Plastic Antibodies - Designing Receptors for the Next Generation of Biosensors - page 121

imprinted with thylakoid D1 protein [ 124 ]. For this purpose MIP and non-imprinted
(NIP) bulk monoliths were prepared in water and extensively ground to produce
MIP NPs. Affinity chromatography on immobilized D1 protein showed that
5-10 kDa MIP NPs had increased binding affinity to this protein as compared to
non-imprinted polymers. In addition, MIP nanoparticles were able to increase the
photosynthetic activity of chloroplasts. However, the yield of the product
synthesized with this method was very low (about 0.2 mg per fraction). Neverthe-
less, this first work represents a milestone in the production of soluble MIP NPs
with biological activity. Haupt and coauthors synthesized water-soluble MIP NPs
able to inhibit the enzymatic activity of trypsin using a precipitation polymerization
approach [ 125 ]. Their method relied on tailor-made “anchoring monomer,”
methacryloylaminobenzamidine (a polymerizable derivative of the trypsin inhibitor
benzamidine), to complex the template with high affinity and locate the synthesis of
the MIP NPs at the surface of the enzyme. The calculated inhibition constant ( K i )
for the MIP NPs was 79 nM which is much lower than the K i value for free
benzamidine (18.9 M), which proved the effectiveness of this imprinting strategy.

Moreover, MIP NPs demonstrated negligible inhibition activity for enzymes such
as chymotrypsin or kallikrein which demonstrated the selectivity of the MIP NPs.
Shea and coauthors recently prepared MIP NPs by precipitation polymerization
imprinted against the bee venom peptide melittin [ 39 ], and applied them as
antidotes in living animals [ 40 ]. The authors optimized the composition of the
polymerization mixture by the creation of a small combinatorial library of acryl-
amide functional monomers including N -isopropylacrylamide and N , N' -methylene-
bisacrylamide (cross-linker). Nanoparticles synthesized under the optimum
conditions had a diameter of 50 nm, which is comparable in size to IgM. The
dissociation constant (25 pM) was similar to that of natural antibodies for melittin
(17 pM). Moreover, only slight cross-reactivity with other proteins was observed.
MIP NPs were tested in vitro on fibrosarcoma cells and did not show any toxic
effect. The authors injected mice with a lethal dose of melittin, followed 20 s later
by an intravenous injection of MIP or non-imprinted NPs. MIP NPs halved the
mortality and reduced the toxic effects of melittin. MIP NPs with adsorbed melittin
were concentrated in the liver while melittin alone was distributed extensively
throughout the body and bloodstream. Although these results are very promising,
the toxicological implications of injecting MIP NPs need to be carefully
investigated to assess the risks deriving from the use of these nanomaterials prior
to their application in humans. In addition, suitable manufacturing protocols should
be developed for large-scale manufacturing of MIP NPs, which would most likely
rely on affinity separation, as in the case of natural antibodies [ 126 , 127 ].

m

4 Conclusions and Outlook

In recent years we have witnessed a growth of activity in the development of
alternative affinity materials to antibodies such as aptamers, engineered scaffold
proteins, and MIP NPs. Recent developments in the synthesis and applications of