Exacerbated hepatotoxicity in in vivo and in vitro non-alcoholic fatty liver models by biomineralized copper sulfide nanoparticles

Copper sulfide nanoparticles (NPs) synthesized through biomineralization have significant commercial potential as photothermal agents, while the safety evaluation of these NPs, especially for patients with non-alcoholic fatty liver (NAFL), remains insufficient. To explore the differential hepatotoxicity of copper sulfide NPs in NAFL conditions, we synthesized large-sized (LNPs, 15.1 nm) and small-sized (SNPs, 3.5 nm) BSA@Cu_2-xS NPs. A NAFL rat model fed with high fat diet (HFD) was successfully established for a 14-day subacute toxicity study by daily repeated administration of BSA@Cu_2-xS NPs. Our findings from serum biochemistry and histopathological examinations revealed that copper sulfide at both sizes NPs induced more pronounced liver damage in NAFL rats than rats fed with normal diet. Transcriptome sequencing analysis showed that LNPs activated inflammation and DNA damage repair pathways in the livers of NAFL rats, while SNPs displayed minimal inflammation. A three-dimensional spheroid model of NAFL developed in our in-house cell spheroid culture honeycomb chips demonstrated that LNPs, but not SNPs, triggered a distinct release of inflammatory factors and increased reactive oxygen species through Kupffer cells. These results highlight that NAFL condition exacerbated the hepatotoxicity of BSA@Cu_2-xS NPs, with SNPs emerging as safer photothermal agents compared to LNPs, suggesting superior potential for clinical applications.