AccuTargetTM GAPDH positive control siRNA and mCherry-siRNA (sense: 5’-GAGGAUAACAUGGCCAUCAUU-3’, antisense: 5’-UGAUGGCCAUGUUAUCCUCUU-3’) was provided by Bioneer Co. (Daejeon, South Korea), and Cy3-labeled siRNA (sense: 5’-GCGACGCUGUCAUCGAUUUUU-3’, antisense: 5’-AAAUCGAUGACAGCGUCGCUU-3’) was synthesized by GenePharma Co., Ltd. (Shanghai, China) in duplex form. All siRNAs were purified by HPLC. All single and fusion peptides were synthesized by GL Biochem, Ltd. (Shanghai, China) with more than 95% purity. Hank’s balanced salt solution (HBSS) was obtained from Life Technologies (CA, USA). Agarose and 10,000X TopRed Nucleic Acid Gel Stain were purchased from GenomicBase (Seoul, South Korea). Tris (Glentham Life Sciences Ltd., Corsham, UK), acetic acid (glacial) (Merck, Hesse, Germany), and EDTA (GenomicBase, Seoul, South Korea) were used for the 1X TAE buffer. Heparin sodium salt (from porcine intestinal mucosa) was purchased from Sigma-Aldrich (MO, USA). 6X DNA loading dye was procured from Biofact Co., Ltd. (Daejeon, South Korea). For cell cultures, we used Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, MA, USA), fetal bovine serum (FBS; PAN Biotech, Bavaria, Germany) and penicillin-streptomycin (Life Technologies, CA, USA). Opti-MEMTM and 0.25% trypsin-EDTA (1X) were obtained from Thermo Fisher Scientific (MA, USA). LipofectamineTM 2000 reagent was purchased from Invitrogen (CA, USA). Hoechst 33342 (Invitrogen, CA, USA), Flamma® 496 Phalloidin (BioActs, Incheon, South Korea) and SiR-actin kit (Cytoskeleton, Inc., CO, USA) were used for fluorescent labeling. 10% neutral buffered formalin solution (Sigma-Aldrich, MO, USA), Triton X-100 (Bio-Rad Laboratories, Inc., CA, USA) and bovine serum albumin (BSA; Generay Biotech Co., Ltd., Shanghai, China) were used for fluorescent imaging. Nuclease-free water was purchased from Integrated DNA Technologies, Inc. (IA, USA). Tri-RNA reagent (Favorgen Biotech Co., Kaohsiung, Taiwan), chloroform (Sigma-Aldrich, MO, USA), isopropanol (Molecular biology grade; Fisher Scientific, NH, USA), and absolute ethanol (Molecular biology grade; Fisher Scientific) were used for the extraction of RNA. ReverTra Ace® qPCR RT Master Mix with gDNA Remover kit and THUNDERBIRD® SYBR® qPCR Mix (TOYOBO Co., Ltd., Osaka, Japan) were procured for cDNA synthesis and quantitative real-time PCR. The CytoTox 96® Non-radioactive cytotoxicity assay kit was obtained from Promega (WI, USA). Chlorpromazine hydrochloride, methyl-β-cyclodextrin, cytochalasin D (from Zygosporium mansonii), and Filipin III (from Streptomyces filipinensis) endocytosis inhibitors were purchased from Merck (Hesse, Germany) for the mechanism study. All cell culture flasks and plates were purchased from NEST Biotechnology Co., Ltd (Wuxi, China). For the in vivo studies, isoflurane (Hana Pharm. Co., Ltd., Hwaseong, South Korea) as an anesthetic and 31-gauge needle insulin syringes (BD, NJ, USA) were used.
Preparation of siRNA/peptide nanocomplexes
GAPDH-siRNA was dissolved in HBSS as 1 μM, and all peptides were dissolved in HBSS or distilled water at 1-2 mg/mL. The sequences of all peptides are summarized in Additional file 1: Table S1. The fusion peptides designed in this study have a GCG linker between SPACE and the oligoarginine peptides. Fusion peptides and siRNAs in HBSS buffer formed the self-assembled nanocomplexes under incubation at room temperature (25°C) for 30 minutes with appropriate nitrogen/phosphate (N/P) ratio. The N/P ratio was derived using the molar ratio of amine groups in the cationic peptides to those of phosphate groups in the RNA.
