Rotational intraperitoneal pressurized aerosol chemotherapy system
For delivering doxorubicin as aerosols, we used our prototype for PIPAC, which sprayed approximately 30-µm sized droplets through the nozzle with a velocity of 5 km/h at a flow rate of 30 ml/min under a pressure of 7 bars equivalent to about 100 psi . The mean diameter of the sprayed region was 18.5 cm, and the penetration depth ranged from 360 to 520 µm, comparable to previous studies using the microinjection pump (Capnopen®; Capnomed, Villingendorf, Germany) [19, 20].
For RIPAC, we added a remote-controlled conical pendulum motion device to our prototype for PIPAC and rotated the nozzle to improve drug delivery. The conical pendulum motion device was composed of a DC motor (12V/1.5A, GM35A-3323, Motorbank, Seoul, South Korea), a 3-D printed rotational stick, two end-stops (PCB-mounted End-stop switch, RepRap, England), and an Arduino Uno. We inserted the nozzle in a 3-D printed rotational stick and locked it with a screw. The angle between the nozzle and the vertical line was determined at 30 degrees by considering a spraying angle of 77.2 degrees. The rotational stick could not rotate in the same direction because the tube line connected between the nozzle and the syringe pump became tangled. Thus, the rotational stick moved clockwise, and when the rotating stick contacted the sensor of the rotating path, it moved counterclockwise to maintain repetitive rotation (Fig. 1) .
We purchased 1% methylene blue and doxorubicin from Sigma-Aldrich (St. Louis, MO, USA) for intraperitoneal chemotherapy. For analyzing the serum and tissue concentrations of doxorubicin, we purchased acetonitrile and methanol from Fisher Scientific (Waltham, MA, USA) and formic acid, acetic acid, and ammonium acetate from Sigma-Aldrich. We purchased 1.5 µg/ml 4’,6-diamidino-2-phenylindole (DAPI) from Sigma-Aldrich to evaluate the penetration depth of doxorubicin.
This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University Hospital before study initiation (No. 18-0051-S1A0) and the investigators complied the protocol of IACUC. We purchased a total of 15 female pigs weighing 40 to 50 kg for this study, which were used to evaluate drug distribution (n = 6), tissue concentrations and penetration depth (n = 6), and pharmacokinetics and safety (n = 3) based on the types of intraperitoneal chemotherapy.
Before intraperitoneal chemotherapy, we applied capnoperitoneum by CO2 insufflation via a Veress needle to each pig, and then inserted two or three 12-mm bladeless trocars (Eagleport®; Dalim Medical Corp., Seoul, South Korea) along the midline of the abdomen, which was used as a passage for inserting the nebulizer and laparoscopic devices (KARL STORZ Endoscopy Korea CO., Ltd., Korea). After inserting the nozzle through the trocar directly down to the ileum, PIPAC and RIPAC were applied using 150 ml of 1% methylene blue to evaluate drug distribution and 3.5 mg of doxorubicin in 50 ml of 0.9% NaCl to evaluate pharmacokinetics, tissue concentrations, and toxicities. After 1% methylene blue and doxorubicin solutions were aerosolized via the nozzle with a velocity of 5 km/h at a flow rate of 30 ml/min under a pressure of 7 bars, capnoperitoneum of 12 mmHg was maintained for 30 minutes (Supplementary Videos 1 and 2).
With PIPAC and RIPAC in each of two pigs, we performed EPIC using 1% methylene blue in an additional two pigs as controls. During EPIC, 1% methylene blue solution was infused at a flow rate of 100 ml/min for 30 minutes, and 1 L of the solution was drained every 10 minutes. Thereafter, the pigs were euthanized and the distribution and intensity of 1% methylene blue in the parietal and visceral peritoneum in the EPIC, PIPAC, and RIPAC pigs were compared with the naked eye.
Tissue concentration and penetration depth
We generated a modified Peritoneal Cancer Index (PCI) using the PCI for patients with PM . The modified PCI included nine parietal regions, including the central, right upper, epigastrium, left upper, left flank, left lower, pelvis, right lower, and right flank regions, and three visceral regions, which included the ileal, jejunal, and gastric regions (Fig. 2). According to the modified PCI, we obtained two specimens of 2×2 cm-sized peritoneal tissue from each region of six pigs after PIPAC and RIPAC.
