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Design of non-steroidal anti-inflammatory drug-based ionic liquids with improved water solubility and drug delivery Guillaume Chantereau, Mukesh Sharma, Atiye Abednejad, Bruno Neves, Gilles Sèbe, Véronique Coma, Mara G. Freire, Carmen Sofia Rocha Freire, and Armando J.D. Silvestre ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.9b02797 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019
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Design of non-steroidal anti-inflammatory drug-
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based ionic liquids with improved water solubility
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and drug delivery
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Guillaume Chantereau1,2, Mukesh Sharma1, Atiye Abednejad1, Bruno M. Neves3, Gilles Sèbe2,
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Véronique Coma2, Mara G. Freire1*, Carmen S. R. Freire1, Armando J. D. Silvestre1*
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1CICECO
– Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810193 Aveiro, Portugal
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2Laboratoire
Berlan, 33607 Pessac Cedex, France
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de Chimie des Polymères Organiques, University of Bordeaux, 16 Avenue Pey-
3Department
of Medical Sciences and Institute of Biomedicine – iBiMED, University of Aveiro,
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3810-193 Aveiro, Portugal
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*Corresponding author:
[email protected] (Mara G. Freire);
[email protected] (Armando J. D.
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Silvestre)
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ABSTRACT
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We here report the synthesis of ionic liquids (ILs) composed of the cholinium cation and
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anions derived from non-steroidal anti-inflammatory drugs (NSAIDs), namely ibuprofen,
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ketoprofen and (S)-naproxen, and their incorporation into bacterial nanocellulose envisaging their
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use in topical drug delivery systems. The chemical structure of the synthesized ILs was confirmed
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by spectroscopic techniques, and thermal analysis confirmed their categorization as ionic liquids
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with melting temperatures below 100°C and resistance to autoclaving. The synthesized ILs display
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an aqueous solubility (at pH 7.4) in the range between 120 and 360 mM, which is up to 100 times
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higher than the solubility of the respective NSAID precursors, thus contributing to improved
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bioavailability. Their incorporation into bacterial cellulose originated transparent and
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homogeneous membranes. Thermogravimetric analysis (stable up to at least 225°C) and
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mechanical assays (with minimum Young’s modulus of 937 MPa, maximum stress of 33 MPa and
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elongation at break of 5.6%) confirmed the suitability of the prepared membranes for application
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as topical drug delivery systems. Furthermore, the re-hydration ability of IL-incorporated
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membranes is 18 to 26 times higher than bacterial cellulose, being valuable to the absorption of
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exudates. Release tests demonstrated a faster and complete release of the IL-based drugs when
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compared with the starting NSAIDs. Finally, it is demonstrated that bacterial cellulose is not
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cytotoxic nor pro-inflammatory, whereas the cytotoxicity and anti-inflammatory properties of IL-
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incorporated BC membranes are similar to those of NSAIDs or ILs, reinforcing their suitability as
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envisioned materials for topical drug release applications.
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Keywords: non-steroidal anti-inflammatory drugs; ionic liquids; cholinium cation; bacterial
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nanocellulose; topical drug delivery
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INTRODUCTION
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Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used
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pharmaceutical ingredients for inflammation relief.1 Their main action is through to the inhibition
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of cyclo-oxygenases, minimizing the production of prostaglandins, responsible for pain and
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inflammation responses.2 However, most NSAIDs are poorly water-soluble3, which is a major
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drawback when envisaging their incorporation into hydrophilic matrices for drug release and
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bioavailability.