Gel retardation assay
The formation of siRNA/peptide nanocomplexes was confirmed by gel retardation assay. Total 10 μL nanocomplexes of 10 pmol siRNA and each peptide were self-assembled with a range of N/P ratios (1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, and 100:1) as mentioned above. After adding 6X loading dye, the 12 μL nanocomplexes were loaded into the 2% agarose gel (w/v) prepared in 1X TAE buffer (40 mM tris, 20 mM acetic acid, 1 mM EDTA, pH 8.6) with 10,000X TopRed Nucleic Acid Gel Stain for visualization. Gel running was performed at 100 V for 30 minutes using the Mupid-2plus electrophoresis system (Optima Inc., Tokyo, Japan). Pictures of the electrophoretic mobility shift of the nanocomplexes were taken by the ChemiDocTM XRS+ System (Bio-Rad, CA, USA).
Size and zeta potential measurement
The size and zeta potential of the nanocomplexes were measured by dynamic light scattering (DLS). Based on the results of the previous gel retardation assay, a 20:1 N/P ratio was determined for the rest of the experiments due to the stable nanocomplex formation of all fusion peptides. 200 pmol siRNA and each peptide were self-assembled at a 20:1 N/P ratio, as described above. After 30 minutes, the nanocomplexes were diluted with HBSS to a final siRNA concentration of 200 nM. 200 nM was chosen as the optimal siRNA concentration based on GAPDH activity assay (Additional file 1: Fig. S2) and used in subsequent experiments. Then, the 200 nM solution was filtered with a 0.45-μm syringe filter (GVS, Bologna, Italy). After vortexing for 30 seconds, 1 mL of the nanocomplexes was loaded into a cuvette (Ratiolab, Hesse, Germany) to measure the size and disposable folded capillary cell (Malvern Panalytical, Ltd., Malvern, UK) to measure the zeta potential. The size and zeta potential of the nanocomplexes were measured using a Zetasizer Nano ZS (Malvern Instruments, Ltd., Worcestershire, UK).
Stability of siRNA in serum
The stability of the siRNA in nanocomplexes was confirmed using agarose gel electrophoresis. 100 pmol of siRNAs and peptides (20:1 N/P ratio) were self-assembled for 30 minutes at room temperature. Then, the nanocomplexes in 10% (v/v) FBS were incubated at 37°C, and 20 μL of each sample were collected at 24, 48, 72, and 96 hours. However, the nanocomplex in 50% (v/v) FBS was incubated at 37°C, and 20-μL samples were collected at 4, 8, 12, 24, and 48 hours. The siRNAs were dissociated from the nanocomplexes using incubation at 37°C for 30 minutes after the addition of 4 μL of 1 mg/mL heparin. After mixing the 6X loading dye to each sample, 24 μL samples were loaded into 2% agarose gel with 1X TAE buffer in Mupid-2plus. Gel running was performed for 30 minutes at 100 V. The remaining siRNA was analyzed by the gel documentation system LSG 1000 (iNtRON Biotechnology, Seongnam, South Korea).
Cellular uptake efficiency using flow cytometry
Human cervical cancer HeLa, human dermal fibroblasts neonatal (HDFn), and immortal keratinocyte cell line HaCaT were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified incubator that contained 5% CO2 (Esco Micro Pte. Ltd., Changi, Singapore). 3.0×105 cells were added to each well of a 6-well plate and incubated at 37°C in a 5% CO2 incubator overnight. After the nanocomplex formation of the final 200 nM Cy3-labeled IL10-siRNA and peptides (20:1 N/P ratio) in serum-reduced Opti-MEMTM, 325 μL of the nanocomplex were added to each well and incubated for 4 hours at 37°C in a 5% CO2 incubator. The wells were washed twice with 1 mL of pre-warmed phosphate-buffered saline (PBS). After 200 μL treatment of 0.25% trypsin-EDTA for 2 minutes, 2 mL of fresh DMEM medium were added. The suspended cells were centrifuged at 360 × g for 5 minutes. After the supernatant was removed, the cells were washed with PBS twice under the same conditions. The final cell pellets were resuspended in ice-cold PBS and analyzed using a flow cytometer (Gallios; Beckman Coulter, CA, USA).
