Shape-Dependent Dissolution and Cellular Uptake of Silver Nanoparticles.

The cellular uptake and dissolution of trigonal silver nanoprisms (edge length 42 ± 15 nm, thickness 8 ± 1 nm) and mostly spherical silver nanoparticles (diameter 70 ± 25 nm) in human mesenchymal stem cells (hMSC's) and human keratinocytes (HaCaT cells) were investigated. Both particles are stabilized by polyvinylpyrrolidone (PVP), with the prisms additionally stabilized by citrate. The nanoprisms dissolved slightly in pure water but strongly in isotonic saline or at pH 4, corresponding to the lowest limit for the pH during cellular uptake. The tips of the prisms became rounded within minutes due to their high surface energy. Afterward, the dissolution process slowed down due to the presence of both PVP stabilizing Ag{100} sites and citrate blocking Ag{111} sites. On the contrary, nanospheres, solely stabilized by PVP, dissolved within 24 h. These results correlate with the finding that particles in both cell types have lost >90% of their volume within 24 h. hMSC's took up significantly more Ag from nanoprisms than from nanospheres, whereas HaCaT cells showed no preference for one particle shape. This can be rationalized by the large cellular interaction area of the plateletlike nanoprisms and the bending stiffness of the cell membranes. hMSC's have a highly flexible cell membrane, resulting in an increased uptake of plateletlike particles. HaCaT cells have a membrane with a 3 orders of magnitude higher Young's modulus than for hMSC. Hence, the energy gain due to the larger interaction area of the nanoprisms is compensated for by the higher energy needed for cell membrane deformation compared to that for spheres, leading to no shape preference.

Lysosomal Dysfunction Caused by Cellular Accumulation of Silica Nanoparticles.

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.

Skin barrier disruptions in tape stripped and allergic dermatitis models have no effect on dermal penetration and systemic distribution of AHAPS-functionalized silica nanoparticles.

The skin is a potential site of entry for nanoparticles (NP) but the role of disease-associated barrier disturbances on the path and extent of skin penetration of NP remains to be characterized. Silica nanoparticles (SiO2-NP) possess promising potential for various medical applications. Here, effects of different skin barrier disruptions on the penetration of N-(6-aminohexyl)-aminopropyltrimethoxysilane (AHAPS) functionalized SiO2-NP were studied. AHAPS-SiO2-NP (55±6 nm diameter) were topically applied on intact, tape stripped or on inflamed skin of SKH1 mice with induced allergic contact dermatitis for one or five consecutive days, respectively. Penetration of AHAPS-SiO2-NP through the skin was not observed regardless of the kind of barrier disruption. However, only after subcutaneous injection, AHAPS-SiO2-NP were incorporated by macrophages and transported to the regional lymph node only. Adverse effects on cells or tissues were not observed. In conclusion, AHAPS-SiO2-NP seem to not cross the normal or perturbed mouse skin. From the clinical editor: Skin is a potential site of entry for nanoparticles; however, it is poorly understood how skin diseases may alter this process. In tape-stripped skin and allergic contact dermatitis models the delivery properties of AHAPS-SiO2 nanoparticles remained unchanged, and in neither case were these NP-s able to penetrate the skin. No adverse effects were noted on the skin in these models and control mice.

Surface functionalization of silica nanoparticles supports colloidal stability in physiological media and facilitates internalization in cells.

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.

Validation of weak biological effects by round robin experiments: cytotoxicity/biocompatibility of SiO2 and polymer nanoparticles in HepG2 cells.

All over the world, different types of nanomaterials with a diversified spectrum of applications are designed and developed, especially in the field of nanomedicine. The great variety of nanoparticles (NPs), in vitro test systems and cell lines led to a vast amount of publications with conflicting data. To identify the decisive principles of these variabilities, we conducted an intercomparison study of collaborating laboratories within the German DFG Priority Program SPP1313, using well-defined experimental parameters and well-characterized NPs. The participants analyzed the in vitro biocompatibility of silica and polymer NPs on human hepatoma HepG2 cells. Nanoparticle mediated effects on cell metabolism, internalization, and inflammation were measured. All laboratories showed that both nanoparticle formulations were internalized and had a low cytotoxicity profile. Interestingly, small variations in nanoparticle preparation, cell handling and the type of culture slide influenced the nanoparticle stability and the outcomes of cell assays. The round robin test demonstrated the importance of the use of clearly defined and characterized NPs and parameters for reproducible results across laboratories. Comparative analyses of in vitro screening methods performed in multiple laboratories are absolutely essential to establish robust standard operation procedure as a prerequisite for sound hazard assessment of nanomaterials.

Overview about the localization of nanoparticles in tissue and cellular context by different imaging techniques.

The increasing interest and recent developments in nanotechnology pose previously unparalleled challenges in understanding the effects of nanoparticles on living tissues. Despite significant progress in in vitro cell and tissue culture technologies, observations on particle distribution and tissue responses in whole organisms are still indispensable. In addition to a thorough understanding of complex tissue responses which is the domain of expert pathologists, the localization of particles at their sites of interaction with living structures is essential to complete the picture. In this review we will describe and compare different imaging techniques for localizing inorganic as well as organic nanoparticles in tissues, cells and subcellular compartments. The visualization techniques include well-established methods, such as standard light, fluorescence, transmission electron and scanning electron microscopy as well as more recent developments, such as light and electron microscopic autoradiography, fluorescence lifetime imaging, spectral imaging and linear unmixing, superresolution structured illumination, Raman microspectroscopy and X-ray microscopy. Importantly, all methodologies described allow for the simultaneous visualization of nanoparticles and evaluation of cell and tissue changes that are of prime interest for toxicopathologic studies. However, the different approaches vary in terms of applicability for specific particles, sensitivity, optical resolution, technical requirements and thus availability, and effects of labeling on particle properties. Specific bottle necks of each technology are discussed in detail. Interpretation of particle localization data from any of these techniques should therefore respect their specific merits and limitations as no single approach combines all desired properties.