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The low water solubility of NSAIDs, in particular ibuprofen, ketoprofen and (S)-naproxen
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(0.07-0.20 mM), can be overcome by various approaches.4 The use of the respective salt forms is
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the simplest strategy. For example, sodium ibuprofen, sodium naproxen and sodium diclofenac
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reach water solubilities of about 102 mM, with the exception of sodium ketoprofen that it is
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considerably less soluble (0.06 mM).4 Koç et al.5 enhanced ibuprofen and ketoprofen water
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solubility up to 101 mM by their encapsulation in polypropylene oxide/polyamidoamine-based
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dendrimers. The preparation of nanosuspensions of ibuprofen with improved dissolution rate and
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lower gastric irritancy has also been described.6
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More recently, ionic liquids (ILs) comprising active pharmaceutical ingredients (APIs) have
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been developed to improve the water solubility of different drugs. This strategy is similar to the
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preparation of more common drug salt forms, yet combining large organic ions instead of inorganic
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ones, leading to lower melting temperatures.7 ILs in particular have raised interest due to their
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negligible vapor pressure, high thermal stability and low flammability.8 Cholinium-based ILs were
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designed for the first time in 1962 with the salicylate counter ion, resulting in improved water
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solubility in comparison to salicylic acid, while preserving the respective anti-inflammatory
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properties.9 Subsequent studies have been performed with cholinium-based ILs comprising APIs
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for improved water solubility, such as cholinium-nalidixate,10 cholinium-sulfasalazine11 and 3 ACS Paragon Plus Environment
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cholinium-ampicillin.8 Recently, Santos et al.12 reported on the synthesis of ibuprofen-based ILs,
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yet combined with different organic cations, such as 4 quaternary ammonium derivatives including
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cholinium, 4 imidazolium derivatives and cetylpiridinum. Yet, the cholinium-ibuprofen organic
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salt they designed had a melting temperature of 104°C and thus do not fit in the ionic liquid
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category.
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The oral administration of NSAIDs is currently the most used route.13 However, this type of
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administration presents several drawbacks, such as possible side-effects in the gastrointestinal
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tract14 and liver damage.2 Therefore, whenever possible, topical administration of anti-
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inflammatory drugs is the most efficient way to reduce most of these side-effects.15 Topical
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delivery can be achieved with creams, gels and patches. Creams and gels are simple to use but lack
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in control of the applied dose and skin surface covered. Patches are generally composed of three
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components: drug-containing matrix (e.g. acrylic-acid polymers, cellulose derivatives, silicone,
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among others), outer membrane (e.g. polyvinyl alcohol, polyurethane, among others) and rate
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controlling layer (e.g. poly(ethylenevinylacetate), polyethylene, poly(4-methyl-1-pentene), among
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others).16,17 First used patches were additionally composed of an adhesive layer; yet, novel products
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tend to favor matrices with inherent adhesiveness. Recently, novel drug-incorporating patches
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composed of a single layer have been proposed. For instance, Tombs et al.18 produced patches
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made of a single poly(ether-urethane)-silicone membrane containing ibuprofen. However, the
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future of topical patches resides in the materials beneficial properties that ideally should permit the
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use of single layer structures, green processing, and be of a biobased and biodegradable nature.
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Cellulose produced by bacteria, known as bacterial nanocellulose (BC), is synthesized by
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several non-pathogenic bacteria of the genus Gluconacetobacter, Sarcina or Agrobacterium.19 BC
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has raised considerable interest in the pharmaceutical field because of its unique properties, namely
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a nanofibrillar structure, high water-holding capacity, moldability and good mechanical 4 ACS Paragon Plus Environment
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properties.19 BC membranes are able to promote a controlled release of drugs, being explored by
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different research groups to develop topical drug delivery systems (TDDS) for different drugs or
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biologically active molecules, e.g. S-propanolol,20 benzalkonium chloride,21 sodium diclofenac22
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and caffeine.23 However, in the case of low water-soluble APIs, such as ibuprofen, organic solvents
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are required to guarantee its incorporation in the BC membranes.24
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In this context, the aim of this work was to synthesize and characterize NSAID-based ionic
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liquids, namely cholinium ibuprofenate, cholinium ketoprofenate and cholinium naproxenate,
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aiming at improving the water solubility of these drugs and incorporate them into BC membranes
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to develop effective TDDS. Briefly, NSAID-based ILs were synthesized and characterized by 1H
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and
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25°C was determined, as well as their cytotoxicity and anti-inflammatory properties. For
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comparison purposes, these characterizations were also performed on the NSAIDs precursor drugs.
