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.

Polymeric nanoparticles of different sizes overcome the cell membrane barrier.

Polymeric nanoparticles have tremendous potential either as carriers or markers in treatment for diseases or as diagnostics in biomedical applications. Finding the optimal conditions for effective intracellular delivery of the payload to the location of interest is still a big challenge. The particles have to overcome the barrier of the cell membrane. Here, we investigated the uptake in HeLa cells of fluorescent polystyrene particles with different size and surface charge. Particles stabilized with the nonionic surfactant Lutensol AT50® (132 nm, 180 nm, 242 nm, 816 nm, 846 nm diameter) were synthesized via dispersion polymerization. Cationic particles (120 nm, 208 nm, 267 nm, 603 nm diameter) were obtained by a combination of miniemulsion and seed dispersion polymerization using cationic surfactant (cetyltrimethylammonium chloride (CTMA-Cl). The particle uptake into HeLa cells was studied by confocal laser scanning microscopy and flow cytometry. Nonionic particles were - independent of their size - taken up by cells only at a barely detectable level, thus aggravating a quantitative comparison. The uptake of positively charged particles was substantially higher and therefore enabling further investigation keeping constant one of these parameters: either material amount or particles number or total interaction surface area. It was found that the uptake rather depends on the total amount of polymeric material present in the media than on the number of particles. The total particle's surface area does not correlate linearly with the uptake, thus indicating that there is no direct dependency between the total surface area and the cellular endocytotic process to overcome the biobarrier "cell membrane." A potentially novel uptake mechanism is found which can be described as an excavator shovel like mechanism. It is a kind of macropinocytosis dependent on actin filaments as well as dynamin, but is clathrin-independent.