Effects of inhalable gene transfection as a novel gene therapy for non-small cell lung cancer and malignant pleural mesothelioma
Inhalable gene drugs
In this study, we prepared three SFD powders in the same way as previously reported15. Briefly, the solution composed of the following ingredients was rapidly frozen by spraying them at 150 kPa and 5 ml/min in liquid nitrogen from the nozzle tip of a spray dryer to generate frozen droplets. The frozen droplets were lyophilized under vacuum for 24 h to obtain the desired SFD powders. Powders were constructed with 50 kDa HA (Kikkoman Biochemifa Co., Tokyo, Japan), L-phenylalanine (Sigma-Aldrich, St. Louis, MO) as dispersing agent, and plasmid DNA (pDNA; Table 1). We used two different tumor suppressor genes that encode p16INK4a (SFD-p16) and human p53 (SFD-p53), driven by the cytomegalovirus promoter, containing 10 µg in 0.5 mg powder. Powder without pDNA was prepared as SFD placebo and 1% indocyanine green (ICG; Fujifilm Wako Pure Chemical Co., Osaka, Japan) was added as fluorescent marker for dry powders in orthotopic lung cancer model in vivo.
Cell lines and cell culture
The human cell line NSCLC A549 (p16INK4a– homozygous deletion), H1299 cells (p53– homozygous partial deletion), and the human cell line MPM H2052 (p16INK4a-homozygous deletion) were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Roswell Park Memorial Institute 1640 (RPMI1640) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin and incubated in 5% CO2 at 37°C. In the in vivo orthotopic lung cancer model, luciferase-expressing A549 cells (A549/Luc), purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan), were cultured under the same conditions as above. .
Air-Liquid Interface (ALI) and Cell Counting
ALI culture was performed in three cell lines. 2.0×105 the cells of each cell line were seeded in the insert of a 24-well Transwell plate (Corning Inc., Corning, NY) and 1 ml of the RPMI1640 culture medium was added. After culturing for 48 h, the medium in each well was replaced with fresh Opti-MEM or RPMI1640, and the previous insert medium was removed. We sprayed 0.5mg of SFD-placebo and SFD-p16 or SFD-p53 on the cell surface using a delivery device prepared by connecting a 1ml syringe (Terumo Co., Tokyo, Japan ) with a three-way stopcock (Top Co., Tokyo, Japan) and then incubated them with ALI for 24 h. Details of the method of administration have been previously reported15. Untreated cells were used as negative controls. Adherent cells were harvested with trypsin/EDTA. These were counted using a hemocytometer after staining with 0.4% trypan blue. The cell growth inhibition rate was calculated as a percentage using the following formula: 100-Ax/An × 100 (where An = number of viable cells of the control and Ax = number of viable cells of the dry powders ).
Real-time polymerase chain reaction (PCR)
After 24 h of transfection in four groups, control, pDNA, SFD-placebo and SFD-p16 or SFD-p53, cells cultured by ALI were collected. The pDNA pool was 10 µg of p16 or p53 naked plasmid DNA in phosphate buffered saline (PBS). Collected cells were lysed with 250 µL of PBS and 750 µL of RNAiso Blood (Takara Bio, Otsu, Japan), and RNA was extracted according to the manufacturer’s protocol. Reverse transcription-polymerase chain reaction (RT-PCR) and qualitative RT-PCR were performed according to the manufacturer’s instructions for the Thunderbird SYBR qPCR/RT Set (Toyobo, Osaka, Japan) in a time-based PCR system real StepOnePlus™ (Applied Biosystems, Foster City, CA, USA). The reaction program was initiated at 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 5 s, and annealing and extension at 60°C for 20 s; and the melting curve was determined at 95°C for 15 s, 60°C for 1 min and 95°C for 15 s. The primer sequences used are listed in Table 2.
