Dopamine receptors D1 and D2 show prognostic significance and potential therapeutic applications for endometrial cancer patients.

OBJECTIVE: Catecholaminergic signaling has been a target for therapy in different type of cancers. In this work, we characterized the ADRß2, DRD1 and DRD2 expression in healthy tissue and endometrial tumors to evaluate their prognostic significance in endometrial cancer (EC), unraveling their possible application as an antitumor therapy. METHODS: 109 EC patients were included. The expression of the ADRß2, DRD1 and DRD2 proteins was evaluated by immunohistochemistry and univariate and multivariate analysis to assess their association with clinic-pathological and outcome variables. Finally, HEC1A and AN3CA EC cell lines were exposed to different concentrations of selective dopaminergic agents alone or in combination to study their effects on cellular viability. RESULTS: ADRß2 protein expression was not associated with clinico-pathological parameters or prognosis. DRD1 protein expression was reduced in tumors samples but showed a significant inverse association with tumor size and stage. DRD2 protein expression was significantly associated with non-endometrioid EC, high grade tumors, tumor size, worse disease-free survival (HR = 3.47 (95%CI:1.35-8.88)) and overall survival (HR = 2.98 (95%CI:1.40-6.34)). The DRD1 agonist fenoldopam showed a reduction of cellular viability in HEC1A and AN3CA cells. The exposure to domperidone, a DRD2 antagonist, significantly reduced cell viability compared to the control. Finally, DRD1 agonism and DRD2 antagonism combination induced a significant reduction in cell viability of the AN3CA cells compared to monotherapy, close to being an additive response than a synergistic effect (CI of 1.1 at 0.5% Fa). CONCLUSION: DRD1 and DRD2 expression levels showed a significant association with clinico-pathological parameters. Both the combined activation of DRD1 and blockage of DRD2 may form an innovative strategy to inhibit tumor growth in EC.

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.

Nano-Based Approved Pharmaceuticals for Cancer Treatment: Present and Future Challenges.

Cancer is one of the main causes of death worldwide. To date, and despite the advances in conventional treatment options, therapy in cancer is still far from optimal due to the non-specific systemic biodistribution of antitumor agents. The inadequate drug concentrations at the tumor site led to an increased incidence of multiple drug resistance and the appearance of many severe undesirable side effects. Nanotechnology, through the development of nanoscale-based pharmaceuticals, has emerged to provide new and innovative drugs to overcome these limitations. In this review, we provide an overview of the approved nanomedicine for cancer treatment and the rationale behind their designs and applications. We also highlight the new approaches that are currently under investigation and the perspectives and challenges for nanopharmaceuticals, focusing on the tumor microenvironment and tumor disseminate cells as the most attractive and effective strategies for cancer treatments.

Human α-Galactosidase A Mutants: Priceless Tools to Develop Novel Therapies for Fabry Disease.

Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme.

Extracellular vesicles from recombinant cell factories improve the activity and efficacy of enzymes defective in lysosomal storage disorders.

In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing alpha-galactosidase A (GLA) or N-sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV-GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR-labelled EVs were localized in brain parenchyma 1 h after intra-arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV-GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies.

Impact of Chemical Composition on the Nanostructure and Biological Activity of α-Galactosidase-Loaded Nanovesicles for Fabry Disease Treatment.

Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems-quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development.

Nanotechnology-based approaches for treating lysosomal storage disorders, a focus on Fabry disease.

