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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. Being these granules formed by a single pure protein species and therefore, chemically homogenous, they act as highly promising time‐sustained drug delivery systems. Despite their enormous clinical potential, the nature of the clustering process and the quality of the released protein have been so far neglected issues. By using diverse polypeptide species and their protein‐only oligomeric nanoscale versions as convenient models, a conformational rearrangement and a stabilization of the building blocks during their transit through the secretory granules, being the released material structurally distinguishable from the original source is proved here. This fact indicates a dynamic nature of secretory amyloids that act as conformational arrangers rather than as plain, inert protein‐recruiting/protein‐releasing granular depots. [ABSTRACT FROM AUTHOR]

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. For that purpose, GWH1, T22, Pt5, and PaD, produced as GFP or human nidogen-based His-tagged fusion proteins, were engineered as self-assembling oligomeric nanoparticles ranging from 10 to 70 nm and further packaged into nanoparticle-leaking submicron granules. Since these materials slowly release functional nanoparticles during their time-sustained unpacking, they are suitable for use as drug depots in vivo. In this context, a particular AMP version (GWH1-NIDO-H6) was selected for in vivo validation in a zebrafish model of a complex bacterial infection. The GWH1-NIDO-H6-secreting protein granules are protective in zebrafish against infection by the multi-resistant bacterium Stenotrophomonas maltophilia, proving the potential of innovative formulations based on nanostructured and slowly released recombinant AMPs in the fight against bacterial infections. [ABSTRACT FROM AUTHOR]

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. Successes in the fabrication and final performance of these materials are expected, irrespective of the protein source. However, this fact has not yet been fully explored and confirmed. By taking the antigenic RBD domain of the SARS-CoV-2 spike glycoprotein as a model building block, we investigated the production of nanoparticles and secretory microparticles out of the versions of recombinant RBD produced by bacteria (Escherichia coli), insect cells (Sf9), and two different mammalian cell lines (namely HEK 293F and Expi293F). Although both functional nanoparticles and secretory microparticles were effectively generated in all cases, the technological and biological idiosyncrasy of each type of cell factory impacted the biophysical properties of the products. Therefore, the selection of a protein biofabrication platform is not irrelevant but instead is a significant factor in the upstream pipeline of protein assembly into supramolecular, complex, and functional materials. [ABSTRACT FROM AUTHOR]

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. In the search for suitable protein agents that might promote endosome escape, we have explored the translocation domain (TD) of the diphtheria toxin as a functional domain in CXCR4-targeted oligomeric nanoparticles designed for cancer therapies. The pharmacological interest of such protein materials could be largely enhanced by improving their proteolytic stability. The incorporation of TD into the building blocks enhances the amount of the material detected inside of exposed CXCR4+ cells up to around 25-fold, in absence of cytotoxicity. This rise cannot be accounted for by endosomal escape, since the lysosomal degradation of the new construct decreases only moderately. On the other hand, a significant loss in the specificity of the CXCR4-dependent cellular penetration indicates the unexpected role of the toxin segment as a cell-penetrating peptide in a dose-dependent and receptor-independent fashion. These data reveal that the diphtheria toxin TD displayed on receptor-targeted oligomeric nanoparticles partially abolishes the exquisite receptor specificity of the parental material and it induces nonspecific internalization in mammalian cells. [ABSTRACT FROM AUTHOR]

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. As a case study, we demonstrate the potential of this methodology to improve the stability of a humanized scaffold protein (a domain of the human nidogen), with the ability to oligomerize into regular nanoparticles usable to deliver payload drugs to tumor cells. Several nidogen mutants suggested by the method showed important and measurable improvements in their structural stability while retaining the functionalities and production yields of the original protein. Then, we propose the procedure developed here as a cost-effective routine tool in the design and optimization of multimeric protein materials prior to any experimental testing. [ABSTRACT FROM AUTHOR]

Advanced medical treatments involving drug delivery require fully biocompatible materials with the ability to release functional drugs in a time‐prolonged way. Ideally, the delivered molecules should be self‐contained as chemically homogenous entities to prevent the use of potentially toxic scaffolds or hold matrices. In nature, peptidic hormones are self‐stored in protein‐only secretory granules formed by the reversible coordination of Zn2+ and histidine residues. Inspired by this concept, an in vitro transversal procedure is developed, analyzed, and comparatively applied for the fabrication of protein‐only secretory granules at the microscale. These materials can be produced from any polyhistidine‐tagged protein using physiological concentrations of Zn2+ as a potent and versatile glue‐like agent. The screening of granules formed by 12 engineered and nonengineered proteins at different Zn2+ concentrations revealed optimal fabrication conditions and the consequent release profiles. Moreover, the functional and structural properties of the delivered protein are fully validated using a drug‐targeting protein platform in a mouse model of human colorectal cancer. In summary, short histidine tags allow the packaging of structurally and functionally dissimilar polypeptides, which supports the proposed fabrication method as a powerful protocol extensible to diverse clinical scenarios in which slow protein drug delivery is required. [ABSTRACT FROM AUTHOR]

