On the pathway of cellular uptake: new insight into the interaction between the cell membrane and very small nanoparticles.

For any living cell the exchange with its environment is vital. Therefore, many different kinds of cargo are able to enter cells via energy-dependent or -independent routes. Nanoparticles are no exemption. It is known that small silica nanoparticles with a diameter below 50 nm are taken up by cells and that their uptake exerts pronounced toxic effects beyond a certain concentration threshold. However, neither the exact uptake mechanism of these particles nor the actual reason for their toxicity has yet been elucidated. In this study we examined the uptake of silica nanoparticles with a diameter of 7, 12 and 22 nm by means of transmission electron microscopy, accompanied by toxicological assays. We show that for every particle diameter tested a different membrane morphology during uptake can be observed and that the amount of particles entering in one event is different for the three sizes. Silica particles with a diameter of 22 nm show single-particle internalization with a membrane wrapped around the particles in the cytosol, whereas 12 nm particles display row-like multi-particle uptake into elongated membrane structures and those with a diameter of 7 nm or less end up in tubular endocytic structures containing many particles. These membrane morphologies proved to be highly reproducible as we found them in five different cell lines. Additionally, we performed ATP and LDH assays to determine particle toxicity. Exceeding a certain concentration threshold the nanoparticles showed a high toxic potential both in the biochemical assay measurements and from morphological findings. We could not find any hint at the induction of apoptosis, neither morphologically nor biochemically. In this regard we discuss membrane damage and consumption as one possible mechanism of toxicity, linking morphological observations to toxicological findings to bridge the gap in understanding the mechanism of toxicity of small nanoparticles.

On the Ultrastructure and Function of Rhogocytes from the Pond Snail Lymnaea stagnalis.

Rhogocytes, also termed "pore cells", occur as solitary or clustered cells in the connective tissue of gastropod molluscs. Rhogocytes possess an enveloping lamina of extracellular matrix and enigmatic extracellular lacunae bridged by cytoplasmic bars that form 20 nm diaphragmatic slits likely to act as a molecular sieve. Recent papers highlight the embryogenesis and ultrastructure of these cells, and their role in heavy metal detoxification. Rhogocytes are the site of hemocyanin or hemoglobin biosynthesis in gastropods. Based on electron microscopy, we recently proposed a possible pathway of hemoglobin exocytosis through the slit apparatus, and provided molecular evidence of a common phylogenetic origin of molluscan rhogocytes, insect nephrocytes and vertebrate podocytes. However, the previously proposed secretion mode of the respiratory proteins into the hemolymph is still rather hypothetical, and the possible role of rhogocytes in detoxification requires additional data. Although our previous study on rhogocytes of the red-blooded (hemoglobin-containing) freshwater snail Biomphalaria glabrata provided much new information, a disadvantage was that the hemoglobin molecules were not unequivocally defined in the electron microscope. This made it difficult to trace the exocytosis pathway of this protein. Therefore, we have now performed a similar study on the rhogocytes of the blue-blooded (hemocyanin-containing) freshwater snail Lymnaea stagnalis. The intracellular hemocyanin could be identified in the electron microscope, either as individual molecules or as pseudo-crystalline arrays. Based on 3D-electron microscopy, and supplemented by in situ hybridization, immunocytochemistry and stress response experiments, we provide here additional details on the structure and hemocyanin biosynthesis of rhogocytes, and on their response in animals under cadmium and starvation stress. Moreover, we present an advanced model on the release of synthesized hemocyanin molecules through the slit apparatus into the hemolymph, and the uptake of much smaller particles such as cadmium ions from the hemolymph through the slit apparatus into the cytoplasm.

Nanoprobing the acidification process during intracellular uptake and trafficking.

