Molecularly imprinted polymers (MIPs) are polymeric networks formed in the presence of a molecular template and the subsequent removal of the template allows the material to exhibit a selective memory toward the imprinted template. Thus, MIPs can be compared to antibodies due to their molecular recognition specificity, but with comparative advantages such as lower cost and greater environmental stability.

Although successfully applied to recognize small molecules, MIPs have faced numerous challenges in recognizing macromolecules such as proteins. Besides the inherent diffusional limitation of macromolecules within polymeric networks, current imprinting strategies are mostly based on synthetic polymers produced with different functional monomers and cross-linking agents. However, these technologies limit their potential range of applications in the biomedical field, such as in sensing, drug delivery and tissue engineering, which require non-toxic and biocompatible products.

Thus, in this work we aim to test the hypothesis of producing biocompatible and biodegradable molecularly imprinted nanoparticles (MINPs) based on the polyelectrolyte complexation (PEC) of natural polymers and using lysozyme (LYZ) as model template. For this purpose, nanoparticles were produced by PEC between positively charged chitosan (CHI) and negatively charged chondroitin sulfate (CS). The size and morphology of the nanoparticles, with and without protein, were characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM). The LYZ incorporation efficiency was assessed by total protein quantification methods.

The protein entrapped within PEC network was then removed by several washing steps thus rendering LYZ imprinted nanoparticles. The impact of molecular imprinting in terms of LYZ adsorption capacity and selectivity will be further investigated in non-competitive and competitive assays using proteins of different molecular weights and isoelectric points such as LYZ, cytochrome c, trypsin inhibitor and BSA. Additionally, depending on the outcomes of current studies, the imprinting of epitopes from large molecular weight proteins will also be considered as template alternatives.

Considering that PEC formation is a reversible process, it may be required to stabilize the MINPs by cross-linking their polyelectrolyte components. Thus, in a second strategy, CHI will be conjugated with norbornene and CS with tetrazine to form covalently cross-linked MINPs using the bioorthogonal inverse electron demand Diels-Alder click reaction.

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).