Publications by authors named "Antonio Villaverde"

222 Publications

Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies.

Int J Mol Sci 2021 Mar 16;22(6). Epub 2021 Mar 16.

Institute for Biotechnology and Biomedicine, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain.

A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories (an engineered form of ) and mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.
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http://dx.doi.org/10.3390/ijms22063020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001920PMC
March 2021

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

J Extracell Vesicles 2021 Mar 12;10(5):e12058. Epub 2021 Mar 12.

Drug Delivery & Targeting CIBBIM-Nanomedicine Vall d'Hebron Institute of Research Universitat Autònoma de Barcelona Barcelona Spain.

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.
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http://dx.doi.org/10.1002/jev2.12058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953474PMC
March 2021

Specific Cytotoxic Effect of an Auristatin Nanoconjugate Towards CXCR4 Diffuse Large B-Cell Lymphoma Cells.

Int J Nanomedicine 2021 5;16:1869-1888. Epub 2021 Mar 5.

Biomedical Research Institute Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain.

Background And Purpose: Around 40-50% of diffuse large-B cell lymphoma (DLBCL) patients suffer from refractory disease or relapse after R-CHOP first-line treatment. Many ongoing clinical trials for DLBCL patients involve microtubule targeting agents (MTAs), however, their anticancer activity is limited by severe side effects. Therefore, we chose to improve the therapeutic window of the MTA monomethyl auristatin E developing a nanoconjugate, T22-AUR, that selectively targets the CXCR4 receptor, which is overexpressed in many DLBCL cells (CXCR4) and associated with poor prognosis.

Methods: The T22-AUR specificity towards CXCR4 receptor was performed by flow cytometry in different DLBCL cell lines and running biodistribution assays in a subcutaneous mouse model bearing CXCR4 DLBCL cells. Moreover, we determined T22-AUR cytotoxicity using cell viability assays, cell cycle analysis, DAPI staining and immunohistochemistry. Finally, the T22-AUR antineoplastic effect was evaluated in vivo in an extranodal CXCR4 DLBCL mouse model whereas the toxicity analysis was assessed by histopathology in non-infiltrated mouse organs and by in vitro cytotoxic assays in human PBMCs.

Results: We demonstrate that the T22-AUR nanoconjugate displays CXCR4-dependent targeting and internalization in CXCR4 DLBCL cells in vitro as well as in a subcutaneous DLBCL mouse model. Moreover, it shows high cytotoxic effect in CXCR4 DLBCL cells, including induction of G2/M mitotic arrest, DNA damage, mitotic catastrophe and apoptosis. Furthermore, the nanoconjugate shows a potent reduction in lymphoma mouse dissemination without histopathological alterations in non-DLBCL infiltrated organs. Importantly, T22-AUR also exhibits lack of toxicity in human PBMCs.

Conclusion: T22-AUR exerts in vitro and in vivo anticancer effect on CXCR4 DLBCL cells without off-target toxicity. Thus, T22-AUR promises to become an effective therapy for CXCR4 DLBCL patients.
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http://dx.doi.org/10.2147/IJN.S289733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944372PMC
March 2021

Title: insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides.

Microb Cell Fact 2021 Feb 2;20(1):30. Epub 2021 Feb 2.

Institute for Biotechnology and Biomedicine, Autonomous University of Barcelona, 08193, Bellaterra, Barcelona, Spain.

Background: Protein aggregation is a biological event observed in expression systems in which the recombinant protein is produced under stressful conditions surpassing the homeostasis of the protein quality control system. In addition, protein aggregation is also related to conformational diseases in animals as transmissible prion diseases or non-transmissible neurodegenerative diseases including Alzheimer, Parkinson's disease, amyloidosis and multiple system atrophy among others. At the molecular level, the presence of aggregation-prone domains in protein molecules act as seeding igniters to induce the accumulation of protein molecules in protease-resistant clusters by intermolecular interactions.

Results: In this work we have studied the aggregating-prone performance of a small peptide (L6K2) with additional antimicrobial activity and we have elucidated the relevance of the accompanying scaffold protein to enhance the aggregating profile of the fusion protein. Furthermore, we demonstrated that the fusion of L6K2 to highly soluble recombinant proteins directs the protein to inclusion bodies (IBs) in E. coli through stereospecific interactions in the presence of an insoluble protein displaying the same aggregating-prone peptide (APP).

Conclusions: These data suggest that the molecular bases of protein aggregation are related to the net balance of protein aggregation potential and not only to the presence of APPs. This is then presented as a generic platform to generate hybrid protein aggregates in microbial cell factories for biopharmaceutical and biotechnological applications.
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http://dx.doi.org/10.1186/s12934-021-01524-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852131PMC
February 2021

In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics.

Pharmaceutics 2020 Dec 17;12(12). Epub 2020 Dec 17.

