Technical design, ex-vivo optimization and technical validation of the ultrasonic nebulizer (USN)
Design of the USN and its corresponding setup for pressurized intraperitoneal aerosol supply
The extra-corporal aerosol generation based on a commercial ultrasonic nebulizer (Fisoneb, Fisons/Medix Electronic/Karapharm, Marseille, France) that was technically modified by FVM Technologies & Consulting (Palaiseau, France). For the operation of the device with CO2 instead of air, the internal ventilator of the device was replaced by a pipe connected to compressed CO2 via a high-pressure regulator to an adjustable flow-rotameter. To obtain a higher drug aerosol output, the electrical power of the 12 VDC power supply was increased from 2 W to 6 W without modification of the ultrasonic probe frequency of 110 kHz.
Aerosol transportation from the outlet of the USN to the inlet to the rat was realized via a silicon tube with an inner diameter of 5 mm and a total length of 305 mm and a catheter (Surflow® I.V. Catheter 14G, ID SR + OX1464C1, Terumo) with an inner diameter of 1.73 mm. Preliminary ex-vivo analyses showed that condensation phenomena due to temperature differences between aerosol stream and the environment led to liquid film formation from the aerosol on the inner tube surface and thus to dripping at the outlet opening of the tube. To minimize particle loss by aerosol condensation and to prevent disturbance of the aerosol flow, the silicon tube was introduced into a heating pipe regulated to 40 °C (FVM Electronic Company, Palaiseau, France).
Ex-vivo optimization of the operating parameters of the USN by 99mTC scintigraphy
A biocompatible capnoperitoneal pressure of 8 to 10 mmHg in rats has been reported already previously [7, 8]. At that pressure the spatial geometry and the volume of the rat capnoperitoneum was determined by means of breathing-triggered computer tomography volumetry (Micro-CT SkyScan 1278, Bruker, Billeric, USA) in five healthy male Wistar rats (Charles River, France) of 250 to 350 g body weight. A median volume of the rat capnoperitonea at a pressure of 8 to 10 mmHg of 130 ml ± 10 ml was observed [14]. Based on these data, we designed artificial rat capnoperitonea phantoms by using cylindrical polyethylene terephthalate cans that were internally coated with a 1 to 2 mm thick agar layer and had an approximately similar geometry dimension of 4.0 × 10.5 cm as the in-vivo rat capnoperitoneum. Optimization of the operating parameters to maximize IP aerosol deposition were determined by real time scintigraphy (Gamma Imager, Biospace, Mesures, France with IRIS Software Aries Nucleaire, France). For this purpose, 99mTc-labelled human plasma albumin scintigraphic studies was chosen due to its low absorption by both lymphatic and blood capillaries in view of the later in-vivo studies.
The freeze-dried radiopharmaceutical was dissolved under vacuum into 3 mL saline. After 5 min, 2 GBq of 99mTc sodium pertechnetate (Cis bio, France) were added. After further 5 min incubation at room temperature, the resulting solution was used to load the liquid chamber of the USN. At a CO2 flow of 2 L/min, an optimum of 99mTc-labelled human plasma albumin aerosol deposition of 30 MBq/1.5 ml inside the rat capnoperitoneum phantoms was determined by real time scintigraphy.
Ex-vivo granulometric analyses of the USN aerosol
Granulometric aerosol analyses were performed by means of laser diffraction spectrometry (LDS). Therefore, a laser diffraction spectrometer (HELOS/KR-H2487, Sympatec GmbH, Germany) according to ISO 13320:2009 [15] was operated for determining volume-weighted droplet size distribution over a size range from 0.5–175 μm [16]. Aerosol characterization was performed at a distance of 5 mm from the aerosol outlet and the laser beam operated as reported in prior studies [10, 11]. For the purpose of the analysis, the USN was operated with a 0.9 wt.-% aqueous sodium chloride solution (B. Braun Melsungen AG, Melsungen, Germany). The time resolution was set to 3 s at a total measurement time of 10 min.
Pressurized intraperitoneal aerosol supply studies and orthotopic PM model in rats
USN and corresponding setup for pressurized intraperitoneal aerosol supply in rats
The setup for pressurized intraperitoneal aerosol supply designed for in-vivo experiments is shown in Fig. 1 and comprises an extra-cavitary aerosol generation based on a modified ultrasonic nebulizer (USN), heated tubing for aerosol transportation and indwelling vein cannulas for aerosol flow supply and outlet to/from the abdominal cavity of the rats.