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. Although the chemical conjugation minimizes the cytotoxic activity of the protein partner in the complexes, the concept of drug combination proposed here is fully feasible and highly promising when considering multiple drug treatments aimed to higher effectiveness or when facing the therapy of cancers with acquired resistance to classical drugs. Image, graphical abstract [ABSTRACT FROM AUTHOR]

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. In this context, the de novo fabrication of artificial IBs (ArtIBs) by simple, cell‐free physicochemical methods, using pure components at defined amounts is proposed here. By this, the resulting functional protein microparticles are intriguing, chemically defined biomimetic materials that replicate relevant functionalities of natural IBs, including mammalian cell penetration and local or remote release of functional ArtIB‐forming protein. In default of severe regulatory issues, the concept of ArtIBs is proposed as a novel exploitable category of biomaterials for biotechnological and biomedical applications, resulting from simple fabrication and envisaging soft developmental routes to clinics. [ABSTRACT FROM AUTHOR]

Oligomerization of antimicrobial peptides into nanosized supramolecular complexes produced in biological systems (inclusion bodies and self-assembling nanoparticles) seems an appealing alternative to conventional antibiotics. In this work, the antimicrobial peptide, GWH1, was N-terminally fused to two different scaffold proteins, namely, GFP and IFN-γ for its bacterial production in the form of such recombinant protein complexes. Protein self-assembling as regular soluble protein nanoparticles was achieved in the case of GWH1-GFP, while oligomerization into bacterial inclusion bodies was reached in both constructions. Among all these types of therapeutic proteins, protein nanoparticles of GWH1-GFP showed the highest bactericidal effect in an in vitro assay against Escherichia coli, whereas non-oligomerized GWH1-GFP and GWH1-IFN-γ only displayed a moderate bactericidal activity. These results indicate that the biological activity of GWH1 is specifically enhanced in the form of regular multi-display configurations. Those in vitro observations were fully validated against a bacterial infection using a mouse mastitis model, in which the GWH1-GFP soluble nanoparticles were able to effectively reduce bacterial loads. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Distinct kinetic features of IB β-Gal at in vitro conditions were described. • IB β-Gal was more efficient than S β-Gal to catalyse lactose hydrolysis. • Protein-protein interactions in IB β-Gal preserved β-Gal activity at extremes pH and T. • The structure-function relationship of D β-Gal was similar to that of S β-Gal. Abstract Bacterial inclusion bodies (IBs) were historically considered one of the major obstacles in protein production through recombinant DNA techniques and conceived as amorphous deposits formed by passive and rather unspecific structures of unfolded proteins aggregates. Subsequent studies demonstrated that IBs contained an important quantity of active protein. In this work, we proved that recombinant β-galactosidase inclusion bodies (IB β-Gal) are functional aggregates. Moreover, they exhibit particular features distinct to the soluble version of the enzyme. The particulate enzyme was highly active against lactose in physiological and in acid pH and also retained its activity upon a pre-incubation at high temperature. IB β-Gal washing or dilution induced the spontaneous release of active enzymes from the supramolecular aggregates. Along this process, we observed a continuous change in the values of several kinetic parameters, including specific activity and Michaelis–Menten constant, measured in the IB β-Gal suspensions. Simultaneously, IB β-Gal turned into a more heterogeneous population where smaller particles appeared. The released protein exhibited secondary structure features more similar to those of the soluble species than to the aggregated enzyme. Concluding, IB β-Gal represents a reservoir and packed source of highly active and stable enzyme. [ABSTRACT FROM AUTHOR]

Despite substantial development of production and purification protocols for heterologous recombinant proteins, some proteins are difficult to produce or, when produced, are accumulated in inclusion bodies (IBs). Nondenaturing protocols can be used to recover the entrapped protein from these protein aggregates. In this chapter, we provide a detailed procedure to analyze the physicochemical properties of one of those proteins produced in prokaryotic expression systems. Serum amyloid A3 (SAA3) was recovered from inclusion bodies (IBs) and its secondary structure associated to thermal stability and size was determined by circular dichroism (CD) and dynamic light scattering (DLS), respectively. These techniques were also applied to evaluate the SAA3 interaction with model membranes. These results show the importance of the structural analysis of proteins released from inclusion bodies under nondenaturing procedures, although similar approaches can be extended to any type of recombinant protein preparation., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

