After precipitation and washing, the fibrils were imaged using fluorescence microscopy and a FITC filter (excitation at 465495 nm) or a TxRed filter (excitation at 540580 nm). of these nanorods with different antibodies allows obtaining a mimic of a bispecific antibody that redirects T lymphocytes Bacitracin to tumoral cells, holding high potential for immunotherapy. Overall, the work illustrates a modular and straightforward strategy to obtain preparative quantities of multivalent antibody-functionalized nanomaterials with multitargeting properties without the need for covalent modification. Keywords:amyloid, dual- or multitargeting, multivalency, antibody, nanorods, nanomaterials == Introduction == Tunable nanomaterials with large surface/volume ratios and multiple functional groups are emerging as novel platforms for diagnosing Bacitracin and treating diseases.1In comparison with small molecules, these materials, including nanotubes, micelles, and proteinpolymer conjugates, exhibit favorable pharmacokinetics2since they can accumulate at higher concentrations and for a longer time at pathological sites, an effect named as enhanced permeability and retention.3Moreover, their superficial functional groups permit the grafting of tailored biomolecules.4The incorporation of specific ligands that target pathogenic cells is expected to minimize the materials toxic side effects and improve their therapeutic efficacy by selective targeting. So far, most of the efforts have been focused on synthetic monofunctional nanomaterials with a single type of ligand intended for a specific target, such as RGD peptides,5monoclonal antibodies,6and other proteins.7However, many diseases are multifactorial, and monospecific conjugates display low effectiveness for their treatment.8In these occasions, multivalency is a requirement, and two or more targets/receptors should be targeted and eventually activated with a single molecular entity.9,10 The concept of dual targeting was initially implemented in the creation of bispecific antibodies (BsAbs), in which each of the two different variable regions targets a distinct antigen or epitope. This allows the simultaneous inhibition of two cell surface receptors and enhances agonism through receptor clustering, blocking two ligands, or recruiting T cells to cancer cells,11resulting in a highly increased targeting and therapeutic efficacy.12However, issues such as low yields,13molecular heterogeneity,14short half-timein vivo,15and toxic side effects16,17have limited the clinical applications of BsAbs. Dual-targeting nanoparticles, conjugating two different small molecules,18peptides,1monoclonal antibodies,19or binding proteins,20are being developed to overcome BsAb limitations and extend their applications. The discovery of functional amyloids21has inspired the building up of functionalized amyloid-based nanomaterials.22These bioactive, biodegradable, and biocompatible peptide or protein-based nanomaterials have been used for biological and biomedical applications, ranging from cancer therapy, bioimaging, or tissue engineering to regenerative medicine.23Self-assembled peptide-based Rabbit Polyclonal to ME3 nanomaterials offer a high surface area versus the volume ratio and constitute stable superstructures with suitable pharmacokinetics. 24Nanomaterials are usually synthesized with a series of complex processes, in which the amyloid scaffold is formed first, and the ligand is covalently conjugated afterward, but this implies a significant inactivation, especially if the ligand has a proteic nature.25Indeed, the major advantage of protein-based materials Bacitracin is the ability to modify their functionalities by simple genetic redesign, as long as the globular domains remain folded and active in the assembled state. Recently, we have been successful in the design of highly ordered amyloid-like nanofibrils containing well-folded and highly Bacitracin functional proteins using a modular approach. In particular, a soft amyloid core (SAC) of the Sup35 yeast prion was used as the driving force for self-assembly, Bacitracin and it was fused using a flexible linker to a globular domain. The fusion protein is produced in a soluble form at high yield, but it can be induced to form a fibrillar structure, sustained by the Sup35-SAC spine, to which the globular-and-folded domains are attached.26Thus, the appended globular protein remains bioactive and accessible. A similar approach has been applied to manufacture functionalized nanofibrils decorated with the Z-domain,27a designed analogue of the B domain fromStaphylococcus aureusprotein A that binds with high affinity to antibodies.28,29It was rationalized that the Z-domain fusion to Sup35-SAC might constitute an optimal strategy to produce amyloid fibrils for dual targeting. Following this hypothesis, we obtained.