Centre d'Étude et de Valorisation de la Diversité Microbienne (CEVDM), Département de Biologie, Université de Sherbrooke, 2500 Boul. Université, Sherbrooke, QC J1K 2R1, Canada.

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 , 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.
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http://dx.doi.org/10.3390/pharmaceutics12121217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766456PMC
December 2020

Developing Protein-Antitumoral Drug Nanoconjugates as Bifunctional Antimicrobial Agents.

ACS Appl Mater Interfaces 2020 Dec 16;12(52):57746-57756. Epub 2020 Dec 16.

Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.

A novel concept about bifunctional antimicrobial drugs, based on self-assembling protein nanoparticles, has been evaluated here over two biofilm-forming pathogens, namely and . 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.
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http://dx.doi.org/10.1021/acsami.0c18317DOI Listing
December 2020

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

Acta Biomater 2021 01 12;119:312-322. Epub 2020 Nov 12.

Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193 Barcelona, Spain; Biomedical Research Institute Sant Pau (IIB Sant Pau), Sant Antoni Mª Claret 167, 08025 Barcelona Spain; Josep Carreras Leukaemia Research Institute (IJC), Barcelona, Spain.

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.
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http://dx.doi.org/10.1016/j.actbio.2020.11.018DOI Listing
January 2021

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

Pharmaceutics 2020 Oct 22;12(11). Epub 2020 Oct 22.

Biomedical Research Institute Sant Pau (IIB Sant Pau), Sant Antoni Mª Claret 167, 08025 Barcelona, Spain.

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). The biodistribution of labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles has been then compared to that of the non-labeled nanoparticle in different CXCR4+ tumor mouse models. We observed that while parental T22-GFP-H6 nanoparticles accumulated mostly and specifically in CXCR4+ tumor cells, labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles showed a dramatic change in the biodistribution pattern, accumulating in non-target organs such as liver or kidney while reducing tumor targeting capacity. Therefore, the use of such labeling molecules should be avoided in target and non-target tissue uptake studies during the design and development of targeted nanoscale drug delivery systems, since their effect over the fate of the nanomaterial can lead to considerable miss-interpretations of the actual nanoparticle biodistribution.
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http://dx.doi.org/10.3390/pharmaceutics12111004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7690626PMC
October 2020

Divalent Cations: A Molecular Glue for Protein Materials.

Trends Biochem Sci 2020 11 3;45(11):992-1003. Epub 2020 Sep 3.

Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain; Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain; CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain. Electronic address:

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. More specifically, we discuss how mechanically stable nanoparticles, fibers, matrices, and hydrogels are generated through their coordination with histidine-rich proteins. We also describe how the rational use of divalent cations combined with simple protein engineering offers unexpected and very simple biochemical approaches to biomaterial design that might address unmet clinical needs in precision medicine.
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http://dx.doi.org/10.1016/j.tibs.2020.08.003DOI Listing
November 2020

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

J Control Release 2020 11 5;327:61-69. Epub 2020 Aug 5.

Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain. Electronic address:

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. Being successful from a functional point of view, IBs pose regulatory concerns to clinical applications because of the mentioned presence of bacterial cell components, including endotoxins. We have here explored the release and activity of the human fibroblast growth factor-2 (hFGF-2) from a novel synthetic material, namely artificial IBs. Being chemically homogenous and compliant with regulatory restrictions, we wondered if these materials would effectively release functional proteins in absence of accompanying bacterial agents. The data provided here fully supports that artificial hFGF-2 IBs act as true and efficient secretory granules and they slowly disintegrate in cell culture to promote wound healing in an in vitro wound healing model. Free from undesired bacterial components, artificial inclusion bodies show promises as delivery agents in regenerative medicine.
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http://dx.doi.org/10.1016/j.jconrel.2020.08.007DOI Listing
November 2020

Nanostructured antimicrobial peptides: The last push towards clinics.

Biotechnol Adv 2020 11 29;44:107603. Epub 2020 Jul 29.

Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain; Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain; CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, Barcelona 08193, Spain.

Peptide drugs hold great potential for the treatment of infectious diseases due to their unconventional mechanisms of action, biocompatibility, biodegradability and ease of synthesis and modification. The increasing rising of bacterial strains resistant to classical antibiotics have pushed the development of new peptide-based antimicrobial therapies. In this context, over the past few years, different approaches have reached a clinical approval. Furthermore, the application of nanotechnological principles to the design of antimicrobial peptide-based composites increases even more the already known benefits of antimicrobial peptides as competent protein drugs. Then, we provide here an overview of the current strategies for antimicrobial peptide discovery and modification and the status of such peptides already under clinical development. In addition, we summarize the innovative formulation strategies for their application, focusing on the controlled self-assembly for the fabrication of antimicrobial nanostructures without the assistance of external nanocarriers, and with emphasis on bioengineering, design of ultra-short peptides and rising insights in bacterial selectivity.
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http://dx.doi.org/10.1016/j.biotechadv.2020.107603DOI Listing
November 2020

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

Acta Biomater 2020 09 27;113:584-596. Epub 2020 Jun 27.

Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), C/Monforte de Lemos 3-5, 28029 Madrid, Spain; Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.actbio.2020.06.033DOI Listing
September 2020

Photobiomodulation: Shining Light on COVID-19.

Photobiomodul Photomed Laser Surg 2020 Jul 9;38(7):395-397. Epub 2020 Jun 9.

Anhembi Morumbi University-UAM, São Paulo, São Paulo, Brazil.

To evaluate the hypothesis that light could reduce the lethality of COVID-19. Most models for projections of the spread and lethality of COVID-19 take into account the ambient temperature, neglecting light. Recent advances in understanding the mechanism of action of COVID-19 have shown that it causes a systemic infection that significantly affects the hematopoietic system and hemostasis, factors extremely dependent of light, mainly in the region of visible and infrared radiation. In the COVID-19 patients hemoglobin is decreasing and protoporphyrin is increasing, generating an extremely harmful accumulation of iron ions in the bloodstream, which are able to induce an intense inflammatory process in the body with a consequent increase in C-reactive protein and albumin. Observing the unsaturation characteristics of the cyclic porphyrin ring allows it to absorb and emit radiation mainly in the visible region. This characteristic can represent an important differential to change this process in the event of an imbalance in this system, through the photobiomodulation to increase the production of adenosine triphosphate (ATP) using red and near-infrared radiation (R-NIR) and vitamin D using ultraviolet B (UVB) radiation. These two compounds have the primary role of activating the defense mechanisms of the immune system, enabling greater resistance of the individual against the attack by the virus. According to the theory of electron excitation in photosensitive molecules, similar to hemoglobin heme, after the photon absorption there would be an increase in the stability of the iron ion bond with the center of the pyrrole ring, preventing the losses of heme function oxygen transport (HbO). The light is also absorbed by cytochrome c oxidase in the R-NIR region, with a consequent increase in electron transport, regulating enzyme activity and resulting in a significant increase of oxygen rate consumption by mitochondria, increasing ATP production. The most favorable range of optical radiation to operate in this system is between R-NIR region, in which cytochrome c oxidase and porphyrin present absorption peaks centered at 640 nm and HbO with absorption peak centered at 900 nm. Based on the mechanisms described earlier, our hypothesis is that light could reduce the lethality of COVID-19.
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http://dx.doi.org/10.1089/photob.2020.4882DOI Listing
July 2020

Engineering Protein Nanoparticles Out from Components of the Human Microbiome.

Small 2020 07 23;16(30):e2001885. Epub 2020 Jun 23.

Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 08193, Spain.

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. Because of their low interactivity with human body components and their tolerated immunogenicity, proteins derived from the human microbiome are appealing and fully biocompatible building blocks for the biofabrication of nonreactive, inert protein materials within the nanoscale. Several phage and phage-like bacterial proteins with natural structural roles are produced in Escherichia coli as polyhistidine-tagged recombinant proteins, looking for their organization as discrete, nanoscale particulate materials. While all of them self-assemble in a variety of sizes, the stability of the resulting constructs at 37 °C is found to be severely compromised. However, the fine adjustment of temperature and Zn concentration allows the formation of robust nanomaterials, fully stable in complex media and under physiological conditions. Then, microbiome-derived proteins show promise for the regulatable construction of scaffold protein nanomaterials, which can be tailored and strengthened by simple physicochemical approaches.
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http://dx.doi.org/10.1002/smll.202001885DOI Listing
July 2020

Recombinant Protein-Based Nanoparticles: Elucidating their Inflammatory Effects In Vivo and their Potential as a New Therapeutic Format.

Pharmaceutics 2020 May 13;12(5). Epub 2020 May 13.

Department of Ruminant Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), 08140 Caldes de Montbui, Spain.

Bacterial inclusion bodies (IBs) are protein-based nanoparticles of a few hundred nanometers formed during recombinant protein production processes in different bacterial hosts. IBs contain active protein in a mechanically stable nanostructured format that has been broadly characterized, showing promising potential in different fields such as tissue engineering, protein replacement therapies, cancer, and biotechnology. For immunomodulatory purposes, however, the interference of the format immunogenic properties-intrinsic to IBs-with the specific effects of the therapeutic protein is still an uncovered gap. For that, active and inactive forms of the catalytic domain of a matrix metalloproteinase-9 (MMP-9 and mutMMP-9, respectively) have been produced as IBs and compared with the soluble form for dermal inflammatory effects in knock-out mice. After protein injections in air-pouches in the mouse model, MMP-9 IBs induce local neutrophil recruitment and increase pro-inflammatory chemokine levels, lasting for at least two days, whereas the effects triggered by the soluble MMP-9 format fade out after 3 h. Interestingly, the IB intrinsic effects (mutMMP-9 IBs) do not last more than 24 h. Therefore, it may be concluded that IBs could be used for the delivery of therapeutic proteins, such as immunomodulating proteins while preserving their stability in the specific tissue and without triggering important unspecific inflammatory responses due to the protein format.
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http://dx.doi.org/10.3390/pharmaceutics12050450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284881PMC
May 2020

Stable anchoring of bacteria-based protein nanoparticles for surface enhanced cell guidance.

