Cellular assays and applied technologies for characterisation of orally administered protein nanoparticles: a...

Cellular assays are essential in pharmaceutical development of protein-loaded nanomedicine. Cell lines provide robust and efficient models to characterise cytotoxicity, cellular uptake, absorption mechanism, intracellular stability, exocytosis mechanism and therapeutic effects of nanomedicine. GI epithelial cells and goblet cells have been employed to examine protein-loaded nanoparticles in vitro. However, the existence of different research protocols hampers the comparison of formulations and obtained results. Although advanced novel microscopy and fluorescent detection techniques are available for facilitating the development of nano-sized formulation, optimised research designs and validated instrument operation procedure are crucial to increase the reliability and validity of research findings. In the current review article, we examined a number of cellular assays, including cellular culture, cytotoxicity assay, cellular uptake assay, transepithelial studies, permeability assays, glucose consumption assays, and exocytosis and endocytosis studies, that have been widely employed for the development of orally administered insulin-loaded nanoparticles. Meanwhile, the role of various technologies, such as CLSM, flow cytometry, ELISA, fluorescence microscopy, microplate reader, and transmission electron microscopy, on visualisation of nanoparticle cellular uptake was evaluated. The following four challenges, including limited nanoparticle diffusion across mucus barrier, unwanted apical exocytosis, P-glycoprotein efflux pumps, endosomal entrapment and lysosomal degradation on protein-loaded nanoparticles, should be addressed in future studies. During formulation optimisation, strategies that can overcome the above hinderance are warranted to maximise oral bioavailability, minimise waste in research funding and facilitate the translation of therapeutic protein-loaded nanomedicine into clinical settings.