Surface Charge Overrides Protein Corona Formation in Determining the Cytotoxicity, Cellular Uptake, and Biodistribution of Silver Nanoparticles

Abstract

Silver nanoparticles (AgNPs) hold great promise in biomedical applications due to their unique properties and potential for specific tissue targeting. However, the clinical translation of nanoparticle-based therapeutics remains challenging, primarily due to an incomplete understanding of how nanoparticle properties influence interactions at the nano-bio interface, as well as the role of surface-adsorbed proteins (i.e., protein corona) in modulating nanoparticle-cell interactions. This study demonstrates that the surface charge has a greater influence than protein corona formation in determining the cytotoxicity, cellular uptake, and biodistribution of AgNPs. Using negatively and positively charged AgNPs, we show that while protein corona formation is essential for ensuring nanoparticle availability for cellular interactions, the adsorption of biomolecules is nonspecific and independent of surface charge. Conversely, the surface charge significantly influences the interactions of AgNPs with cells. Positively charged nanoparticles exhibit enhanced cellular uptake, preferential accumulation in lysosomes, and pronounced mitochondrial damage compared to their negatively charged counterparts, resulting in greater cytotoxic effects. This effect is particularly evident in human breast cancer cells, where negatively charged nanoparticles show minimal uptake and cytotoxicity. These findings demonstrate that surface charge is the primary factor governing nanoparticle-cell interactions rather than protein corona formation. Nonetheless, the protein corona plays a critical role in stabilizing nanoparticles in physiological environments.

Keywords: Biodistribution; Cell uptake; Cytotoxicity; Nanoparticles; Protein corona; Surface charge.

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Schematic representation of (a) negatively (AgNPs-cit and AgNPs-PSS) and (b) positively (AgNPs-PAH and AgNPs-PDDA) charged AgNPs. (c) STEM image of (top left) AgNPs-cit, and TEM images of (top right) AgNPs-PSS, (bottom left) AgNPs-PAH, and (bottom right) AgNPs-PDDA stained with phosphotungstic acid (see the Experimental Section). Scale bar: 200 nm STEM and 10 nm TEM. (d) Physical properties of (top) size (^ahydrodynamic diameter) and (bottom) surface charge (^b ζ-potential) as a function of surface coating. (e) UV–vis absorption spectra of AgNPs in deionized water as a function of surface coating.

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(a) Cell viability of NIH-3T3, MCF7, and Caco2 cells treated for 24 and 48 h with AgNPs (20 μg/mL) as a function of surface coating (AgNPs-cit and AgNPs-PSS negatively charged; AgNPs-PAH and AgNPs-PDDA positively charged). Data represent the mean ± SD and are plotted as a percentage in reference to control samples (ctrl). At least seven independent experiments, each with 10 biological replicates, were carried out, and statistical analyses were performed using ANOVA followed by Tukey’s test. **p < 0.01 and ***p < 0.001 denote significant differences with respect to the control. (b) Flow cytometric analysis of Annexin V and PI staining of NIH-3T3, MCF7, and Caco2 cells treated for 24 (NIH-3T3 and Caco2) or 48 (MCF7) h with AgNPs (20 μg/mL) as a function of surface coating (AgNPs-cit and AgNPs-PSS negatively charged; AgNPs-PAH and AgNPs-PDDA positively charged). At least three independent experiments, each with three biological replicates, were carried out.

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(a) Cellular uptake of silver by NIH-3T3, MCF7, and Caco2 cells after 16 h of exposure to 20 μg/mL AgNPs as a function of surface coating. Data are presented as mean ± SD. At least three independent experiments, each with three biological replicates, were carried out. (b) Fluorescence micrographs of NIH-3T3, MCF7, and Caco2 cells labeled specifically for actin (red) and the nucleus (blue) and treated with AgNPs (20 μg/mL) as a function of surface coating after 48 h of incubation (scale bar: 50 μm). Scale bar in insets: 200 μm.

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TEM images of ultrathin sections of NIH-3T3 (a–h), MCF7 (i–p), and Caco2 (q–z) cells treated for 6 h with 20 μg/mL citrate- (first row), PSS- (second row), PAH- (third row), and PDDA-coated (fourth row) AgNPs. Legend: nucleus (N), nucleoli (n), cytoplasm (cy), and mitochondria (m). Black arrows indicate the presence of nanoparticles. Scale bar in insets: 500 nm (b and h) and 1 μm (n).

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(a) Hydrodynamic diameter and (b) ζ-potential of AgNPs before (water) and after incubation in growth media: fibroblast (NIH-3T3) medium and cancer cell (MCF7 and Caco2) medium. Data are presented as mean ± SD and based on at least five independent measurements. (c) SDS-PAGE of biomolecules recovered from AgNPs after 24 h of incubation with fibroblast (NIH-3T3) medium and cancer cell (MCF7 and Caco2) medium. The molecular weight ladder is shown in lane 1.

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Bioinformatics classification of corona proteins: molecular weight (MW), isoelectric point (pI), and physiological function distribution of proteins recovered from AgNPs after 24 h of incubation with (a–c) fibroblast medium and (d–f) cancer cell medium. Surface charge of NPs: “n” denotes negatively charged NPs (AgNPs-cit and AgNPs-PSS) and “p” positively charged NPs (AgNPs-PAH and AgNPs-PDDA). (g–i) Distribution of proteins shared by the protein corona formed after 24 h of incubation with cancer cell medium on negatively charged NPs (AgNPs-cit and AgNPs-PSS) and positively charged NPs (AgNPs-PAH and AgNPs-PDDA).