Observation of cellular uptake using fluorescence microscopy
HeLa cells were seeded into a 24-well plate at the number of 1.0×105 cells per well and incubated overnight at 37°C in a 5% CO2 incubator. After the nanocomplex formation of the final 200 nM Cy3-labeled IL10-siRNA and peptides (20:1 N/P ratio), 160 μL of the nanocomplexes were applied to the cells in Opti-MEMTM for 4 hours. After washing twice with 200 μL of pre-warmed PBS, the cells were fixed using 200 μL of 10% formalin solution for 10 minutes. Then, the cells were treated serially with 200 μL of 0.1% Triton X-100 in PBS (0.1% PBST) for 10 minutes, then 200 μL of 2% BSA in 0.1% PBST at room temperature for 30 minutes. The cells were incubated in the Hoechst 33342 dye solution for 10 minutes in the absence of light. After washing twice with 200 μL of pre-warmed PBS, the cells were incubated in the Phalloidin dye solution at room temperature for 1 hour. After washing twice with 200 μL of pre-warmed PBS, the cells were observed at 200× magnification by a fluorescence microscope (Ti-E; Nikon, Tokyo, Japan).
Cellular internalization observed using confocal microscopy
The cellular internalization of the siRNA/peptide nanocomplex was investigated using confocal images. HeLa cells of 2.0×104 were incubated in a 35-mm confocal dish (SPL Life Sciences Co., Ltd., Pocheon, South Korea) for 24 hours. After a 30-minute incubation of the final 50 nM Cy3-labeled siRNA and fluorescein (FITC)-labeled S-R15 (20:1 N/P ratio), the nanocomplex was applied to the cells for 4 hours. The nucleus and actin were stained using 5 μg/mL Hoechst 33342 and 100 nM SiR-actin kit, respectively. The intracellular localization and co-localization of siRNA and S-R15 were confirmed using fluorescence and a confocal microscope (Ti2; Nikon, Tokyo, Japan). Both of them were analyzed at the single-molecule level using super-resolution radial fluctuation (SRRF). Bright-field and fluorescence images were acquired at 900× magnification. ImageJ software was used to merge the fluorescence images of Cy3, FITC, and SiR-actin .
Gene knockdown evaluation by quantitative RT-PCR
GAPDH mRNA expression reduced by nanocomplex was checked using quantitative real-time PCR. HeLa cells were seeded into a 24-well plate at a density of 1.0×105 cells per well. After overnight incubation at 37°C in a 5% CO2 incubator, 160 μL nanocomplex with the final 200 nM siRNA at 20:1 N/P ratio was applied to the cells in Opti-MEMTM for 5 hours. As a positive control, LipofectamineTM 2000 reagent was used according to the provided protocols. After incubation for 5 hours, the media were replaced with 500 μL of fresh supplemented DMEM and incubated for an additional 19 hours. After washing three times with nuclease-free water, the total RNA of the cells was purified using Tri-RNA reagent following the manufacturer’s protocol. The concentration and purity of purified RNA were measured using the Take3 plate as a nanodrop mode of a microplate reader (Epoch 2; BioTek Instruments, Inc., VT, USA). 100 ng of RNA were transcribed reversely to cDNA using ReverTra Ace® qPCR RT Master Mix with a gDNA Remover kit according to the manufacturer’s protocol. The primer sequences of GAPDH and beta-actin that were used are provided in Additional file 1: Table S3. After mixing 10 ng of cDNA, primers and THUNDERBIRD® SYBR® qPCR Mix according to the manufacturer’s protocol, the PCR reaction was performed following the three-step cycle (Pre-denaturation; 95°C for 60 seconds, Denaturation; 95°C for 15 seconds, Annealing; 55°C for 30 seconds, Extension; 72°C for 60 seconds). GAPDH mRNA expression was analyzed using the QuantStudio 3 real-time PCR system (Applied Biosystems, CA, USA). The GAPDH mRNA expression was normalized by β-actin mRNA expression. The relative expression level was calculated using the ΔΔCt method.
Endocytosis pathway study
In a 6-well plate, HeLa cells were seeded at a density of 4.0×105 cells per well. After replacing the medium with Opti-MEMTM, the cells were treated with each inhibitor for 30 minutes, i.e., Chlorpromazine (10 μg/mL), Methyl-β-cyclodextrin (5 mM), Cytochalasin D (1 μM), and Filipin III (1 μg/mL) . The nanocomplex of the final 200 nM Cy3-labeled siRNA and FITC-labeled S-R15 was self-assembled as described above. After adding 325 μL of nanocomplex to the inhibitor-treated cells, the cells were incubated for 4 hours at 37°C in a 5% CO2 incubator. Cells with the fluorescence of Cy3 and FITC were analyzed using flow cytometry, as mentioned above.