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ILs and NSAIDs were incorporated into BC membranes and characterized in terms of thermal and
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mechanical properties. The release of the ILs and original NSAIDs from the BC membranes was
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then investigated. Finally, the cytotoxicity and anti-inflammatory properties of the IL-loaded
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membranes were determined to confirm their potential for topical drug delivery applications.
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13C
NMR, FTIR, TGA and DSC. Furthermore, their solubility in PBS aqueous solutions at
EXPERIMENTAL SECTION Materials
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Cholinium bicarbonate, 80% in water, racemic ibuprofen ≥98% (Ibu), Dulbecco’s
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Modification of Eagles Medium (DMEM) c and fetal bovine serum (FBS) were purchased from
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Sigma Aldrich, USA. Racemic ketoprofen 98% (Ket) and (S)-naproxen 99% (Nap) were supplied
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by Alfa Aesar, Germany. Raw 264.7 macrophages (TIB-71) were acquired from American Type
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Culture Collection, Manassas, USA. All chemicals were used as received.
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Synthesis of NSAID-based ionic liquids
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NSAID-based ionic liquids (ILs) ([Ch][NSAID]), namely cholinium ibuprofenate
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([Ch][Ibu]), cholinium ketoprofenate ([Ch][Ket]) and cholinium naproxenate ([Ch][Nap]), were
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synthesized by simple metathesis reactions as reported in detail elsewhere.27 Briefly, 1:1.05 molar
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ratio of cholinium bicarbonate and each NSAID were mixed, by the addition of 11.50, 12.93 and
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11.71 g of ibuprofen, ketoprofen and (S)-naproxen, respectively, into 10 g of cholinium
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bicarbonate, 80% in water. Synthesized ILs were washed three times with ethyl acetate to remove
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unreacted NSAIDs. Excess solvent and water were removed under reduced pressure using a rotary
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evaporator (60 °C, 30 min) and a nitrogen vacuum line (60°C, 72h, ca 0.01 mbar). Dried ILs were
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finally collected (water content below 2.12 wt%; determined by Karl-Fischer Titration (Metrohm
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831 Karl Fisher coulometer); see the Supporting Information) and stored in closed vials at 4°C up
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to characterization and use. The optical rotation of [Ch][Nap] was measured using a polarimeter
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JASCO P-2000 at 589 nm, demonstrating that its specific rotation was -9.6 ± 0.3°.
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Bacterial cellulose production
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BC membranes were prepared by growing the bacterial strain Gluconacetobacter sacchari28
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following a previously described protocol.29 After 6 days of incubation, BC membranes were
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withdrawn from the culture medium and treated in alkaline conditions, at 90°C, prior to washing
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with distilled water. This step was repeated until neutral pH was reached. Wet BC membranes were
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stored in distilled water at 4°C until use.
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Incorporation of NSAIDs and ILs into bacterial cellulose
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Wet BC membranes were cut in 2 x 2 cm² rectangular pieces (99.33 ± 0.20% water content).
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For the incorporation of NSAIDs, BC membranes were submitted to a solvent exchange with
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ethanol (5 times 1h soaking in ethanol) and then circa 50% of their ethanol content was drained.
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BC membranes were then soaked in 1 mL of ethanol solutions containing 10 mg.mL-1 ibuprofen, 6 ACS Paragon Plus Environment
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ketoprofen or (S)-naproxen, as controls. In the case of ILs, the solvent exchange step was not
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necessary due to their high water solubility. 2x2 cm² BC samples were pressed until circa 50% of
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their water content was drained. The concentration was adapted to obtain BC-IL membranes with
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10 mg of NSAID, namely 15.0, 14.1 and 14.5 mg.mL-1 for [Ch][Ibu], [Ch][Ket] and [Ch][Nap],
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respectively.
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After complete absorption of the solutions, membranes were dried at 40°C. Dried membranes
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were kept in a desiccator until use. After the recovery of the dried membranes, sample holders were
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rinsed with 4 mL of adequate solvent and the content of IL or NSAID in the solvent was determined
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by UV-Vis spectroscopy (see the Supporting Information).
