Biofunctionalized nanomaterials have been extensively studied as a tool for a wide range of applications in biomedical fields. Despite many existing strategies to conjugate proteins to colloidal particles, determining the grafting efficiency that is, the amount of protein conjugated to the surface of a nanoparticle (NP)remains challenging. Formulations for biomedical applications are subjected to strict constraints, and a lack of precise characterization can prevent otherwise promising formulations to be explored further. Here, we propose a simple approach to precisely measure the grafting efficiency of biological molecules on the surface of three types of widely used NPs: polymeric NPs, inorganic NPs, and metallic NPs. This approach relies on the simultaneous hydrolysis of the grafted protein and the NP degradation in acidic conditions, followed by a spectrophotometric quantification of primary amines in solution. This strategy can be applied to any type of protein and does not require any labeling agent. It can be performed in a high-throughput manner as a routine experiment and only requires a conventional oven and a microplate reader.
Long-term delivery is a successful strategy used to reduce the adverse effects of monoclonal antibody (mAb)-based treatments. Macroporous hydrogels and affinity-based strategies have shown promising results in sustained and localized delivery of the mAbs. Among the potential tools for affinity-based delivery systems, the de novo designed Ecoil and Kcoil peptides are engineered to form a high-affinity, heterodimeric coiled-coil complex under physiological conditions. In this study, we created a set of trastuzumab molecules tagged with various Ecoil peptides and evaluated their manufacturability and characteristics. Our data show that addition of an Ecoil tag at the C-termini of the antibody chains (light chains, heavy chains, or both) does not hinder the production of chimeric trastuzumab in CHO cells or affect antibody binding to its antigen. We also evaluated the influence of the number, length, and position of the Ecoil tags on the capture and release of Ecoil-tagged trastuzumab from macroporous dextran hydrogels functionalized with Kcoil peptide (the Ecoil peptide-binding partner). Notably, our data show that antibodies are released from the macroporous hydrogels in a biphasic manner; the first phase corresponding to the rapid release of residual, unbound trastuzumab from the macropores, followed by the affinity-controlled, slow-rate release of antibodies from the Kcoil-functionalized macropore surface.
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