Biocompatibility of the fusion peptides
The biocompatibility of the fusion peptides was assessed using CytoTox 96® non-radioactive cytotoxicity assay according to the manufacturer’s protocol. Human dermal fibroblasts neonatal (HDFn) cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 humidified incubator (Esco Micro Pte., Ltd., Changi, Singapore). HDFn cells were seeded on a 96-well plate at a density of 8.0×103 cells per well. After incubation overnight, serially-diluted fusion peptides were added to cells at concentrations of 1, 0.5, 0.25, 0.125, and 0.0625 mg/mL. After incubation for 5 hours, 50 μL aliquots of each well were transferred to each empty well of a 96-well plate. Then, 50 μL of CytoTox 96® reagent were added to each well. Cells were incubated for 30 minutes at room temperature in light-free conditions. After adding 50 μL of stop solution, absorbance was measured at 490 nm using a microplate reader.
In vivo siRNA retention effect
All animal experiments were performed according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Incheon National University (INU-ANIM-2020-01). HeLa cells cultured over 90% confluency were prepared to 6.0×106 - 1.0×107 cells in 100 μL of fresh DMEM (w/o FBS). Five-week-old BALB/c nude mice (Orientbio, Inc., Seongnam, South Korea) were anesthetized with 1.5 - 2% isoflurane in pure oxygen gas. Then, the cells were injected subcutaneously into both sides of the backs of the mice with a 1 mL insulin syringe. The formation of massive tumors was confirmed after 10 days. On day 10, the tumor-xenografted mice were anesthetized, and 29.78 μL of 1 μg free Cy3-labeled siRNA were injected intratumorally into the left tumor of a mouse. Then, 29.78 μL of 1 μg Cy3-labeled siRNA in the S-R11 nanocomplex (20:1 N/P ratio, as mentioned above) were injected intratumorally into the right tumor. The fluorescence intensity of Cy3 was visualized every hour up to 4 hours after injection using an in vivo fluorescence imaging system (FOBI, CELLGENTEK Co., Ltd., Cheongju, South Korea). The area, mean of intensity, and integrated density (area × mean of intensity) were quantified from the fluorescence images using NEOimage software.
In vivo gene silencing effect
In vivo gene silencing effect of nanocomplex was assessed using mCherry fluorescence imaging. For the transient expression of mCherry, 32 μg of pmCherry-N1 vector were transfected into HEK293T cells cultured in T175 flask over 80% confluency. As a transfection reagent, Lipofector-EXT (AptaBio, Yongin, South Korea) were used according to the manufacturer’s protocols. After incubation for 5 hours in 20 mL of Opti-MEMTM, the cell media were replaced with 40 mL of fresh DMEM and the cells were cultured in the 5% CO2 incubator at 37oC for another 2 days. The pmCherry-N1-transfected cells were harvested and prepared at 5.0×106 cells/50 μL. Then, 4 μg of mCherry-siRNA in the S-R11 nanocomplex (20:1 N/P ratio, prepared as mentioned above) were added to the pmCherry-N1 transfected cells. The cells with free siRNAs and cells with the siRNA nanocomplex were injected subcutaneously on the left and right back of BALB/c nude mice under 1.5 - 2% isoflurane anesthesia, respectively. The fluorescence images of mCherry were taken on day 0 and 1 using an in vivo fluorescence imaging system and quantitative analysis of four mice images was carried out as previously mentioned. The relative integrated intensity was calculated by that integrated intensity at day 1 was divided by that at day 0.
Histological analysis of skin tissues
The potential safety was explored using histological analysis of the nanocomplex-injected skin tissues. The siRNA/S-R11 nanocomplex was injected intradermally into three spots of hairless back of anesthetized five-week-old BALB/c mice. The S-R11 nanocomplex (20:1 N/P ratio) was prepared as mentioned above. After 6 days, the mice were euthanized and the harvested skin tissues were fixed in 10% neutral buffered formalin. Then, the tissues were dehydrated, paraffin embedded, and sectioned to 4 μm thickness. After the deparaffinization, the tissues were stained with standard hematoxylin and eosin (H&E), and observed using a Leica DM1000 LED microscope (Leica Microsystems, Hesse, Germany) under 4X and 20X magnifications (scale bar = 100 μm).
The quantitative data was presented as mean ± standard deviation. The statistical significance of the differences was evaluated using a p-value less than 0.05, 0.01, and 0.001 calculated by a t-test.