Lysosomal storage disorders (LSDs) are a group of rare diseases in which the defect of a lysosomal protein results in a pathogenic accumulation of nonmetabolized products within the cells. The main treatment for LSDs is enzyme replacement therapy (ERT), consisting in the exogenous administration a recombinant protein to replace the defective one. Although several diseases such as Gaucher, Fabry, and Pompe are treated following this approach, ERT is limited to LSDs without severe neuronal affectation because recombinant enzymes do not cross the blood-brain barrier. Moreover, ERT shows additional drawbacks, including enzyme low half-life, poor bioavailability, and immunogenic responses. In this scenario, nanotechnology-based drug delivery systems (DDS) have been proposed as solution to overcome these limitations and improve the efficacy of ERT. The present review summarizes distinct approaches followed by our group and collaborators on the use of DDS for restoring lysosomal enzymes in disease-affected cells. During the last decade, we have been exploring different synthetic nanoparticles, from electrolytic complexes, to liposomes and aggresomes, for the delivery of α-galactosidase A (GLA) enzyme. Studies were mainly conducted on Fabry disease models, but results can be also extrapolated to other LSDs, as well as to other diseases treated with alternative therapeutic proteins. The advantages and disadvantages of different DDS, the difficulties from working with very labile and highly glycosylated enzymes and the relevance of using appropriate targeting moieties is thoroughly discussed. Finally, the use of natural DDS, namely extracellular vesicles (EVs) is also introduced. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Cardiovascular Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Bacterial Inclusion Bodies: Discovering Their Better Half.

Bacterial inclusion bodies (IBs) are functional, non-toxic amyloids occurring in recombinant bacteria showing analogies with secretory granules of the mammalian endocrine system. The scientific interest in these mesoscale protein aggregates has been historically masked by their status as a hurdle in recombinant protein production. However, progressive understanding of how the cell handles the quality of recombinant polypeptides and the main features of their intriguing molecular organization has stimulated the interest in inclusion bodies and spurred their use in diverse technological fields. The engineering and tailoring of IBs as functional protein particles for materials science and biomedicine is a good example of how formerly undesired bacterial byproducts can be rediscovered as promising functional materials for a broad spectrum of applications.

Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs.

In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4+ cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals' endocrine system.

In vivo architectonic stability of fully de novo designed protein-only nanoparticles.

The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein-protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids.

Sheltering DNA in self-organizing, protein-only nano-shells as artificial viruses for gene delivery.

By recruiting functional domains supporting DNA condensation, cell binding, internalization, endosomal escape and nuclear transport, modular single-chain polypeptides can be tailored to associate with cargo DNA for cell-targeted gene therapy. Recently, an emerging architectonic principle at the nanoscale has permitted tagging protein monomers for self-organization as protein-only nanoparticles. We have studied here the accommodation of plasmid DNA into protein nanoparticles assembled with the synergistic assistance of end terminal poly-arginines (R9) and poly-histidines (H6). Data indicate a virus-like organization of the complexes, in which a DNA core is surrounded by a solvent-exposed protein layer. This finding validates end-terminal cationic peptides as pleiotropic tags in protein building blocks for the mimicry of viral architecture in artificial viruses, representing a promising alternative to the conventional use of viruses and virus-like particles for nanomedicine and gene therapy. FROM THE CLINICAL EDITOR: Finding efficient gene delivery methods still represents a challenge and is one of the bottlenecks to the more widespread application of gene therapy. The findings presented in this paper validate the application of end-terminal cationic peptides as pleiotropic tags in protein building blocks for "viral architecture mimicking" in artificial viruses, representing a promising alternative to the use of viruses and virus-like particles for gene delivery.

Intracellular CXCR4⁺ cell targeting with T22-empowered protein-only nanoparticles.

BACKGROUND: Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. RESULTS: Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4⁺ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. CONCLUSION: Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents.

Packaging protein drugs as bacterial inclusion bodies for therapeutic applications.

A growing number of insights on the biology of bacterial inclusion bodies (IBs) have revealed intriguing utilities of these protein particles. Since they combine mechanical stability and protein functionality, IBs have been already exploited in biocatalysis and explored for bottom-up topographical modification in tissue engineering. Being fully biocompatible and with tuneable bio-physical properties, IBs are currently emerging as agents for protein delivery into mammalian cells in protein-replacement cell therapies. So far, IBs formed by chaperones (heat shock protein 70, Hsp70), enzymes (catalase and dihydrofolate reductase), grow factors (leukemia inhibitory factor, LIF) and structural proteins (the cytoskeleton keratin 14) have been shown to rescue exposed cells from a spectrum of stresses and restore cell functions in absence of cytotoxicity. The natural penetrability of IBs into mammalian cells (reaching both cytoplasm and nucleus) empowers them as an unexpected platform for the controlled delivery of essentially any therapeutic polypeptide. Production of protein drugs by biopharma has been traditionally challenged by IB formation. However, a time might have arrived in which recombinant bacteria are to be engineered for the controlled packaging of therapeutic proteins as nanoparticulate materials (nanopills), for their extra- or intra-cellular release in medicine and cosmetics.

