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 clathrincoated 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.
9.
10. 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-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.
14. • 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.
17. 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.
25. 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.
30. Transport across the Blood-Brain-Barrier
Cell
migration
Passive CarrierCarrierdiffusion mediated mediated
efflux
influx
Receptor- Adsorptive- Opening of
mediated mediated
the tight
transcytosis transcytosis junctions
+ +
Lipid-soluble
amphiphilic
Lipid-soluble drugs
Glucose
Transferrin Histone
non-polar
Amino acids
Insulin
Avidin
Amines
Cationised
Monocarboxylates
albumin
Nucleosides
Small peptides
Polar
31.
32. 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
35. Laurdan fluorescence emission wavelength
after interaction with negatively charged NPs (0-400 is
the NP/lipid ratio)
36. Adhesion and internalization
-direct visualization using electron microscopy
-extent of degradation of metabolizable markers
e.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 +Inulin
BSA
Aminoacids
+TCA
Precipitate
(Adhesion)
Precipitat
(internalization)
39. 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
40. 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.
41. 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.
42. 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.
43. • 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 γemitters by scintigraphy under a γ-camera. Isotopes that
are being used for nuclear medicine imaging are
technetium [99mTc] and gallium [67Ga].