Antibody-Functionalized Inorganic NPs: Mimicking Nature for Targeted Diagnosis and Therapy - Bioinspired Approaches for Human-Centric Technologies - page 13

acids included into the antibody structure, thus allowing the attachment of the
antibody to the nanoparticle on the Fc region and thus having the right orientation.
On the Fc region of the antibody, also the carbohydrate chains placed on the C2H
chains can be considered as possible anchoring points for the reaction with amino-
or hydrazine-functionalized nanoparticles. Indeed, upon a mild oxidation of the
sugar chains, the aldehyde-activated carbohydrates can be coupled to the amino-
bearing nanoparticles via condensation reaction (Puertas et al. 2010 ) and a further
reduction step. This method however requires a modification of the antibody, but at
least the coupling chemistry takes place farther away from the antigen binding site.
Among the different sites on the antibody which have been exploited for the
coupling to nanoparticles, also the thiol (-SH) chemistry of the cysteine residues of
the antibody protein chains can be considered suitable anchoring points. The thiol
groups in a protein contribute to the ternary structure via the sulfur-sulfur (S-S)
bridge formation of each of the protein subunits, and they join also the light and the
heavy chains and the antibody fragments at the hinge region. The latter can be
selectively cleaved to thiol groups by reducing agents such as mercaptoetha-
nolamine or dithiothreitol and thus can be coupled to nanoparticles which carry
functional groups reactive toward SH moieties (via, for instance, maleimide chem-
istry to cite one example). In this case however, a pre-modification of the protein is
required. This type of chemistry has been shown to be suitable not only with whole
antibodies but also with antibody fragments, as, for instance, the Fc or the scFc
fragments (see Fig. 1.1 ). In one interesting study, the comparison of tumor targeting
when using anti-HER2 either whole or half-chain antibody or scFv fragment
targeted iron oxide nanocrystals suggested that the longer period of accumulation
of the scFv-functionalized iron oxide nanocrystals makes them ideal for breast
cancer. A conclusion of the work was that the advantage of using a fragment with
respect to the whole antibody resides mainly in the reduced size of the final scFv-
iron oxide conjugates: the smaller size and thus the better stability in physiological
conditions are likely the reasons why a higher tumor targeting in vivo could be
achieved (Fiandra et al. 2013 ).

The Use of an Adapter Complex (Fig. 1.2c ) Alternatively, the antibody can be
attached to the nanoparticle via spacer (bio)molecules. The most common approach
is based on the use of streptavidin-biotin or avidin-biotin couple, as the binding
affinities of the biotin-(strept)avidin are among the highest values known for pro-
teins ( K

10 15 M). In this case, either the biotin or the streptavidin
needs to be attached to the nanoparticles, and the biotin derivatization of the
antibody is also required. The streptavidin can work as the bridging molecule
placed in between the biotinylated nanoparticles and the biotinylated antibody
(streptavidin has four binding sites for protein), or it can be directly attached to
the nanoparticles and thus directly link the antibody previously modified with
biotin. In both cases, a modification of the antibody with biotin is required, either
to the SH chemistry of the hinge region or via the carbohydrate moieties of the Fc
region (Park et al. 2011 ; Cho et al. 2007 ).

10 14

¼