Iron oxide nanoparticles stabilized with dendritic polyglycerols as selective MRI contrast agents.

Monodisperse small iron oxide nanoparticles functionalized with dendritic polyglycerol (dPG) or dendritic polyglycerol sulfate (dPGS) are prepared. They are highly stable in aqueous solutions as well as physiological media. In particular, oleic acid capped iron oxide particles (core diameter = 11 ± 1 nm) were modified by a ligand exchange process in a one pot synthesis with dPG and dPGS bearing phosphonate as anchor groups. Dynamic light scattering measurements performed in water and different biological media demonstrate that the hydrodynamic diameter of the particles is only slightly increased by the ligand exchange process resulting in a final diameter of less than 30 nm and that the particles are stable in these media. It is also revealed by magnetic resonance studies that their magnetic relaxivity is reduced by the surface modification but it is still sufficient for high contrast magnetic resonance imaging (MRI). Additionally, incubation of dPGS functionalized iron oxide nanoparticles with human umbilical vein endothelial cells showed a 50% survival at 85 nM (concentration of nanoparticles). Surface plasmon resonance (SPR) studies demonstrate that the dPGS functionalized iron oxide nanoparticles inhibit L-selectin ligand binding whereas the particles containing only dPG do not show this effect. Experiments in a flow chamber with human myelogenous leukemia cells confirmed L-selectin inhibition of the dPGS functionalized iron oxide nanoparticles and with that the L-selectin mediated leukocyte adhesion. These results indicate that dPGS functionalized iron oxide nanoparticles are a promising contrast agent for inflamed tissue probed by MRI.

AHAPS-functionalized silica nanoparticles do not modulate allergic contact dermatitis in mice.

Allergic contact dermatitis (ACD) is a common skin disease in people and may become a potential site of exposure to nanoparticles (NP). Silica nanoparticles (SiO2-NP) possess a promising potential for various medical and non-medical applications, including normal and diseased skin as target organs. However, it has been shown that negatively charged SiO2-NP may act as proinflammatory adjuvant in allergic diseases. The effect of topical SiO2-NP exposure on preexisting ACD has not been studied to date although this reflects a common in vivo situation. Of particular interest are the potential effects of positively charged N-(6-aminohexyl)-aminopropyltrimethoxysilane (AHAPS)-functionalized SiO2-NP which are promising candidates for delivery systems, including gene delivery into the skin. Here, the effects of such AHAPS-functionalized SiO2-NP (55 ± 6 nm in diameter) were studied in an oxazolone-induced ACD model in SKH1 mice and compared to ACD mice treated with vehicle only. The clinical course of the disease was assessed by monitoring of the transepidermal water loss (TEWL) and the erythema. In histologic and morphometric analyses, the distribution of particles, the degree of inflammation, epidermal thickness, and the inflammatory infiltrate were characterized and quantified by standard and special histological stains as well as immunohistochemistry for CD3+ lymphocytes. To assess possible systemic effects, serum immunoglobulin E (IgE) was determined by enzyme-linked immunosorbent assay. Following administration of AHAPS-SiO2-NP for five consecutive days, no effects were observed in all clinical, histologic, morphometric, and molecular parameters investigated. In conclusion, positively charged AHAPS-SiO2-NP seem not to affect the course of ACD during exposure for 5 days.

PVP-coated, negatively charged silver nanoparticles: A multi-center study of their physicochemical characteristics, cell culture and in vivo experiments.

PVP-capped silver nanoparticles with a diameter of the metallic core of 70 nm, a hydrodynamic diameter of 120 nm and a zeta potential of -20 mV were prepared and investigated with regard to their biological activity. This review summarizes the physicochemical properties (dissolution, protein adsorption, dispersability) of these nanoparticles and the cellular consequences of the exposure of a broad range of biological test systems to this defined type of silver nanoparticles. Silver nanoparticles dissolve in water in the presence of oxygen. In addition, in biological media (i.e., in the presence of proteins) the surface of silver nanoparticles is rapidly coated by a protein corona that influences their physicochemical and biological properties including cellular uptake. Silver nanoparticles are taken up by cell-type specific endocytosis pathways as demonstrated for hMSC, primary T-cells, primary monocytes, and astrocytes. A visualization of particles inside cells is possible by X-ray microscopy, fluorescence microscopy, and combined FIB/SEM analysis. By staining organelles, their localization inside the cell can be additionally determined. While primary brain astrocytes are shown to be fairly tolerant toward silver nanoparticles, silver nanoparticles induce the formation of DNA double-strand-breaks (DSB) and lead to chromosomal aberrations and sister-chromatid exchanges in Chinese hamster fibroblast cell lines (CHO9, K1, V79B). An exposure of rats to silver nanoparticles in vivo induced a moderate pulmonary toxicity, however, only at rather high concentrations. The same was found in precision-cut lung slices of rats in which silver nanoparticles remained mainly at the tissue surface. In a human 3D triple-cell culture model consisting of three cell types (alveolar epithelial cells, macrophages, and dendritic cells), adverse effects were also only found at high silver concentrations. The silver ions that are released from silver nanoparticles may be harmful to skin with disrupted barrier (e.g., wounds) and induce oxidative stress in skin cells (HaCaT). In conclusion, the data obtained on the effects of this well-defined type of silver nanoparticles on various biological systems clearly demonstrate that cell-type specific properties as well as experimental conditions determine the biocompatibility of and the cellular responses to an exposure with silver nanoparticles.