Imaging Genetic Information - Nanotechnology for Biomedical Imaging and Diagnostics - page 381

13.5.2 Bioreductively releasable strand displacement probe nanoparticle

Another approach to a strand displacement-based detection system was reported that
could detect miR-221, a miRNA (Fig. 13.10) [142]. In this system, the antisense
strand displacement probe was designed to be bioreductively released from the NP
and then transfer the fluorescent antisense strand to the target mRNA. The NP con-
sisted of a silica-coated cobalt ferrite core with a carboxylated surface that was linked
via a disulfide bond to a quenched strand displacement turn-on probe and also to a
pretargeting aptamer ligand. The strand displacement probe consisted of an antisense
strand linked to cy 5.0 fluorophore that was hybridized to a shorter sense strand
linked to a quencher, BHQ2 on the 5′-end. The DNA aptamer AS 1411 that targets
nucleolin was used for pretargeting and to promote cellular internalization, presum-
ably by endocytosis. There is some uncertainty about exactly how AS 1411 facilitates
internalization and the role of nucleolin in the process [143, 144]. Nonetheless, miR-
221-specific fluorescence enhancement was observed in cell culture and in mouse
xenografts. For this NP to have worked, some feature must have facilitated the escape
of the NP and/or the strand displacement probe from the endosomal pathway by
which it is presumed to have entered. There were no specific auxiliaries attached to
the NP for this purpose, though it is possible that the remaining surface carboxylates
were functioning as a pH sponge. Because miRNAs are ultimately sequestered in a
RISc complex and presumably inaccessible, the strand displacement probe must
have been able to intercept the miRNA somewhere along the maturation pathway, or
while in the RISc complex, but this remains to be elucidated. It is remarkable that
this NP system was able to work in vivo in a mouse since there was no other func-
tionality to impart stealth properties to the NP and prevent its uptake by the mononu-
clear phagocyte system (mPS). Apparently, some fraction of the NP must have been
able to accumulate in the xenograft, presumably by the enhanced permeability and
retention (ePR) effect.

SS

Targeting
aptamer

miRNA

HS

SH

fIgure 13.10 Targeted miRNA-sensing NP. This nanoparticle antisense imaging system
makes use of an aptamer to pretarget an miRNA-sensing quenched strand displacement probe
to a nucleolin-bearing cell, which then internalizes the particle into the cytoplasm. Because of
the reducing environment, the disulfide bond is cleaved freeing the probe to bind to the miRNA
target.