New In Vitro Coculture Model for Evaluating Intestinal Absorption of Different Lipid Nanocapsules
Standard models used for evaluating the absorption of nanoparticles like Caco-2 ignore the presence of vascular endothelium, which is a part of the intestinal multi-layered barrier structure. Therefore, a coculture between the Caco-2 epithelium and HMEC-1 (Human Microvascular Endothelial Cell type 1) on a Transwell® insert has been developed.
The model has been validated for (a) membrane morphology by transmission electron microscope (TEM); (b) ZO-1 and β-catenin expression by immunoassay; (c) membrane integrity by trans-epithelial electrical resistance (TEER) measurement; and (d) apparent permeability of drugs from different biopharmaceutical classification system (BCS) classes. Lipid nanocapsules (LNCs) were formulated with different sizes (55 and 85 nm) and surface modifications (DSPE-mPEG (2000) and stearylamine). Nanocapsule integrity and particle concentration were monitored using the Förster resonance energy transfer (FRET) technique.
The result showed that surface modification by DSPE-mPEG (2000) increased the absorption of 55-nm LNCs in the coculture model but not in the Caco-2. Summarily, the coculture model was validated as a tool for evaluating the intestinal absorption of drugs and nanoparticles. The new coculture model has a different LNCs absorption mechanism suggesting the importance of intestinal endothelium and reveals that the surface modification of LNCs can modify the in vitro oral absorption.
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Article information: Kaeokhamloed, N.; Roger, E.; Béjaud, J.; Lautram, N.; Manero, F.; Perrot, R.; Abbara, C.; Briet, M.; Legeay, S. New In Vitro Coculture Model for Evaluating Intestinal Absorption of Different Lipid Nanocapsules. Pharmaceutics 2021, 13, 595. https://doi.org/10.3390/pharmaceutics13050595
Materials: Caco-2 and HMEC-1 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Sodium chloride, sodium tetraphenylborate, ethyl acetate, dichloromethane, methanol, triton X-100, Trizma® (base), anti-β-catenin rabbit mAb, anti-TJP1 (tight junction protein-1; a.k.a. ZO-1, zonula occludens-1) rabbit mAb, collagen type 1 (calf skin), 4′,6-diamidino-2-phenylindole (DAPI), Dulbecco’s modified Eagle’s medium (DMEM D6429, with 4500 mg/L glucose, l-glutamine, sodium pyruvate, and sodium bicarbonate), dimethyl sulfoxide (DMSO), epidermal growth factor, hydrocortisone HCl powder for injection, Osmium tetroxide (OsO4), Epon™ 812 resin, metoprolol tartrate, propranolol HCl, naproxen, atenolol and furosemide, and stearylamine were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Hank’s balanced salted solution (HBSS), MCDB 131 medium (Gibco 10372-019), penicillin-streptomycin solution, goat anti-rabbit IgG Alexa Flour® 488, DiI (1,10-dioctadecyl-3,3,30,30-tetramethyl-indocarbocyanine perchlorate), and DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-dicarbocyanine perchlorate) were purchased from Thermofisher (Villebon-sur-Yvette, France). Amphotericin B, Phosphate-buffered saline (PBS), and l-glutamine were purchased from PAA Laboratories (Toronto, ON, Canada). Fetal bovine serum (FBS) was purchased from Biowest (Nuaillé, France). Paraformaldehyde 32% and glutaraldehyde 25% were purchased from Electron Microscopy Science (Hatfield, PA, USA). Nonessential amino acid (NEAA) was purchased from Lonza (Verviers, Belgium). Ultrapure water was obtained from a Milli-Q® Advantage A10 System (Merck Millipore, Darmstadt, Germany). Costar® Transwell® (12-well, polycarbonate membrane filters, 0.4 µm pore size, 1.12 cm2 growth area) and T75 cell culture flasks were purchased from Costar (New York, NY, USA). Captex® 8000 (glyceryl tricaprylate) was kindly provided by Abitec Corporation (Columbus, OH, USA). Lipoid® S75-3 (phosphatidylcholine and phosphatidylethanolamine mixture) was purchased from Lipoid GmbH (Steinhausen, Switzerland); Kolliphor® HS-15 (PEG 660 and polyethylene glycol 660 hydroxystearate mixture) from BASF (Ludwigshafen, Germany); and DSPE-mPEG(2000) (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)) from Avanti Polar Lipids (Alabaster, AL, USA).