Note that the background signals were approximately 1000 RLU/mg of protein for Huh7 cells and 300 RLU/mg of protein for HeLa cells, respectively.

On the other hand, peptides 4, 5, and 6 achieved significant transfection levels at N/P ratios = 8, 4, and 2, respectively. Maximal transfection efficiencies of peptides 4−6 were higher than that of the R9 peptide. pDNA complexed with longer peptides and at higher N/P ratios was efficiently delivered into the cultured cells. In order to gain further insight on the transfection abilities of the peptides, peptide/pDNA complexes at an N/P ratio = 8 and jetPEI/pDNA were transfected into Huh-7 cells (Figure 6a) and HeLa cells (Figure 6b) with various postincubation times (0, 24, 48, and 72 h). Commercially available reagent jetPEI was used as a positive control. Peptides 1−3/pDNA complexes showed almost no transfection efficiencies at all postincubation times with both cell types. Transfection abilities of the R9 peptide and jetPEI tended to decrease with an increase in postincubation times. E


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Figure 7. Intracellular distribution of Cy5−pDNA (magenta) with peptides (green) and jetPEI with HeLa cells. Acidic late endosomes/lysosomes and nuclei were stained with LysoTracker Red (red) and Hoechst 33342 (blue).

On the other hand, peptides 4−6 showed increasing transfection efficiencies even at a 72 h postincubation. In the comparison of peptides with jetPEI, the results obtained were different according to the cell types. Transfection efficiencies of peptides 4−6 were better with Huh-7 cells at longer postincubation times (≥24 h) but less for HeLa cells than jetPEI. The transfection profiles of all samples were often not consistent between Huh-7 cells and HeLa cells, which may have been due to differences of pDNA complexes in cellular uptake mechanisms, leakiness from endosomes to cytosols, and so on, among the cell lines. The cytotoxicity of peptide/pDNA complexes prepared at high N/P ratios (N/P ratios = 8 and 16) was evaluated with Huh-7 cells and HeLa cells (Figure S3). Negligible cytotoxicities of peptide/pDNA complexes were detected in HeLa cells (Figure S3a), but longer peptide/ pDNA complexes at an N/P ratio = 16 showed slight cytotoxicities against Huh-7 cells (Figure S3b). Note that jetPEI exhibited less than 20 and 30% cell viabilities with Huh7 cells and HeLa cells, respectively. CLSM Observation. The intracellular distribution of Cy5− pDNA (magenta) complexed with peptides (green) (N/P ratio = 8) and jetPEI was observed in HeLa cells using CLSM (Figures 7 and S4). Acidic late endosomes/lysosomes and nuclei were stained with LysoTracker Red (red) and Hoechst 33342 (blue), respectively. Furthermore, the fluorescence intensity of Cy5−pDNA in a single HeLa cell (Figure 8a), colocalization ratio of Cy5−pDNA with LysoTracker Red (Figure 8b), and un-colocalization ratio of Cy5−pDNA with peptides (Figure 8c) were quantified from the CLSM images. The amounts of Cy5−pDNA in the cells increased with an increase in the peptide chain length but was higher for jetPEI than for peptides. Colocalization ratios of Cy5−pDNA with LysoTracker Red were not so different among the peptides. On the other hand, jetPEI exhibited a much lower colocalization ratio than peptides, suggesting that the peptide/pDNA complexes here did not have an active endosomal escape mechanism. The amounts of Cy5−pDNA localizing without

peptides were significantly lower for peptides 5 and 6 than for peptides 1−4 and R9 peptide. pDNA appeared to be protected by longer peptides for a long time in the cells.

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DISCUSSION In the present study, we designed peptides composed of Arg− Arg−Aib repeating sequences with different peptide chain lengths, (Arg−Arg−Aib)n (n = 1−6), in order to develop Aib CPP foldamers for pDNA delivery. Several types of effective CPP foldamers, including β-peptides,31,32 γ-peptides,33 and peptoids,34 have been reported and some of them could deliver pDNA into the living cells efficiently.35 First, CD spectra measurements of synthesized peptides were performed to examine their peptide secondary structures. Peptides 2−6 exhibited characteristic spectra of right-handed (P) helical structures (Figure 2), indicating that Aib acted as a strong helix inducer. Helical foldamers were reported to have a good cellpenetrating ability.36,37 Furthermore, we have recently reported that dAA-containing peptides adopting a stable helical structure showed much higher cell-penetrating ability than those adopting a random structure.38 Indeed, the cellular uptake amount of peptide 3 was similar to that of R9 peptide (Figure 3), although peptide 3 had three fewer Arg residues than the R9 peptide, probably because of the stable helical structure of peptide 3. Peptides 4−6 showed better cellpenetrating abilities than the R9 peptide, and thus, the peptide chain length of Aib-containing peptides had a big influence on their cell-penetrating abilities. These results were consistent with previous reports on oligoarginine CPPs,34,39 in which the number of Arg resides was critical for their cell-penetrating abilities. pDNA transfection abilities of Aib CPP foldamers were strongly dependent on the peptide chain length (Figure 5). Complexes among shorter peptides, peptides 1−3, and pDNA exhibited almost no transfection efficiencies under all the conditions tested (two cell lines; all N/P ratios; all postincubation times). Physicochemical properties of peptides 1− F


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Figure 8. Quantified data from the results of CLSM observations using HeLa cells. Error bars represent the standard deviation, n = 18. (a) Fluorescence intensity of Cy5−pDNA in a cell. (b) Colocalization ratio of Cy5−pDNA with LysoTracker Red. (c) Un-colocalization ratio of Cy5− pDNA with peptides.