J Mater Chem B 2020 06 13;8(23):5080-5088. Epub 2020 May 13.

Department of Molecular Nanoscience and Organic Materials, Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus UAB, Bellaterra 08193, Spain.

In tissue engineering, biological, physical, and chemical inputs have to be combined to properly mimic cellular environments and successfully build artificial tissues which can be designed to fulfill different biomedical needs such as the shortage of organ donors or the development of in vitro disease models for drug testing. Inclusion body-like protein nanoparticles (pNPs) can simultaneously provide such physical and biochemical stimuli to cells when attached to surfaces. However, this attachment has only been made by physisorption. To provide a stable anchoring, a covalent binding of lactic acid bacteria (LAB) produced pNPs, which lack the innate pyrogenic impurities of Gram-negative bacteria like Escherichia coli, is presented. The reported micropatterns feature a robust nanoscale topography with an unprecedented mechanical stability. In addition, they are denser and more capable of influencing cell morphology and orientation. The increased stability and the absence of pyrogenic impurities represent a step forward towards the translation of this material to a clinical setting.
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http://dx.doi.org/10.1039/d0tb00702aDOI Listing
June 2020

Selective delivery of T22-PE24-H6 to CXCR4 diffuse large B-cell lymphoma cells leads to wide therapeutic index in a disseminated mouse model.

Theranostics 2020 6;10(12):5169-5180. Epub 2020 Apr 6.

Biomedical Research Institute Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Pau, Barcelona Spain.

: Novel therapeutic strategies are urgently needed to reduce relapse rates and enhance survival in Diffuse Large B-Cell Lymphoma (DLBCL) patients. CXCR4-overexpressing cancer cells are good targets for therapy because of their association with dissemination and relapse in R-CHOP treated DLBCL patients. Immunotoxins that incorporate bacterial toxins are potentially effective in treating haematological neoplasias, but show a narrow therapeutic index due to the induction of severe side effects. Therefore, when considering the delivery of these toxins as cancer therapeutics, there is a need not only to increase their uptake in the target cancer cells, and their stability in blood, but also to reduce their systemic toxicity. We have developed a therapeutic nanostructured protein T22-PE24-H6 that incorporates exotoxin A from which selectively targets lymphoma cells because of its specific interaction with a highly overexpressed CXCR4 receptor (CXCR4) in DLBCL. : T22-PE24-H6 cytotoxicity and its dependence on the CXCR4 receptor were evaluated in DLBCL cell lines using cell viability assays. Different experiments (mitochondrial membrane potential, Western Blot, Annexin V and DAPI staining) were conducted to determine T22-PE24-H6 cell death mechanisms. imaging and therapeutic effect studies were performed in a disseminated DLBCL mouse model that mimics organ infiltration in DLBCL patients. Finally, immunohistochemistry and histopathology analyses were used to evaluate the antineoplastic effect and systemic toxicity. : T22-PE24-H6 induced selective cell death of CXCR4 DLBCL cells by activating the apoptotic pathway. In addition, repeated T22-PE24-H6 intravenous administration in a CXCR4 DLBCL-disseminated mouse model showed a significant reduction of lymphoma burden in organs clinically affected by DLBCL cells (lymph nodes and bone marrow). Finally, we did not observe systemic toxicity associated to the nanoparticle treatment in non-DLBCL-infiltrated organs. : We have demonstrated here a potent T22-PE24-H6 antineoplastic effect, especially in blocking dissemination in a CXCR4 DLBCL model without associated toxicity. Thereby, T22-PE24-H6 promises to become an effective alternative to treat CXCR4 disseminated refractory or relapsed DLBCL patients.
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http://dx.doi.org/10.7150/thno.43231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196303PMC
April 2020

An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia dissemination.

J Hematol Oncol 2020 04 15;13(1):36. Epub 2020 Apr 15.

Biomedical Research Institute Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Pau, 08041, Barcelona, Spain.

Background: Current acute myeloid leukemia (AML) therapy fails to eliminate quiescent leukemic blasts in the bone marrow, leading to about 50% of patient relapse by increasing AML burden in the bone marrow, blood, and extramedullar sites. We developed a protein-based nanoparticle conjugated to the potent antimitotic agent Auristatin E that selectively targets AML blasts because of their CXCR4 receptor overexpression (CXCR4+) as compared to normal cells. The therapeutic rationale is based on the involvement of CXCR4 overexpression in leukemic blast homing and quiescence in the bone marrow, and the association of these leukemic stem cells with minimal residual disease, dissemination, chemotherapy resistance, and lower patient survival.

