Female 6–10-week-old CD (Sprague Dawley; SD) rats (Charles River Japan Inc., Kanagawa, Japan) and BALB/c mice (CLEA Japan Inc., Tokyo, Japan) were used in the study. All animals were maintained under controlled conditions (temperature, 21.0–24.5°C; humidity, 45 ± 15%; ventilation, 8–15 times/h; light/dark cycle, 12 h) in a pathogen-free room. Animals received ad libitum food and water and were handled according to the approved protocols of the Animal Committee of Osaka University (Suita, Japan) and the Ethics Committee for animal experiments of the Safety Research Institute for Chemical Compounds Co. Ltd. (Sapporo, Japan).
Optimization of Intradermal Injection Conditions
To determine the suitable ignition powder mass for rats, the animals were anesthetized. India ink (Kaimei & Co., Ltd. Saitama, Japan) was diluted twice with distilled water before injecting 30 μL via PJI (DAICEL Corporation, Osaka, Japan) into the right flank using various doses of ignition powder (15, 35, 55, 75, or 90 mg) with 40 mg smokeless powder. The mice were also injected with 10 μL of diluted India ink (diluted 10 times with distilled water before injection) into the right flank area using 15, 25, 35, or 45 mg of ignition powder with 40 mg smokeless powder. The ignition powder mass affects the distribution depth and smokeless powder affects the injection volume of India ink . After injection, the tissues were collected and fixed with 10% formalin (Nacalai Tesque, Inc., Tokyo, Japan) for histopathological analysis. The target injection area is illustrated in Supplementary Fig. 1.
Intra-Nuclear Plasmid DNA Delivery by Pyro-Drive Jet Injector
Cy3-labeled plasmid DNA (Cy3-p) at 0.5 μg/μL (Takara Bio Inc., Shiga, Japan) was injected into the right flank area with the PJI or needle syringe using anesthetized rats (30 μL) and mice (10 μL). A combination of 35 mg ignition powder and 40 mg smokeless powder was used for rats, whereas a combination of 25 mg ignition powder and 40 mg smokeless powder was used for mice. Needle syringes of 27G and 30G were used for rats and mice, respectively. The diameter of the PJI injection nozzle was roughly equivalent to 30G for both rats and mice. After injection, the tissue was excised from an approximate 1-cm2 region and immediately frozen in OCT compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan) and sectioned at − 20°C, which is the optimal cutting temperature. To analyze plasmid DNA delivery, sectioned skin samples were stained with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, MA, USA) and observed using a fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). When the Cy3 and DAPI fluorescence signal overlap was > 50%, Cy3 was considered to be introduced into the nuclear directory after injection.
Evaluation of Skin Injury after Plasmid DNA Injection
To assess the influence of plasmid DNA injection in the intradermal region, pGL3 firefly luciferase expression plasmid (Luc plasmid) DNA (1 μg/μL) (Promega Corporation, WI, USA) was dorsally injected (30 μg) using the PJI and needle syringe (27G) into the right flank area of rats, and dorsally injected (10 μg) using the PJI and needle syringe (30G) in mice. Combinations of 35 mg ignition powder and 40 mg smokeless powder for rats, and 25 mg ignition powder and 40 mg smokeless powder for mice were used. After injection, skin samples were collected at 0, 6, and 24 h. The excised skin sections were fixed with 10% formalin and embedded in paraffin. Histological examinations were performed based on hematoxylin–eosin (HE) staining. HE staining procedures were subsequently performed on deparaffinized sections.
Gene Expression and Kinetic Analysis of Injected Plasmid DNA
Time Course of Gene Expression
To compare gene expression between PJI and needle syringe methods, the Luc plasmid (1 μg/μL) was dorsally injected into rats (n = 4; 30 μg/animal) and mice (n = 6; 10 μg/animal) on the right and left flank areas using the PJI and needle syringes. Combinations of 35 mg ignition powder and 40 mg smokeless powder for rats, and 25 mg ignition powder and 40 mg smokeless powder for mice were used. After injection, the plasmid DNA-injected skin regions were punched out with a 5-mm (in diameter) biopsy punch (Kai Industries Co Ltd., Seki, Japan) every 24 h for 72 h. After skin sample collection, a luciferase assay was detected using a Luciferase assay kit (Promega) according to the manufacturer’s instructions. Relative light units (RLU) were measured using a lumitester C-110 (Kikkoman Biochemifa Company, Tokyo, Japan).
Quantitative Analysis of Injected Plasmid DNA
The injection conditions for the Luc plasmid (30 μg) in SD rats (n = 3) were the same as those for the gene expression experiments described previously herein. After the tissue samples were collected, total DNA was purified using a NucleoSpin DNA purification kit (Macherey-Nagel, Germany) according to the manufacturer’s instructions. Luciferase gene copy number was determined using a luciferase gene specific Taq-man probe (ID Mr0398758_mr) and an Applied Biosystems 7900HT PCR System (Thermo Fisher Scientific).