By means of a simple puncture into the abdominal cavity, a catheter (Surflow® I.V. Catheter 14G, ID SR + OX1464C1, Terumo) with an inner diameter of 1.73 mm was used for aerosol supply into the rat’s abdomen. Correct puncture of the abdominal cavity was checked by aspiration and saline sip test. The capnoperitoneum was then established and analogous to the insertion of the aerosol inlet catheter, two further catheters (Surflow® I.V. Catheter 14G, ID SR + OX1464C1, Terumo) were inserted into the abdominal cavity of the rat. One catheter was connected to a HEPA filter capsule (ID: 1602051, TSI Inc., Shoreview, USA) to separate any particulate contamination from the outlet aerosol flow of the abdomen. Immediately before the inlet of the absolute filter, a pressure control unit (FVM Technologies & Consulting, Palaiseau, France) was placed to adjust and regulate the IP pressure to 8 to 10 mmHg in order to set a stable and reproducible capnoperitonea. The third catheter (Surflow® I.V. Catheter 16G, ID: SR + OX1651C1, Terumo) was laterally positioned into the abdomen to monitor the actual intraperitoneal CO2 pressure (Testo 510 differential pressure meter, Testo S.a.r.l, Forbach, France). The three catheters were anatomically positioned similarly in all experiments.
Orthotopic human colon cancer peritoneal metastasis (PM) model
For the development of an orthotopic peritoneal metastasis (PM) model of colorectal cancer, 12 male immunosuppressed 250 to 300 g RNU rats (Crl: NIH-Foxn1rnu, Charles River, France) previously irradiated at 5 Gy with Co-60, received an IP administration of HCT116-Luc2 human colon cancer cells (Perkin Elmer, France) that express stable luciferase gene for bioluminescence imaging. HCT116-Luc2 cells were cultured at 37 °C and 5% CO2 in Mc Coy’s 5a (modified) medium (Gibco® 26,600–023, Thermo Fischer Scientific) supplemented with 10% fetal bovine serum (Gibco®) and 1% Penicillin-Streptomycin (Gibco®). Each rat received 1 × 107 HCT116-Luc2 cells, suspended in 2 mL phosphate buffered saline (Gibco®) under gas anesthesia (2% air/isoflurane mixture, Iso-Vet, Piramal Healthcare) IP. To improve the homogeneity of metastatic patterns, two separate IP injections laterally into the abdominal cavity of 1 mL each containing 5 × 106 cells were performed.
Aerosol and liquid IP deposition and spatial distribution studies in rats
Single photon emission computed tomography (SPECT) imaging with IP 99mTc-labelled human plasma albumin administration, analog radiopharmaceutical preparation as described above was performed in a total 15 healthy male Wistar rats (Charles River, France) of 250 to 350 g body weight.
First, the radiopharmaceutical was administered IP in five rats under 2% isoflurane anesthesia using the USN for pressurized intraperitoneal aerosol supply (Group 1). The drug chamber of the USN was loaded with 185 MBq of 99mTc labelled human plasma albumin in NaCl 0.9 wt.-% to a total volume of 10 ml. The drug was then nebulized during 10 min into a stable capnoperitoneum of 8 to 10 mmHg which was maintained for another 20 min. Whole body counting (Dose Calibrator, Capintec, Inc., Florham Park, NJ, USA) showed decay corrected median dose of 30 ± 0.2 MBq of radiopharmaceutical deposited IP in the animals corresponding to 15 wt.-% (1.5 mL) of the total 10 ml 99mTc labelled human plasma albumin loaded in the liquid drug chamber of the USN. To assess the effect of the 8 to 10 mmHg capnoperitoneum on the spatial distribution pattern, simple liquid IP injection of equivalent doses/volume (30 MBq 99mTc labelled human plasma albumin in 1.5 ml) were performed either with (Group 2) or without (Group 3) capnoperitoneum. The chronology of the experiments performed was as follows:
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Group 1; (n = 5): IP drug aerosol supply with the USN at a CO2 flow rate of 2 L/min for 10 min (IP deposited dose/volume of 30 MBq/1.5 mL) into a constant capnoperitoneum of 8 to 10 mmHg pressure which was maintained for another 20 min after the end of nebulizing under anesthesia
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Group 2; (n = 5): manual liquid IP injection by means of a small-caliber puncture syringe of 30 MBq/1.5 mL into a constant capnoperitoneum of 8 to 10 mmHg pressure which was maintained for 30 min under anesthesia
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Group 3; (n = 5): manual liquid IP injection by means of a small-caliber puncture syringe of 30 MBq/1.5 ml without capnoperitoneum followed by 30 min of anesthesia
SPECT imaging started 30 min after the administration of the radiopharmaceutical without capnoperitoneum in all three groups and was performed in a dedicated, multiplexed multi-pinhole small animal SPECT/CT imaging device (NanoSPECT/CT, Mediso, Budapest, Hungary). The system was equipped with 4 collimators (nine pinholes, aperture 1.5 mm). The detection energy window for 99mTc was set to 140 keV ± 10%. Helical scanning mode was used and 24 projections of 30 s were acquired resulting in 21 min scans. During the scan, the rats were kept under 2.0% isoflurane (Iso-Vet, Piramal Healthcare, UK) anesthesia in air carrier at 0.5 L/min and maintained at 37 °C. The imaged data were reconstructed (HiSPECT NG software. Scivis GmbH, Göttingen, Germany) and analyzed with image analysis software (InVivo Scope, Bioscan Inc., USA).