Ex vivo treatment in nude mice with subcutaneous transplantation of lung cancer cells
Cultured A549 cells (2.5 × 107) were collected and sprinkled with approximately 0.5 mg each of SFD-p16 or SFD-placebo after the medium was sufficiently removed. Approximately 30 min later, the cells (2.5 × 107) were mixed with 50 μL of Matrigel and were transplanted subcutaneously into the back of BALB/c naked/naked nude mice (5 week old males) purchased from CLEA Japan, Inc. (Tokyo, Japan). One week later, the tumor was removed and fixed with 4% paraformaldehyde (PFA) for 3 days before measuring the weight and size of the tumor. Next, we calculated the tumor volume (mm3) as (width2× length)/2. Animal experiments with subcutaneously transplanted mice were performed according to Hyogo College of Medicine animal experiment regulations and ARRIVE guidelines.
In vivo treatment in nude mice with subcutaneous transplantation of lung cancer cells
Cultured A549 cells (2.5 × 107) mixed with 50 μL of Matrigel were injected subcutaneously into the back of BALB/c naked/naked mice (5 week old males). After confirmation of tumor formation 1 week later, the skin of the mice was incised and approximately 0.5 mg of SFD-p16 and SFD-placebo were sprayed on the tumor by releasing 0.25 ml of compressed air in a syringe. The skin was then sutured. One week later, the tumor was removed and fixed with 4% PFA for 3 days before measuring tumor weight and size. Next, we calculated the tumor volume (mm3) as (width2 × length)/2.
Oral Inhalation Treatment of an Inhalable Gene Drug in Orthotopic Transplant Mice
Animal experiments on orthotopically transplanted lung cancer mice were approved by the Animal Experimentation and Welfare Committee of Meijyo University and performed in accordance with the Guidelines for the Care and Use of laboratory animals approved by Meijo University Faculty of Pharmacy. This study was also carried out in accordance with the ARRIVE recommendations. BALB/c naked/naked mice (6 week old males) were purchased from Japan SLC (Hamamatsu, Japan).
Each mouse was anesthetized with isoflurane upon injection of A549/Luc cells (5.0 × 106) through the retro-orbital venous sinus. After the luminescence intensity, determined using an in vivo imaging system (IVIS; IVIS-SPECTRUM, Caliper Life Sciences, Hopkinton, MA, USA), in the lungs reached 5.0 ×105photons/s (on day 4 following inoculation), the treatments began.
Administration of powder for inhalation, SFD-p16 or SFD-placebo, was performed as previously described.34. Briefly, on day 4 after A549/Luc inoculation, each mouse was anesthetized with a mixture of three anesthetics (0.3 mg/kg body weight [BW] medetomidine, 4.0 mg/kg BW midazolam and 5.0 mg/kg BW butorphanol), and a 4 cm PE-60 polyethylene cannula was inserted into the trachea through the mouth. The tip of the delivery device was inserted into the cannula, and the powder was dispersed at a dose of 0.5 mg/mouse by releasing 0.25 mL of compressed air into a syringe. Another group of mice was left untreated throughout the experiment.
After pulmonary administration, fluorescence and luminescence were detected using IVIS, as described in our previous reports35.36. ICG fluorescence was observed to confirm successful delivery of the gene powder to the lungs. To detect luminescence corresponding to luciferase activity, luciferin as a luciferase substrate was administered intranasally (75 mg/kg body weight) 10 min before the point of detection, and the time of exposure was set at 1 min.
To quantify luminescence intensities in mouse lungs, the region of interest was fitted to a rectangle with a width of 3 cm and a height of 1 cm. The luminescence intensity (total flux [photon/s]) was determined as A549/Luc cell growth.
Statistical analyzes were performed using JSTAT software ver.22.0 J and BellCurve for Excel ver. 3.21. Results are expressed as mean ± standard deviation (SD) unless otherwise specified. Mean values were compared using one-way analysis of variance (ANOVA) followed by Bonferroni’s or Dunnett’s multiple comparison tests and a one-tailed Student’s t-test, as indicated in the figure legends. The significance thresholds were set at pp