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For tensile tests, 7x7 cm² membranes were prepared with adapted NSAID- or IL-volumes of
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the solutions.
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Characterization techniques
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1H
and 13C NMR spectra of [Ch][NSAID] ILs were recorded using a Bruker Avance 300 at
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300.13 MHz and 75.47 MHz, respectively, using deuterated water (D2O) as solvent. 13C solid-state
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cross-polarized magic-angle spinning nuclear magnetic resonance spectra of BC and BC-
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[Ch][NSAID] membranes were recorded on a Bruker Avance 400 spectrometer. Samples were
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packed into a zirconia rotor sealed with Kel-F caps and spun at 7 kHz. The acquisition parameters
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were: 4 µs 90° pulse width, 2 ms contact time, and 4 s dead time delay.
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The determination of NSAIDs and [Ch][NSAID] concentrations in ethanol and phosphate
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buffer solutions at pH 7.4 (PBS, 0.1M), was carried out by UV-vis spectroscopy, using a Thermo
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Scientific Evolution 600 spectrophotometer. Selected maximum wavelengths in PBS were 221,
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230, 259, 260, 230 and 230 nm for ibuprofen, [Ch][Ibu], ketoprofen, [Ch][Ket], (S)-naproxen and
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[Ch][Nap], respectively. The wavelengths used for their quantification in ethanol were 218, 251
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and 271 nm for ibuprofen, ketoprofen and (S)-naproxen, respectively. An excess of each compound 7 ACS Paragon Plus Environment
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was added to the PBS or ethanol solution and allowed to equilibrate at 25°C under 750 rpm for
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72h. After equilibration, solutions were centrifuged during 8 min at 8000 rpm. Supernatant was
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recovered and centrifuged again. Saturated aqueous solutions were diluted by successive 10-fold
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dilutions and their concentration was determined by UV-Vis spectroscopy as described.
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Experiments were carried out in duplicate, and the average value and corresponding standard
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deviation are given.
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FTIR spectra of NSAIDs, [Ch][NSAID] ILs and BC membranes were obtained on a Perkin
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Elmer spectrometer equipped with a single horizontal Golden Gate ATR cell. 32 scans were
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acquired in the 4000–800 cm-1 range, with a resolution of 4 cm-1. Spectra were obtained in triplicate
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and averaged.
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Glass transition and melting temperatures of [Ch][NSAID] ILs were measured in a power
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compensation differential scanning calorimeter, PERKIN ELMER model Pyris Diamond DSC,
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using hermetically sealed aluminum crucibles with a constant flow of nitrogen (50 mL.min-1).
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Samples of about 15 mg were used in each experiment. Glass transitions (Tg) and melting
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temperatures (Tmelt) were taken as onset temperatures.
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The decomposition temperatures of NSAIDs, [Ch][NSAID] and BC membranes were
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determined by TGA conducted with a Shimadzu TGA 50 analyzer equipped with a platinum cell.
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Samples were heated at a constant rate of 10°C.min−1, from room temperature up to 800 °C, under
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a nitrogen flow of 20 mL.min−1. Thermal decomposition temperatures (Tdec) were taken as the
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maximum of the derivative of TG curves and initiation of the decomposition temperatures (T5%)
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were taken as onset temperatures. Residue at 800°C was calculated in terms of weight percentage
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of the dried material.
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The surface morphology of BC, BC-NSAID and BC-[Ch][NSAID] membranes was assessed
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by SEM by cutting an adequate size membrane sample, while for cross-section images membranes 8 ACS Paragon Plus Environment
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were broken after immersion in liquid nitrogen. Samples were then covered with carbon and
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analyzed using a Hitachi SU-70 microscope at 4 kV and 10 mm focal distance.
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Tensile assays of BC, BC-NSAID and BC-[Ch][NSAID] membranes were performed using
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an Instron 5944 testing machine with Bluehill 3 software in tensile mode with a 1kN load cell.
