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. 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.

Probing the Biosafety of Implantable Artificial Secretory Granules for the Sustained Release of Bioactive Proteins.

Among bio-inspired protein materials, secretory protein microparticles are of clinical interest as self-contained, slow protein delivery platforms that mimic secretory granules of the human endocrine system, in which the protein is both the drug and the scaffold. Upon subcutaneous injection, their progressive disintegration results in the sustained release of the building block polypeptides, which reach the bloodstream for systemic distribution and subsequent biological effects. Such entities are easily fabricated in vitro by Zn-assisted cross-molecular coordination of histidine residues. Using cationic Zn for the assembly of selected pure protein species and in the absence of any heterologous holding material, these granules are expected to be nontoxic and therefore adequate for different clinical uses. However, such presumed biosafety has not been so far confirmed and the potential protein dosage threshold not probed yet. By selecting the receptor binding domain (RBD) from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein as a model protein and using a mouse lab model, we have explored the toxicity of RBD-made secretory granules at increasing doses up to ∼100 mg/kg of animal weight. By monitoring body weight and biochemical blood markers and through the histological scrutiny of main tissues and organs, we have not observed systemic toxicity. Otherwise, the bioavailability of the material was demonstrated by the induction of specific antibody responses. The presented data confirm the intrinsic biosafety of artificial secretory granules made by recombinant proteins and prompt their further clinical development as self-contained and dynamic protein reservoirs.

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. 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.

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. In that way, targeting drugs to specific cell receptors allows a selective intracellular delivery process, avoiding potential side effects of the payload. However, once internalized, the endo-lysosomal route taken by the engulfed material usually results in full degradation, preventing their adequate subcellular localization, bioavailability and subsequent therapeutic effect. Thus, entrapment into endo-lysosomes is a main bottleneck in the efficacy of protein-drug nanomedicines. Promoting endosomal escape and preventing lysosomal degradation would make this therapeutic approach clinically plausible. In this review, we discuss the mechanisms intended to evade lysosomal degradation of proteins, with the most relevant examples and associated strategies, and the methods available to measure that effect. In addition, based on the increasing catalogue of peptide domains tailored to face this challenge as components of protein nanocarriers, we emphasize how their particular mechanisms of action can potentially alter the functionality of accompanying protein materials, especially in terms of targeting and specificity in the delivery process.

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. 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.

Protein scaffolds in human clinics.

Fundamental clinical areas such as drug delivery and regenerative medicine require biocompatible materials as mechanically stable scaffolds or as nanoscale drug carriers. Among the wide set of emerging biomaterials, polypeptides offer enticing properties over alternative polymers, including full biocompatibility, biodegradability, precise interactivity, structural stability and conformational and functional versatility, all of them tunable by conventional protein engineering. However, proteins from non-human sources elicit immunotoxicities that might bottleneck further development and narrow their clinical applicability. In this context, selecting human proteins or developing humanized protein versions as building blocks is a strict demand to design non-immunogenic protein materials. We review here the expanding catalogue of human or humanized proteins tailored to execute different levels of scaffolding functions and how they can be engineered as self-assembling materials in form of oligomers, polymers or complex networks. In particular, we emphasize those that are under clinical development, revising their fields of applicability and how they have been adapted to offer, apart from mere mechanical support, highly refined functions and precise molecular interactions.

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. 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.

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. The capability of these scaffolds for the selective delivery of Monomethyl auristatin E has been comparatively evaluated in a disseminated mouse model of human, CXCR4+ acute myeloid leukemia. Monomethyl auristatin E is an ultra-potent anti-mitotic drug used against a range of hematological neoplasias, which because of its high toxicity is not currently administered as a free drug but as payload in antibody-drug conjugates. The protein nanoconjugates generated here offer a collective strength of simple manufacturing process, high proteolytic and structural stability and multivalent ligand receptor interactions that result in a highly efficient and selective delivery of the payload drug and in a potent anticancer effect. The approach shown here stresses this class of human scaffold proteins as promising alternatives to antibodies for targeted drug delivery in the rapidly evolving drug development landscape.

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. Although initially used almost exclusively in protein production, H6 and related His-rich peptides are progressively proving a broad applicability in novel utilities including enzymatic processes, advanced drug delivery systems and diagnosis, through a so far unsuspected adaptation of their binding capabilities. In this context, the coordination of histidine residues and metals confers intriguing functionalities to His-rich sequences useable in the forward-thinking design of protein-based nano- and micro-materials and devices, through strategies that are comprehensively presented here.

Ion-dependent slow protein release from in vivo 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 in vivo disintegration pattern of a set of such depots, upon subcutaneous administration in mice. These microparticles were fabricated with cationic forms of either Zn, Ca, Mg, or Mn, which abound in the mammalian body. By using a CXCR4-targeted fluorescent protein as a reporter building block we categorized those cations regarding their ability to persist in the administration site and to sustain a slow release of functional protein. Ca2+ and specially Zn2+ have been observed as particularly good promoters of time-prolonged protein leakage. The released polypeptides result is available for selective molecular interactions, such as specific fluorescent labeling of tumor tissues, in which the protein reaches nearly steady levels.

