This document discusses various modes of interaction between nanoparticles (NPs) and cells, including adhesion and cellular uptake via receptor-mediated or non-receptor mediated endocytosis. It describes specific endocytosis pathways like clathrin-mediated endocytosis, caveolae-mediated uptake, macropinocytosis, phagocytosis, and pinocytosis. Methods to determine the intracellular fate and trafficking of NPs are also outlined, such as using appropriate markers, electron microscopy, and fluorescence techniques. The challenges of transporting NPs across biological barriers like the blood-brain barrier are also addressed.

1. INTERACTION WITH CELLS
AND
INTRACELLULAR TRAFFIC

2. Modes of NP-cell interaction:
1-Adhesion
2-Cellular uptake

3. Adhesion

4. Cellular uptake

5. Cellular uptake
• Receptor-mediated
• Non-receptor mediated
Chlatrin
mediated
Caveolin
mediated
Chlatrin and
caveolin-indipendent

6. Receptor-mediated uptake
• Via chlatrin coated pits
• Important only for targeted NPs

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.

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 are enriched
of the cholesterol-binding protein caveolin (Vip21),
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.

12. transcytosis

13. pinocytosis

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.

16. phagocytosis

17. phagocytosis
Phagocytosis (literally, cell-eating) is the process by which
cells bind and internalize particulate matter larger than
around 0.75 (750nm) μm in diameter, such as small-sized
dust particles, cell debris, micro-organisms ,
aggregates of 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.

19. endocytosis

21. LDL (NP)
transcytosis

22. NP-cell interaction is affected by NP corona

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.

26. STRUCTURE OF THE
BLOOD-BRAIN-BARRIER

27. Scanning
Electron
Micrograph
Cast of Rat
Thalamus
Bar =50mm

29. Ideal properties to reach the brain

30. Transport across the Blood-Brain-Barrier
+ +
Passive
diffusion
Carrier-mediated
efflux
Carrier-mediated
influx
Receptor-mediated
transcytosis
Adsorptive-mediated
transcytosis
Opening of
the tight
junctions
Lipid-soluble
non-polar
Lipid-soluble
amphiphilic
drugs Glucose
Transferrin
Insulin
Amino acids
Amines
Monocarboxylates
Nucleosides
Small peptides
Histone
Avidin
Cationised
albumin
Polar
Cell
migration

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

33. Adhesion

35. Laurdan fluorescence is sensitive to bilayer fluidity.
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.
• Disadvantage: there may be routes of
internalization which do not involve
lysosomal or other degradation,
BSA +Inulin
BSA Aminoacids
+TCA
Precipitat
Precipitate
(Adhesion) (internalization)

37. Electron microscopy Sub-cellular localization
1d
1m
3m
lyso/phagosomes
lyso/endosomes
Nature Nanotechnology, vol 3, 2008

38. • Fusion
Possible for liposomes

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, Confocal microscopy
• 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 : [3H]-inulinin 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.

43. CONFOCAL MICROSCOPY WITH DUALLY_LABELED NP

44. • 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].