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  2. 2. Modes of NP-cell interaction:1-Adhesion2-Cellular uptake
  3. 3. Adhesion
  4. 4. Cellular uptake
  5. 5. Cellular uptake
  6. 6. Receptor-mediated uptake• Via chlatrin coated pits• Important only for targeted NPs
  7. 7. pathways* Clathrin-mediated endocytosis is mediated by small (approx. 200nm in diameter) vesicles that have a morphologically characteristic crystalline coat made up of a complex of proteins that mainly associate with the cytosolic protein clathrin. Clathrin-coated vesicles (CCVs) are found in virtually all cells and form from domains of the plasma membrane termed clathrin- coated pits. Coated pits can concentrate a large range of extracellular molecules that are different receptors responsible for the receptor-mediated endocytosis of ligands, e.g. low density lipoprotein, transferrin, growth factors, antibodies and many others.
  8. 8. Caveolae-mediated uptake• Caveolae are the most common reported non-clathrin coated plasma membrane buds, which exist on the surface of many, but not all cell types. They consist of the cholesterol-binding protein caveolin (Vip21) with a bilayer enriched in cholesterol and glycolipids. Caveolae are small (approx. 50 nm in diameter) flask-shape pits in the membrane that resemble the shape of a cave (hence the name caveolae). They can constitute approximately a third of the plasma membrane area of the cells of some tissues, being especially abundant in smooth muscle, type I pneumocytes, fibroblasts, adipocytes, and endothelial cells. Uptake of extracellular molecules is also believed to be specifically mediated via receptors in caveolae.
  9. 9. pinocytosis
  10. 10. • Pinocytosis (literally, cell-drinking). This process is concerned with the uptake of solutes and single molecules such as proteins.• Macropinocytosis, which usually occurs from highly ruffled regions of the plasma membrane, is the invagination of the cell membrane to form a pocket, which then pinches off into the cell to form a vesicle (0.5-5µm in diameter) filled with large volume of extracellular fluid and molecules within it. The filling of the pocket occurs in a non-specific manner. The vesicle then travels into the cytosol and fuses with other vesicles such as endosomes and lysosomes.
  11. 11. phagocytosis
  12. 12. phagocytosisPhagocytosis (literally, cell-eating) is the process by which cells bind and internalize particulate matter larger than around 0.75 µm in diameter, such as small-sized dust particles, cell debris, micro-organisms and even apoptotic cells, which only occurs in specialized cells. These processes involve the uptake of larger membrane areas than clathrin-mediated endocytosis and caveolae pathway. The membrane folds around the object (engulfs), and the object is sealed off into a large vacuole known as a phagosome.
  13. 13. endocytosis
  14. 14. transcytosis LDL (NP)
  15. 15. NP-cell interaction is affected by NP corona
  16. 16. Blood-brain barrierBBB controls the passage of molecules from blood intobrain. The permeability of this physical barrier isrestricted to lipophylic molecules, actively transportedcompounds or small soluble molecules (< 500 Da). ForNP it is not known to what extent they can bedistributed in the brain following systemic or oraladministration.
