In Vivo Feasibility of Electrostatic Precipitation as an Adjunct to Pressurized Intraperitoneal Aerosol Chemotherapy (ePIPAC)

This was an exploratory experimental in vivo study in a large animal model comparing the effect of ePIPAC (3 animals) versus PIPAC (3 animals) versus 1 control animal (ePIPAC, no stain).

The experiment was performed in compliance with the German Animal Protection Law (TierSchG 2006) and was authorized by the competent authority, State of Thuringia. ARRIVE guidelines were implemented. Seven German nonsyngeneic landrace pigs (5 females, 2 males) weighing 31.5 ± 4.5 kg were operated on by qualified surgeons under the supervision of a veterinarian. The sample size was determined in order to obtain experimental results in triplicate plus a negative control. Animals were randomly assigned to the experimental groups. Procedures were performed under general anesthesia adhering to strict protocols. The animals were euthanized under narcosis at the end of the procedure and immediately necropsied. The primary outcome measure was DT01 concentration in tissue and peritoneal fluid at the end of the procedures (quantitative measurement). Secondary outcomes were the homogeneity of blue staining within the peritoneal cavity (qualitative measurement) and the toluidine blue concentration in the peritoneal fluid at the end of the procedure (semiquantitative measurement).

DT01 are noncoding small DNA fragments designed to bait and hijack the enzyme complexes that repair DNA double-strand breaks, diverting them away from their primary objective, the double-strand breaks on chromosomes.15,18 DT01 administration with PIPAC has previously been validated.8 In this study, 30 mg toluidine blue and 6 mg Cy5-labeled DT01 were diluted in 1000 ml NaCl 0.9 % solution. A volume of 150 ml solution was administered via an aerosolizer (Capnopen, Capnomed GmbH, Germany) to each animal (n = 6). The negative control animal received 150 ml NaCl 0.9 %.

PIPAC was applied as described previously.9 After insufflation of a 12 mm Hg capnoperitoneum with a Veress needle, 2 balloon safety trocars (Kii 5 and 12 mm; Applied Medical, Germany) were inserted into the abdominal wall. The Capnopen was connected to an intravenous high-pressure injector (Arterion 7; Medrad, Germany) and inserted into the abdomen. The pressurized aerosol was applied via aerosolizer and injector. Flow rate was 30 ml/min, and maximal upstream pressure was 200 psi. The therapeutic capnoperitoneum was maintained for 30 min. The aerosol was exsufflated and the trocars removed. Identical conditions were used for the ePIPAC subjects (n = 3), with the additional use of the Ultravision technology. The system was activated at the point of completion of aerosol generation and the electric current was maintained for 30 min. This negative control animal received NaCl 0.9 % through ePIPAC under the above conditions.

The Ultravision system integrates the following components: a generator unit (voltage 7500–9500 V, current ≤10 µA), an active cable terminating in an atraumatic stainless steel brush electrode (Ionwand) that is responsible for the electrostatic charging of aerosol particles, and a return electrode with a solid patient return plate (Fig. 1). The Ionwand emits a stream of electrons, resulting in the creation of negative gas ions. The gas ions collide with particulate matter, passing on the negative charge. The return electrode confers a weak positive charge on the subject, which results in the electrostatic attraction of the negatively charged aerosol particles to the tissue surfaces of the contained space—that is, the peritoneum.

Toluidine blue distribution was assessed qualitatively as described previously.7 Immediately after the procedure, peritoneum was sampled via biopsy; peritoneal fluid was sampled, and droplets were distributed onto filter paper to visualize the intensity of the blue stain. The tissue and peritoneal fluid samples were snap-frozen in liquid nitrogen and processed at the Institut Curie/Orsay for blinded analysis. The quantitation of DT01 in the peritoneal fluid was performed by a hybridization enzyme-linked immunosorbent assay (ELISA) using a biotin-conjugated capture oligonucleotide probe (300 μl) and a digoxigenin-conjugated detection probe (300 μl), with sequences complementary to the DT01 sequence (Exiqon, USA) in 96-well plates. Then samples were incubated with an anti-digoxigenin horseradish peroxidase–conjugated antibody (1:10,000; Roche, USA), and detection was performed by the addition of 3,3′,5,5′-tetramethylbenzidine substrate (100 μl). Absorbance was measured at 450 and 560 nm, and quantity of DT01 was calculated from calibration standards over a working range of 25–1000 ng/ml using a 4-parameter logistic curve. Because the ELISA failed to produce reliable quantification in tissues, we used fluorescent quantification, a reliable technique for assessing molecule distribution.19 Peritoneal tissue samples were defrosted, then crushed in phosphate-buffered saline–ethylenediaminetetraacetic acid. Fifty microliters of each extract was formed into aliquots in a 96-well plate, and fluorescence was measured with a Typhoon scanner (GE Healthcare, USA). The quantity of DT01 was calculated from a standard curve performed using peritoneal extract from untreated groups, with Cy5-labeled DT01 concentrations ranging from 0 to 1 µg/ml.