The influence of the surface functionalization of silica particles on their colloidal stability in physiological media is studied and correlated with their uptake in cells. The surface of 55 ± 2 nm diameter silica particles is functionalized by amino acids or amino- or poly(ethylene glycol) (PEG)-terminated alkoxysilanes to adjust the zeta potential from highly negative to positive values in ethanol. A transfer of the particles into water, physiological buffers, and cell culture media reduces the absolute value of the zeta potential and changes the colloidal stability. Particles stabilized by L-arginine, L-lysine, and amino silanes with short alkyl chains are only moderately stable in water and partially in PBS or TRIS buffer, but aggregate in cell culture media. Nonfunctionalized, N-(6-aminohexyl)-3-aminopropyltrimethoxy silane (AHAPS), and PEG-functionalized particles are stable in all media under study. The high colloidal stability of positively charged AHAPS-functionalized particles scales with the ionic strength of the media, indicating a mainly electrostatical stabilization. PEG-functionalized particles show, independently from the ionic strength, no or only minor aggregation due to additional steric stabilization. AHAPS stabilized particles are readily taken up by HeLa cells, likely as the positive zeta potential enhances the association with the negatively charged cell membrane. Positively charged particles stabilized by short alkyl chain aminosilanes adsorb on the cell membrane, but are weakly taken up, since aggregation inhibits their transport. Nonfunctionalized particles are barely taken up and PEG-stabilized particles are not taken up at all into HeLa cells, despite their high colloidal stability. The results indicate that a high colloidal stability of nanoparticles combined with an initial charge-driven adsorption on the cell membrane is essential for efficient cellular uptake.

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. Nanoparticles (NPs)2 are widely used as components of drugs or cosmetics and hold great promise as tools in biomedicine to improve the detection and treatment of diseases (1). For example, nanoparticle technology has enabled improvements in cancer treatment, ranging from improved efficacy of drug delivery (2) to enhanced immunogenicity of cancer vaccines (3). Moreover, NPs are used as biosensors and biomarkers (4, 5) or for DNA/drug delivery (6). Hence, it is necessary to understand the mechanisms of interaction of NPs with living cells and tissues to assess the biological consequences associated with their application in biomedicine (7). At present, the risks associated 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.Upon cellular application, NPs initially interact with the plasma membrane, often followed by their internalization into the cell interior (10 -12) via clathrin-dependent as well as clathrin-independent endocytosis routes (i.e. via caveolae), which may require the membrane-severing GTPase dynamin (13,14). Due to the questionable specificity of many commonly used pharmacological tools toward these pathways (15) the precise mechanisms of cellular uptake of NPs often have remained elusive. After cell entry NPs are delivered to the endolysosomal system (16), where they may accumulate. Lysosomes play essential roles in cell physiology ranging from the degradation of malfunctional or aggregated proteins (e.g. via autophagy) or lipids to nutrient signaling and cellular growth control (17). For example, internalized growth factors such as EGF are sorted to la...

Endosomal membrane traffic serves crucial roles in cell physiology, signaling, and development. Sorting between endosomes and the trans-Golgi network (TGN) is regulated among other factors by the adaptor AP-1, an essential component of multicellular organisms. Membrane recruitment of AP-1 requires phosphatidylinositol 4-phosphate [PI(4)P], though the precise mechanisms and PI4 kinase isozyme (or isozymes) involved in generation of this PI(4)P pool remain unclear. The Wnt pathway is a major developmental signaling cascade and depends on endosomal sorting in Wnt-sending cells. Whether TGN/endosomal sorting modulates signaling downstream of Frizzled (Fz) receptors in Wnt-receiving cells is unknown. Here, we identify PI4-kinase type 2β (PI4K2β) as a regulator of TGN/endosomal sorting and Wnt signaling. PI4K2β and AP-1 interact directly and are required for efficient sorting between endosomes and the TGN. Zebrafish embryos depleted of PI4K2β or AP-1 lack pectoral fins due to defective Wnt signaling. Rescue experiments demonstrate requirements for PI4K2β-AP-1 complex formation and PI4K2β-mediated PI(4)P synthesis. Furthermore, PI4K2β binds to the Fz-associated component Dishevelled (Dvl) and regulates endosomal recycling of Fz receptors and Wnt target gene expression. These data reveal an evolutionarily conserved role for PI4K2β and AP-1 in coupling phosphoinositide metabolism to AP-1-mediated sorting and Wnt signaling.

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