For quantitative spatial distribution analysis 99mTc labelled human plasma albumin, SPECT/CT full imaging along the main body axis (250 images of 92 × 92 pixels) were stored in Digital Imaging and Communications in Medicine (DICOM®) files. A custom computer program (source code available on GitHub at https://github.com/grezniczek/SPECTalyzer) was used to process these files. First, the 100 consecutive slices that contain the highest combined signal intensities were identified. Then, data points were ordered from highest (top) to lowest signal value and the number of data points (from the top) were counted that were needed for their cumulative signal to exceed a given percentage (30%) of the total cumulative signal in the 100 slices. This number, the TVE30 (top voxels exceeding 30%), is represented as the fraction of the total number of data points. Thus, higher TVE30 values correspond to a more uniform distribution. SigmaPlot14 (Systat Software Inc., San Jose, CA) was used for statistical analysis. The Shapiro-Wilk test was used to assert normal distribution of experimental data. Paired t-test was used to compare within experimental groups. All reported p-values are two-tailed.
Monitoring of PM induction and growth
Monitoring of tumor proliferation was performed in-vivo by bioluminescence imaging (BLI), 2-deoxy-2-[fluorine-18]fluoro-D-glucose PET imaging (18F-FDG-PET) and necropsy. Prior bio-luminescence imaging (Ivis® Lumina II, Perkin Elmer, France), rats received an injection of 40 mg Luciferin (Perkin Elmer, France) 4 min before induction of anesthesia (2% air/isoflurane mixture (Iso-Vet, Piramal Healthcare)). The rodents were imaged once a week over 3 weeks at both posterior and anterior faces. Regions of interest were drawn on the anterior and posterior surface of animals in the abdomen and quantified via the bioluminescence signal (Living Image 4.4, Perkin Elmer, France) to document the evolution of PM. Results were expressed in numbers of emitted photons per second.
Five animals were selected on the basis of BLI to undergo 18F-FDG-PET examination on day 17 (D17) post HCT116-Luc2 cell injection. For PET, the radiopharmaceutical fluorine-18-fluordeoxyglucose (18F-FDG, AAA, France) with an activity of approximately 35 MBq was injected into the tail vein of the rats, which had free access to fresh water but not to food for 12 h before the examination. After 1 hour of tracer distribution in the body, animals were placed in the bed of a micro-PET scanner (eXplore VISTA, Sedecal, Spain). Therefore, the rats were anesthetized with an air/isoflurane mixture (5% for induction and 2% in steady state; Iso-Vet, Piramal Healthcare) and maintained at 37 °C during the examination. For a whole-body image, a sequence of 3 scans with 3 bed positions was acquired. Each bed position was scanned for 15 min that cumulates in a total scanning time of 45 min. Radionuclide decay was automatically corrected between each bed position. The energy window was set to 250–700 keV. Images were reconstructed and visualized by MMWKS Image Software (4.7 Build 452). 3D FORE/2D OSEM algorithms were used for image reconstruction.
Thirty days post induction of the human colon cancer cells, all rats were sacrificed by an overdose of anesthesia and then autopsied to characterize metastases and to compare their anatomical locations to foci observed by BLI and 18F-FDG-PET.
Feasibility of repetitive applications of pressurized intraperitoneal aerosol
The technical feasibility and handling of USN and the corresponding minimally invasive operative setup for repeated intraperitoneal pressurized aerosol administration in rats were explored on day 7 (D7), day 14 (D14), day 21 (D21), and day 28 (D28) after intraperitoneal instillation of tumor cells immediately following BLI imaging (see Fig. 1). The drug chamber of the USN was loaded with 10 ml 0.9 wt.-% sterile aqueous sodium chloride solution for aerosol generation.