Methods: Monomethyl Auristatin E (MMAE) was conjugated with the CXCR4 targeted protein nanoparticle T22-GFP-H6 produced in E. coli. Nanoconjugate internalization and in vitro cell viability assays were performed in CXCR4+ AML cell lines to analyze the specific antineoplastic activity through the CXCR4 receptor. In addition, a disseminated AML animal model was used to evaluate the anticancer effect of T22-GFP-H6-Auristatin in immunosuppressed NSG mice (n = 10/group). U of Mann-Whitney test was used to consider if differences were significant between groups.

Results: T22-GFP-H6-Auristatin was capable to internalize and exert antineoplastic effects through the CXCR4 receptor in THP-1 and SKM-1 CXCR4+ AML cell lines. In addition, repeated administration of the T22-GFP-H6-Auristatin nanoconjugate (9 doses daily) achieves a potent antineoplastic activity by internalizing specifically in the leukemic cells (luminescent THP-1) to selectively eliminate them. This leads to reduced involvement of leukemic cells in the bone marrow, peripheral blood, liver, and spleen, while avoiding toxicity in normal tissues in a luminescent disseminated AML mouse model.

Conclusions: A novel nanoconjugate for targeted drug delivery of Auristatin reduces significantly the acute myeloid leukemic cell burden in the bone marrow and blood and blocks its dissemination to extramedullar organs in a CXCR4+ AML model. This selective drug delivery approach validates CXCR4+ AML cells as a target for clinical therapy, not only promising to improve the control of leukemic dissemination but also dramatically reducing the severe toxicity of classical AML therapy.
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http://dx.doi.org/10.1186/s13045-020-00863-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160905PMC
April 2020

Nanostructured recombinant protein particles raise specific antibodies against the nodavirus NNV coat protein in sole.

Fish Shellfish Immunol 2020 Apr 24;99:578-586. Epub 2020 Feb 24.

Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, 08193, Barcelona, Spain; Department of Cell Biology, Animal Physiology and Immunology, Universitat Autònoma de Barcelona, 08193, Barcelona, Spain. Electronic address:

Nervous necrosis virus (NNV) reassortant strains RGNNV/SJNNV have emerged as a potent threat to the Mediterranean marine aquaculture industry, causing viral encephalopathy and retinopathy (VER) in Senegalese sole (Solea senegalensis). In this study, a cheap and practical vaccine strategy using bacterial inclusion bodies made of the coat protein of a virulent reassortant strain of this betanodavirus was devised. The nanostructured recombinant protein nanoparticles, VNNV-C, were administered without adjuvant to two groups of juvenile sole, one by intraperitoneal injection and the other by oral intubation. Specific antibodies were raised in vivo against the NNV coat protein via both routes, with a substantial specific antibody expansion in the injected group 30 days post homologous prime boost. Expression levels of five adaptive immune-related genes, cd8a, cd4, igm, igt and arg2, were also quantified in intestine, spleen and head kidney. Results showed cd4 and igm were upregulated in the head kidney of injected fish, indicating activation of an adaptive systemic response, while intubated fish exhibited a mucosal response in the intestine. Neither route showed significant differential expression of cd8a. The specific antibody response elicited in vivo and the lack of any signs of toxicity over the 6-week study period in young fish (n = 100), evidences the potential of the nanoparticle as a vaccine candidate.
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http://dx.doi.org/10.1016/j.fsi.2020.02.029DOI Listing
April 2020

The Biological Potential Hidden in Inclusion Bodies.

Pharmaceutics 2020 Feb 15;12(2). Epub 2020 Feb 15.

Department of Ruminant Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), 08140 Caldes de Montbui, Spain.

Inclusion bodies (IBs) are protein nanoclusters obtained during recombinant protein production processes, and several studies have demonstrated their potential as biomaterials for therapeutic protein delivery. Nevertheless, IBs have been, so far, exclusively sifted by their biological activity in vitro to be considered in further protein-based treatments in vivo. Matrix metalloproteinase-9 (MMP-9) protein, which has an important role facilitating the migration of immune cells, was used as model protein. The MMP-9 IBs were compared with their soluble counterpart and with MMP-9 encapsulated in polymeric-based micelles (PM) through ionic and covalent binding. The soluble MMP-9 and the MMP-9-ionic PM showed the highest activity values in vitro. IBs showed the lowest activity values in vitro, but the specific activity evolution in 50% bovine serum at room temperature proved that they were the most stable format. The data obtained with the use of an air-pouch mouse model showed that MMP-9 IBs presented the highest in vivo activity compared to the soluble MMP-9, which was associated only to a low and a transitory peak of activity. These results demonstrated that the in vivo performance is the addition of many parameters that did not always correlate with the in vitro behavior of the protein of interest, becoming especially relevant at evaluating the potential of IBs as a protein-based nanomaterial for therapeutic purposes.
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http://dx.doi.org/10.3390/pharmaceutics12020157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076398PMC
February 2020

Artificial Inclusion Bodies for Clinical Development.

