Mesoporous silica- and silicon-based materials ... - Helda - Helsinki.fi

4.1.3 Cell culture (IV)
Human colon adenocarcinoma Caco-2 cells were obtained from American Type Culture
Collection. The cells were grown in a medium consisting of Dulbecco’s Modified Eagle
Medium (4.5 g/l glucose) supplemented with 10% Fetal Bovine Serum (heat inactivated at
56 °C for 30 min), 1% Non Essential Amino Acids, 1% L-glutamine (200 mM), penicillin
(100 IU/ml), and streptomycin (100 µg/ml). The cultures were maintained at 37 °C in an
incubator at an atmosphere of 5% CO2 and 95% relative humidity. The growth medium
was changed three times a week during cell growth and differentiation. The cells were
seeded at 6.8 × 10 4 cells/cm 2 onto polycarbonate filter membranes (pore size 0.4 µm,
growth area 1.1 cm 2 ) in 12-Transwell inserts. Cells from passage numbers 31–42 were
used in the experiments at ages ranging from 21 to 28 days.
4.1.4 Dissolution media and other chemicals (I-IV)
Buffered Hank’s balanced salt solution (HBSS) was used as a medium in drug release
studies (I, IV). Biological buffers HEPES (4-(2-Hydroxyethyl)piperazine-1ethanesulfonic
acid) or MES [2-(N-Morpholino)ethanesulfonic acid] at 10 mM
concentration were used to buffer the HBSS. 0.2M HCl / 0.2 M KCl was the buffer used at
pH 1.2 (II). In the experiments including a pH change from 1.2 to 6.8, 0.1 M HCl solution
was used as the initial medium at pH 1.2, which was further raised to pH 6.8 by adding 0.2
M Na3PO4 solution (II). At pH 5.5, 0.1 M phosphate buffer was used (III).
Solvents used in the sample manufacture and analysis were EtOH (I-IV), phosphoric
acid (I-IV), acetonitrile (I, IV), dichloromethane (III), methanol (IV), dimethylsulfoxide
(DMSO) (IV) and octanol (IV). D-[ 14 C]mannitol solution was used to assess the Caco-2
monolayer integrity after the permeation studies. Excipients in tablet formulations (III)
were Avicel PH 102 (microcrystalline cellulose), Ac–Di–Sol (croscarmellose sodium),
Aerosil 200 (colloidal silica), Kollidon 30 (polyvinylpyrrolidone), Pharmatose 200 M
(lactose monohydrate) and magnesium stearate.
4.2 Loading of mesoporous microparticles (I-IV)
Immersion loading method (I-IV)
In the immersion loading method the drug was first dissolved in an appropriate solvent at
the following concentrations: ibuprofen (I) 800 mg/ml in EtOH, IMC 180 mg/ml and 250
mg/ml (II) or 17 mg/ml (III) in EtOH, and furosemide (IV) 600 mg/ml in DMSO. The
loading was performed at room temperature, except for IMC loading in publication II,
where heated EtOH at 68±2 °C was used as a solvent. The mesoporous particles were
added and the suspension stirred for at least 1 h. After the loading, the suspension was
filtered and the loaded particles were dried in an oven. The drying temperature varied
between 60 and 120 °C depending on the solvent.
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