Biomedical Engineering Reference
In-Depth Information
Another essential component when designing MRSws is the attachment of
an appropriate targeting group, so as to endow proper binding selectivity and
sensitivity upon the nanoparticles. Because the iron oxide nanocrystal is entrapped
within an aminated polysaccharide coating, many common chemical crosslinking
strategies can be employed. However, a specifi c bioconjugation method has
been shown to greatly infl uence the performance of targeted SPIO nanosensors
by altering the number and nature of the targeting groups per nanoparticle
[81]. Several different types of bioconjugation strategies have been used to
activate the nanoparticles. For example, a team led by E. Y. Sun demonstrated
the use a CLIO variant termed a magnetofl uorescent nanoparticle, which consisted
of two fl uorescein isothiocyanate (FITC) molecules attached to amino-CLIO
nanoparticles, to demonstrate the rapid development of nanoparticle libraries.
For these particles, the
R
1
was 21 m
M
− 1
s
− 1
and
R
2
62 m
M
− 1
s
− 1
, while the FITC
absorbed at 494 nm with an extinction coeffi cient of 73 m
M
− 1
c m
− 1
[82] . Small
molecules were attached via a variety of reactive handles to the
62 free
amines per nanoparticle. The small-molecule reactive handles included anhydride,
amino, hydroxyl, carboxyl, thiol, and epoxy. Each of the resulting conjugates
had unique functionality in terms of MRSw biosensor response and macrophage
uptake, demonstrating that the specifi city and selectivity of a nanoparticle
conjugate is determined by the surface functionality [82]. Other crosslinkers
that have been to date used include Pierce Biotech (Rockford, IL, USA) heterobi-
functional crosslinking agents such as
N
- succinimidyl 3 - (2 - pyridyldithio) - propio-
nate (SPDP) and
N
- succinimidyl -
S
- acetylthioacetate ( SATA ), generic activating
and crosslinking agents such as succinimidyl iodoacetate, 1 - ethyl - 3 - [3 - dimethyl-
aminopropyl]carbodiimide hydrochloride (EDC or EDAC),
N
- hydroxysulfosuccin-
imide ( Sulfo - NHS ), and antibody - specifi c coupling reagents such as protein G.
In another series of studies, a team led by E.Y. Sun demonstrated the use of
azide-alkyne reactions (known as “click chemistry”) for the attachment of targeting
groups. Sun's group demonstrated that stable alkyne- or azido-functionalized
CLIO nanoparticles could be generated for click chemistry attachment to a
variety of appropriately functionalized small molecules [83]. Unfortunately,
the details of bioconjugation methods are beyond the scope of this chapter; thus,
the reader is referred to the original data (as cited) and to more comprehensive
sources [84] .
Regardless of the specifi c coupling method used, a number of critical issues
must be considered in particle design. These include the activity and number of
targeting groups attached on each nanoparticle - an issue was explored by a team
led by D. Hogeman at MGH. In these studies, it was shown that nonselective
oxidative coupling of the protein transferrin led to an inferior biosensor perfor-
mance when compared to that coupled with the heterobifunctional linker, SPDP
[81]. The SPDP linker led to a fourfold increase in the number of transferrin
molecules per nanoparticle, and also preserved the activity of the transferrin
protein, leading to an increased binding affi nity for their cellular target. The
reduced affi nity of transferrin when coupled to nanoparticles via oxidative cou-
pling most likely arose from the nonselective nature of the coupling, which may
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