Chemicals, culture media, and drug solutions
Artesunate (Chemical Abstract Service CAS number: 88495-63-0; IPCA #19003AS6RII [1 kg]) was purchased from IPCA Laboratories Limited (Mumbai, India) through Hepartex (Saint-Cloud, France). Krebs-ringer buffer (KRB) was prepared by dissolving 6.8 g NaCl, 0.4 g KCl, 0.14 g NaH2PO4ּ H2O, 2.1 g NaHCO3, 3.575 g HEPES, 1.0 g D-glucose, 0.2 g MgSO4ּ 7H2O, and 0.26 g CaCl2ּ 2H2O in 1L of distilled sterile water. HEPES, D-glucose, and NaHCO3, were purchased from Carl Roth GmbH (Karlsruhe, Germany). All other KRB reagents, 0.25% (w/v) trypsin-EDTA, Trypan blue stain (0.4%), phosphate buffer solution (PBS) tablets, fluorescein sodium salt (NaF), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Dimethyl sulfoxide (DMSO), were purchased from Sigma-Aldrich Merck (Saint-Louis, Missouri, USA). Culture medium included minimum essential medium (MEM), 10% (v/v) heat-inactivated sterile foetal bovine serum (FBS), 1% (w/v) L-Glutamine, 1% (v/v) non-essential amino-acids (NEAA), and 20 µg/ml gentamicin. MEM, heat-inactivated FBS, L-glutamine, and NEAA, were purchased from Sigma-Aldrich Merck (Saint-Louis, Missouri, USA). Nasal epithelial cells RPMI 2650 (RMPI 2650 ECACC 88031602) were purchased from the European Collection of Authenticated Cell Cultures (ECACC, Porton, Wiltshire, England). Falcon® Cell-culture flasks, Flacon® 96-well plates and Corning Costar® 12-well plates were purchased from Corning (Glendale, Arizona, USA). ThinCert® tissue-culture inserts for 12-Well plates (polyethylene terephthalate membrane, 1.13 cm², 0.4 mm pore size) were purchased from Greiner Bio-One (Kremsmünster, Austria). Artesunate and artesunate-d4 standards for mass spectrometry (MS) analysis were purchased from Alsachim (Illkirch-Graffenstaden, France). Ultrapure water was obtained from Biosolve (Dieuze, France) and Thermo Fischer Scientific (Massachusetts, USA). Mass spectrometry-grade (MS-grade) acetonitrile was obtained from Biosolve (Dieuze, France). MS-grade ammonium acetate, acetic acid and methanol were obtained from (Thermo Fischer Scientific (Massachusetts, USA). Formic acid and ammonium formiate were purchased from Sigma Aldrich (Saint-Louis, Missouri, USA).
MTT stock solution was prepared in PBS at a final concentration of 5 mg/ml. Artesunate stock solutions (30 mg/ml) for permeation and MTT tests were prepared into a 5% (w/v) NaHCO3 aqueous solution followed by dilutions into adequate media. Artesunate for MTT assays was diluted into culture medium to final concentrations of 16, 12, 6, 3, 1.5, and 0.75 µg/ml. The donor for artesunate permeation studies was either a 0.75 µg/ml artesunate solution in KRB or a 20 µg/mg powder mixture of artesunate and corn starch. Corn starch is an inert excipient that was used to dilute the artesunate content in the powder formulation, increase the weight of the formulation, and thus allow its reproducible weighing for the permeation study [35, 36]. The donor solution for NaF permeation tests was prepared by dissolving NaF into KRB (25 µg/ml). All molecules’ solutions were filtered before use (0.22 µm filter).
