No animal was used in this study.
MS2 VLP Plasmid construction and expression system
MS2 VLP expression vector was prepared by amplifying the MS2 Coat and a part of maturase DNA sequences from the pMS27 Plasmid (BCCM/LMBP plasmid Collection, Cabri, Belgium) (Table 1)18. The amplified sequence was inserted in-frame into pACYCDuet plasmid (Novagen, Gibbstown, NJ, USA) through BamH1 and HindIII restriction enzyme (Thermo Fisher Scientific Inc., USA) sites. The oligonucleotide sequence encoding the shRNA sequence was designed by siRNA Wizard ™software (Invivogen, USA) (Table 1). The synthesized shRNA (Macrogen Inc., South Korea) fused to four repeats of MS2 bacteriophage pac sequence (Macrogen Inc., South Korea) was sub-cloned into the other multiple cloning sites of pACYCDuet plasmid through BglII and Kpn1 restriction enzyme (Thermo Fisher Scientific Inc., USA) sites (Fig. 4e). This sequence (shRNA-4pac, Table 1) was used as a control RNA sequence with the ability to be packed into VLP in each step, plasmid construction was verified by Sanger sequencing (Macrogen Inc., South Korea) and was named the SC Duet.
The SC Duet plasmid was introduced into the BL21(DE3) strain of E. coli Bacteria as a prokaryotic expression system. The Ecoli BL21(DE3)-SC Duet was cultured in a terrific broth medium at 37 °C, supplemented with 34 µg/ml chloramphenicol (pACYCDuet antibiotic selection). The expression of the sequences was induced by 1 mM IPTG (isopropyl β-d-1-thiogalactopyranoside, Thermo Fisher Scientific Inc., USA) at an OD 600 = 0.6 for 16 h at 22 °C. Bacteria were precipitated by centrifugation (6000 g, 20 min, 4 °C), and the derived precipitate was resuspended in the appropriate buffer (in 1/5th of the initial bacterial culture volume). Lysozyme treatment (0.05 mg ml-1) was performed for 30 min at room temperature followed by sonication (total time: 3 min, 51 W). Cell debris was collected by centrifugation (13500 g, 20 min, 4 °C). The supernatant containing VLPs was filtered through a 0.22 µm membrane (Jet Bio-Filtration Co, China) and stored at 4 °C for further examination2. To eliminate cell debris, the suspension was filtered through a 0.1 µm membrane (Sartorius, Germany) and then MS2 VLPs were purified according to the polyethylene glycol (PEG) precipitation method44. In brief, the precipitation of VLPs was performed by adding PEG (MW: 6000, Sigma Aldrich, USA) at 10% of the final concentration (w/v) and 500 mM NaCl. The suspension was incubated at 4 °C for 18 h and subsequently was centrifuged at 10000 g for 1 h, 4 °C. The pellet containing MS2 VLPs was resuspended in the buffer used in the first step of purification (in 1/10th of the initial resuspension volume). Eventually, this MS2 VLP suspension was filtered through a 0.22 µm membrane (Jet Bio-Filtration Co, China). All MS2 VLP samples were kept at 4 °C. Solutions of 1 mM, and 100 mM NaNO3 (Merck & Co., Inc. USA), 1 M Tris (Sigma Aldrich, USA) buffer, and HEPES Buffer (Biowest, France) were prepared fresh, one day before experiments.
MS2 VLPs disassembly and Western blot analysis
To confirm the expression of VLP coat proteins, purified MS2 VLPs were disassembled by diluting 1:2 in 20 mM cold glacial acetic acid for 1 h at 4 °C. To precipitate the free nucleic acids, samples were centrifugated at 4 °C (16,000g, 20 min). Then, we assessed the expression of coat protein by SDS-PAGE (12.5% acrylamide gel). Proteins were transferred to a 0.45 µm nitrocellulose membrane (Bio-Rad Laboratories, Inc, USA). The membrane was blocked with 5% non-fat dry milk overnight at 4 °C and then incubated with a polyclonal Anti-Enterobacteria Phage MS2 coat protein antibody (1:7000 dilution, Millipore Sigma Co. USA) for 3 h at room temperature. Incubation with HRP-labeled secondary antibody (1/18,000 dilution, Abcam, USA) for 1 h was performed to confirm the presence of 14 and 28 k Da bands. The membrane was developed with Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific Inc., USA). Signals were detected using the Chemiluminescence Detector system (G:BOX Chemi XT Analyser, SYNGENE, Eur.).