Many nanoparticular drug delivery approaches rely on a detailed knowledge of the acidification process during intracellular trafficking of endocytosed nanoparticles (NPs). Therefore we produced a nanoparticular pH sensor composed of the fluorescent pH-sensitive dual wavelength dye carboxy seminaphthorhodafluor-1 (carboxy SNARF-1) coupled to the surface of amino-functionalized polystyrene NPs (SNARF-1-NP). By applying a calibration fit function to confocal laser scanning microscopy (CLSM) images, local pH values were determined. The acidification and ripening process of endo/lysosomal compartments containing nanoparticles was followed over time and was found to progress up to 6h to reach an equilibrium pH distribution (maximum pH5.2 [±0.2]). The SNARF-1-NP localization in endo/lysosomal compartments was confirmed by transmission electron microscopy (TEM) and quantitative co-localization analysis with fluorescent endolysosomal marker Rab-proteins by confocal laser scanning microscopy (CLSM). The herein described nanoparticular pH-sensor is a versatile tool to monitor dynamic pH processes inside the endolysosomal compartments. FROM THE CLINICAL EDITOR: In this interesting article, the authors elegantly designed a nanoparticular pH sensor with fluorescence probe with the capability to measure intracellular and intravesicular pH changes. The application of this method would enable the further understanding of nanoparticle uptake and intracellular physiology.

Heparin-based nanocapsules as potential drug delivery systems.

Herein, the synthesis and characterization of heparin-based nanocapsules (NCs) as potential drug delivery systems is described. For the synthesis of the heparin-based NCs, the versatile method of miniemulsion polymerization at the droplets interface was achieved resulting in narrowly distributed NCs with 180 nm in diameter. Scanning and transmission electron microscopy images showed clearly NC morphology. A highly negative charge density for the heparin-based NCs was determined by measuring the electro-kinetic potential. Measuring the activated clotting time demonstrated the biological intactness of the polymeric shell. The ability of heparin-based NCs to bind to antithrombin (AT III) was investigated using isothermal titration calorimetry and dynamic light scattering experiments. The chemical stability of the NCs was studied in physiological protein-containing solutions and also in medically interesting fluids such as sodium chloride 0.9%, Ringer's solution, and phosphate buffer saline using dynamic light scattering and measuring the fluorescence intensity. The impressive uptake of NCs in different cells was confirmed by fluorescence-activated cell sorting, confocal laser scanning microscopy, and transmission electron microscopy. The low toxicity of all types of NCs was demonstrated.

Mass spectrometry and imaging analysis of nanoparticle-containing vesicles provide a mechanistic insight into cellular trafficking.

Rational design of nanocarriers for drug delivery approaches requires an unbiased knowledge of uptake mechanisms and intracellular trafficking pathways. Here we dissected these processes using a quantitative proteomics approach. We isolated intracellular vesicles containing superparamagnetic iron oxide polystyrene nanoparticles and analyzed their protein composition by label-free quantitative mass spectrometry. The proteomic snapshot of organelle marker proteins revealed that an atypical macropinocytic-like mechanism mediated the entry of nanoparticles. We show that the entry mechanism is controlled by actin reorganization, atypical macropinocytic signaling, and ADP-ribosylation factor 1. Additionally, our proteomics data demonstrated a central role for multivesicular bodies and multilamellar lysosomes in trafficking and final nanoparticle storage. This was confirmed by confocal microscopy and cryo-TEM measurements. By quantitatively analyzing the protein composition of nanoparticle-containing vesicles, our study clearly defines the routes of nanoparticle entry, intracellular trafficking, and the proteomic milieu of a nanoparticle-containing vesicle.

Drug delivery without nanoparticle uptake: delivery by a kiss-and-run mechanism on the cell membrane.

Nearly all concepts of nanocarriers as drug delivery devices rely on intracellular uptake. Instead, we demonstrate an alternative concept for rapid and specific delivery of cargo by nanoparticles to TIP47+/ADRP+ lipid droplets. The model can serve as a novel strategy for the non-invasive delivery of drugs by releasing hydrophobic cargo, in our case a model dye, through a kiss-and-run mechanism between nanoparticles and the cell membrane.