Molecularly imprinted polymers (MIPs) are polymers formed in the presence of a target molecule called template and the subsequent removal of the template allows the MIPs to rebind selectively to the template. They have the potential to be low-cost and robust alternatives to natural recognitive biomolecules such as antibodies and receptors.

Current imprinting strategies are mostly based on synthetic polymers produced with different functional monomers and cross-linking agents. However, these strategies might find limitations in biomedical applications that requires non-toxic and biocompatible materials.

In this work, we have produced molecularly imprinted nanoparticles (MINPs) using natural origin polymers namely, chitosan (CHI) and chondroitin sulfate (CS), as imprinting matrix and lysozyme (LYZ) as template. CHI and CS were chosen based on the diversity of specific chemical interactions that can be established between the functional groups of the polymers (amino/acetoamido groups in CHI and carboxylic and sulfate groups in CS, besides the hydroxyl groups of the polysaccharides backbone) and the template, but also due to the natural affinity existing between LYZ and CS in mammalian organisms [1].

MINPs were produced by polyelectrolyte complexation (PEC) in the presence of LYZ. To stabilize the molecularly imprinted PEC, CHI was conjugated with norbornene and CS with tetrazine allowing the occurrence of a bioorthogonal inverse electron demand Diels-Alder click reaction and the formation of covalently cross-linked MINPs.

The produced MINPs (379±27 nm) were characterized by different physico-chemical techniques. MINPs have shown increased LYZ adsorption capacity compared to the non-imprinted nanoparticles (NIPs), resulting in an imprinting factor (IF) of about 2.5. MINPs selectivity was challenged against other proteins covering a wide range of molecular weights (bovine serum albumin, 66.5 KDa, and insulin, 5.8 KDa). Remarkably, in non-competitive assays, both NIPs and MINPs have shown similar protein binding capacity (IF ≈ 1), irrespective of their molecular weight. The selectivity of the MINPs in competitive assays will be evaluated to further validate the imprinting concept here proposed.

Acknowledgments: Miranda and Domingues thank Fundação para a Ciência e a Tecnologia (FCT) the grants (SFRH/BPD/110868/2015) and (SFRH/BPD/112459/2015). Authors thank Recognize project (UTAP-ICDT/CTM-BIO/0023/2014) and project NORTE-01-0145-FEDER-000021 supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).

[1] Moss et al Arch Biochem Biophys 348 (1997) 49-55