Molecularly imprinted nanoparticles (MINPs) are nanoparticles formed in the presence of a molecular template and the subsequent removal of the template allows the MINPs to exhibit a selective memory toward that template. MINPs with selective affinity for a specific protein biomarker could be used as an environmentally robust and cost effective biomaterial for different biomedical applications including biosensing, targeted drug delivery and tissue engineering and regenerative medicine.

The aim of this work was to develop biocompatible, biodegradable and stable MINPs by covalently cross-linking two natural polymers, chitosan (CHI) and chondroitin sulfate (CS), and using lysozyme (LYZ) as model template. Recently, different polymers (e.g. alginate) have been modified with tetrazine and norbornene functional groups to allow the formation of covalently crosslinked hydrogels using the bioorthogonal inverse electron demand Diels-Alder click reaction.[1,2] This rapid and specific click reaction is irreversible and hydrogels have been formed capable of encapsulating cells without damaging them. Thus, in this work we have conjugated CHI with norbornene (CHI-N) and CS with tetrazine (CS-T) using carbodiimide chemistry. The degree of substitution of norbornene and tetrazine groups was determined by NMR analysis. CHI-N and CS-T solutions were mixed together to allow the Diels-Alder click reaction to occur and to form the covalently cross-linked nanoparticles. The size and morphology of the formed nanoparticles, with and without protein, were characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM). The LYZ incorporation efficiency was assessed by a total protein quantification method. The LYZ template was then extracted from the nanoparticles by alternating washes in water and 10% acetic acid, thus rendering MINPs. These were also characterized by DLS and SEM and the remaining LYZ entrapped was also quantified. 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. Additionally, depending on the outcomes of current studies, the imprinting of epitopes from large molecular weight proteins will also be considered as template alternatives.

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. Desai, et al. Biomaterials 50, 30, 2015

2. Koshy, et al. Advanced Healthcare Materials 5, 541, 2016.