Biomedical Engineering Reference
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some membrane-bound receptors can attach. Binding of ligand to these receptors
triggers assembly of further clathrin molecules to form a vesicle around the particle
which is pinched off to become completely internalized. The clathrin coat is lost
and the vacuole becomes an early endosome. As in phagocytosis, acidification,
receptor recycling and fusion with endosomes occur. Another vesicular endocytotic
pathway has been described more recently: the caveolae pathway. Caveolae are
flask-shaped invaginations in the membrane coated with the protein caveolin. The
membrane composition is different from the bulk composition, rich in cholesterol
and resembling that of lipid rafts; some receptors are particularly associated with
these areas, particularly those which have a GPI anchor (Anderson 1998 ). The
vesicles formed after pinching off of the caveolae by the protein dynamin are not
acidified and do not fuse with lysosomes. Finally, macropinocytosis involves the
actin-driven formation of membrane ruffles which collapse and fuse with the
plasma membrane to enclose a vesicle, internalizing a droplet of the extracellular
medium without any specific receptor. Unlike clathrin- and caveolin-coated vesi-
cles which are about 200 nm in diameter, vesicles formed by macropinocytosis can
be as large as 5 mm (Swanson and Watts 1995 ), and thus presents a mechanism of
uptake for larger-sized particles.
The endocytic pathway taken by drug delivery systems is usually determined by
the use of specific inhibitors, such as cytochalasin B for clathrin-mediated endocy-
tosis, filipin for caveolae-mediated endocytosis and amiloride for macropinocyto-
sis. Cholesterol depletion is also used to detect caveolae-mediated processes. Thus,
Rejman et al. ( 2004 ) were able to reveal the influence of size on the mechanism of
uptake of fluorescent polystyrene nanoparticles by non phagocytic B16 cells. While
particles smaller than 200 nm were internalized by clathrin-coated pits, larger par-
ticles from 200 to 500 nm in diameter were preferentially taken up by caveolae.
A variant of the endocytic pathway is transcytosis. In this pathway, vesicles
formed by endocytosis do not fuse with lysosomes but cross the cell, fuse with the
plasma membrane in another region of the cell and release their contents into the
extracellular medium. In particular, receptor-mediated transcytosis is a mechanism
of carrying macromolecules across endothelial cells. For example, transcytosis of
insulin, IgG, LDL and iron bound to the transport protein transferrin cross the
endothelial cells of the blood-brain barrier by transcytosis. This pathway can be
exploited for drug delivery to the brain. The group of Pardridge has reported results
using monoclonal antibodies targeting either the transferrin receptor or the insulin
receptor conjugated to long-circulating liposomes (Pardridge 2010a ). Jallouli et al.
( 2007 ) observed that neutral or cationic polysaccharide nanoparticles of 60 nm in
diameter without any surface modification underwent transcytosis by the caveolae
pathway across a model of the blood-brain barrier. PLGA nanoparticles coated with
transferrin followed the same route in this model (Chang et al. 2009 ). On the other
hand, PEGylated poly (alkylcyanoacrylate)-based nanoparticles were taken up by
rat brain endothelial cells by a clathrin-dependent pathway (Kim et al. 2007 ). This
uptake was found to be via LDL receptors, since Apolipoprotein E is adsorbed
onto PEGylated nanoparticles. These results illustrate the complexity of uptake
mechanisms for drug delivery systems.
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