Adv Sci (Weinh) 2020 Feb 27;7(3):1902420. Epub 2019 Nov 27.

Institut de Biotecnologia i de Biomedicina Universitat Autònoma de Barcelona Bellaterra 08193 Barcelona Spain.

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.
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http://dx.doi.org/10.1002/advs.201902420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001620PMC
February 2020

Aggregation-prone peptides modulate activity of bovine interferon gamma released from naturally occurring protein nanoparticles.

N Biotechnol 2020 Jul 3;57:11-19. Epub 2020 Feb 3.

Institute for Biotechnology and Biomedicine, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain; Department of Genetics and Microbiology, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain; Bioengineering, Biomaterials and Nanomedicine Networking Biomedical Research Centre (CIBER-BBN), Bellaterra, Barcelona, Spain. Electronic address:

Efficient protocols for the production of recombinant proteins are indispensable for the development of the biopharmaceutical sector. Accumulation of recombinant proteins in naturally-occurring protein aggregates is detrimental to biopharmaceutical development. In recent years, the view of protein aggregates has changed with the recognition that they are a valuable source of functional recombinant proteins. In this study, bovine interferon-gamma (rBoIFN-γ) was engineered to enhance the formation of protein aggregates, also known as protein nanoparticles (NPs), by the addition of aggregation-prone peptides (APPs) in the generally recognized as safe (GRAS) bacterial Lactococcus lactis expression system. The L6K2, HALRU and CYOB peptides were selected to assess their intrinsic aggregation capability to nucleate protein aggregation. These APPs enhanced the tendency of the resulting protein to aggregate at the expense of total protein yield. However, fine physico-chemical characterization of the resulting intracellular protein NPs, the protein released from them and the protein purified from the soluble cell fraction indicated that the compactability of protein conformations was directly related to the biological activity of variants of IFN-γ, used here as a model protein with therapeutic potential. APPs enhanced the aggregation tendency of fused rBoIFN-γ while increasing compactability of protein species. Biological activity of rBoIFN-γ was favored in more compacted conformations. Naturally-occurring protein aggregates can be produced in GRAS microorganisms as protein depots of releasable active protein. The addition of APPs to enhance the aggregation tendency has a positive impact in overall compactability and functionality of resulting protein conformers.
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http://dx.doi.org/10.1016/j.nbt.2020.02.001DOI Listing
July 2020

Antiedematous Effect Promoted by Occlusion of Legs with Compressive Socks Containing Infrared-Emitting Ceramic Particulates.

Photobiomodul Photomed Laser Surg 2020 Jan;38(1):51-56

Biomedical Engineering Center, Anhembi Morumbi University (UAM), São José dos Campos, São Paulo, Brazil.

Published literature reports significant improvements in pathological conditions, such as pain, blood dyscrasias, and cellulite, after using topical occlusive accessories containing particulate ceramic materials. In this study, we investigated whether the use of a topical occluding garment made with synthetic fibers embedded with powdered ceramic materials could be beneficial to patients suffering from edema of lower limb extremities. The cohort comprised 30 volunteers of both genders who were divided into two study groups. The bioceramic (BC) group wore compressive socks made of fabrics with embedded ceramic powder for 8 h a day for 28 days. The placebo group wore compressive socks of the same material without ceramic powder. The efficacy of the treatment was quantified through weekly plethysmographic measurements. In addition, pain relief was evaluated through a visual analog scale (VAS). Data showed a statistically significant reduction in the edema volume for the BC group compared with the placebo group: 78.9 ± 10.7 mL versus 41.3 ± 5.6 mL,  = 0.003. Further, there was also greater pain relief for the BC group when compared with the placebo group, with a pain decrease of 7.0 ± 0.2 U versus 3.3 ± 0.2 U, respectively, on the VAS from 0 to 10 ( = 0.024). The topical occlusive therapy with compressive socks containing infrared-emitting ceramic particulate in its fabrics showed that they were more beneficial than the placebo garment in the treatment of edema of the inferior member extremities as well as in relief of associated pain.
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http://dx.doi.org/10.1089/photob.2019.4709DOI Listing
January 2020

Nanostructured toxins for the selective destruction of drug-resistant human CXCR4 colorectal cancer stem cells.

J Control Release 2020 04 10;320:96-104. Epub 2020 Jan 10.

Amsterdam UMC, Univ of Amsterdam, LEXOR, Center for Experimental and Molecular Medicine, Cancer Center Amsterdam, Meibergdreef 9, 1105AZ Amsterdam, Netherlands; Oncode Institute, Meibergdreef 9, 1105AZ Amsterdam, Netherlands.