Transmission electron microscopy
The MS2 VLPs were diluted at 1:4 in the buffer. 10 µl of samples were dropped onto carbon-coated copper grids and then were negatively stained by 2% uranyl acetate. After drying, VLPs were observed by transmission electron microscopy (Leo912AB, Germany) at 120 kV at 4000×, 10,000×, 25,000×, 31,500×, 50,000×, 63,000×, and 80,000× magnifications.
Particle size, polydispersity index evaluation, Zeta potential evaluation, and electrophoretic mobility
MS2 VLPs were diluted 1:4 in selected buffers and solutions. Particle size and polydispersity index (PDI) were measured by particle size analyzer (Vasco3-Cordouan, France) based on Dynamic Light Scattering (DLS), 657 nm at 25 °C. Samples were analyzed by cumulants analysis of 5 consecutive measurements.
Zeta potential for those samples which were not stored in Buffer was estimated by Zeta potential detector (Zeta compact-CAD, France) and reported as zeta potential (mV) line plot at 25 °C. To ensure reliability, it has been performed at least three times.
Electrophoretic mobility (denoted as µ) of MS2 VLPs was measured at 23 ± 1 °C using Zeta compact-CAD (France) as a function of buffer containing different electrolytes at pH 7 and 8 for PEG-precipitated VLPs after filtering via a 0.1 µm membrane to remove all the aggregated particles from the suspension. VLPs were diluted at 1:10 in selected buffers and solutions. Experiments were carried out at least three times.
pH assessment
The pH of all solutions and buffers were measured by a pH meter (Mettler Toledo Co., Switzerland), one day before the experiment. Evaluations were done in triplicates at 0 h, 3 h, 12 h, 24 h, 7 days, and 1-month time points.
Protein spectrophotometry and MS2 VLPs measurement
Protein concentration was measured by absorbance at 280 nm (A280)-1 Abs in 1 cm (1 mg ml−1) (Epoch2™ BioTek, Agilent Technologies, USA). Measurements were performed three times at different time points (1, 2, 3, 4, 7, and 12 months). According to the protein concentration and the molecular mass of each VLP particle (14 kDa per coat protein × 180 coat proteins per VLP = 2520 kDa), the number of VLPs in 100 mM NaNO3 solution and NaNO3-Tris buffer (pH:8) was calculated:
Each VLP molecular mass = 2520 kDa = 4.18e−15 mg.
Number of VLPs (in each milliliter) = protein concentration of VLP suspension (mg ml−1)/4.18e−15.
The stability of MS2 VLP containing shRNA-pac in serum
To determine MS2 VLP’s stability in biological conditions, samples were mixed with fetal bovine serum (Biowest, France) (1:1), incubated in 37 °C for 2, 4, 6, 8, 10, 12, 24, and 48 h, and then stored in − 80 °C for further RNA isolation, reverse transcription, and PCR. Furthermore, MS2 VLPs were also incubated in different serum concentrations (10, 30, 50%) for 1 h at 4 °C and particle size and PDI were evaluated diluting 1:4 with additional buffer.
RNA extraction, reverse transcription, and polymerase chain reaction
300 ul of MS2 VLP suspension in different solutions or buffers (after purification by PEG precipitation method) were subjected to RNA isolation using the Total RNA Isolation Kit (DENAzist Asia Co., Iran) according to the manufacturer’s instructions. The quantity of RNA was determined by a spectrophotometer (spectroEpoch2™ BioTek, Agilent Technologies, USA). A total of 1 µg RNA was used for cDNA synthesis using random hexamer and MMLV reverse transcriptase (Thermo Fisher Scientific Inc., USA). To identify and check the integrity of shRNA-4pac RNA, polymerase chain reactions were carried out by Taq DNA Polymerase Master Mix (Ampliqon, Denmark) and designed primers (Table 1). Polymerase chain reaction steps were 94 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, 15 s at 70 °C, 15 s at 72 °C, and a final extension period of 10 min at 72 °C. PCR products were run on 2% agarose gel.