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. In this context, the de novo fabrication of artificial IBs (ArtIBs) by simple, cell-free physicochemical methods, using pure components at defined amounts is proposed here. By this, the resulting functional protein microparticles are intriguing, chemically defined biomimetic materials that replicate relevant functionalities of natural IBs, including mammalian cell penetration and local or remote release of functional ArtIB-forming protein. In default of severe regulatory issues, the concept of ArtIBs is proposed as a novel exploitable category of biomaterials for biotechnological and biomedical applications, resulting from simple fabrication and envisaging soft developmental routes to clinics., Competing Interests: E.V., R.M., and A.V. are cofounders of NANOLIGENT, devoted to develop antitumoral drugs based on proteins. J.M.S., H.L‐L., P.A., E.V., R.M. and A.V. are co‐inventors in a patent covering the use of ArtIBs., (© 2019 The Authors. Published by WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

The membrane pore-forming activities of the antimicrobial peptide GWH1 have been evaluated in combination with the CXCR4-binding properties of the peptide T22, in self-assembling protein nanoparticles with high clinical potential. The resulting materials, of 25 nm in size and with regular morphologies, show a dramatically improved cell penetrability into CXCR4 + cells (more than 10-fold) and enhanced endosomal escape (the lysosomal degradation dropping from 90% to 50%), when compared with equivalent protein nanoparticles lacking GWH1. These data reveal that GWH1 retains its potent membrane activity in form of nanostructured protein complexes. On the other hand, the specificity of T22 in the CXCR4 receptor binding is subsequently minimized but, unexpectedly, not abolished by the presence of the antimicrobial peptide. The functional combination T22-GWH1 results in 30% of the nanoparticles entering cells via CXCR4 while also exploiting pore-based uptake. Such functional materials are capable to selectively deliver highly potent cytotoxic drugs upon chemical conjugation, promoting CXCR4-dependent cell death. These data support the further development of GWH1-empowered cell-targeted proteins as nanoscale drug carriers for precision medicines. This is a very promising approach to overcome lysosomal degradation of protein nanostructured materials with therapeutic value.

Under the unmet need of efficient tumor-targeting drugs for oncology, a recombinant version of the plant toxin ricin (the modular protein T22-mRTA-H6) is engineered to self-assemble as protein-only, CXCR4-targeted nanoparticles. The soluble version of the construct self-organizes as regular 11 nm planar entities that are highly cytotoxic in cultured CXCR4 + cancer cells upon short time exposure, with a determined IC50 in the nanomolar order of magnitude. The chemical inhibition of CXCR4 binding sites in exposed cells results in a dramatic reduction of the cytotoxic potency, proving the receptor-dependent mechanism of cytotoxicity. The insoluble version of T22-mRTA-H6 is, contrarily, moderately active, indicating that free, nanostructured protein is the optimal drug form. In animal models of acute myeloid leukemia, T22-mRTA-H6 nanoparticles show an impressive and highly selective therapeutic effect, dramatically reducing the leukemia cells affectation of clinically relevant organs. Functionalized T22-mRTA-H6 nanoparticles are then promising prototypes of chemically homogeneous, highly potent antitumor nanostructured toxins for precise oncotherapies based on self-mediated intracellular drug delivery., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The kinetics and the thermodynamics of melanin concentrating hormone (MCH) adsorption, penetration, and mixing with membrane components are reported. MCH behaved as a surface active peptide, forming stable monolayers at a lipid-free air-water interface, with an equilibrium spreading pressure, a collapse pressure, and a minimal molecular area of 11 mN/m, 13 mN/m, and 140 A (2), respectively. Additional peptide interfacial stabilization was achieved in the presence of lipids, as evidenced by the expansion observed at pi > pi sp in monolayers containing premixtures of MCH with zwitterionic or charged lipids. The MCH-monolayer association and dissociation rate constants were 9.52 x 10 (-4) microM (-1) min (-1) and 8.83 x 10 (-4) min (-1), respectively. The binding of MCH to the dpPC-water interface had a K d = 930 nM at 10 mN/m. MCH penetration in lipid monolayers occurred even up to pi cutoff = 29-32 mN/m. The interaction stability, binding orientation, and miscibility of MCH in monolayers depended on the lipid type, the MCH molar fraction in the mixture, and the molecular packing of the monolayer. This predicted its heterogeneous distribution between different self-separated membrane domains. Our results demonstrated the ability of MCH to incorporate itself into biomembranes and supports the possibility that MCH affects the activity of mechanosensitive membrane proteins through mechanisms unrelated with binding to specific receptors.