Cell Culture
Nasal epithelial cells RPMI 2650 were used between passage 11 and 25 for all experiments. They were routinely cultured into 25 cm² polystyrene cell-culture flasks under standard conditions (humid atmosphere at 37°C and 5% CO2). For cell passaging, cells were washed with PBS at 37°C and then detached with 0.25% (w/v) trypsin-EDTA at 37°C seven days after previous seeding. Cell viability was assessed using a standard trypan blue staining procedure. After counting, cells were seeded in a new flask at a \(4\times {10}^{4} \text{c}\text{e}\text{l}\text{l}\text{s}/\text{c}\text{m}²\) seeding density. Culture medium was changed every two to three days. Multilayer cell culture was performed into tissue-culture inserts according to the protocol previously described by Reich and Becker [37]. Briefly, \(2\times {10}^{5} \text{c}\text{e}\text{l}\text{l}\text{s}/\text{c}\text{m}²\) were seeded on permeable ThinCert® insert membranes and cultured under liquid-covered conditions (LCC) for eight days. After this period, the inserts were lifted at the air-liquid interface (ALI) and cultured for 14 more days to allow the formation of multiple cell layers and tights junctions [37, 38]. Transepithelial electrical resistance (TEER) was measured every two to three days during ALI culture. Experiments were performed in triplicate during at least three independent assays.
Transepithelial Electrical Resistance (Teer)
TEER was measured using a Millicell® ERS-2 Voltohmmeter (Merck, Darmstadt, Germany) and the STX01 chopstick electrode according to the manufacturer’s instructions (Fig. 1). Briefly, culture medium or KRB was added to the apical and basolateral compartments of the cell culture to final volumes of 1 ml and 1.5 ml, respectively. Cultures were then left to equilibrate at 37°C for 30 minutes before measurements. TEER readings were corrected by subtracting blank filters values and normalized to the surface area of the membrane (1.13 cm²). Cell cultures having TEER values of at least 60 Ω.cm² were used for permeation assays [39].
Mtt Cytotoxicity Assay
A MTT cytotoxicity assay was performed to assess artesunate cytotoxicity on epithelial cells RPMI 2650 [40, 41]. Briefly, the cells were seeded at a density of \(1.5\times {10}^{4} \text{c}\text{e}\text{l}\text{l}\text{s}/\text{c}\text{m}²\) in 96-well plates. After 24 hours, artesunate drug dilutions were added and cells were incubated for 24 hours. Following drug exposure, cells were gently washed twice with 100 µl PBS at 37°C, before adding 110 µl of MTT stock solution diluted in culture medium (final MTT concentration : ~ 0.5 mg/ml). After four hours of incubation, the MTT solution was discarded and 200 µl of DMSO was added to each well. After one hour of incubation, absorbances were read at 485 nm using a Tristar2 LB 942 Multimode Microplate Reader (Berthold Technologies, Germany). Negative (cells without xenobiotics) and positive (cells with 25% v/v DMSO) controls were included. Results were expressed as cell viability (%) relative to negative control (100 % viability). Acording to ISO 10993-5:2009, artesunate dilutions with cell viability percentages above 80% were considered as non-cytotoxic [42, 43]. Experiments were performed in triplicate during three independent assays.
In vitro permeation studies
The permeation of the sodium salt of fluorescein (NaF) was used to validate the human nasal mucosa model derived from the culture of nasal epithelial cells RPMI 2650. Before the permeation test, multilayer cultures were rinsed with preheated KRB (37°C) and their TEER were measured. In another 12-Well plate, 0.5 ml of donor solution was added into the donor chamber and 1.5 ml of KRB (37°C) in the receptor chamber. The plates were incubated for one hour under orbital shaking (37°C, 5% CO2, humid atmosphere, 100 rpm). Samples (100 µl) were collected from the receptor chamber at fixed time intervals and immediately replaced with fresh KRB (37°C). Permeated NaF was quantified into each sample using a Tristar2 LB 942 Multimode Microplate Reader (Berthold Technologies, Germany) with excitation and emission wavelengths of 485 and 535, respectively. Experiments were performed with five replicates during two independent assays. The apparent permeability coefficient of NaF (Papp) was calculated with the following FDA approved equation and expressed in cm/s [39]:
$${P}_{app}=\left(\frac{V\times {C}_{f}}{{C}_{i}\times A\times t}\right)$$
V: volume of the acceptor chamber (cm3); Cf : drug concentration in the acceptor chamber at the end of the experiment (g/l or mol/l); Ci: initial drug concentration in the donor chamber (g/l or mol/l); A: surface area of the membrane (cm²); t: duration of the experiment (s).