Cell culture and Immunocytochemistry
To confirm the VLP entrance, Neuro2A cells (Mouse Neuroblastoma cell line, ATCC) were cultured in Dulbecco’s modified minimal essential medium (DMEM, low glucose) (Gibco: Thermo Fisher Scientific, USA) containing 10% (v/v) of FBS (Gibco: Thermo Fisher Scientific, USA) and 1% penicillin/streptomycin (Biowest, France) at 37 °C, 5% CO2 and 95% humidity. Cells were grown on poly D-lysine (Sigma Aldrich, USA) coated coverslips in four groups (1 h and 4 h after VLP treatment, sham (without VLP), and blank negative control (with VLP treatment and without primary antibody). Briefly, cells were treated with 0.05 µg/ml of VLP suspension 24 h after seeding. The cells were fixed by 2% paraformaldehyde (w/v) for 10 min at room temperature. Subsequently, after washing with cold PBS, the cells were permeabilized with 0.25% Triton X-100 in PBS) for 10 min at room temperature and blocked with PBS containing 1% BSA. Cells were then incubated with primary antibody (anti- Enterobacteria Phage MS2 coat protein, 1:5000, Millipore Sigma Co. USA) for 3 h at room temperature. The coverslips were washed with cold PBS and then incubated for 1 h at room temperature in darkness with a secondary antibody (Monkey anti-rabbit Cy3 conjugated, 1:200, Jackson ImmunoResearch Laboratories Inc., USA). The coverslips were mounted on slides after washing with cold PBS. Stained cells were visualized using fluorescent microscopy (Nikon, Japan).
Flow cytometry
The quantitative analysis of VLP entrance to Neuro2A cells was performed by flow cytometry. Cells were cultured as previously described in the immunocytochemistry section. Neuro2A cells were treated with 0.05 µg/ml of VLP suspension 24 h after seeding and prepared for flow cytometry in four groups (1 h and 4 h after VLP treatment, sham (without VLP), and blank negative control (with VLP treatment and without primary antibody). Following the cell incubation with VLP (1 and 4 h) and washing with PBS to remove additional VLPs, the cells were trypsinized and subjected to flow cytometry. Followed by Fixing with 4% paraformaldehyde (w/v) for 30 min at room temperature, permeabilizing by 0.2% Triton X-100 (15 min), and blocking with 0.5% BSA and 2% FBS for 30 min on ice. Subsequently, the cells were incubated with primary antibody solution (anti- Enterobacteria Phage MS2 coat protein, 1:500, Millipore Sigma Co. USA) for 3 h at room temperature and then were incubated for 1 h at room temperature in darkness with a secondary antibody (Monkey anti-rabbit Cy3 conjugated, 1:400, Jackson ImmunoResearch Laboratories Inc., USA). Interval of all steps the cells were washed with 0.5% FBS-PBS (cold, 3 times) and were centrifuged at room temperature (1500 rpm, 4 min). the cells were diluted in PBS and analyzed with BD ™Accuri C6 Flow Cytometer (USA). Results were analyzed using FlowJo software (Version 7.6.1, USA) and were shown as an intensity plot (Fig. 3).
Statistical analysis
Data analysis was performed by GraphPad Prism (version 8.0.2, GraphPad Software Inc., CA). Results were analyzed using the Mann–Whitney and Kruskal Wallis test analysis.
Equipment and setting
All charts of pictures were set and labeled in Adobe photoshop (2020) and were saved in TIFF format. TEM microscopic and cell images were used without any change. Imaging for ICC experiments was done by Nikon fluorescent microscope in the same condition for all shots at 10× magnification (NIS-Elements F 4.60.00 64-bit software). Agarose gels are full-length versions and just a little brightness and contrast were changed for the entire gels. A full-length original version of the blot was used. All graphs were designed with GraphPad Prism (version 8.0.2, GraphPad Software Inc., CA).