Biological activities of histidine-rich peptides; merging biotechnology and nanomedicine.

Histidine-rich peptides are commonly used in recombinant protein production as purification tags, allowing the one-step affinity separation of the His-tagged proteins from the extracellular media or cell extracts. Genetic engineering makes feasible the post-purification His-tag removal by inserting, between the tag and the main protein body, a target site for trans-acting proteases or a self-proteolytic peptide with regulatable activities. However, for technical ease, His tags are often not removed and the fusion proteins eventually used in this form. In this commentary, we revise the powerful biological properties of histidine-rich peptides as endosomolytic agents and as architectonic tags in nanoparticle formation, for which they are exploited in drug delivery and other nanomedical applications. These activities, generally unknown to biotechnologists, can unwillingly modulate the functionality and biotechnological performance of recombinant proteins in which they remain trivially attached.

Engineered biological entities for drug delivery and gene therapy protein nanoparticles.

The development of genetic engineering techniques has speeded up the growth of the biotechnological industry, resulting in a significant increase in the number of recombinant protein products on the market. The deep knowledge of protein function, structure, biological interactions, and the possibility to design new polypeptides with desired biological activities have been the main factors involved in the increase of intensive research and preclinical and clinical approaches. Consequently, new biological entities with added value for innovative medicines such as increased stability, improved targeting, and reduced toxicity, among others have been obtained. Proteins are complex nanoparticles with sizes ranging from a few nanometers to a few hundred nanometers when complex supramolecular interactions occur, as for example, in viral capsids. However, even though protein production is a delicate process that imposes the use of sophisticated analytical methods and negative secondary effects have been detected in some cases as immune and inflammatory reactions, the great potential of biodegradable and tunable protein nanoparticles indicates that protein-based biotechnological products are expected to increase in the years to come.

Biomedical applications of distally controlled magnetic nanoparticles.

Nano-sized magnetic particles are increasingly being used across a wide spectrum of biomedical fields. Upon functionalization to enable specific binding, magnetic particles and their targets can be conveniently positioned in vitro and in vivo by the distal application of magnetic fields. Furthermore, such particles can be magnetically heated after reaching their in vivo targets, thus inducing localized cell death that has a considerable therapeutic value in, for instance, cancer therapy. In this context, innovative biomedical research has produced novel applications that have exciting clinical potential. Such applications include magnetically enhanced transfection, magnetically assisted gene therapy, magnetically induced hyperthermia and magnetic-force-based tissue engineering, and the principles and utilities of these applications will be discussed here.

Modular protein engineering in emerging cancer therapies.

An urgent need of more effective and personalized treatments for cancer and other genetic diseases is becoming a generalized claim. This is pushing forward experimental alternative approaches based on targeted nanoconjugates, which are designed to be specifically directed against target cells. These constructs, although suitable to carry conventional chemical drugs, are specifically appropriate to deliver expressible or antisense DNA molecules, silencing RNAs or functional proteins as novel biopharmaceuticals. In this new scenario, the specificity and adequateness of director moieties to target cells is fundamental to achieve successful therapies. In this regard, natural or modified proteins or short peptides offer appropriate tools to functionalize vehicles for targeted drug delivery. Besides, conventional protein engineering allows combining, by recombinant DNA technologies, different active peptides in single-chain polypeptides with modular architecture. This offers intriguing possibilities for the development of multifunctional and smart drug vehicles at the nanoscale. In this review we first discuss the pharmacological applications of recombinant proteins, the procedures to identify, obtain and engineer functional and multifunctional polypeptides for target drug delivery and the potential applications of such constructs in emerging cancer therapies. For that, we discuss in detail the molecular traits in the biology of cancer that are critical for the identification and selection of suitable targets for protein-based drug delivery.