Current therapies fail to eradicate colorectal Cancer Stem Cells (CSCs). One of the proposed reasons for this failure is the selection, by chemotherapy exposure, of resistant cells responsible for tumor recurrence. In this regard, CXCR4 overexpression in tumor associates with resistance and poor prognosis in colorectal cancer (CRC) patients. In this study, the effectiveness of engineered CXCR4-targeted self-assembling toxin nanoparticles has been explored in the selective killing of CXCR4 human colon-CSCs compared to 5-Fluorouracil and Oxaliplatin, both classical CRC chemotherapeutic agents. To assess this, 3D spheroid colon-CSCs cultures directly derived from CRC patients and CRC-CSC spheroid-derived tumor mouse models were developed. In these animal models, nanostructured toxins show highly selective induction of pyroptosis in the absence of apoptosis, thus having a great potential to overcome tumor resistance, since the same tumor models show resistance to chemotherapeutics. Results set the basis for further development of more efficient therapies focused on selective CXCR4 CSCs elimination activating non-apoptotic mechanisms and represent a pre-clinical proof of concept for the use of CSCs-targeted nanostructured toxins as protein drugs for CRC therapy.
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http://dx.doi.org/10.1016/j.jconrel.2020.01.019DOI Listing
April 2020

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

Adv Mater 2020 Feb 27;32(7):e1907348. Epub 2019 Dec 27.

CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), C/Monforte de Lemos 3-5, 28029, Madrid, Spain.

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. These proteins are functionalized with a peptidic ligand of CXCR4, a chemokine receptor overexpressed in metastatic cancer stem cells that confers high selective cytotoxicity in vitro and in vivo. In the mouse models of human colorectal cancer, time-deferred anticancer activity is detected after the subcutaneous deposition of 500 µg of PE24-based amyloids, which promotes a dramatic arrest of tumor growth in the absence of side toxicity. In addition, long-term prevention of lymphatic, hematogenous, and peritoneal metastases is achieved. These results reveal the biomedical potential and versatility of bacterial inclusion bodies as novel tunable secretory materials usable in delivery, and they also instruct how therapeutic proteins, even with high functional and structural complexity, can be packaged in this convenient format.
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http://dx.doi.org/10.1002/adma.201907348DOI Listing
February 2020

Engineering a Nanostructured Nucleolin-Binding Peptide for Intracellular Drug Delivery in Triple-Negative Breast Cancer Stem Cells.

ACS Appl Mater Interfaces 2020 Feb 21;12(5):5381-5388. Epub 2020 Jan 21.

CIBER de Bioingeniería , Biomateriales y Nanomedicina (CIBER-BBN) , C/ Monforte de Lemos 3-5 , 28029 Madrid , Spain.

Five peptide ligands of four different cell surface receptors (nucleolin, CXCR1, CMKLR1, and CD44v6) have been evaluated as targeting moieties for triple-negative human breast cancers. Among them, the peptide F3, derived from phage display, promotes the fast and efficient internalization of a genetically fused green fluorescent protein (GFP) inside MDA-MB-231 cancer stem cells in a specific receptor-dependent fashion. The further engineering of this protein into the modular construct F3-RK-GFP-H6 and the subsequent construct F3-RK-PE24-H6 resulted in self-assembling polypeptides that organize as discrete and regular nanoparticles. These materials, 15-20 nm in size, show enhanced nucleolin-dependent cell penetrability. We show that the F3-RK-PE24-H6, based on the exotoxin A (PE24) as a core functional domain, is highly cytotoxic over target cells. The combination of F3, the cationic peptide (RK), and the toxin domain PE24 in such unusual presentation appears as a promising approach to cell-targeted drug carriers in breast cancers and addresses selective drug delivery in otherwise difficult-to-treat triple-negative breast cancers.
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http://dx.doi.org/10.1021/acsami.9b15803DOI Listing
February 2020

Self-assembling as regular nanoparticles dramatically minimizes photobleaching of tumour-targeted GFP.

Acta Biomater 2020 02 6;103:272-280. Epub 2019 Dec 6.

CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), C/ Monforte de Lemos 3-5, 28029 Madrid, Spain; Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain. Electronic address:

Fluorescent proteins are useful imaging and theranostic agents, but their potential superiority over alternative dyes is weakened by substantial photobleaching under irradiation. Enhancing protein photostability has been attempted through diverse strategies, with irregular results and limited applicability. In this context, we wondered if the controlled oligomerization of Green Fluorescent Protein (GFP) as nanoscale supramolecular complexes could stabilize the fluorophore through the newly formed protein-protein contacts, and thus, enhance its global photostability. For that, we have here analyzed the photobleaching profile of several GFP versions, engineered to self-assemble as tumour-homing nanoparticles with different targeting, size and structural stability. This has been done under prolonged irradiation in confocal laser scanning microscopy and by small-angle X-ray scattering. The results show that the oligomerization of GFP at the nanoscale enhances, by more than seven-fold, the stability of fluorescence emission. Interestingly, GFP nanoparticles are much more resistant to X-ray damage than the building block counterparts, indicating that the gained photostability is linked to enhanced structural resistance to radiation. Therefore, the controlled oligomerization of self-assembling fluorescent proteins as protein nanoparticles is a simple, versatile and powerful method to enhance their photostability for uses in precision imaging and therapy. STATEMENT OF SIGNIFICANCE: Fluorescent protein assembly into regular and highly symmetric nanoscale structures has been identified to confer enhanced structural stability against radiation stresses dramatically reducing their photobleaching. Being this the main bottleneck in the use of fluorescent proteins for imaging and theranostics, this protein architecture engineering principle appears as a powerful method to enhance their photostability for a broad applicability in precision imaging, drug delivery and theranostics.
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http://dx.doi.org/10.1016/j.actbio.2019.12.003DOI Listing
February 2020

High-Throughput Cell Motility Studies on Surface-Bound Protein Nanoparticles with Diverse Structural and Compositional Characteristics.