Model DNA Vaccination Experiment
Ovalbumin (OVA) Gene Expression
Dosages of either 3 μg (0.1 μg/μL), 10 μg (0.33 μg/μL), or 30 μg (1 μg/μL) pOVA (pcDNA3-OVA; pcDNA3-OVA was a gift from Sandra Diebold & Martin Zenke; Addgene plasmid no. 64599, http://n2t.net/addgene:64599, RRID = Addgene_64,599; Addgene, MA, USA) were injected into the right flank area of SD rats using the PJI and needle syringe (n = 3 for each device) . Twenty-four hours after injection, the plasmid DNA-injected skin region was punched out with a 5-mm (in diameter) biopsy punch and total protein was extracted using the same methods as in the luciferase assay protocol. OVA and total protein were quantified using an OVA ELISA Kit (ITEA Inc., Tokyo, Japan) and Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, CA, USA), respectively, according to the manufacturers’ instructions.
OVA-Specific Antibody Production by pOVA Injection
Dosages of 10 μg pOVA (0.3 μg/μL, 30 μL/injection), 60 μg pOVA (1 μg/μL, 30 μL × 2 injections/rat), and 120 μg pOVA (1 μg/μL, 30 μL × 4 injections/rat) were injected into the right flank area of SD rats (n = 4) a total of three times over a 2-week period (at weeks 0, 2, and 4) using the PJI, and 120 μg pOVA (1 μg/μL, 30 μL × 4 injections/rat) was injected into the right flank area of SD rats (n = 4) a total of three times over a 2-week period (at weeks 0, 2, and 4) using a needle syringe (27G). Serum was collected before injection, and then again every 2 weeks from 0 to 8 weeks.
To compare antibody production, levels were compared after pOVA injection by the PJI and those induced by OVA protein delivered via the needle syringe (27G). For this, 10 μg pOVA, (0.3 μg/μL, 30 μL/injection) and 120 μg pOVA (1 μg/μL, 30 μL × 4 injections/rat) were injected by the PJI, and 60 μg OVA (1 μg/μL, 30 μL × 2 injections/rat) was injected using the needle syringe to SD rats (n = 4 for 60 μg OVA group and n = 5 for other groups) a total of three times over a 2-week period. Serum was collected before injection, and then again every 2 weeks from 0 to 8 weeks. To assess antibody production stability, 60 μg of pOVA (1 μg/μL, 30 μL × 2 injections/rat) was injected by the PJI using two different quantities of ignition powder, 35 or 90 mg (n = 3), into the right flank area a total of three times over a 2-week period (0, 2, and 4 weeks), and serum was collected before every injection until 6 weeks (0, 2, 4, and 6 weeks). A total of 60 μL of 10 mM Tris–1 mM EDTA (TE) solution (Qiagen N.V., PL, NLD) was injected by the PJI as a negative control for all experiments. In the mouse model, 10 μg (0.5 g/μL), 3.3 μg (0.17 g/μL), and 1 μg (0.05 g/μL) pOVA were injected into the right flank area of Balb/c mice (n = 4) a total of three times over a 2-week period (at weeks 0, 2, and 4) using the PJI and a needle syringe (30G). Serum was collected before injection, and then every 2 weeks from 0 to 8 weeks. A TE solution was injected with the PJI as a negative control. Anti-OVA specific antibody levels were analyzed by ELISA.
OVA Antibody ELISA
Serum was collected from rats on days 0, 14, and 28 before pOVA injection and on day 42 for all vaccination studies, as well as on day 56 for the antibody production stability study. The collected serum samples were stored at − 80°C until analysis. The recombinant OVA protein (Wako Pure Chemicals Industries Ltd., Tokyo, Japan) was coated onto ELISA plates at 10 μg/mL in a carbonate buffer incubated overnight at 4°C. Serum samples were diluted from 10- to 31,250-fold in PBS containing 5% skim milk and then incubated on plates at 4°C overnight. After serum culture, HRP anti-rat IgG (GE Healthcare Life Sciences, PA, USA) was incubated with the samples for 3 h at room temperature (24°C), and color development was performed with the peroxidase chromogenic substrate 3,3′-5,5′-tetramethyl benzidine (Sigma-Aldrich Co., St Louis, MI, USA). Absorbance was detected using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm. OVA antibody ELISA for mouse serum was performed according to the rat serum ELISA procedure save for the detection antibody. HRP anti-mouse IgG (Promega Corporation, WI, USA) was employed for the mouse model.
The cut-off value was calculated using the OD450 value from ELISA using the serum obtained from the TE-injected group. The average OD450 value was applied as follows: cut-off value = OD450 average × 2 and the antibody titer was equal to the dilution fold at the cut-off point, calculated as the antibody titer of serum samples using two-point calibration.
A non-parametric Shirley–Williams test and one-way analysis of variance, followed by Dunnett’s test were used to evaluate statistical significance using BellCurve for Excel (Social Survey Research Information Ltd., Tokyo, Japan). P values less than 0.05 were considered statistically significant (*p < 0.05, **p < 0.01).