3/pDNA complexes supported the results of transfection experiments. From the results of fluorescence intensities (Figure S2) and zeta potentials (Figure 4) of peptide/pDNA solutions, peptide 1 did not interact with pDNA at all. Zeta potentials of peptides 2 and 3 increased with an increase in N/ P ratios but did not reach positive values even at an N/P ratio = 8, indicating their incomplete complexation. On the other hand, the fluorescence intensities of peptide 4 were quenched by N/P ratios of 2, and the zeta potential of peptide 4/pDNA solution was positive already at an N/P ratio = 2. There appeared to be a critical peptide length and/or critical Arg number for complexation of peptides with pDNA between peptide 3 (three Aib and six Arg) and peptide 4 (four Aib and eight Arg). Among peptides 4−6, peptide/pDNA complexes with sizes of approximately 150 nm and zeta potentials over +15 mV (i.e., N/P ratios = 8 and 16 for peptide 4, N/P ratios

= 2, 4, 8, 16 for peptides 5 and 6) showed significantly better transfection efficiencies (Figure 5). Maximal transfection efficiencies of peptides 4−6 were better than those of R9 peptide. Peptide 6/pDNA complexes had a good transfection ability even at an N/P ratio = 2. As the peptides became longer, the transfection abilities of the peptides improved and reached maximal levels at low N/P ratios. In general, stability and transfection efficiency of polyplexes between polycations and pDNA are dependent on the length of the polycations,40,41 which is consistent with our results in the current study. Furthermore, pDNA complexes with the R16 peptide, which had four more Arg residues than the peptide 6, and at an N/P ratio = 2, was reported to show very low transfection efficiency.42 Aib peptide foldamers have obviously better transfection abilities than oligoarginines. Cell viabilities of cultured cells treated with peptide/pDNA complexes were G



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partially affected by peptide chain length and N/P ratios; however, the cytotoxicities of peptide/pDNA were much less than with jetPEI/pDNA complex (Figure S3). Figure 6 revealed that Aib CPP foldamers had prolonged transfection abilities. Luciferase gene expression by peptides 4−6/pDNA complexes was more likely to increase with postincubation times up to 72 h. On the other hand, the transfection efficiencies of R9 peptide/pDNA and jetPEI/pDNA complexes decreased or remained the same with increasing postincubation times. Peptides containing dAAs were reported to acquire high resistance to enzymatic degradation compared with peptides composed of only natural α-amino acids,20,22 in which a half-life of the peptide containing dAAs in medium containing 10% serum was over 2 days. Aib CPP foldamers, peptides 4−6, might remain intact in medium containing 10% serum and in cells and protect encapsulated pDNA against proteases for a long time, resulting in prolonged high transfection abilities. To gain further insights into the good and prolonged transfection abilities of Aib CPP foldamers, CLSM observation was carried out using HeLa cells with staining late endosomes/ lysosomes, which had abundant proteases (Figures 7, S4, and 8). The cellular uptake amount of Cy5−pDNA was roughly estimated from the obtained images (Figure 8a). The results were similar to those of Figure 3, that is, cellular uptake amounts of peptides (Figure 3) and pDNA (Figure 8a) increased with an increase in the peptide chain length. Colocalization ratios of Cy5−pDNA with late endosomes/ lysosomes were similar among the peptides (Figure 8b). Aib CPP foldamers did not have effective endosomal escaping abilities like jetPEI, however, which may have contributed to prolonged transfection abilities of peptides 4−6 because of the slow release of peptide/pDNA complexes from acidic organelles. There was a difference in the un-colocalization ratio of Cy5−pDNA with peptides between longer peptides (peptides 5 and 6) and shorter peptides (peptides 1−4). pDNA delivered by longer peptides distributed in the cells with peptides more than those delivered by shorter peptides, implying that longer peptides protected pDNA for a long time in the cells. Taken together, longer peptide/pDNA complexes were stable, internalized into the cultured cells efficiently, and protected encapsulated pDNA against protease digestion in acidic organelles as well as in the cultured medium for a long time, resulting in the good and prolonged transfection efficiencies, compared with shorter peptides and R9 peptide/ pDNA complexes. This study demonstrated that Aib CPP foldamers were very useful tools as pDNA delivery.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b01451. RP-HPLC charts and MALDI-TOF MS spectra of peptides 1−6. Fluorescence intensities of peptide/ pDNA complex solutions. Cell viabilities of Huh-7 cells and HeLa cells treated with peptide/pDNA complexes. CLSM observation of peptide/pDNA complexes (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-95-819-2424; Fax: +81-95-819-2424; E-mail: [email protected]. (M.O.) ORCID

Makoto Oba: 0000-0002-3691-3608 Takuma Kato: 0000-0002-2561-8582 Masakazu Tanaka: 0000-0002-6929-4380 Author Contributions

This manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported in part by JSPS KAKENHI Grant Numbers JP25713008 (for M.O.) and JP17H03998 (for M. T.), by a Grant from the Takeda Science Foundation (for M.O.), and by a Grant from the Terumo Foundation for Life Sciences and Arts (for M.O.).

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REFERENCES

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CONCLUSION

We prepared peptides composed of (Arg−Arg−Aib)n (n = 1− 6) repeating sequences and evaluated them as a pDNA delivery tool. The peptide chain length critically affected a peptide secondary structure, cell-penetrating ability, and pDNA transfection efficiency. Longer peptides adopted a helical structure (n ≥ 2), internalized into the cultured cells effectively (n ≥ 3), and exhibited good pDNA transfection efficiency (n ≥ 4). These results reveal that Aib CPP foldamers are promising for pDNA delivery tools and may be helpful for designing novel CPP foldamers. H


Article

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Plasmid DNA Delivery Using Cell-Penetrating Peptide Foldamers

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