Editorial: High added-value nanoparticles: Rethinking and recycling cell protein waste

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Endosomal escape of protein nanoparticles engineered through humanized histidine-rich peptides

Altres ajuts: EU COST Action CA 17140. AV received an ICREA ACADEMIA awardPoly-histidine peptides such as H6 (HHHHHH) are used in protein biotechnologies as purification tags, protein-assembling agents and endosomal-escape entities. The pleiotropic properties of such peptides make them appealing to design protein-based smart materials or nanoparticles for imaging or drug delivery to be produced in form of recombinant proteins. However, the clinical applicability of H6-tagged proteins is restricted by the potential immunogenicity of these segments. In this study, we have explored several humanized histidine-rich peptides in tumor-targeted modular proteins, which can specifically bind and be internalized by the target cells through the tumoral marker CXCR4. We were particularly interested in exploring how protein purification, self-assembling and endosomal escape perform in proteins containing the variant histidine-rich tags. Among the tested candidates, the peptide H5E (HEHEHEHEH) is promising as a good promoter of endosomal escape of the associated full-length protein upon endosomal internalization. The numerical modelling of cell penetration and endosomal escape of the tested proteins has revealed a negative relationship between the amount of protein internalized into target cells and the efficiency of cytoplasmic release. This fact demonstrates that the His-mediated, proton sponge-based endosomal escape saturates at moderate amounts of internalized protein, a fact that might be critical for the design of protein materials for cytosolic molecular delivery

Editorial: High added-value nanoparticles: Rethinking and recycling cell protein waste

Extracellular vesicles; Biomedicine; NanobiotechnlogyVesículas extracelulares; Biomedicina; NanobiotecnologíaVesícules extracel·lulars; Biomedicina; Nanobiotecnologi

Towards Protein-Based Viral Mimetics for Cancer Therapies

High resistance and recurrence rates, along with elevated drug clearance, compel the use of maximum tolerated drug doses in cancer therapy, resulting in high-grade toxicities and limited clinical applicability. Promoting active drug accumulation in tumor tissues would minimize such issues and improve therapeutic outcomes. A new class of therapeutic drugs suitable for the task has emerged based on the concept of virus-mimetic nanocarriers, or 'artificial viruses.' Among the spectrum of materials under exploration in nanocarrier research, proteins offer unparalleled structural and functional versatility for designing viruslike molecular vehicles. By exhibiting 'smart' functions and biomimetic traits, protein-based nanocarriers will be a step ahead of the conventional drug-protein conjugates already in the clinics in ensuring efficient delivery of passenger antitumor drugs

CXCR4(+)-targeted protein nanoparticles produced in the food-grade bacterium Lactococcus lactis

Lactococcus lactis is a Gram-positive (endotoxin-free) food-grade bacteria exploited as alternative to Escherichia coli for recombinant protein production. We have explored here for the first time the ability of this platform as producer of complex, self-assembling protein materials. Materials & methods: Biophysical properties, cell penetrability and in vivo biodistribution upon systemic administration of tumor-targeted protein nanoparticles produced in L. lactis have been compared with the equivalent material produced in E. coli. Results: Protein nanoparticles have been efficiently produced in L. lactis, showing the desired size, internalization properties and biodistribution. Conclusion: In vitro and in vivo data confirm the potential and robustness of the production platform, pointing out L. lactis as a fascinating cell factory for the biofabrication of protein materials intended for therapeutic applications.Award-winningPostprint (published version

BBB-targeting, protein-based nanomedicines for drug and nucleic acid delivery to the CNS

The increasing incidence of diseases affecting the central nervous system (CNS) demands the urgent development of efficient drugs. While many of these medicines are already available, the Blood Brain Barrier and to a lesser extent, the Blood Spinal Cord Barrier pose physical and biological limitations to their diffusion to reach target tissues. Therefore, efforts are needed not only to address drug development but specially to design suitable vehicles for delivery into the CNS through systemic administration. In the context of the functional and structural versatility of proteins, recent advances in their biological fabrication and a better comprehension of the physiology of the CNS offer a plethora of opportunities for the construction and tailoring of plain nanoconjugates and of more complex nanosized vehicles able to cross these barriers. We revise here how the engineering of functional proteins offers drug delivery tools for specific CNS diseases and more transversally, how proteins can be engineered into smart nanoparticles or 'artificial viruses' to afford therapeutic requirements through alternative administration routes

