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The coordination between histidine-rich peptides and divalent cations supports the formation of nano- and micro-scale protein biomaterials, including toxic and non-toxic functional amyloids, which can be adapted as drug delivery systems. Among them, inclusion bodies (IBs) formed in recombinant bacteria have shown promise as protein depots for time-sustained protein release. We have demonstrated here that the hexahistidine (H6) tag, fused to recombinant proteins, impacts both on the formation of bacterial IBs and on the conformation of the IB-forming protein, which shows a higher content of cross-beta intermolecular interactions in H6-tagged versions. Additionally, the addition of EDTA during the spontaneous disintegration of isolated IBs largely affects the protein leakage rate, again protein release being stimulated in His-tagged materials. This event depends on the number of His residues but irrespective of the location of the tag in the protein, as it occurs in either C-tagged or N-tagged proteins. The architectonic role of H6 in the formation of bacterial IBs, probably through coordination with divalent cations, offers an easy approach to manipulate protein leakage and to tailor the applicability of this material as a secretory amyloidal depot in different biomedical interfaces. In addition, the findings also offer a model to finely investigate, in a simple set-up, the mechanics of protein release from functional secretory amyloids.

Altres ajuts: to EU COST Action CA 17140 Functional amyloids produced in bacteria as nanoscale inclusion bodies are intriguing but poorly explored protein materials with wide therapeutic potential. Since they release functional polypeptides under physiological conditions, these materials can be potentially tailored as mimetic of secretory granules for slow systemic delivery of smart protein drugs. To explore this possibility, bacterial inclusion bodies formed by a self-assembled, tumor-targeted Pseudomonas exotoxin (PE24) are administered subcutaneously in mouse models of human metastatic colorectal cancer, for sustained secretion of tumor-targeted therapeutic nanoparticles. These proteins are functionalized with a peptidic ligand of CXCR4, a chemokine receptor overexpressed in metastatic cancer stem cells that confers high selective cytotoxicity in vitro and in vivo. In the mouse models of human colorectal cancer, time-deferred anticancer activity is detected after the subcutaneous deposition of 500 µg of PE24-based amyloids, which promotes a dramatic arrest of tumor growth in the absence of side toxicity. In addition, long-term prevention of lymphatic, hematogenous, and peritoneal metastases is achieved. These results reveal the biomedical potential and versatility of bacterial inclusion bodies as novel tunable secretory materials usable in delivery, and they also instruct how therapeutic proteins, even with high functional and structural complexity, can be packaged in this convenient format.

Cell‐selective targeting is expected to enhance effectiveness and minimize side effects of cytotoxic agents. Functionalization of drugs or drug nanoconjugates with specific cell ligands allows receptor‐mediated selective cell delivery. However, it is unclear whether the incorporation of an efficient ligand into a drug vehicle is sufficient to ensure proper biodistribution upon systemic administration, and also at which extent biophysical properties of the vehicle may contribute to the accumulation in target tissues during active targeting. To approach this issue, structural robustness of self‐assembling, protein‐only nanoparticles targeted to the tumoral marker CXCR4 is compromised by reducing the number of histidine residues (from six to five) in a histidine‐based architectonic tag. Thus, the structure of the resulting nanoparticles, but not of building blocks, is weakened. Upon intravenous injection in animal models of human CXCR4+ colorectal cancer, the administered material loses the ability to accumulate in tumor tissue, where it is only transiently found. It instead deposits in kidney and liver. Therefore, precise cell‐targeted delivery requires not only the incorporation of a proper ligand that promotes receptor‐mediated internalization, but also, unexpectedly, its maintenance of a stable multimeric nanostructure that ensures high ligand exposure and long residence time in tumor tissue. Fil: López Laguna, Hèctor. Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina; España. Universitat Autònoma de Barcelona; España Fil: Sala, Rita. Hospital de la Santa Creu i Sant Pau; España. Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina; España Fil: Sanchez, Julieta Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina; España. Universitat Autònoma de Barcelona; España Fil: Álamo, Patricia. Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina; España. Hospital de la Santa Creu i Sant Pau; España Fil: Unzueta, Ugutz. Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina; España. Hospital de la Santa Creu i Sant Pau; España Fil: Sánchez Chardi, Alejandro. Universitat Autònoma de Barcelona; España Fil: Serna, Naroa. Universitat Autònoma de Barcelona; España. Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina; España Fil: Sánchez-García, Laura. Universitat Autònoma de Barcelona; España. Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina; España Fil: Voltà Durán, Eric. Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina; España. Universitat Autònoma de Barcelona; España Fil: Mangues, Ramón. Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina; España. Hospital de la Santa Creu i Sant Pau; España Fil: Villaverde Corrales, Antonio. Universitat Autònoma de Barcelona; España. Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina; España Fil: Vázquez, Esther. Centro de Investigación Biomédica en Red. Bioingeniería, Biomateriales y Nanomedicina; España. Universitat Autònoma de Barcelona; España