Artesunate permeation was evaluated in a similar way as described above for NaF. Aliquots of solution and powder formulations, equivalent to 0.357 µg and 200 µg of artesunate, respectively, were added into the donor chamber. Each permeation test lasted for 4 hours and 200 µl samples were regularly collected from the acceptor chamber. TEER measurements were performed immediately before (TEERi), after (TEER4h), and 24 hours after (TEER24h) test initiation. Artesunate permeation tests were performed in triplicate during three independent tests.
Artesunate in vitro deposition study
In vitro deposition of artesunate nasal powder was characterized in an adult male nasal cast with chemical quantification (Fig. 2). The nasal cast model used was designed from Computed Tomography images (CT scan) of a plastinated head model [44] and was previously validated as a predictive model for nasal aerosol deposition [45, 46].
Six unidose (UDSp) devices supplied by Aptar Pharma (Le Vaudreuil, France) were filled with 10 ± 0.5 mg of artesunate powder. One device per nostril was manually actuated into the nasal cast with a fixed insertion depth of 1.5 cm, delivery angle (horizontal plane) of 45° and angle from the centre wall of 4°, after being humidified for 10 minutes at 3 lpm with a AMGH nebuliser (DTF, Saint-Etienne, France) (Fig. 3). The delivered dose was assessed by weighing each device before and after testing. The different regions of interest (Nose, Olfactory Zone, Turbinates, Nasal Floor, Nasopharynx and Lungs) were rinsed with a PBS solution at pH 8 and frozen at -80°C to ensure artesunate remained stable.
Analytical Methods
For the permeation study, artesunate quantification was performed with an ultra-high performance liquid chromatography system (AcquityTM, Waters, Massachusetts, USA) coupled to a tandem mass spectrometer (XEVO-TQ-MS, Waters). Chromatographic separation was performed using an Ethylene Bridged Hybrid (BEH) C18 column (1.7µm, 2.1 x 100 mm, Waters). The mobile phase consisted of 10 mM ammonium formiate in water (pH = 3) and 0.1% (v/v) formic acid in acetonitrile. Calibrators were prepared in KRB at concentrations ranging from 0.5 to 25 000 ng/ml. Before quantification, 20 µL of acetonitrile containing artesunate-d4 internal standard (at a concentration of 2.5 µg/mL) were added to 180 µL of each sample.
For the nasal deposition study, the samples were, firstly, thawed in a water bath at room temperature (20°C). One millilitre (5 x 200 µl) of each sample was deposited on five filter membranes mounted on hydrophobic supports (OmniporeTM 10.0 µm, 47 mm). The membranes were thereafter left to dry overnight at room temperature. Artesunate was eluted from the membranes with 5 ml of methanol for each sample and the resulting solutions were analysed for drug determination. Artesunate quantification was performed using an Ultra-High Performance Liquid Chromatography system (Ultimate 3000, Thermo Fischer Scientific, Massachusetts, USA) coupled to a quadrupole-time-of-flight mass spectrometer (Impact II, Bruker, Massachusetts, USA). Chromatographic separation was performed using a Luna Omega Polar C18 column (1.6 µm, 2.1 x 50 mm, Phenomenex, California, USA). The mobile phase consisted of solvent (A), 1 mM ammonium acetate in ultra-pure water and 0.05% (v/v) acetic acid and solvent (B) 1 mM ammonium acetate in methanol and 0.05% (v/v) acetic acid. The lower limit of quantification (LOQ) of artesunate was set at 0.05 µg/g of solution. The drug deposition in each region of the nasal cast was expressed as the fraction (%) of the actual dose (100%) delivered with the device.
Statistical analysis
All data management was performed with Microsoft Office Excel and GraphPad Prism 9.2.0 software. Statistical analyses, including one sample Wilcoxon tests and Mann-Whitney tests were performed with the significance threshold of α = 0.05. Data are presented as the mean ± standard deviation (SD) of at least two replicates.