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Samples were strips of 70×10 mm² and the gauge length 30 mm. At least 5 strips were tested for
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each sample. The corresponding stress–strain curves permitted to determine Young’s modulus
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(slope at 0.05% elongation), maximum stress and elongation at break. Experiments were carried
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out in quintuplicate and the average value and corresponding standard deviation are presented.
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Re-hydration tests were conducted for BC, BC-NSAID and BC-IL membranes. Membranes
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were weighed and soaked in individual containers with 200 mL PBS (pH 7.4; 0.1 M) aqueous
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solutions at room temperature, for 24h. Samples were regularly taken out, the excess of water was
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gently removed, and membranes were weighted and re-immersed again. The absorbed aqueous
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solution, in g per g of dried material, was calculated according to eq 1:
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𝑃𝐵𝑆 𝐴𝑏𝑠𝑜𝑟𝑝𝑡𝑖𝑜𝑛 =
𝑤𝑤𝑒𝑡 ― 𝑤𝑑𝑟𝑦 𝑤𝑑𝑟𝑦
(1)
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where wdry and wwet are the weight of dried and wet samples, respectively. Experiments were carried
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out in triplicate and the average value and corresponding standard deviation are presented.
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BC-NSAID and BC-[Ch][NSAID] membranes were placed in closed flasks, containing 200
200
mL of PBS, under magnetic stirring and protected from light. The release of NSAIDs and ILs was
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then evaluated. At determined time intervals (during 24h), 2 mL of solution was withdrawn, and
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the same volume of fresh buffer was added to maintain the volume constant. NSAID or IL content
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in each aliquot was determined by UV-Vis spectroscopy as previously described. The IL or NSAID
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content at each time was plotted as a cumulated percentage release, determined according to eq 2:
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𝑛 ― 12 × 𝐶𝑘 200
𝐶𝑐𝑢𝑚𝑢𝑙 = 𝐶𝑛 + ∑𝑘 = 0
(2) 9
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where Cn is the IL or NSAID concentration at time n. The mass of NSAID or IL leaching out from
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the sample was calculated and divided by the initial mass incorporated to get the released ratio
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(wt%). Experiments were carried out in triplicate and the average value and corresponding standard
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deviation are presented.
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The effect of NSAIDs, [Ch][NSAID], BC-NSAID and BC-[Ch][NSAID] on Raw 264.7
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macrophage viability was assessed by the resazurin assay. Cells were seeded in 96-well plates and
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incubated during 12h to allow attachment, being then exposed for 24h to concentrations ranging
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from 1 µM to 4 mM. In experiments with membranes, cells were plated in 6 well plates, let to
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stabilize overnight, and then membranes containing equivalent amounts of NSAIDs or ILs samples
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were placed into contact with cell cultures. The detailed protocol used is given elsewhere.27
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Experiments were carried out in triplicate and the average value and corresponding standard
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deviation are presented. To calculate the EC50 values, dose-response curves were fitted with the
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non-linear least squares method using a linear logistic model.
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The potential anti-inflammatory activity of NSAIDs, [Ch][NSAID] ILs, BC, BC-NSAIDs
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and BC-[Ch][NSAID] membranes was tested by analyzing their capacity to inhibit
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lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in
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macrophages. NO production was evaluated according to a previously described method.26 Cells
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were incubated with the culture medium (control), non-toxic concentrations of NSAIDs (ibuprofen:
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250 M; ketoprofen: 1 mM; (S)-naproxen: 1 mM), equivalent concentrations of [Ch][Ibu],
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[Ch][Ket], [Ch][Nap] and with respective membrane-conditioned media for 24h. PGE2 was
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quantified in cell supernatants using an ELISA kit according to manufacturer instructions (R&D
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Systems, Minneapolis, USA).
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Multiple group comparisons were made by One-Way ANOVA analysis, with a Dunnett´s
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Multiple Comparison post-test. Statistical analysis was performed using GraphPad Prism, version
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6.01 (GraphPad Software, San Diego, CA, USA). Significance levels are as follows: ****p
