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227
soluble APIs with specific counter-ions is an excellent approach to up-
grade conventional pharmaceuticals using an IL-based strategy [16].
This prodrug technique can eliminate the effect of drug polymorphisms
or crystallinity, which is often responsible for reducing therapeutic
efficiency, bioavailability and thermal stability [15–17]. Recently, Rogers
groups demonstrated the successful application of an IL formulation to
deliver the poorly soluble drug sulfasalazine and reported that solubility
increased 4000 times and bioavailability was 2.5-fold higher compared
with that of free drug [18]. Yoshiura et al. [19] described an IL-based
microemulsion for the delivery of MTX and demonstrated that MTX
penetration through pig skin increased dramatically. Moniruzzaman
et al. [20] reported a novel imidazolium-based IL-in-oil microemulsion,
in which MTX dissolved more efficiently than in water. However, an
MTX-based API-IL, either as an anion or as a cation, has not previously
been reported.
from Wako Pure Chemical Industries Ltd. The coupling constants (J)
are reported in Hertz (Hz). The DeltaV software package (version
5.0.5.1, JEOL) was used to process the spectra. The purities of the synthe-
sized MTX-IL moieties were determined using the following equation:
h
X
i
X
Purity ð%Þ ¼
I ðproductÞ=
I ðtotalÞ ꢀ 100
ð1Þ
where, I represents the relative area of each signal [22].
2.4. Fourier transform infrared analysis
The Fourier transform infrared (FT-IR) spectra of the MTX-ILs were
recorded using a Perkin Elmer spectrometer (Frontier FT/IR, Waltham,
MA) in the range 400–4000 cm−1 with an accumulation of 20 scans.
In the present study, we explored the use of the IL-API approach to
solve the deficiencies of the solid drug MTX, by creating an MTX
anion-based IL, and report the synthesis, characterization, solubility
(both in aqueous and physiological fluids) and in vitro antitumor
activity of the MTX-IL moieties.
2.5. Thermogravimetric analysis
Thermogravimetric and derivative thermogravimetric analysis
(TGA/DTG) was performed using a Hitachi High-Technologies TG/DTA
7300 (Tokyo, Japan) under a flow of nitrogen. Samples weighing
approximately 10 to 20 mg were analyzed in an aluminum crucible
and were heated from 30 to 550 °C using a constant heating rate of
5 °C min−1 with a 30 min isothermal step at 70 °C.
2. Experimental
2.1. Materials and methods
2.6. Differential scanning calorimetry
MTX (anhydrous, N98% purity) was purchased from Wako Chemicals
Ltd. (Osaka, Japan). L-Proline, L-aspartic acid, tetramethylammonium
hydroxide (15% in water), and tetrabutylphosphonium hydroxide
(40% in water) were obtained from Wako Chemicals Ltd. (Osaka, Japan).
L-Phenylalanine with a high level of purity (N98.0%) was purchased
from Kishida Chemical Co. Ltd. (Osaka, Japan), and 1-ethyl-3-
methylimidazolium chloride was purchased from TCI America (Tokyo,
Japan). The concentrations of ammonium, phosphonium, choline and
imidazolium were determined via titration. All other reagents, solvents
and materials were of analytical grade and were used without any further
purification.
Differential scanning calorimetric (DSC) measurements were carried
out using a Hitachi High-Technologies DSC X7000 (Tokyo, Japan) to
characterize the thermal behavior of the drug moieties. Standard alumi-
num pans containing 4 to 6 mg samples were crimped with an alumi-
num lid using a press (T Zero sample press), and heated from −50 to
300 °C at a rate of 10 °C min−1 under constant nitrogen at 30 mL/min.
An empty pan, sealed in the same way as the sample, was used as a
reference.
2.7. Powder X-ray diffraction (p-XRD)
Gibco minimum essential media (MEM), Opti-MEM, fetal bovine
serum, and antibiotic-antimycotic were purchased from Thermo Fisher
Scientific (Waltham, MA, USA). Dulbecco's phosphate buffered saline
(PBS) and 0.25% trypsin/1 mM ethylenediaminetetraacetic acid
were obtained from Nacalai Tesque (Kyoto, Japan). A WST-8 (2-(2-
methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-
tetrazolium monosodium salt) cell counting kit was obtained from
Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). The HeLa
cell line was provided by the RIKEN cell bank (Tsukuba, Japan).
For structural analysis, p-XRD analyses were performed using a
high-resolution X-ray diffractometer (Rigaku, Smartlab 9 kW, Tokyo,
Japan) with monochromatized and Ni-filtered Cu Kα radiation (λ =
1.5418 Å), operating at 40 kV and 30 mA. The data were collected
using the 2θ range of 5–50° with a scanning speed of 2°/min with 0.02°.
2.8. Partitioning coefficient determination
Approximately 2 to 3 mg of free MTX-ILs were added to a mixture of
5 mL of Milli-Q-water and 5 mL of octanol. The mixtures were allowed
to mix overnight at room temperature with constant shaking in the
dark. Then, the solution was centrifuged at 10,000g for 60 min to
separate the layers. The concentration of free MTX in each layer was
quantified through UV spectroscopy (JASCO V-750, Japan) using
known concentrations of a standard at 302 nm. The partition coefficient,
log Po/w, was determined using the following equation:
2.2. General synthetic procedure for cations and MTX-IL moieties
The desired cations were synthesized in accordance with our previ-
ously reported procedure [2,21]. The synthetic route for the preparation
of amino acid esters (AAEs) is outlined in Scheme S1 and involves a re-
action between amino acids and thionyl chloride (mole ratio of thionyl
chloride: amino acid = 1.5:1) in ethanol and neutralization using an
ammonium solution (two equivalents) as a base [2]. The synthetic
route for the other cations outlined in scheme S2 involves adding an
equimolar amount of silver oxide to the respective chloride salt of the
cations [21]. Finally, MTX anion-containing IL-API moieties were syn-
thesized as shown in Scheme S3, through the ionization of a hydropho-
bic MTX hydrate and hydroxide salts of the desired cations in methanol
at 40 °C for 2 h (see supporting information for details).
log Po=w ¼ log ðSoluteoctanol=Solutewater
Þ
ð2Þ
where, Soluteoctanol and Solutewater represent the amount of free MTX in
octanol and water, respectively.
2.9. Media for solubility studies
2.3. NMR measurements
Milli-Q-water, PBS, simulated intestinal fluid (SIF) and simulated
gastric fluid (SGF) were used as dissolution media to perform a solubil-
ity study of the MTX-IL moieties. PBS (pH = 7.4) was purchased from
Nacalai Tesque Inc. (Kyoto, Japan). SIF (pH = 6.8, without pancreatic
enzyme) and SGF (pH = 1.2, without pepsin enzyme) were prepared
The 1H NMR spectra were recorded using a JEOL ECZ400S 400 MHz
spectrometer (Tokyo, Japan) in deuterated dimethyl sulfoxide
(2.5 ppm)/ methanol (3.3 ppm). The NMR solvents were obtained