Delivery to Intracellular Targets by Nanosized Particles - Intracellular Delivery: Fundamentals and Applications - page 77

hydrophilic polymers at the surface of the particle can reduce protein absorption
and in this way hinder binding to the cell surface. This is a useful property when
the aim is to reduce clearance by phagocytic cells, but will also reduce binding to
target cells. Specific binding to the target can be achieved by the attachment of a
ligand to the particle surface. Many different types of molecule have been proposed
as targeting ligands for liposomes and nanoparticles. Antibodies are obvious candi-
dates because of their extreme specificity; however saccharides and other small
molecules may also be employed. The efficiency of ligand-mediated binding will
depend on the orientation of the ligand on the surface. For example, when lipo-
somes are covered with PEG, the ligand should be attached to the distal end of the
PEG chain, to allow unhindered interaction with the receptor (Mercadal et al.
1999 ). It should also be noted that the binding of a ligand to a receptor will not
necessarily lead to internalization; this depends on the nature of the receptor and
also on the size of the particle carrying the ligand (Allen and Moase 1996 ).
When the particle is a liposome, there are several theoretical possibilities after
binding to the cell surface: internalization of the intact particle by endocytotic pro-
cesses; fusion of the liposome membrane with the cell membrane, leading to deliv-
ery of its contents directly to the cytoplasm; exchange of lipids between the
liposome membrane and the plasma membrane. A very early study by Pagano and
Huang ( 1975 ) using Chinese hamster V79 cells and liposomes prepared from diol-
eylphosphatidylcholine and cholesterol gave evidence for the last two mechanisms.
However, subsequent studies have shown that liposome-cell fusion is quite rare
unless specific fusogenic peptides are included (see for example Fattal et al. 1994 ;
Pecheur et al. 1997 ). Koning et al. ( 2002 ) exploited lipid exchange between lipo-
somes immobilized at the surface of colon carcinoma cells by means of a specific
monoclonal antibody to deliver a lipophilic prodrug of 5-fluorodeoxyuridine.
In the case of nanoparticles, fusion with the cell membrane is not possible.
Transfer of lipophilic cargo to the cell can occur after binding to the cell surface,
as shown by Mosqueira et al. ( 2001 ) with a fluorescent marker within the oily
phase of non PEGylated nanocapsules. However, most interactions take place by
endocytotic mechanisms, including phagocytosis, clathrin- or caveolin-mediated
endocytosis and macropinocytosis.
Phagocytosis (“cell-eating”) is restricted to specialized cells of the immune sys-
tem: monocytes and neutrophils in the blood and macrophages and dendritic cells
in the tissues. The physiological role of phagocytosis is to clear foreign bodies and
to present antigens from them to other actors in the immune response. The size of
particles that can be engulfed by macrophages reaches several microns; senescent
red blood cells, for example. Binding to the phagocyte surface is greatly increased
by opsonization with plasma proteins, among which immunoglobulins, fibronectin
and complement component C3b play important roles. The phagocyte membrane
carries receptors for these proteins, as well as receptors for saccharides (mannose/
fucose and galactose), apolipoproteins and the less specific scavenger receptors.
Pseudopodia grow out around the particle and a “zip fastener” effect of binding to
a number of receptors on the same particle encloses it within a vacuole known as a
phagosome. This vacuole is subsequently acidified and fuses with lysosomes