Intracellular trafficking of a dynein-based nanoparticle designed for gene delivery

The success of viruses in the delivery of the viral genome to target cells relies on the evolutionary selection of protein-based domains able to hijack the intermolecular interactions through which cells respond to intra- and extracellular stimuli. In an effort to mimic viral infection capabilities during non-viral gene delivery, a modular recombinant protein named T-Rp3 was recently developed, containing a DNA binding domain, a dynein molecular motor interacting domain, and a TAT-derived transduction domain. Here, we analyzed at the microscopic level the mechanisms behind the cell internalization and intracellular trafficking of this highly efficient modular protein vector. We found that the protein has the ability to self-assemble in discrete protein nanoparticles resembling viral capsids, to bind and condense plasmid DNA (pDNA), and to interact with eukaryotic cell membranes. Confocal and single particle tracking assays performed on living HeLa cells revealed that the T-Rp3 nanoparticles promoted an impressive speed of cellular uptake and perinuclear accumulation. Finally, the protein demonstrated to be a versatile vector, delivering siRNA at efficiencies comparable to Lipofectamine™. These results demonstrate the high potential of recombinant modular proteins with merging biological functions to fulfill several requirements needed to obtain cost-effective non-viral vectors for gene-based therapies

Biofabricació de nanopartícules proteiques funcionals mitjançant l'ús de cues d'histidina

Investigadors del grup de Nanobiotecnologia del IBB-UAB en col·laboració amb CIBER-BBN i ICTS-Nanbiosis-PPP han desenvolupat una nova tècnica de fabricació de nanopartícules proteiques amb funcionalitats úniques i útils a la biomedicina i la biotecnologia. El procediment de síntesi, eficient i econòmic, es podueix mitjançant l'ús de pèptids d'histidina i de cations divalents.Investigadores del grupo de Nanobiotecnología del IBB-UAB en colaboración con CIBER-BBN e ICTS-Nanbiosis-PPP han desarrollado una nueva técnica de fabricación de nanopartículas proteicas con funcionalidades únicas y útiles en la biomedicina y la biotecnología. El procedimiento de síntesis, eficiente y económico, se produce mediante el uso de péptidos de histidina y de cationes divalentes.Researchers from the IBB-UAB Nanobiotechnology group in collaboration with CIBER-BBN and ICTS-Nanbiosis-PPP have developed a new technique for the fabrication of protein nanoparticles with unique and useful functionalities in biomedicine and biotechnology. The efficient and economical synthesis procedure is performed using histidine peptides and divalent cations

Insights on the emerging biotechnology of histidine-rich peptides

Altres ajuts: Acord transformatiu CRUE-CSICIn 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

Protein-based therapeutic killing for cancer therapies

Altres ajuts: CIBER-BBN (project NANOPROTHER) to A.V.; the Marató de TV3 foundation (TV32013-3930); CIBER-BBN (NanoMets 3) to R.M. in support of our research on cell-targeted antitumor drugs. N.S. by a predoctoral fellowship from the Gobierno de Navarra, R.D.O. by an overseas predoctoral fellowship from Conacyt (Gobierno de Méjico, 2016), U.U. received a Sara Borrell postdoctoral fellowship from ISCIII, and A.V. an Institución Catalana de Investigación y Estudios Avanzados (ICREA) ACADEMIA award.The treatment of some high-incidence human diseases is based on therapeutic cell killing. In cancer this is mainly achieved by chemical drugs that are systemically administered to reach effective toxic doses. As an innovative alternative, cytotoxic proteins identified in nature can be adapted as precise therapeutic agents. For example, individual toxins and venom components, proapoptotic factors, and antimicrobial peptides from bacteria, animals, plants, and humans have been engineered as highly potent drugs. In addition to the intrinsic cytotoxic activities of these constructs, their biological fabrication by DNA recombination allows the recruitment, in single pharmacological entities, of diverse functions of clinical interest such as specific cell-surface receptor binding, self-activation, and self-assembling as nanoparticulate materials, with wide applicability in cell-targeted oncotherapy and theragnosis