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Kanaki, et al.
compared to hypodermic needle injections . Despite its Previous studies by our groups have shown that
[12]
advantages, the coating process remains challenging due Gem’s limitations can be overcome with different
to limited quantity of the drug coating, the uniformity strategies. Initially, different targeting peptides were
of the coating, material waste, and the precision in drug developed and used to generate novel targeted compounds
dosing. Several technologies have been used in the past, (peptide-Gem conjugate analogs that specifically bound
such as dip coating [7,13] , gas jet drying [14,15] , and spray to receptors known to be overexpressed in cancer cells).
coating [16,17] with noted limitations [18,19] . Such analogs provided us with improved efficacy,
In the last decade, three-dimensional (3D) printing pharmacokinetic advantages, and improved toxicity in
technologies have emerged as promising tools for both comparison to equimolar Gem dosing [30-32] .
optoelectronics, and biomedical applications . Through As an extension of the peptide-drug conjugate work,
[20]
printing methodologies, a plethora of materials can be used another concept, the metronomic approach (MTR) based
with various geometries to achieve a layer-by-layer building on the daily low-dose administration of Gem, was recently
[33]
of 3D structures. The 3D printing approaches can be used evaluated . An oral pro-drug of Gem (OralGem) was
to coat MNs by transferring small microdroplets of the API chosen for this work in non-small cell lung cancer animal
solution onto the MNs, thus forming uniform layers. models. We showed that MTR chemotherapy resulted in
The most noticeable techniques among 3D low circulating and sustained levels of Gem that could
printing technologies are droplet-based bioprinting potentially lead to the efficacy with less toxicity in
(inkjet printing, thermal, piezoelectric, and electrostatic comparison to MTD treatments. However, the continuous
printing), extrusion bioprinting, and laser-induced forward low-dose supplementation of Gem that is essential for
transfer (LIFT). For the coating of MNs for transdermal MTR dosing schemes remains a challenge. For example,
applications, inkjet printing has been used before [21-23] ; OralGem is a prodrug of Gem with its own toxicity and
[34]
however, it has limitations with high viscosity materials, dosing limitations due to its first-pass effect .
due to excessive force required to eject highly viscous In the effort to search for treatments that provide the
drops. Moreover, inkjet printing is also associated with optimum efficacy to safety window for cancer patients, a
nozzle clogging [24,25] . In contrast, LIFT is a digital, novel concept is presented in this paper. We employed LIFT
[26]
high-resolution, non-destructive, contactless (nozzle- bioprinting of Gem for coating polymethylmethacrylate
free) bio-printing technique, which employs single laser (PMMA) MNs with different drug amounts and evaluated
pulses to propel the bioink under transfer toward the the transdermal delivery in mice. Our approach produced
receiving substrate with high precision. Therefore, LIFT reproducible, accurate, and uniform coatings of the drug
is a promising method used to improve drug coating on on the MN arrays, and also yielded dose-proportional
MNs with uniform coating layers. concentrations of Gem in mice, following in vivo
Anticancer chemotherapy drugs (doxorubicin, transdermal application of the coated MNs.
paclitaxel, methotrexate, etoposide, cisplatin, and
gemcitabine [Gem]) are typically associated with 2. Materials and methods
non-specific systemic toxicity that leads to patient 2.1. Materials
discomfort and even treatment secession. Gem has
been shown to be efficacious against colon, pancreatic, Gem hydrochloride was purchased from Carbosynth
ovarian, breast, head and neck, and lung cancers, in Limited (Compton, Berkshire, UK). Ammonium acetate,
combination with various anticancer agents . However, formic acid, and glycerol (≥99.5%) were purchased from
[27]
its poor pharmacokinetics creates a need for alternative Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Munich,
[28]
approaches for Gem delivery, including encapsulation Germany). Acetonitrile (ACN, liquid chromatography
in nanocarriers (e.g., liposomes, dendrimers, carbon [LC-MS] grade) was purchased from Fisher Scientific
nanotubes, hydrogel ) or transdermal patches . (Fisher Scientific, Loughborough, UK). Water (LC-MS
[21]
[29]
Gem’s pharmacokinetic and efficacy limitations can grade) was purchased from Carlo Erba (Carlo Erba,
be attributed to rapid deactivation and formation of Milan, Italy). Ketamin was purchased from Richter
the inactive metabolite 2′,2′-difluoro-2′-deoxyuridine pharma ag (Austria). Xylazine was purchased from
(dFdU) by cytidine deaminase. As Gem is administered Neocell Pharmaceuticals.
based on the classical maximum tolerated dose approach 2.2. Gem dispersion preparation
(MTD), initial high doses lead to efficacy. However, such
high doses are associated with significant side effects. Gem solutions with concentrations of 10, 37.5, and
Furthermore, plasma concentrations of Gem decline 75 mg/mL were prepared by dissolving Gem in a
rapidly. Patient recovery after treatment necessitates long H O: Glycerol solution (90:10 v: v).
2
intervals between doses (3 – 4 weeks, that often lead to Solubility feasibility experiments were conducted
disease progression. to identify the proper solution mixture for the described

International Journal of Bioprinting (2022)–Volume 8, Issue 3 137