INTERACTION WITH CELLS

AND

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 amorphologically characteristic crystalline coat made upof a complex of proteins that mainly associate with thecytosolic protein clathrin. Clathrin-coated vesicles(CCVs) are found in virtually all cells and form fromdomains of the plasma membrane termed clathrin-coated pits. Coated pits can concentrate a large rangeof extracellular molecules that are different receptorsresponsible for the receptor-mediated endocytosis ofligands, e.g. low density lipoprotein, transferrin,growth factors, antibodies and many others.

Caveolae are the most common reported non-clathrincoated plasma membrane buds, which exist on the surface of many, but not all cell types. They are enrichedof 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 namecaveolae). They can constitute approximately a third ofthe plasma membrane area of the cells of some tissues, being especially abundant in smooth muscle, type I pneumocytes, fibroblasts, adipocytes, and endothelialcells. Uptake of extracellular molecules is also believed tobe specifically mediated via receptors in caveolae.

Caveolae-mediated uptake

transcytosis

pinocytosis

• Pinocytosis (literally, cell-drinking). This process isconcerned with the uptake of solutes and single moleculessuch as proteins.

• Macropinocytosis, which usually occurs from highly ruffledregions 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) filledwith large volume of extracellular fluid and moleculeswithin it. The filling of the pocket occurs in a non-specificmanner. The vesicle then travels into the cytosol and fuseswith other vesicles such as endosomes and lysosomes.

phagocytosis

phagocytosis

Phagocytosis (literally, cell-eating) is the process by whichcells bind and internalize particulate matter larger thanaround 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 processesinvolve the uptake of larger membrane areas thanclathrin-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 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.

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

HistoneAvidinCationisedalbumin

Polar

Cellmigration

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 in the same Nps.

The entrance in the lysosomal pathway, possiblyfollowed by NP degradation, is the commonestintracellular fate of NPs

Adhesion

Laurdan fluorescence is sensitive to bilayer fluidity.

emission wavelength after interaction with negativelycharged NPs (0-400 is the NP/lipid ratio)

Adhesion and internalization

-direct visualization using electron microscopy

-extent of degradation of metabolizablemarkers

e.g. labeled [125I]-BSA, is hydrolysable in lysosomes and degraded to amino acids. The intact protein (adhesion) isdistinguished from hydrolysis products(internalization) by its acid precipitability.

Parallel experiments using a non-metabolizable marker (e.g. [125I]-polyvinylpyrrolidone, [3H]-inulin) can giveindependent estimate of total uptake.

• 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

Possible for liposomes

fusion, adsorption and endocytosis

• The classic method of monitoring fusion ofNPs with cells is that of fluorescencedequenching of carboxyfluorescein (CF).

• CF fluorescence is quenched whenconcentrated 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 afterfusion of NPs with the plasmamembrane.:The cell will display a strong diffuse fluorescencewith a dark area in the region of the nucleus,.

Endocytosis: punctatefluorescence restricted tothe secondary lysosomaland 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). Theseagents interfere with phagocytosis but not with fusion.

• use of fluorescent phospholipid analogues, where punctatelysosomal localization can be differentiated visually from diffuse plasma membrane fluorescence. Another complication in this case, however, would be the possibility of adsorption of liposomes, whichis difficult to distinguish from fusion. A possible solution in this case would be the use of photobleaching studies, where the mobility ofadsorbed 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 oflysosomal delivery . It is a colourlesssubstrate for lysosomal alkalinephosphatase and is converted to the free indole strongly colored precipitate localized within the lysosomes.

• Formation of the dye is extremelyspecific to lysosomes, even afterexocytosis or subsequent extrusion oflysosomal contents into the cytoplasm.

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 (whetherinside 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 trappedwithin the cell. Thus, measurement of the ratio of the twoisotopes retainedwithin the cell will give an indication of the extent to which NPs havebeenbroken 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 equalto the glomerular filtration rate and its radiolabeled form has often beenused as a marker for in vivo studies. Any material remaining in the bloodafter a long period of time must therefore still be in NP form.

CONFOCAL MICROSCOPY WITH DUALLY_LABELED NP

• 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 (preferablyradiolabeled) in each of the individual organs. However, thishas 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 thatare being used for nuclear medicine imaging are technetium [99mTc] and gallium [67Ga].