Polyelectrolyte multilayer (PEM) capsules are carrier vehicles with great potential for biomedical applications. With the future aim of designing biocompatible, effective therapeutic delivery systems (e.g., for cancer), the pathway of internalization (uptake and fate) of PEM capsules was investigated. In particular the following experiments were performed: (i) the study of capsule co-localization with established endocytic markers, (ii) switching-off endocytotic pathways with pharmaceutical/chemical inhibitors, and (iii) characterization and quantification of capsule uptake with confocal and electron microscopy. As result, capsules co-localized with lipid rafts and with phagolysosomes, but not with other endocytic vesicles. Chemical interference of endocytosis with chemical blockers indicated that PEM capsules enter the investigated cell lines through a mechanism slightly sensitive to electrostatic interactions, independent of clathrin and caveolae, and strongly dependent on cholesterol-rich domains and organelle acidification. Microscopic characterization of cells during capsule uptake showed the formation of phagocytic cups (vesicles) to engulf the capsules, an increased number of mitochondria, and a final localization in the perinuclear cytoplasma. Combining all these indicators we conclude that PEM capsule internalization in general occurs as a combination of different sequential mechanisms. Initially, an adsorptive mechanism due to strong electrostatic interactions governs the stabilization of the capsules at the cell surface. Membrane ruffling and filopodia extensions are responsible for capsule engulfing through the formation of a phagocytic cup. Co-localization with lipid raft domains activates the cell to initiate a lipid-raft-mediated macropinocytosis. Internalization vesicles are very acidic and co-localize only with phagolysosome markers, excluding caveolin-mediated pathways and indicating that upon phagocytosis the capsules are sorted to heterophagolysosomes.
We study the spreading and contraction behavior of platelets in microfluidic flow.
A five-meal modified Mediterranean-type diet with two daily portion-controlled sweet snacks was effective for weight management in a self-help setting for overweight and grade 1 obese subjects. Fat modification through canola oil, walnuts and walnut oil improved blood lipids even at 12 months.
Encapsulating reacting biological or chemical samples in microfluidic droplets has the great advantage over single-phase flows of providing separate reaction compartments. These compartments can be filled in a combinatoric way and prevent the sample from adsorbing to the channel walls. In recent years, small-angle X-ray scattering (SAXS) in combination with microfluidics has evolved as a nanoscale method of such systems. Here, we approach two major challenges associated with combining droplet microfluidics and SAXS. First, we present a simple, versatile, and reliable device, which is both suitable for stable droplet formation and compatible with in situ X-ray measurements. Second, we solve the problem of "diluting" the sample signal by the signal from the oil separating the emulsion droplets by multiple fast acquisitions per droplet and data thresholding. We show that using our method, even the weakly scattering protein vimentin provides high signal-to-noise ratio data.
X-ray imaging of intact biological cells is emerging as a complementary method to visible light or electron microscopy. Owing to the high penetration depth and small wavelength of X-rays, it is possible to resolve subcellular structures at a resolution of a few nanometers. Here, we apply scanning X-ray nanodiffraction in combination with time-lapse bright-field microscopy to nuclei of 3T3 fibroblasts and thus relate the observed structures to specific phases in the cell division cycle. We scan the sample at a step size of 250 nm and analyze the individual diffraction patterns according to a generalized Porod's law. Thus, we obtain information on the aggregation state of the nuclear DNA at a real space resolution on the order of the step size and in parallel structural information on the order of few nanometers. We are able to distinguish nucleoli, heterochromatin, and euchromatin in the nuclei and follow the compaction and decompaction during the cell division cycle.
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