Md. R. Islam, et al.
International Journal of Pharmaceutics 582 (2020) 119335
lipophilic systems (Liu et al., 2009; Saffari et al., 2016) have also been
reported to significantly enhance skin permeation of various drugs.
Transdermal delivery of sparingly soluble drugs, especially those
that are insoluble in water and other conventional solvents, is a great
challenge. In recent years, ionic liquids (ILs), which have a set of ions
and exist as a liquid at room temperature or below 100 °C (Roger and
Seddon, 2003), have been widely used to increase the solubility of such
drugs (Chowdhury et al., 2019a; Huang et al., 2019; Moniruzzaman
et al., 2010a, 2010b). The drug permeation enhancing capacity of ILs is
another important factor that must be taken into consideration (Sidat
et al., 2019; Wang et al., 2018).
Although application of ILs in the pharmaceutical industry is sig-
nificantly increasing, the two important parameters of ILs, cytotoxicity
and degradability, are still now in under debate (Constable et al., 2007).
Even though in the past few years, the uses of ILs in the pharmaceutical
applications have been increased, most of them have not been exposed
as ecologically and geologically friendly. It has been reported that the
commonly used imidazolium, quinolinium, pyridinium, and fluor-
inates-based ILs exhibited as non-biodegradable and toxic to the en-
vironment and ecology (Adawiyah et al., 2016; Gomes et al., 2019;
Sivapragasam et al., 2019). It has also been reported that imidazolium-
based ILs showed higher toxicity toward cultured human keratinocytes
cell line (HaCat) (Santos de Almeida et al., 2017). It is well confirmed
that imidazolium-based ILs are not suitable because of their high toxi-
city and low biodegradability (Adawiyah et al., 2016; Amde et al.,
2. Materials and methods
2.1. Materials
ACV, IPM (purity ≥ 98.0%), and EtOH (purity > 99.9%) were
purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
Choline chloride ([Ch][Cl]), silver oxide (Ag O), glycine, and L-serine
2
were purchased from Sigma-Aldrich (Tokyo, Japan) and L-alanine was
purchased from Wako Pure Chemicals Ltd. (Osaka, Japan). Opti-MEM,
fetal bovine serum, Gibco minimum essential medium (MEM), and
antibiotic–antimycotic solution were also purchased from Wako Pure
Chemicals Ltd. Dulbecco’s phosphate-buffered saline (D-PBS) and
ethylene diamine tetraacetic acid/trypsin (1 mM ethylenediaminete-
traacetic acid and 0.25% trypsin) solution were obtained from Nacalai
Tesque (Kyoto, Japan). 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-
5-(2,4-disulfophenyl)-2H-tetrazolium
(WST-8)
and
3-[4,5-di-
methylthiazol-2-yl]-2,5-di-phenyl-tetrazolium bromide (MTT) were
provided by Dojindo Molecular Technologies, Inc. (Kumamoto, Japan).
Milli-Q water was used in all of the experiments. All of the other re-
agents were analytical grade.
Female balb/c mice were obtained from CLEA Japan, Inc. Mice were
accommodated under natural conditions (25 ± 2 °C, 60 ± 10% re-
lative humidity) for one week prior to the experiments. Animals were
nurtured and controlled as per guide line of ‘Animal Ethics Committee’
of Kyushu University, Japan. Mammalian fibroblast cell line L-929 was
supplied by the RIKEN cell bank (Tsukuba, Japan). Reconstructed
human epidermal model (LabCyte EPI-MODEL-12) was supplied by
Japan Tissue Engineering Co., Ltd. (Gamagori, Japan).
