Paradoxical cell targeting of calreticulin-empowered, protein-only nanoparticles.

Surface-exposed calreticulin (CRT) serves as a crucial cell damage-associated molecular pattern for immunogenic apoptosis, by generating an "eat me" signal to macrophages. Aiming at precision immunotherapies we intended to artificially label tumoral cells in vivo with a recombinant CRT, in a targeted way. For that, we have constructed a CRT fusion protein intended to surface attach CXCR4 cancer cells, to stimulate their immunological destruction.

Hybrid Micro-/Nanoprotein Platform Provides Endocrine-like and Extracellular Matrix-like Cell Delivery of Growth Factors.

Protein materials are versatile tools in diverse biomedical fields. Among them, artificial secretory granules (SGs), mimicking those from the endocrine system, act as mechanically stable reservoirs for the sustained release of proteins as oligomeric functional nanoparticles. Only validated in oncology, the physicochemical properties of SGs, along with their combined drug-releasing and scaffolding abilities, make them suitable as smart topographies in regenerative medicine for the prolonged delivery of growth factors (GFs).

Efficient Delivery of Antimicrobial Peptides in an Innovative, Slow-Release Pharmacological Formulation.

Both nanostructure and multivalency enhance the biological activities of antimicrobial peptides (AMPs), whose mechanism of action is cooperative. In addition, the efficacy of a particular AMP should benefit from a steady concentration at the local place of action and, therefore, from a slow release after a dynamic repository. In the context of emerging multi-resistant bacterial infections and the urgent need for novel and effective antimicrobial drugs, we tested these concepts through the engineering of four AMPs into supramolecular complexes as pharmacological entities.

High-precision targeting and destruction of cancer-associated PDGFR-β stromal fibroblasts through self-assembling, protein-only nanoparticles.

The need for more effective and precision medicines for cancer has pushed the exploration of new materials appropriate for drug delivery and imaging, and alternative receptors for targeting. Among the most promising strategies, finding suitable cell surface receptors and targeting agents for cancer-associated platelet derived growth factor receptor β (PDGFR-β) stromal fibroblasts is highly appealing. As a neglected target, this cell type mechanically and biologically supports the growth, progression, and infiltration of solid tumors in non-small cell lung, breast, pancreatic, and colorectal cancers.

Endosomal escape for cell-targeted proteins. Going out after going in.

Protein-based nanocarriers are versatile and biocompatible drug delivery systems. They are of particular interest in nanomedicine as they can recruit multiple functions in a single modular polypeptide. Many cell-targeting peptides or protein domains can promote cell uptake when included in these nanoparticles through receptor-mediated endocytosis.

The Diphtheria Toxin Translocation Domain Impairs Receptor Selectivity in Cancer Cell-Targeted Protein Nanoparticles.

Protein-based materials intended as nanostructured drugs or drug carriers are progressively gaining interest in nanomedicine, since their structure, assembly and cellular interactivity can be tailored by recruiting functional domains. The main bottleneck in the development of deliverable protein materials is the lysosomal degradation that follows endosome maturation. This is especially disappointing in the case of receptor-targeted protein constructs, which, while being highly promising and in demand in precision medicines, enter cells via endosomal/lysosomal routes.

An In Silico Methodology That Facilitates Decision Making in the Engineering of Nanoscale Protein Materials.

Under the need for new functional and biocompatible materials for biomedical applications, protein engineering allows the design of assemblable polypeptides, which, as convenient building blocks of supramolecular complexes, can be produced in recombinant cells by simple and scalable methodologies. However, the stability of such materials is often overlooked or disregarded, becoming a potential bottleneck in the development and viability of novel products. In this context, we propose a design strategy based on in silico tools to detect instability areas in protein materials and to facilitate the decision making in the rational mutagenesis aimed to increase their stability and solubility.

A diphtheria toxin-based nanoparticle achieves specific cytotoxic effect on CXCR4 lymphoma cells without toxicity in immunocompromised and immunocompetent mice.

High rates of relapsed and refractory diffuse large B-cell lymphoma (DLBCL) patients and life-threatening side effects associated with immunochemotherapy make an urgent need to develop new therapies for DLBCL patients. Immunotoxins seem very potent anticancer therapies but their use is limited because of their high toxicity. Accordingly, the self-assembling polypeptidic nanoparticle, T22-DITOX-H6, incorporating the diphtheria toxin and targeted to CXCR4 receptor, which is overexpressed in DLBCL cells, could offer a new strategy to selectively eliminate CXCR4 DLBCL cells without adverse effects.

The Poly-Histidine Tag H6 Mediates Structural and Functional Properties of Disintegrating, Protein-Releasing Inclusion Bodies.

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.

Engineering non-antibody human proteins as efficient scaffolds for selective, receptor-targeted drug delivery.

Self-assembling non-immunoglobulin scaffold proteins are a promising class of nanoscale carriers for drug delivery and interesting alternatives to antibody-based carriers that are not sufficiently efficient in systemic administration. To exploit their potentialities in clinics, protein scaffolds need to be further tailored to confer appropriate targeting and to overcome their potential immunogenicity, short half-life in plasma and proteolytic degradation. We have here engineered three human scaffold proteins as drug carrier nanoparticles to target the cytokine receptor CXCR4, a tumoral cell surface marker of high clinical relevance.

