Toxicological evaluation of nanoparticles (NPs) requires the utilization of in vitro techniques due to their number and diverse properties. Cell culture systems are often lacking in their ability to perform comparative toxicity assessment due to dosimetry issues and capacity to simulate in vivo environments. Upon encountering a physiological environment, NPs become coated with biomolecules forming a biocorona (BC), influencing function, biodistribution, and toxicity. Disease-induced alterations in the biological milieu can alter BC formation. This study evaluates the role of low-density lipoprotein (LDL) in altering macrophage responses to iron oxide (Fe₃O₄) NPs. BCs were formed by incubating Fe₃O₄ NPs in serum-free media, or 10% fetal bovine serum with or without LDL present. Following exposures to a normalized dose (25 μg/mL), macrophage association of Fe₃O₄ NPs with a LDL-BC was enhanced. TNF-α mRNA expression and protein levels were differentially induced due to BCs. Cell surface expression of SR-B1 was reduced following all Fe₃O₄ NPs exposures, while only NPs with an LDL-BC enhanced mitochondrial membrane potential. These findings suggest that elevations in LDL may contribute to distinct BC formation thereby influencing NP-cellular interactions and response. Further, our study highlights challenges that may arise during the in vitro evaluation of disease-related variations in the NP-BC.