  18. 18. ScanningElectronMicrographCast of RatThalamusBar =50 m
  19. 19. Ideal properties to reach the brain
  20. 20. Transport across the Blood-Brain-BarrierCell Passive Carrier- Carrier- Receptor- Adsorptive- Opening ofmigration diffusion mediated mediated mediated mediated the tight efflux influx transcytosis transcytosis junctions + + Lipid-soluble amphiphilic Lipid-soluble drugs Glucose Transferrin Histone Polar non-polar Amino acids Insulin Avidin Amines Cationised Monocarboxylates albumin Nucleosides Small peptides
  21. 21. HOW TO DETERMINE THE INTRACELLULAR FATE OF NPs-appropriate markers should be used to avoidmisinterpretations due to artifacts.-it is advisable to conduct studies using several markers inthe same Nps.The entrance in the lysosomal pathway, possiblyfollowed by NP degradation, is the commonestintracellular fate of NPs
  22. 22. Adhesion
  23. 23. Laurdan fluorescence emission wavelengthafter interaction with negatively charged NPs (0-400 isthe NP/lipid ratio)
  24. 24. Adhesion and internalization-direct visualization using electron microscopy-extent of degradation of metabolizable markers BSA +Inuline.g. labeled [125I]-BSA, is hydrolysable in lysosomes and degraded to amino acids. The intact protein (adhesion) is distinguished from hydrolysis products (internalization) by its acid precipitability.Parallel experiments using a non-metabolizable marker (e.g. [125I]-polyvinylpyrrolidone, [3H]- inulin) can give independent estimate of total uptake. Inulin in its free form has an elimination rate equal to the glomerular filtration rate and its radiolabeled form has often been used as a marker for in vivo studies. Any material remaining in the blood after a long period of time must therefore still be in NP form. BSA Aminoacids• Disadvantage: there may be routes of internalization which do not involve lysosomal +TCA or other degradation, Precipitate Precipitat (Adhesion) (internalization)
  25. 25. Electron microscopy Sub-cellular localization lyso/phagosomes 1d 1m lyso/endosomes 3m Nature Nanotechnology, vol 3, 2008
  26. 26. • Fusion
  27. 27. fusion and endocytosis• The classic method of monitoring fusion of NPs with cells is that of fluorescence dequenching of carboxyfluorescein (CF).• CF fluorescence is quenched when concentrated inside NPs.• Adsorbed NPs will not fluoresce• After fusion, CF is diluted into the cell and fluorescence is dequenched (increases)Fusion: CF is released in the cytoplasm after fusion of NPs with the plasmamembrane.:The cell will display a strong diffuse fluorescence with a dark area in the region of the nucleus,. Endocytosis: punctate fluorescence restricted to the secondary lysosomal and endocytic vacuoles
  28. 28. Other indications of the mechanism are:• treatment of cells with metabolic inhibitors, known to inhibit fusion of lysosomes with the phagosome, (cytochalasin B, sodium azide and deoxyglucose, ammonium chloride or chloroquine). These agents interfere with phagocytosis but not with fusion.• use of fluorescent phospholipid analogues, where punctate lysosomal localization can be differentiated visually from diffuse plasma membrane fluorescence. Another complication in this case, however, would be the possibility of adsorption of liposomes, which is difficult to distinguish from fusion. A possible solution in this case would be the use of photobleaching studies, where the mobility of adsorbed lipids is lower than that of lipids incorporated into the membrane by fusion.
  29. 29. lysosomal and cytoplasmic localization• 5-bromo, 4-chloro, 3-indolyl phosphate (BCIP) is a very sensitive indicator of lysosomal delivery . It is a colourless substrate for lysosomal alkaline phosphatase and is converted to the free indole strongly colored precipitate localized within the lysosomes.• Formation of the dye is extremely specific to lysosomes, even after exocytosis or subsequent extrusion of lysosomal contents into the cytoplasm.
  30. 30. intact and degraded NPs• E.M.• AFM• X rays• Double radiolabel technique. The two labels are 22Na and 51Cr/EDTA and the assay is based on the fact that sodium and chromium ions are processed differently by the cell. As long as the NPs remain intact (whether inside or outside a cell) the ratio of the two labels will remain the same. However, if the NPs release their contents inside a cell, then the fates of the two labels will be very different:Sodium ions are rapidly excreted from the cell by Na+/K+ pumps, while 51Cr/EDTA has no suchmethod of exit and remains trapped within the cell. Thus, measurement of the ratio of the twoisotopes retained within the cell will give an indication of the extent to which NPs have beenbroken down. If NPs remain intact inside the cell, the ratio of the isotopes will be identicalInulin in its free form has an elimination rate equal to the glomerular filtration rate and has radiolabeled form has often been used as a marker for in vivo studies. Any material remaining in the blood after a long period of time must therefore still be in NP form.
  31. 31. • Whole body distribution• The tissue distribution of NPs throughout the whole body in experimental systems can clearly be determined by measuring the concentration of markers (preferably radiolabeled) in each of the individual organs. However, this has the disadvantage that only one of a few time points can be obtained and it cannot be applied in clinical situations.• Continuous monitoring of NPs components can be carried out by viewing the distribution of Positron (PET) or γ- emitters by scintigraphy under a γ-camera. Isotopes that are being used for nuclear medicine imaging are technetium [99mTc] and gallium [67Ga].