Ion-dependent slow protein release from disintegrating micro-granules.

Through the controlled addition of divalent cations, polyhistidine-tagged proteins can be clustered in form of chemically pure and mechanically stable micron-scale particles. Under physiological conditions, these materials act as self-disintegrating protein depots for the progressive release of the forming polypeptide, with potential applications in protein drug delivery, diagnosis, or theragnosis. Here we have explored the disintegration pattern of a set of such depots, upon subcutaneous administration in mice.

Biofabrication of functional protein nanoparticles through simple His-tag engineering.

We have developed a simple, robust, and fully transversal approach for the fabrication of functional multimeric nanoparticles with potential biomedical applications, validated here by a set of diverse and unrelated polypeptides. The proposed concept is based on the controlled coordination between Zn ions and His residues in His-tagged proteins. This approach results in a spontaneous and reproducible protein assembly as nanoscale oligomers that keep the original functionalities of the protein building blocks.

Insights on the emerging biotechnology of histidine-rich peptides.

In the late 70's, the discovery of the restriction enzymes made possible the biological production of functional proteins by recombinant DNA technologies, a fact that largely empowered both biotechnological and pharmaceutical industries. Short peptides or small protein domains, with specific molecular affinities, were developed as purification tags in downstream processes to separate the target protein from the culture media or cell debris, upon breaking the producing cells. Among these tags, and by exploiting the interactivity of the imidazole ring of histidine residues, the hexahistidine peptide (H6) became a gold standard.

Biparatopic Protein Nanoparticles for the Precision Therapy of CXCR4 Cancers.

The accumulated molecular knowledge about human cancer enables the identification of multiple cell surface markers as highly specific therapeutic targets. A proper tumor targeting could significantly avoid drug exposure of healthy cells, minimizing side effects, but it is also expected to increase the therapeutic index. Specifically, colorectal cancer has a particularly poor prognosis in late stages, being drug targeting an appropriate strategy to substantially improve the therapeutic efficacy.

Self-Assembled Nanobodies as Selectively Targeted, Nanostructured, and Multivalent Materials.

Nanobodies represent valuable tools in advanced therapeutic strategies but their small size (∼2.5 × ∼ 4 nm) and limited valence for interactions might pose restrictions for applications, especially regarding their modest capacity for multivalent and cooperative interaction. In this work, modular protein constructs have been designed, in which nanobodies are fused to protein domains to provide further functionalities and to favor oligomerization into stable self-assembled nanoparticles.

Design and engineering of tumor-targeted, dual-acting cytotoxic nanoparticles.

The possibility to conjugate tumor-targeted cytotoxic nanoparticles and conventional antitumoral drugs in single pharmacological entities would open a wide spectrum of opportunities in nanomedical oncology. This principle has been explored here by using CXCR4-targeted self-assembling protein nanoparticles based on two potent microbial toxins, the exotoxin A from Pseudomonas aeruginosa and the diphtheria toxin from Corynebacterium diphtheriae, to which oligo-floxuridine and monomethyl auristatin E respectively have been chemically coupled. The resulting multifunctional hybrid nanoconjugates, with a hydrodynamic size of around 50 nm, are stable and internalize target cells with a biological impact.

Fluorescent Dye Labeling Changes the Biodistribution of Tumor-Targeted Nanoparticles.

Fluorescent dye labeling is a common strategy to analyze the fate of administered nanoparticles in living organisms. However, to which extent the labeling processes can alter the original nanoparticle biodistribution has been so far neglected. In this work, two widely used fluorescent dye molecules, namely, ATTO488 (ATTO) and Sulfo-Cy5 (S-Cy5), have been covalently attached to a well-characterized CXCR4-targeted self-assembling protein nanoparticle (known as T22-GFP-H6).

A refined cocktailing of pro-apoptotic nanoparticles boosts anti-tumor activity.

A functional 29 amino acid-segment of the helix α5 from the human BAX protein has been engineered for production in recombinant bacteria as self-assembling, GFP-containing fluorescent nanoparticles, which are targeted to the tumoral marker CXCR4. These nanoparticles, of around 34 nm in diameter, show a moderate tumor biodistribution and limited antitumoral effect when systemically administered to mouse models of human CXCR4 colorectal cancer (at 300 μg dose). However, if such BAX nanoparticles are co-administered in cocktail with equivalent nanoparticulate versions of BAK and PUMA proteins at the same total protein dose (300 μg), protein biodistribution and stability in tumor is largely improved, as determined by fluorescence profiles.

Engineering Protein Nanoparticles Out from Components of the Human Microbiome.

Nanoscale protein materials are highly convenient as vehicles for targeted drug delivery because of their structural and functional versatility. Selective binding to specific cell surface receptors and penetration into target cells require the use of targeting peptides. Such homing stretches should be incorporated to larger proteins that do not interact with body components, to prevent undesired drug release into nontarget organs.

Engineering Secretory Amyloids for Remote and Highly Selective Destruction of Metastatic Foci.

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.