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.

细胞检测对于载蛋白纳米药物的药物开发至关重要。细胞系提供了强大而有效的模型来表征纳米医学的细胞毒性、细胞摄取、吸收机制、细胞内稳定性、胞吐作用机制和治疗效果。胃肠道上皮细胞和杯状细胞已被用于体外检测载有蛋白质的纳米颗粒. 然而，不同研究方案的存在阻碍了配方和获得结果的比较。尽管先进的新型显微镜和荧光检测技术可用于促进纳米级配方的开发，但优化的研究设计和经过验证的仪器操作程序对于提高研究结果的可靠性和有效性至关重要。在当前的评论文章中，我们检查了许多细胞检测，包括细胞培养、细胞毒性检测、细胞摄取检测、跨上皮研究、渗透性检测、葡萄糖消耗检测以及胞吐作用和内吞作用研究，这些研究已广泛用于开发口服胰岛素负载纳米颗粒。同时，各种技术的作用，如CLSM、流式细胞术、ELISA、评估了荧光显微镜、酶标仪和透射电子显微镜对纳米颗粒细胞摄取的可视化。以下四个挑战，包括有限的纳米颗粒扩散穿过粘液屏障、不需要的顶端胞吐作用、P-糖蛋白外排泵、内体截留和蛋白质负载纳米颗粒上的溶酶体降解，应在未来的研究中解决。在配方优化过程中，需要克服上述障碍的策略，以最大限度地提高口服生物利用度，最大限度地减少研究资金浪费，并促进将治疗性蛋白质负载纳米药物转化为临床环境。包括有限的纳米颗粒扩散穿过粘液屏障、不需要的顶端胞吐作用、P-糖蛋白外排泵、内体截留和蛋白质负载纳米颗粒上的溶酶体降解，应在未来的研究中解决。在配方优化过程中，需要克服上述障碍的策略，以最大限度地提高口服生物利用度，最大限度地减少研究资金浪费，并促进将治疗性蛋白质负载纳米药物转化为临床环境。包括有限的纳米颗粒扩散穿过粘液屏障、不需要的顶端胞吐作用、P-糖蛋白外排泵、内体截留和蛋白质负载纳米颗粒上的溶酶体降解，应在未来的研究中解决。在配方优化过程中，需要克服上述障碍的策略，以最大限度地提高口服生物利用度，最大限度地减少研究资金浪费，并促进将治疗性蛋白质负载纳米药物转化为临床环境。