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INTRACELLULAR TRAFFIC

Modes of NP-cell interaction:

1-Adhesion

2-Cellular uptake

Adhesion

Cellular uptake

Cellular uptake

• Receptor-mediated• Non-receptor mediated

Chlatrinmediated

Caveolinmediated Chlatrin and

caveolin-indipendent

Receptor-mediated uptake

• Via chlatrin coated pits• Important only for targeted NPs

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.

• 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-shaped 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.

Caveolae-mediated uptake

transcytosis

pinocytosis

• 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.

phagocytosis

phagocytosis

Phagocytosis (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 , nanoparticles 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.

endocytosis

LDL (NP)transcytosis

NP-cell interaction is affected by NP corona

Blood-brain barrier

BBB controls the passage of molecules from blood into brain. The permeability of this physical barrier is restricted to lipophylic molecules, actively transported compounds or small soluble molecules (< 500 Da). For NP it is not known to what extent they can be distributed in the brain following systemic or oral administration.

STRUCTURE OF THE BLOOD-BRAIN-BARRIER

ScanningElectronMicrograph

Cast of RatThalamus

Bar =50mm

Ideal properties to reach the brain

Transport across the Blood-Brain-Barrier

+ +

Passivediffusion

Carrier-mediatedefflux

Carrier-mediatedinflux

Receptor-mediatedtranscytosis

Adsorptive-mediatedtranscytosis

Opening of the tightjunctions

Lipid-solublenon-polar

Lipid-solubleamphiphilicdrugs Glucose

Amino acidsAminesMonocarboxylatesNucleosidesSmall peptides

TransferrinInsulin

HistoneAvidinCationised albumin

Polar

Cellmigration

HOW TO DETERMINE THE INTRACELLULAR FATE OF NPs

-appropriate markers should be used to avoid misinterpretations due to artifacts.-it is advisable to conduct studies using several markers in the same Nps.

The entrance in the lysosomal pathway, possibly followed by NP degradation, is the commonest intracellular fate of NPs

Adhesion

Laurdan fluorescence emission wavelength after interaction with negatively charged NPs (0-400 is the NP/lipid ratio)

Adhesion and internalization -direct visualization using electron microscopy-extent of degradation of metabolizable markerse.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.

• Disadvantage: there may be routes of internalization which do not involve lysosomal or other degradation,

BSA Aminoacids

+TCA

Precipitate(Adhesion) (internalization)

BSA +Inulin

Precipitat

Electron microscopy Sub-cellular localization1d

1m

3m

lyso/phagosomes

lyso/endosomes

Nature Nanotechnology, vol 3, 2008

• Fusion

fusion, adsorption 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

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.

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.

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:

Intact NP in cells. 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 identical

Intact NP in blood : Inulin 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.

• 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].