Page 37 - IJB-9-6

International Journal of Bioprinting Transdermal delivery of printed cisplatin

The donor substrate consisted of a transparent glass coated (5000 IU/mL), which were then placed immediately on ice
with a Ti film laser absorbing interlayer, onto which 5 μL of and centrifuged for 10 min at 3000 rpm. Plasma was then
the cisplatin solution (1 mg/mL in saline) were drop cast. stored in a -80°C freezer until the time of analysis. Mice
The receiver substrate was the MN patch onto which the were then euthanized.
cisplatin solution was transferred. For sample preparation, mouse plasma (50 μL)
The printing process has been described previously . was placed in low-retention Eppendorf tubes and was
[29]
Briefly, by focusing the laser beam onto the donor substrate, spiked with 5 μL of cisplatin working solutions for the
a high-pressure vapor pocket is produced at the interface preparation of standards. A DDTC solution (1% DDTC in
of the deposited API solution and the Ti layer as a result of 0.1N NaOH) was then added (15 μL), and samples were
the donor’s Ti layer absorbing the laser pulse. For each laser vortexed and incubated in a water bath (40°C) for 30 min
beam pulse, an nL droplet of cisplatin solution is printed for the formation of the Pt-DDTC complex. Following the
onto the MN substrates as a result of this high-pressure incubation step, 500 μL of cold ΑCN with internal standard
vapor pocket expanding and propelling the supernatant (IS, midazolam, 1 ng/mL) was added to the precipitate of
fluid into a dynamic jet that drives the API solution to the proteins. Subsequently, samples were vortexed for 2 min
receiver substrate at a high-impact velocity. The donor– and centrifuged at 13,000 rpm for 10 min. The supernatant
receiver substrate distance was kept at 700 µm. On the containing the Pt-DDTC complex and IS was transferred
MNs substrate, the laser transfer produced a continuous into glass tubes, and evaporation to dryness followed (50°C)
cisplatin film (5 × 5 mm) of 5 µL of the API solution. The for approximately 60 min. Samples were then reconstituted
LIFT process was repeated two more times on the same in 150 μL of H 0:ΑCN:FA (80:20:0.1%), centrifuged for
2
MN patch to achieve a total nominal amount of 15 µL of 1 min, and then transferred into 96-well plates for liquid
cisplatin solution. The coated MN patches’ morphology chromatography tandem mass spectrometry (LC-MS/MS)
was determined by scanning electron microscopy (SEM; analysis .
[32]
FESEM Nova NanoSEM 230, by FEI Europe, Eindhoven,
Netherlands). 2.7. Detection and quantification of cisplatin via
2.5. Preparation of cisplatin stock and standard LC-MS/MS
solutions The amount of cisplatin in standard and unknown samples
Stock solutions of cisplatin were prepared by dissolving was determined via a LC-MS/MS methodology, previously
[32]
approximately 3 mg of the compound in saline at a final reported by Agilent with slight modifications, that
concentration of 1 mg/mL. For preparation of cisplatin quantifies cisplatin after derivatization with a DDTC
working solutions, serial dilutions in ΑCN:H 0 (1:1, v/v) reagent and formation of a Pt-DDTC complex. For the
2
were prepared in the concentration ranges of 50 ng/mL to setup of the bioanalytical assay and sample analyses, a
25 μg/mL. The internal standard midazolam was prepared Triple Quad 5500+ LC-MS/MS System – QTRAP (AB
by serially diluting Dormixal 15 mg/3 mL in ΑCN:H 0 SCIEX LLC, CA, USA) was used. Chromatographic
2
(1:1, v/v) to 100 ng/mL. All stock and working solutions separation of the analyte of interest was accomplished via
were stored at 4°C until the day of sample preparation in a dC18 column (Waters, Atlantis, 2.1 × 50 mm, 3 μΜ) at a
mouse plasma. flow rate of 0.3 mL/min. Mobile phases included solution
A (100% H 0, 0.1% formic acid [FA]), solution Β (100%
2
2.6. Pharmacokinetic studies ΑCN), and needle wash (ΑCN:MeOH:H 0, 1:1:1), and
2
Cisplatin was administered in mice transdermally or injection volume for each sample was 10 μL. Elution of
intraperitoneally for evaluation of its pharmacokinetic the analyte was achieved by applying a gradient system
properties per dosing route. For transdermal described as follows: T = 0–0.5 min for 95% A and 5% Β,
administration of cisplatin in mice, MN patches with 15 μg Τ = 1.0 min for 25% Α and 75% Β, Τ = 1.5 min for 10%
of LIFT-printed cisplatin were prepared. Furthermore, for A and 90% B, Τ = 2.5–4.0 min for 5% A and 95% B, Τ =
intraperitoneal dosing of the platinum-based compound, 5.0 min for 95% A and 5% B. The transitions monitored
a solution of 600 μg/mL in saline was prepared. Four via multiple reaction monitoring were m/z 492.5/422.1
mice were used for administration per dosing route, and for cisplatin at 2.67 min and m/z 326.1/291.1 at 2.44 min
all mice were equally dosed with 60 μg of cisplatin in for midazolam, respectively. Additionally, transitions m/z
MN or administered with cisplatin intraperitoneally. Α 640.4/116.1 and 640.4/492.0 for cisplatin and 326.1/209.1
serial cheek bleeding protocol was performed in mice at for midazolam, respectively, were used for confirmation
2, 4, 24, and 72 h after MN application or intraperitoneal of the bioanalytical result. The mass spectrometry (MS)
injection. The collected blood samples were placed in system was operated using positive electrospray ionization
heparinized Eppendorf tubes, containing 10 μL heparin mode (ESI). The applied bioanalytical methodology

Volume 9 Issue 6 (2023) 29 https://doi.org/10.36922/ijb.0048