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Cristescu, et al.
removed. Thus, the pharmacologic agent forms ultrasonically cleaned using ethanol, air dried,
most of the molecules deposited onto the substrate. then sterilized by exposing to germicidal UV-C
In this study, MAPLE was used to deposit radiation from a VL-115UV UV lamp (Vilber,
antiproliferative rapamycin-PVP thin films on Marne-la-Vallée, France) before deposition.
glass and silicon (Si) substrates. Alamar Blue and Rapamycin-PVP thin films were characterized
PicoGreen assays were performed to measure using atomic force microscopy (AFM) and
the metabolic health and DNA content of L929 attenuated total reflectance Fourier-transform
mouse fibroblasts as a measure of viability and infrared (ATR-FTIR) spectroscopy. The AFM
proliferation, respectively. The results of this study micrographs were collected in tapping mode using
indicate that the MAPLE-deposited rapamycin- a hard tapping tip (~300 kHz Fo, k ~ 40 N/m)
PVP thin films successfully reduced cell viability using an Asylum Research MFP-3D instrument
and proliferation. (Goleta, CA). Thirty-two scans with a resolution
of 4 cm were collected using Thermo Fisher
−1
2 Materials and methods Nicolet iS50 FTIR spectroscopy bench with built-
Homogenous solutions consisting of in diamond attenuated total reflection attachment.
polyvinylpyrrolidone (PVP), rapamycin, and Cell viability and proliferation were measured
dimethyl sulfoxide (DMSO) were prepared at using Alamar Blue (Thermo Scientific, Grand
room temperature by dissolving 40 mg of PVP in Island, NY) and PicoGreen (Thermo Scientific,
4 mL DMSO, and then adding 1 mg rapamycin. Grand Island, NY) assays, respectively. All
Before each laser deposition, 3.5 mL of the freshly reagents were purchased from Thermo Scientific
prepared solution was dropped using a syringe unless otherwise noted. L929 mouse fibroblasts
in a copper holder and frozen in liquid nitrogen (ATCC, Manassas, VA) were cultured using
(77 K) for 30 min. The cryogenic process was RPMI media with 10% v/v fetal bovine serum in
accomplished by plunging the target in a Dewar T-150 flasks. The medium used in the plate assays
vessel, which was filled with liquid nitrogen. After contained an additional 100 IU/mL penicillin and
freezing, the target holder was rapidly mounted streptomycin (ATCC, Manassas, VA); antibiotics
inside the deposition chamber. are necessary since UV sterilization would damage
All MAPLE thin films were fabricated using the rapamycin-PVP thin films.
a 248 nm wavelength KrF* excimer laser source Three each of the unsterilized MAPLE-
(25 ns pulse duration, 10 Hz repetition rate). The deposited rapamycin-PVP-coated glass samples
laser was operated at the fluence of 200 mJ/cm per and 10-mm square glass coverslips (Ted Pella,
2
pulse for 101,000 laser pulses. Both the target and Redding, CA) were placed into a 24-well plate and
substrate were rotated at a rate of 50 Hz during the seeded with 3.1 × 10 cells/cm using 0.5 mL of
2
4
coating process, while the laser beam was swept cell suspension. The plate was centrifuged at 50 g
over the entire target surface at an incident angle for 1 min then incubated overnight at 37°C, 5.0%
of 45°. The target was maintained at cryogenic CO , 95% RH for cell attachment.
2
temperatures using direct contact with a cooling Samples and control coverslips were transferred
device connected to a liquid nitrogen reservoir by to a fresh 24-plate and 0.5 mL of 10% v/v Alamar
copper heat pipes. A laser beam homogenizer was Blue in-media was added; wells containing only
used to produce a top-hat energy distribution and 10% v/v Alamar Blue in-media were used as the
increase the deposition area on the substrate. All assay blank. The plate was then incubated for 3 h.
depositions were performed at a 10 Pa background 100 µl of solution from each well were added to a
−1
pressure; a 5 cm substrate-to-target separation 96-well plate in triplicate and read in a fluorescence
distance was used to deposit the MAPLE coatings. plate reader (SpectraMAX Gemini EM, Molecular
One-side polished Si <100> and 10 × 10 mm Devices, San Jose, CA) using 570 nm excitation
2
borosilicate optical glass pieces were used as and 585 nm emission wavelengths. The remaining
substrates in this study. The substrates were dye solution was aspirated from the 24-well plate,

International Journal of Bioprinting (2020)–Volume 6, Issue 1 107