For tissue concentrations, all tissue specimens were stored at -80 ℃ and homogenized with a solvent consisting of a 1:1 mixture of methanol and 1% acetic acid equivalents to twice the weight of the tissue specimens. Then, the homogenized tissues were mixed with 1 ml of ethanol and vortexed for 30 minutes, and held overnight in a refrigerator. After that, the mixture was centrifuged at 14,000 rpm for 10 minutes, and the supernatants were dried in a SpeedVac for 180 minutes at 45 ℃. The samples were reconstituted to 50 µl, vortexed with 150 µl of acetonitrile with 50 ng/ml of daunorubicin as the internal standard for 30 seconds, and centrifuged at 13,000 rpm for 5 minutes The supernatant (5 µl) was injected into HPLC for analysis.
To investigate the penetration depth of doxorubicin, we rinsed all tissue specimens with 0.9% NaCl solution to clean doxorubicin off the surface and then froze them in liquid nitrogen. We prepared cryosections with a thickness of 7 µm from three different specimen areas and applied DAPI. Thereafter, we estimated the depth of concentrated diffusion (DCD) and the depth of maximal diffusion (DMD) of doxorubicin in 12 regions by confocal laser scanning microscopy using a Leica TCS SP8 (Leica Mikrosysteme GmbH, Hessen, Germany) and compared them between the PIPAC and RIPAC treatments. In this study, we defined DCD as the distance between the luminal surface and the surface where positive doxorubicin staining was most accumulated, and DMD as the distance between the luminal surface and the innermost depth at which positive doxorubicin staining was visualized.
Pharmacokinetics and toxicities
For evaluating the pharmacokinetics of RIPAC using doxorubicin, we collected blood from three pigs a total of 11 times as follows: before RIPAC, after 15 minutes, after 30 minutes, after 45 minutes, after 1 hour, after 1.25 hours, after 1.5 hours, after 1.75 hours, after 2 hours, after 24 hours, and after 48 hours. Then, 50 µl of serum and 100 µl of 0.1% formic acid acetonitrile with 15 ng/ml of daunorubicin as the internal standard were vortexed for 30 minutes. The mixtures were centrifuged at 14,000 rpm for 10 minutes, and 5 µl of the supernatants were injected into HPLC for analysis.
To investigate renal and hepatic toxicities, we collected blood from three pigs a total of six times as follows: before RIPAC, immediately after RIPAC, and after one to four days. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), bilirubin, alkaline phosphatase (ALP), creatinine, and C-reactive protein (CRP) were measured in the serum.
Liquid chromatography and tandem mass spectrometry
We analyzed the serum and tissue concentrations of doxorubicin by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity (Agilent, Santa Clara, CA, USA), followed by tandem mass spectrometry (MS/MS) using API4000QTRAP (Applied Biosystems, Waltham, MA, USA). For the HPLC analysis, a Gemini 5 µm C18, 50 × 2.0 mm analytical column (Phenomenex, Torrance, CA, USA) was used. The mobile phase consisted of 5 mM ammonium acetate and 0.1% acetic acid acetonitrile with a flow rate of 0.3 ml/min and a 25 ℃ column temperature over 7.5 minutes.
The MS/MS was equipped with a positive ionization mode with Turbo Spray, and multiple reaction monitoring was used for quantification. The nebulizer and desolvation gas pressure was 50 psi, both using nitrogen. MS/MS was conducted under a needle voltage of 5000 V and a set temperature of 400 ℃. The acquisition delay was 0 seconds with a pause time of 5 msec.
We performed a pharmacokinetic study for RIPAC with doxorubicin, based on a non-compartmental model using R software for pharmacokinetic analysis. To characterize the pharmacokinetic analysis, the peak serum concentration (Cmax, ng/ml) and the time to the peak serum concentration (Tmax, hour) were identified. Then, the area under the curve (AUC, ng/ml × hour) of the individual pharmacokinetic curve was calculated using the linear trapezoidal rule from zero to the time of the last observed positive concentration. The continuous variables were analyzed by the Kruskal-Wallis test and the Mann-Whitney U test with the Bonferroni correction in SPSS version 22 software (IBM Corp., Armonk, NY, USA, RRID:SCR_002865). In this study, a significant P-value was defined as P ≤ 0.05 because of the non-parametric tests.