Intracellular Bacteria and Protozoa - Intracellular Delivery: Fundamentals and Applications - page 781

6.2.2 Targeted Delivery to Erythrocytes

The first studies from the end of 1980s and early 1990s showed that chloroquine
loaded in surface grafted anti-erythrocyte antibodies liposomes markedly increased
its efficacy against both chloroquine-susceptible and chloroquine-resistant P. berghei
infections in mice (Agrawal et al. 1987 ; Chandra et al. 1991 ). Later, a more elabo-
rated approach employed F(ab´) 2 fragments of a mouse monoclonal antibody (MAb
F10), raised against cell membranes isolated from P. berghei -infected mouse eryth-
rocytes were covalently attached to the liposome surface (60 nm, egg PC: chol:
gangliosides, 20:20: 4 M ratio) (Owais et al. 1995 ). Double radiolabelled liposomes
by attaching 125 I-labelled F(ab) 2 fragments to the liposome surface, and entrapping
[ 14 C]inulin in the aqueous compartment were i.v. administered to healthy and
P. berghei -infected BALB/c mice, and its distribution was determined. MAb F10-
liposomes did not significantly bind to the erythrocytes in healthy animals, but it
readily recognized these cells in P. berghei -infected mice. MAb F10-liposomes
containing chloroquine given once at 5.0 mg/kg effectively controlled not only the
chloroquine-susceptible but also the resistant P. berghei infections. On the other
hand upon two doses on days 4 and 6 post-infection, 75-90% of the animals sur-
vived the chloroquine-resistant P. berghei infections and no parasites were detect-
able in their blood even on day 30 post-treatment. Only 40-50% survived when
liposomes were conjugated to an antibody non specifically targeted against eryth-
rocytes, that recognized infected erythrocytes.
These studies were discontinued, owed probably to the structural complexity of
the carrier construct that made difficult it's further scaling up.

6.2.3 Targeted Delivery to Hepatocytes

More recently, two different strategies could successfully target hepatocytes. The first
of them was based on the use of reconstituted artificial chylomicron emulsion made of
commercially available lipids (Dierling and Cui 2005 ). Lipoproteins are endogenous
particles that transport lipids through the blood to various cell types, where they are
recognised and taken up via specific receptors. For example, the remnant receptor and
the asialoglycoprotein receptor (ASGPr), which are uniquely localised on hepatocytes,
recognise chylomicrons and lactosylated high density lipopoteins (HDL), respectively.
Being endogenous, lipoproteins are biodegradable, do not trigger immune reactions,
and are not recognised by the reticuloendothelial system. Primaquine loaded into a
chylomicron emulsion, was significantly retained by the emulsion and was more
stable than free primaquine in the presence of serum. This lipophilized-primaquine
was i.v. administered to mice and showed an enhanced accumulation in liver, as
compared to free primaquine. The antimalarial activity was not determined.
The second strategy employed LUV grafted to sporozoite recognizement
peptides. The liver specificity of this construct is attributed to two major surface
proteins on the sporozoite, circumsporozoite protein (CSP) and thrombospondin-
related anonymous protein (Ménard 2000 ; Mota and Rodriguez 2002 ). The CSP
contains two conserved amino acid sequences (termed regions I and II) that are