Page 38 - IJB-9-6

International Journal of Bioprinting Transdermal delivery of printed cisplatin

and linearity of cisplatin was tested in a concentration MN patches, with each containing 15 μg of cisplatin, were
range of 10–1000 ng/mL in mouse plasma with a limit of pressed with the thumb for approximately 2 min and then
quantification (LOQ; at 10 ng/mL). taped with 3M™ Steri-Strip™ Reinforced Adhesive Skin
Closures (Digas, Athens, Greece) for 60 min. The vehicle
2.8. Cell expansion cohort was treated daily with saline solution containing
H1437 cells were originally purchased from the American 12.5% DMSO and 12.5% Kolliphor® EL orally, and every
Type Culture Collection (ATCC). Cells were grown 5 days with saline intraperitoneally. After 15 days, the mice
in Dulbecco’s Modified Eagle’s Medium (DMEM) were sacrificed with cervical dislocation, and the tumors
supplemented with 10% fetal bovine serum and penicillin/ were dissected, weighed, and photographed.
streptomycin in a humidified chamber with 5% CO .
2
2.10. Cisplatin uptake measurement with mass
2.9. Efficacy study cytometry-CyTOF
NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ mice (NSG mice; Frozen murine lung tumor tissue specimens were thawed
Stock No.: 005557) [33,34] were purchased from the Jax in ice-cold dissociation solution (PBS supplemented with
repository (Bar Harbor, ME, USA) and bred in individually 2 mM EDTA). Tissues were further dissected into small
3
ventilated cages under specific pathogen-free conditions, pieces with an average size of approximately 27–30 mm
under veterinarian supervision, in full compliance with (3 × 3 × 3 mm). Then, using a culture dish filled with
Federation of Laboratory Animal Science Associations dissociation solution, tissue was pressed through a 100-μm
recommendations in the Animal House Facility of the nylon mesh with the plunger of a 5-mL syringe to prepare
Biomedical Research Foundation of the Academy of Athens a cell suspension. During this step, the nylon mesh was in
(BRFAA, Greece). All procedures for care and treatment of contact with the surface of the liquid in the culture dish.
animals were approved by the Institutional Committee on The mesh was then rinsed two or three times with ice-
Ethics of Animal Experiments and the Greek Ministry of cold dissociation solution. Resulting suspension was then
Agriculture (Protocol #1392861, 28/12/22). filtered through a 40-μm nylon mesh and adjusted on top
of a 50-mL polypropylene Falcon tube, and dissociation
H1437 and H1437 KMT2C/KD cells were grown solution was added to a final volume of 15 mL followed by
in DMEM supplemented with 10% fetal bovine serum centrifugation at 300 × g for 7 min. Cells were then washed
and penicillin/streptomycin. Two million of cells were twice with 5 mL of Maxpar Cell Staining Buffer (CSB)
injected subcutaneously in the right flank of approximately from Standard BioTools Inc. (SB; South San Francisco,
6-week-old male NSG mice. Tumor dimensions were CA, USA; formerly known as Fluidigm) and centrifuged at
measured with a caliper. When tumors grew to a diameter 300 × g for 5 min. Following washing, cells were fixed with
of approximately 0.3 cm, the mice were randomly assigned freshly prepared 2%, filtered, methanol-free formaldehyde
to groups. No exclusion criterion was applied. Four groups solution (prepared from 16% stock solution; Sigma-
of at least 8 mice for either H1437 or the KMT2C/KD Aldrich) for 10 min at room temperature. Fixed cells were
derivative were used for in vivo administration of olaparib, then centrifuged at 800 × g for 5 min and stained with
olaparib/cisplatin (Olap/C-IP), olaparib/cisplatin-MNs DNA intercalator solution (1:1000 dilution of 125 μM
(Olap/C-MN), and vehicle. Cisplatin was administered to Cell-ID™ Intercalator-Ir) in Maxpar Fix and Perm buffer
mice either transdermally or intraperitoneally. The efficacy (all from SB, USA) and incubated at 4°C overnight. The
of the vehicle cohort, Olap/C-IP cohort, and Olap/C- following day, cells were washed twice with CSB buffer and
MN cohort was compared with one another. Cisplatin cell acquisition solution (SB, USA). Immediately before
dosing was 3 mg/kg of cisplatin either intraperitoneally the acquisition, cells were re-suspended in cell acquisition
or transdermally, i.e., 60 μg/dose. Furthermore, 50 mg/ solution supplemented with EQ Four Element Calibration
kg olaparib in saline solution containing 12.5% Beads (EQ4 beads from SB), diluted 1:10, at a final cell
dimethylsulfoxide (DMSO) and 12.5% Kolliphor® EL concentration 1 × 10 cells/mL. Acquisition was performed
6
(Sigma-Aldrich) was administered orally on a daily basis. on a Helios™ mass cytometry system (SB). To maximize data
This is the typical amount of olaparib administered in mice quality, the acquisition rate on the Helios was maintained
for therapeutic purposes by most groups. at a rate of <400 events/s. Acquired data were normalized

For transdermal application of cisplatin, mice were using EQ4 beads (SB method) with CyTOF software
anesthetized with a ketamine:xylazine mix (90 mg/kg (version 10.7.1014). Normalized fcs files were analyzed
ketamine and 10 mg/kg xylazine) as previously with bivariate dot plots and histograms in FlowJo™ v10.8
described [29,35] . MN application onto the skin of mice was Software (BD Life Sciences, Franklin Lakes, NJ, USA).
previously described . Briefly, the opposite (left) non- Graphs and statistical analysis were performed using
[29]
tumor-bearing flank of the mouse was shaved, and four GraphPad Prism 9 (version 9.2.0 for Windows, GraphPad

Volume 9 Issue 6 (2023) 30 https://doi.org/10.36922/ijb.0048