Abstract
Nanoparticles (NPs) are widely used as components of drugs or cosmetics and hold great promise for biomedicine, yet their effects on cell physiology remain poorly understood. Here we demonstrate that clathrin-independent dynamin 2-mediated caveolar uptake of surface-functionalized silica nanoparticles (SiNPs) impairs cell viability due to lysosomal dysfunction. We show that internalized SiNPs accumulate in lysosomes resulting in inhibition of autophagy-mediated protein turnover and impaired degradation of internalized epidermal growth factor, whereas endosomal recycling proceeds unperturbed. This phenotype is caused by perturbed delivery of cargo via autophagosomes and late endosomes to SiNP-filled cathepsin B/L-containing lysosomes rather than elevated lysosomal pH or altered mTOR activity. Given the importance of autophagy and lysosomal protein degradation for cellular proteostasis and clearance of aggregated proteins, these results raise the question of beneficial use of NPs in biomedicine and beyond.
Highlights
14170 JOURNAL OF BIOLOGICAL CHEMISTRY with the biomedical application of NPs at the cellular and organismic levels remain incompletely understood [1]
To unravel the mechanism of silica nanoparticles (SiNPs) uptake, cells were treated with small interfering RNAs to deplete them of endogenous clathrin heavy chain, a key essential component of clathrin-mediated endocytosis, flotillin 1, an integral membrane protein thought to contribute to clathrin-independent fluid-phase endocytosis via the CLIC/GEEC pathway [23, 24], or of caveolin, the main structural component of caveolae that undergoes dynamin-mediated fission [25]
We conclude that intralysosomal accumulation of SiNPs leads to the accumulation of p62-containing LC3-positive autophagosomes that apparently fail to undergo further lysosomal degradation as further explored in detail below. These results indicate that cellular accumulation of SiNPs impairs degradation of cytoplasmic proteins targeted for lysosomal degradation via autophagy, they do not address whether growth factors internalized from the plasma membrane via endosomes can be degraded efficiently in SiNP-filled lysosomes
Summary
14170 JOURNAL OF BIOLOGICAL CHEMISTRY with the biomedical application of NPs at the cellular and organismic levels remain incompletely understood [1]. Among the phenotypic changes reported to be associated with the biomedical application of NPs are cellular stress responses (i.e. redox imbalance, oxidative stress), DNA damage, and altered gene expression [8, 9] Which of these phenotypes can be considered a direct consequence of cellular NP association or uptake and the underlying molecular mechanisms have remained in many cases unknown. We further show that intralysosomal accumulation of SiNPs severely perturbs autophagy-mediated protein turnover and degradation of internalized epidermal growth factor due to impaired cargo delivery via autophagosomes or late multivesicular endosomes to SiNP-filled lysosomes. Consistent with these results we show that lysosomal SiNP accumulation reduces the metabolic activity of cells it does not induce cell death due to apoptosis or necrotic cell lysis. Scheme illustrating the synthesis of AHAPS-functionalized SiNPs using the microemulsion and Stöber technique (see “Experimental Procedures”)
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