2015). Interestingly, ILs containing organic (e.g., acetate, phosphate,
carboxylate, amino acid), linear long chain (e.g., hexanoate, octanoate,
and oleate) anions, and amino acid ester, choline, piperidinium, and
pyrrolidinium cations are considered as readily biodegradable and non-
toxic (Ali et al., 2019; Amde et al., 2015; De Santis et al., 2015; Hou
et al., 2013). These important properties of ILs have opened a new
avenue for selecting suitable biocompatible ILs for pharmaceutical
purposes. It is interesting to note that because of the tunable properties,
non-toxic ILs can be synthesized using biocompatible, biodegradable,
and pharmaceutically acceptable organic cations and anions. The ILs
composed of choline and amino acid (AA) are considered as the most
safest and biocompatible IL (Chowdhury et al., 2019b; Nurunnabi et al.,
2.2. Selection and synthesis of the ILs
All of the ILs containing choline as the cation and an AA as the anion
were synthesized following a well-established method in our laboratory
(Chowdhury et al., 2018). In brief, the choline AA ([Ch][AA]) ILs were
synthesized by the two-step metathesis method (Fig. S1). In the first
step, 5.0 g of [Ch][Cl] was mixed with Milli-Q water and stirred. An
2
019). Choline is known as source of micronutrients and performs a
excess amount of Ag
stirring at room temperature and the solution was allowed to stand in
the dark. After 2 h, the excess Ag O was removed by centrifugation
2
O was then added to [Ch][Cl] with vigorous
vital role in the development of brain and memory development, and
AAs are the most generous classes of organic molecules in universe, are
generally regarded as safe (GRAS) and biocompatible (De Santis et al.,
2
(3500 rpm, 30 min) followed by filtration to obtain [Ch][OH] (choline
hydroxide) aqueous solution. In the second step, the [Ch][OH] filtrate
was neutralized with an aqueous solution of the AA by continuous
stirring and allowed to stand for 24 h at room temperature. The solvent
was removed by rotary evaporation (EYELA, NVC-2200, New York,
USA) at 40 °C. An acetonitrile/methanol (9:1 v/v) mixture was then
added to precipitate the unreacted AA, which was filtered off. The fil-
trate solution was evaporated again to remove the solvent and then
freeze-dried for 48 h to completely evaporate the remaining solvent.
Finally, successful synthesis of the ILs was confirmed by 1H NMR, ele-
mental analysis, and the Karl Fischer water content.
2015; Hou et al., 2013; Zeisel and Da Costa, 2009).
In this study, we developed potent biocompatible IL-based ternary
systems containing isopropyl myristate (IPM), a pharmaceutically ap-
proved solvent, and ethanol (EtOH), a pharmaceutically approved co-
solvent. Three ILs with choline as the cation and glycine, alanine, and
serine as the anion, which were referred to as [Ch][Gly], [Ch][Ala], and
[
Ch][Ser], respectively, were used to investigate the solubility of a
sparingly soluble drug (acyclovir, ACV). These ILs were biocompatible
and nontoxic because of the biological sources of their cation and an-
ions (Hou et al., 2013; Yazdani et al., 2016). Because the ILs were not
able to transfer ACV into skin because of their strong hydrophilic
nature, IPM and EtOH were incorporated in the ternary system. Here,
IPM acted as a skin penetration enhancer (Eichner et al., 2017;
Panchagnula et al., 2005; Zidan et al., 2017) and EtOH acted as a
common solvent to overcome the problem associated with the mis-
cibility between IL and IPM. Skin permeation of ACV using the IL-based
ternary (IL–EtOH–IPM) systems and its effect on the skin barrier func-
tion were investigated using Yucatan micropig (YMP) full thickness
skin. In addition, the in vitro cytotoxicity and skin irritation (bio-
compatibility) of the ILs and IL–EtOH–IPM systems were evaluated
using mammalian fibroblast cells (L-929) and a reconstructed human
epidermal model (LabCyte EPI-MODEL-12), respectively. Finally, in
vivo histological analysis was performed to confirm the safety of the
proposed ternary systems.
2
2.3. Solubility of ACV in the ILs and IL–EtOH and IL–H O mixtures
The optimum solubility of ACV in the ILs and IL–EtOH and IL–H
mixtures was determined using different weight ratios of the ILs and
EtOH or H O (Table 1 and Fig. 1). In brief, an excess amount of ACV
was added to each IL, IL–EtOH, and IL–H O system and they were then
2
O
2
2
stirred at 25 °C for 24 h. The undissolved amounts of ACV were sepa-
rated by filtration using a syringe driven filter (Millipore, 0.45 μm
diameter). The ACV content in the filtrated solution was determined
using an ultraviolet (UV) spectrophotometer at 252 nm with suitable
dilution with methanol (MeOH) according to our previous report
(Moniruzzaman et al., 2010b).
2