ACS Biomater Sci Eng 2019 Oct 23;5(10):5470-5480. Epub 2019 Sep 23.

Department of Molecular Nanoscience and Organic Materials, Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus UAB, 08193 Bellaterra, Spain.

Eighty areas with different structural and compositional characteristics made of bacterial inclusion bodies formed by the fibroblast growth factor (FGF-IBs) were simultaneously patterned on a glass surface with an evaporation-assisted method that relies on the coffee-drop effect. The resulting surface patterned with these protein nanoparticles enabled to perform a high-throughput study of the motility of NIH-3T3 fibroblasts under different conditions including the gradient steepness, particle concentrations, and area widths of patterned FGF-IBs, using for the data analysis a methodology that includes "heat maps". From this analysis, we observed that gradients of concentrations of surface-bound FGF-IBs stimulate the total cell movement but do not affect the total net distances traveled by cells. Moreover, cells tend to move toward an optimal intermediate FGF-IB concentration (i.e., cells seeded on areas with high IB concentrations moved toward areas with lower concentrations and vice versa, reaching the optimal concentration). Additionally, a higher motility was obtained when cells were deposited on narrow and highly concentrated areas with IBs. FGF-IBs can be therefore used to enhance and guide cell migration, confirming that the decoration of surfaces with such IB-like protein nanoparticles is a promising platform for regenerative medicine and tissue engineering.
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http://dx.doi.org/10.1021/acsbiomaterials.9b01085DOI Listing
October 2019

Targeting Antitumoral Proteins to Breast Cancer by Local Administration of Functional Inclusion Bodies.

Adv Sci (Weinh) 2019 Sep 24;6(18):1900849. Epub 2019 Jul 24.

Institut de Biotecnologia i de Biomedicina Universitat Autònoma de Barcelona Bellaterra 08193 Barcelona Spain.

Two structurally and functionally unrelated proteins, namely Omomyc and p31, are engineered as CD44-targeted inclusion bodies produced in recombinant bacteria. In this unusual particulate form, both types of protein materials selectively penetrate and kill CD44 tumor cells in culture, and upon local administration, promote destruction of tumoral tissue in orthotropic mouse models of human breast cancer. These findings support the concept of bacterial inclusion bodies as versatile protein materials suitable for application in chronic diseases that, like cancer, can benefit from a local slow release of therapeutic proteins.
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http://dx.doi.org/10.1002/advs.201900849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755514PMC
September 2019

Collaborative membrane activity and receptor-dependent tumor cell targeting for precise nanoparticle delivery in CXCR4 colorectal cancer.

Acta Biomater 2019 11 5;99:426-432. Epub 2019 Sep 5.

CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), C/Monforte de Lemos 3-5, 28029 Madrid, Spain; Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain. Electronic address:

By the appropriate selection of functional peptides and proper accommodation sites, we have generated a set of multifunctional proteins that combine selectivity for CXCR4 cell binding and relevant endosomal escape capabilities linked to the viral peptide HA2. In particular, the construct T22-GFP-HA2-H6 forms nanoparticles that upon administration in mouse models of human, CXCR4 colorectal cancer, accumulates in primary tumor at levels significantly higher than the parental T22-GFP-H6 HA2-lacking version. The in vivo application of a CXCR4 antagonist has confirmed the prevalence of the CXCR4 tumor tissue selectivity over unspecific cell penetration, upon systemic administration of the material. Such specificity is combined with improved endosomal escape, what overall results in a precise and highly efficient tumor biodistribution. These data strongly support the functional recruitment as a convenient approach to generate protein materials for clinical applications. More precisely, they also support the unexpected concept that enhancing the unspecific membrane activity of a protein material does not necessarily compromise, but it can even improve, the selective cell targeting offered by an accompanying functional module. STATEMENT OF SIGNIFICANCE: We have shown here that the combination of cell-penetrating and tumor cell-targeting peptides dramatically enhances precise tumor accumulation of protein-only nanoparticles intended for selective drug delivery, in mouse models of human colorectal cancer. This fact is a step forward for the rational design of multifunctional protein nanomaterials for improved cancer therapies.
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http://dx.doi.org/10.1016/j.actbio.2019.09.002DOI Listing
November 2019