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

We have developed a simple, robust, and fully transversal approach for the a-la-carte 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 Zn2+ 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. The assembly of these materials is not linked to particular polypeptide features, and it is based on an environmentally friendly and sustainable approach. The resulting nanoparticles, with dimensions ranging between 10 and 15 nm, are regular in size, are architecturally stable, are fully functional, and serve as intermediates in a more complex assembly process, resulting in the formation of microscale protein materials. Since most of the recombinant proteins produced by biochemical and biotechnological industries and intended for biomedical research are His-tagged, the green biofabrication procedure proposed here can be straightforwardly applied to a huge spectrum of protein species for their conversion into their respective nanostructured formats.

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. In this study, we have explored the potential of the human albumin-derived peptide, EPI-X4, as a suitable ligand to target colorectal cancer via the cell surface protein CXCR4, a chemokine receptor overexpressed in cancer stem cells. To explore the potential use of this ligand, self-assembling protein nanoparticles have been generated displaying an engineered EPI-X4 version, which conferred a modest CXCR4 targeting and fast and high level of cell apoptosis in tumor CXCR4+ cells, in vitro and in vivo. In addition, when EPI-X4-based building blocks are combined with biologically inert polypeptides containing the CXCR4 ligand T22, the resulting biparatopic nanoparticles show a dramatically improved biodistribution in mouse models of CXCR4+ human cancer, faster cell internalization and enhanced target cell death when compared to the version based on a single ligand. The generation of biparatopic materials opens exciting possibilities in oncotherapies based on high precision drug delivery based on the receptor CXCR4.

Developing Protein-Antitumoral Drug Nanoconjugates as Bifunctional Antimicrobial Agents.

A novel concept about bifunctional antimicrobial drugs, based on self-assembling protein nanoparticles, has been evaluated here over two biofilm-forming pathogens, namely Pseudomonas aeruginosa and Staphylococcus aureus. Two structurally different antimicrobial peptides (GWH1 and PaDBS1R1) were engineered to form regular nanoparticles of around 35 nm, to which the small molecular weight drug Floxuridine was covalently conjugated. Both the assembled peptides and the chemical, a conventional cytotoxic drug used in oncotherapy, showed potent antimicrobial activities that were enhanced by the combination of both molecules in single pharmacological entities. Therefore, the resulting prototypes show promises as innovative nanomedicines, being potential alternatives to conventional antibiotics. The biological performance and easy fabrication of these materials fully support the design of protein-based hybrid constructs for combined molecular therapies, expected to have broad applicability beyond antimicrobial medicines. In addition, the approach taken here validates the functional exploration and repurposing of antitumoral drugs, which at low concentrations perform well as unexpected biofilm-inhibiting agents.

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. This fact leads to a potent and faster destruction of tumor tissues when compared to individual pro-apoptotic factors. The analysis and interpretation of the boosted effect, from both the structural and functional sides, offers clues for the design of more efficient nanomedicines and theragnostic agents in oncology based on precise cocktails of human proteins. STATEMENT OF SIGNIFICANCE: Several human pro-apoptotic peptides (namely BAK, BAX and PUMA) have been engineered as self-assembling protein nanoparticles targeted to the tumoral marker CXCR4. The systemic administration of the same final amounts of those materials as single drugs, or as combinations of two or three of them, shows disparate intensities of antitumoral effects in a mouse model of human colorectal cancer, which are boosted in the triple combination on a non-additive basis. The superiority of the combined administration of pro-apoptotic agents, acting at different levels of the apoptotic cascade, opens a plethora of possibilities for the development of effective and selective cancer therapies based on the precise cocktailing of pro-apoptotic nanoparticulate agents.

Effect of cultivation conditions on ß-estradiol removal in laboratory and pilot-plant photobioreactors by an algal-bacterial consortium treating urban wastewater.

The use of microalgal consortia for urban wastewater treatment is an increasing trend, as it allows simultaneous nutrient removal and biomass production. Emerging contaminants proposed for the list of priority substances such as the hormone 17ß-estradiol are commonly found in urban wastewater, and their removal using algal monocultures has been accomplished. Due to the inherent potential of algae-based systems, this study aimed to assess the capability of native photobioreactor biomass to remove 17ß-estradiol under indoor and outdoor conditions. At the same time, the microbial community changes in regular and bioaugmented operations with Scenedesmus were assessed. The results show that almost complete removal (>93.75%) of the hormone 17ß-estradiol can be attained in the system under favourable seasonal conditions, although these conditions greatly influence biomass concentrations and microbial diversity. Even under the harsh conditions of low temperatures and solar irradiation, the established consortium removed more than 50% of the pollutant in 24 h. While species from genus Chlorella were stable during the entire operation, the microbial diversity analysis revealed that assorted and evenly distributed populations stimulate the removal rates. Bioaugmentation assays proved that the input of additional biomass results in higher overall removal and decreases the yield per mg of biomass.