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Exploiting different mechanisms of action, a novel triple drug delivery system of 5-fluorouracil, curcumin, and piperine co-loaded human serum albumin nanoparticles (5FU-CUR-PIP-HSA-NPs) was developed via the self\u2010assembly method for suppressing breast tumor. Both hydrophobic and hydrophilic drugs were successfully encapsulated in the HSA NPs with a high drug loading efficiency (DLE) of 10%. Successful clinical translation of nanomedicines, however, is a challenging process requiring considerable preclinical <jats:italic>in vitro<\/jats:italic> and <jats:italic>in vivo<\/jats:italic> animal tests. The aim of this study was to develop a homemade preclinical 3D culture model in the standard 96-well plates in a cost and time-effective novel approach for the rapid generation of homogenous compact tumor spheroids for disease modeling, and anticancer therapeutic\/nanomedicine screening. The knowledge of drug screening can be enhanced by employing such a model in a high-throughput manner. Accordingly, to validate the formulated drug delivery system and investigate the cellular uptake and cytotoxicity effect of the nanoformulation, 3D tumor spheroids were employed. The practicality of the nanomedicine system was substantiated in different tests. The <jats:italic>in vitro<\/jats:italic> uptake of the NPs into the tight 3D tumor spheroids was facilitated by the semi-spherical shape of the NPs with a proper size and surface charge. 5FU-CUR-PIP-HSA-NPs demonstrated high potency of migration inhibition as a part of successful anti-metastatic therapy as well. The remarkable differences in 2D and 3D cytotoxicities emphasize the importance of employing 3D tumor models as an intermediate step prior to <jats:italic>in vivo<\/jats:italic> animal experiments for drug\/nanomedicine screening.<\/jats:p>","DOI":"10.1371\/journal.pone.0282064","type":"journal-article","created":{"date-parts":[[2023,2,17]],"date-time":"2023-02-17T18:40:39Z","timestamp":1676659239000},"page":"e0282064","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":20,"title":["Rapid generation of homogenous tumor spheroid microtissues in a scaffold-free platform for high-throughput screening of a novel combination 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