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).

Structural Stabilization of Clinically Oriented Oligomeric Proteins During their Transit through Synthetic Secretory Amyloids.

Developing time-sustained drug delivery systems is a main goal in innovative medicines. Inspired by the architecture of secretory granules from the mammalian endocrine system it has generated non-toxic microscale amyloid materials through the coordination between divalent metals and poly-histidine stretches. Like their natural counterparts that keep the functionalities of the assembled protein, those synthetic structures release biologically active proteins during a slow self-disintegration process occurring in vitro and upon in vivo administration.

Nanoparticle-Based Secretory Granules Induce a Specific and Long-Lasting Immune Response through Prolonged Antigen Release.

Developing prolonged antigen delivery systems that mimic long-term exposure to pathogens appears as a promising but still poorly explored approach to reach durable immunities. In this study, we have used a simple technology by which His-tagged proteins can be assembled, assisted by divalent cations, as supramolecular complexes with progressive complexity, namely protein-only nanoparticles and microparticles. Microparticles produced out of nanoparticles are biomimetics of secretory granules from the mammalian hormonal system.

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.

Protein features instruct the secretion dynamics from metal-supported synthetic amyloids.

Hexahistidine-tagged proteins can be clustered by divalent cations into self-containing, dynamic protein depots at the microscale, which under physiological conditions leak functional protein. While such protein granules show promise in clinics as time-sustained drug delivery systems, little is known about how the nature of their components, that is, the protein and the particular cation used as cross-linker, impact on the disintegration of the material and on its secretory performance. By using four model proteins and four different cation formulations to control aggregation, we have here determined a moderate influence of the used cation and a potent impact of some protein properties on the release kinetics and on the final fraction of releasable protein.

Recombinant Proteins for Assembling as Nano- and Micro-Scale Materials for Drug Delivery: A Host Comparative Overview.

By following simple protein engineering steps, recombinant proteins with promising applications in the field of drug delivery can be assembled in the form of functional materials of increasing complexity, either as nanoparticles or nanoparticle-leaking secretory microparticles. Among the suitable strategies for protein assembly, the use of histidine-rich tags in combination with coordinating divalent cations allows the construction of both categories of material out of pure polypeptide samples. Such molecular crosslinking results in chemically homogeneous protein particles with a defined composition, a fact that offers soft regulatory routes towards clinical applications for nanostructured protein-only drugs or for protein-based drug vehicles.

Enhanced recombinant protein capture, purity and yield from crude bacterial cell extracts by N-Lauroylsarcosine-assisted affinity chromatography.

Background: Recombinant proteins cover a wide range of biomedical, biotechnological, and industrial needs. Although there are diverse available protocols for their purification from cell extracts or from culture media, many proteins of interest such as those containing cationic domains are difficult to purify, a fact that results in low yields of the final functional product. Unfortunately, this issue prevents the further development and industrial or clinical application of these otherwise interesting products.

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.

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.

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.

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.

Divalent Cations: A Molecular Glue for Protein Materials.

Among inorganic materials, divalent cations modulate thousands of physiological processes that support life. Their roles in protein assembly and aggregation are less known, although they are progressively being brought to light. We review the structural roles of divalent cations here, as well as the novel protein materials that are under development, in which they are used as glue-like agents.

Release of functional fibroblast growth factor-2 from artificial inclusion bodies.

Growth factors are required for cell proliferation and differentiation under physiological conditions but especially in the context of regenerative medicine. The time-prolonged administration of those factors has been explored using different sustained drug delivery systems. These platforms include natural materials such as bacterial inclusion bodies (IBs) that contain chaperones and other bacterial components that might favour protein release.

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.

Artificial Inclusion Bodies for Clinical Development.

Bacterial inclusion bodies (IBs) are mechanically stable protein particles in the microscale, which behave as robust, slow-protein-releasing amyloids. Upon exposure to cultured cells or upon subcutaneous or intratumor injection, these protein materials secrete functional IB polypeptides, functionally mimicking the endocrine release of peptide hormones from secretory amyloid granules. Being appealing as delivery systems for prolonged protein drug release, the development of IBs toward clinical applications is, however, severely constrained by their bacterial origin and by the undefined and protein-to-protein, batch-to-batch variable composition.

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.

Assembly of histidine-rich protein materials controlled through divalent cations.

Nanostructured protein materials show exciting biomedical applications, since both structure and function can be genetically programmed. In particular, self-assembling histidine-rich proteins benefit from functional plasticity that allows the generation of protein-only nanoparticles for cell targeted drug delivery. However, the rational development of constructs with improved functions is limited by a poor control of the oligomerization process.