Epitope-imprinted polymers: Design principles of synthetic binding partners for natural biomacromolecules

Abstract

Molecular imprinting (MI) has been explored as an increasingly viable tool for molecular recognition in various fields. However, imprinting of biologically relevant molecules like proteins is severely hampered by several problems. Inspired by natural antibodies, the use of epitopes as imprinting templates has been explored to circumvent those limitations, offering lower costs and greater versatility. Here, we review the latest innovations in this technology, as well as different applications where MI polymers (MIPs) have been used to target biomolecules of interest. We discuss the several steps in MI, from the choice of epitope and functional monomers to the different production methods and possible applications. We also critically explore how MIP performance can be assessed by various parameters. Last, we present perspectives on future breakthroughs and advances, offering insights into how MI techniques can be expanded to new fields such as tissue engineering.

Fig. 1.. Rationale of epitope imprinting concept.

Fig. 2.. Major steps in the process of epitope imprinting.

Each one presents an array of options that must be carefully considered to optimize MIP efficacy considering the target application.

Fig. 3.. Parameters commonly used to assess MIP performance.

Fig. 4.. Epitope selection strategies.

Peptide epitopes (blue box) are by far the most commonly used type of templates in the field, although emerging strategies using saccharides or small molecules (yellow box) have also been shown to be potential alternatives. Complementary strategies have been increasingly applied for rational peptide selection (red box). Whether they can be applied to aid the selection of other types of epitope remains to be demonstrated. a.a., amino acid.

Fig. 5.. Construction of conformational epitopes using apamin as molecular scaffold.

Adapted with permission (48, 71). Copyright 2015 (A) and 2020 (B), Wiley.

Fig. 6.. Bulk molecular imprinting method and representative applications.

(A) Bulk imprinting procedure; bulk MIPs can further be processed into microparticles/nanoparticles by grinding and sieving (dashed arrow). (B) Scanning electron microscopy micrographs of imprinted (MIP C) and nonimprinted (NIP C) poly(2-hydroxyethyl methacrylate-co-N-methacryloyl-l-aspartic acid) cryogels for IgG purification. Reproduced with permission (74). Copyright 2015, Elsevier. (C) Atomic force microscopy (AFM) images of MIP-coated (above) and bare (below) gold chips for human albumin detection. Adapted with permission (111). Copyright 2016, Elsevier.

Fig. 7.. Surface imprinting on thin flat films.

(A) Surface MI procedure, allowing the creation of surface-accessible binding sites for the target molecule. (B) Generation of an epitope-imprinted biointerface for dynamic cell adhesion and harvesting. Adapted with permission (69). Copyright 2017, the authors.

Fig. 8.. Surface imprinting on NP thin coatings.

(A) MI procedure with immobilized templates on the surface of an NP. (B to E) Transmission electron microscopy images of (B) magnetic carbon nanotubes (MCNTs), (C) silica-coated MCNTs, (D) MCNTs coated with methacryloxypropyl trimethoxysilane, and (E) MIP-coated MCNTs. Adapted with permission (221). Copyright 2018, Wiley.

Fig. 9.. Inverse microemulsion MI and representative applications.

(A) Scheme depicting the process of surface imprinting of polymeric NPs by inverse microemulsion polymerization. (B) MINPs against cancer biomarker p32, carrying the chemotherapeutic drug methylene blue, tendentially accumulated in tumoral tissues and drastically reduced tumor growth in mice. Adapted with permission (71). Copyright 2015, Wiley. (C) Tissue culture plastic coverslips coated with MINPs (but not NINPs) can sequester and retain target transforming growth factor–β3 (TGF-β3). Left: AFM images of coated coverslips. Right: fluorescent immunolabeling of TGF-β3. Scale bars, 250 nm (left), 100 mm (right). (D) ASC cell pellets cultured with MINPs (left) grew to larger sizes and produced more collagenous matrix than pellets cultured with NINPs (right). Adapted with permission (48). Copyright 2020, Wiley. Scale bars, 150 mm (lower magnification images), 50 mm (insets).

Fig. 10.. Solid phase imprinting method.

(A) Solid-phase epitope imprinting procedure. The affinity purification steps allow removal of low-affinity particles and unreacted monomers by eluting at the same temperature as polymerization (blue arrow), followed by recovery of high-affinity MINPs at a different temperature (red arrow). (B) Confocal microscopy images of cancer cell line cultures with low (left) and high (right) expression of epidermal growth factor receptor (EGFR). MINPs against EGFR (green) accumulate significantly only in cells with high expression. Adapted with permission (77). Copyright 2018, American Chemical Society. (C) MINPs against β2 microglobulin, a marker of senescent cells, preferentially accumulate in older mice. Adapted with permission (169). Copyright 2019, The Royal Society of Chemistry. DAPI, 4′,6-diamidino-2-phenylindole.