RESUMO
Most nanomaterial-based medicines are intravenously applied since oral administration comprises challenging-related biological obstacles, such as interactions with distinct digestive fluids and their transport through the intestinal barrier. Moreover, there is a lack of nanoparticle-based studies that faithfully consider the above-cited obstacles and boost oral-administered nanomedicines' rational design. In this study, the physicochemical stability of fluorescent model silica nanoparticles (f-SiO2NPs) passing through all simulated gastrointestinal fluids (salivary, gastric, and intestinal) and their absorption and transport across a model human intestinal epithelium barrier are investigated. An aggregation/disaggregation f-SiO2NPs process is identified, although these particles remain chemically and physically stable after exposure to digestive fluids. Further, fine imaging of f-SiO2NPs through the absorption and transport across the human intestinal epithelium indicates that nanoparticle transport is time-dependent. The above-presented protocol shows tremendous potential for deciphering fundamental gastrointestinal nanoparticles' evolution and can contribute to rational oral administration-based nanomedicine design.
Assuntos
Líquidos Corporais , Nanopartículas , Humanos , Mucosa Intestinal , Trato Gastrointestinal , Administração OralRESUMO
The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 µM and 2 µM respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Intestinos/fisiologia , Fígado/fisiologia , Farmacocinética , Acetaminofen/farmacocinética , Acetaminofen/toxicidade , Animais , Células CACO-2 , Núcleo Celular/metabolismo , Células HT29 , Humanos , Dispositivos Lab-On-A-Chip , Fígado/citologia , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , Sobrevivência de Tecidos/efeitos dos fármacosRESUMO
This study describes the characterization of pharmacokinetic (PK) properties of acetaminophen (APAP) in the Two-Organ-Chip platform (2-OC), a two-chamber device able to cultivate 3D tissues under flow. The APAP intestinal absorption and hepatic metabolism were emulated by human intestine and liver equivalents respectively. The intestinal barrier was produced using Caco-2 and HT-29â¯cells. The liver spheroids were produced with HepaRG and HHSTeC cells. Cell viability and toxicity were assessed by MTT assay, histology, confocal immunohistochemistry, and multiparametric high content analysis. Gene expression of intestine and liver equivalents were assessed by real-time PCR. Three assemblies of Microphysiological System (MPS) were applied: Intestine 2-OC, Liver 2-OC, and Intestine/Liver 2-OC. The oral administration was emulated by APAP placement over the apical side of the intestinal barrier and the intravenous routes were mimic by the application in the medium. Samples were analyzed by HPLC/UV. APAP 12⯵M or 2⯵M treatment did not induce cytotoxicity for the intestinal barrier (24â¯h time-point) or for the liver spheroids 12â¯h time-point), respectively. All preparations showed slower APAP absorption than reported for humans: Peak time (Tmax)â¯=â¯12â¯h for Intestine 2-OC and 6â¯h for Intestine/Liver 2-OC in both static and dynamic conditions, against reported Tmax of 0,33 to 1,4â¯h after oral administration to humans. APAP metabolism was also slower than reported for humans. The APAP half-life (T1/2) was 12â¯h in the dynamic Liver 2-OC, against T1/2â¯=â¯2⯱â¯0,4â¯h reported for humans. Samples taken from the Liver 2-OC static preparation did not show APAP concentration decrease. These findings show the MPS capability and potential to emulate human PK properties and highlight the critical role of mechanical stimulus over cell functionality, especially by demonstrating the clear positive influence of the microfluidic flow over the liver equivalents metabolic performance.