Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles

Serna, Naroa; Céspedes, María Virtudes; Saccardo, Paolo; Xu, Zhongjie; Unzueta, Ugutz; Álamo, Patricia; Pesarrodona, Mireia; Sánchez-Chardi, Alejandro; Roldán, Mónica; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio; Ferrer-Miralles, Neus

doi:10.1016/j.nano.2016.01.004

Cited by 28 publications

(26 citation statements)

References 40 publications

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“…Because of the easy protein engineering, building blocks might also contain functional peptides such as cell‐targeting agents, endosomolytic agents or nuclear localization signals, in the form of fused stretches with modular organization. To explore this innovative concept, we have applied a nanoarchitectonic principle based on the addition, to a core protein, of a cationic N‐terminal domain plus a C‐terminal polyhistidine . It is known that these end‐terminal tags and the resulting charge distribution in the whole fusion promote self‐assembling and oligomerization of monomeric proteins as robust toroid nanoparticles, stable in plasma and with high cellular penetrability if empowered with cell‐targeting peptides .…”

Section: Introductionmentioning

confidence: 99%

Peptide‐Based Nanostructured Materials with Intrinsic Proapoptotic Activities in CXCR4⁺ Solid Tumors

Serna

Céspedes

Sánchez‐García

et al. 2017

Adv Funct Materials

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Protein materials are gaining interest in nanomedicine because of the unique combination of regulatable function and structure. A main application of protein nanoparticles is as vehicles for cell-targeted drug delivery in the form of nanoconjugates, in which a conventional or innovative drug is associated to a carrier protein. Here, a new nanomedical approach based on self-assembling protein nanoparticles is developed in which a chemically homogeneous protein material acts, simultaneously, as vehicle and drug. For that, three proapoptotic peptidic factors are engineered to self-assemble as protein-only, fully stable nanoparticles that escape renal clearance, for the multivalent display of a CXCR4 ligand and the intracellular delivery into CXCR4 + colorectal cancer models. These materials, produced and purified in a single step from bacterial cells, show an excellent biodistribution upon systemic administration and local antitumoral effects. The design and generation of intrinsically therapeutic protein-based materials offer unexpected opportunities in targeted drug delivery based on fully biocompatible, tailor-made constructs.

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Section: Introductionmentioning

confidence: 99%

Peptide‐Based Nanostructured Materials with Intrinsic Proapoptotic Activities in CXCR4⁺ Solid Tumors

Serna

Céspedes

Sánchez‐García

et al. 2017

Adv Funct Materials

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“…In previous studies, we have developed a protein engineering platform to promote the self-assembly of modular GFP constructs, based on the combination of end-terminal cationic stretches and polyhistidines [17,18]. Driven by electrostatic interactions and with a strong involvement of the histidine-rich tail, these peptides promote the formation of stable oligomers of defined average size in the nanoscale irrespective of the amino acid sequence and origin of the core protein placed in between.…”

Section: Introductionmentioning

confidence: 99%

“…When displaying appropriate peptide ligands of cell surface cancer markers CXCR4 or CD44 (T22 and A5G27 respectively) they specifically accumulate in primary tumor and metastasis in colorectal and mammary cancer models respectively [20,21], being suited for antitumoral drug delivery. The same platform has been used to construct fluorescent nanoparticles that cross the blood-brain barrier and target the brain [18].…”

Section: Introductionmentioning

confidence: 99%

Intrinsic functional and architectonic heterogeneity of tumor-targeted protein nanoparticles

et al. 2017

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Self-assembling proteins are gaining attention as building blocks for application-tailored nanoscale materials. This is mostly due to the biocompatibility, biodegradability, and functional versatility of peptide chains. Such a potential for adaptability is particularly high in the case of recombinant proteins, which are produced in living cells and are suitable for genetic engineering. However, how the cell factory itself and the particular protein folding machinery influence the architecture and function of the final material is still poorly explored. In this study we have used diverse analytical approaches, including small-angle X-ray scattering (SAXS) and field emission scanning electron microscopy (FESEM) to determine the fine architecture and geometry of recombinant, tumor-targeted protein nanoparticles of interest as drug carriers, constructed on a GFP-based modular scheme. A set of related oligomers were produced in alternative Escherichia coli strains with variant protein folding networks. This resulted in highly regular populations of morphometric types, ranging from 2.4 to 28 nm and from spherical- to rod-shaped materials. These differential geometric species, whose relative proportions were determined by the features of the producing strain, were found associated with particular fluorescence emission, cell penetrability and receptor specificity profiles. Then, nanoparticles with optimal properties could be analytically identified and further isolated from producing cells for use. The cell’s protein folding machinery greatly modulates the final geometry reached by the constructs, which in turn defines the key parameters and biological performance of the material.Peer ReviewedPostprint (author's final draft

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“…So, rapid and cheap methods such as ours may be applied for the adequate characterization of complex molecules and their future commercialization. Although, in this study, we have evaluated the efficiency of the newly developed protocols with protein‐based aggregates, the study of peptides and proteins in a wide plethora of presentations at nanoscale such as self‐assembling nanoparticles, bioinspired proteosomes, dendrimers, micelles, scaffolds, nanocomposites, coiled‐coils, and vault nanocapsules, among others, could be substantially improved with these two new approaches. Although more specific studies are necessary, the results presented here focused on the use of general methods as a first screening of samples but with the mind in specific protocols to obtain best possible localization of peptides and proteins in a wide variety of samples.…”

Section: Resultsmentioning

confidence: 99%

Improving Biomaterials Imaging for Nanotechnology: Rapid Methods for Protein Localization at Ultrastructural Level

Cano‐Garrido

García‐Fruitós

Villaverde

et al. 2018

Biotechnology Journal

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The preparation of biological samples for electron microscopy is material- and time-consuming because it is often based on long protocols that also may produce artifacts. Protein labeling for transmission electron microscopy (TEM) is such an example, taking several days. However, for protein-based nanotechnology, high resolution imaging techniques are unique and crucial tools for studying the spatial distribution of these molecules, either alone or as components of biomaterials. In this paper, we tested two new short methods of immunolocalization for TEM, and compared them with a standard protocol in qualitative and quantitative approaches by using four protein-based nanoparticles. We reported a significant increase of labeling per area of nanoparticle in both new methodologies (H = 19.811; p < 0.001) with all the model antigens tested: GFP (H = 22.115; p < 0.001), MMP-2 (H = 19.579; p < 0.001), MMP-9 (H = 7.567; p < 0.023), and IFN-γ (H = 62.110; p < 0.001). We also found that the most suitable protocol for labeling depends on the nanoparticle's tendency to aggregate. Moreover, the shorter methods reduce artifacts, time (by 30%), residues, and reagents hindering, losing, or altering antigens, and obtaining a significant increase of protein localization (of about 200%). Overall, this study makes a step forward in the development of optimized protocols for the nanoscale localization of peptides and proteins within new biomaterials.

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Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles

Cited by 28 publications

References 40 publications

Peptide‐Based Nanostructured Materials with Intrinsic Proapoptotic Activities in CXCR4⁺ Solid Tumors

Peptide‐Based Nanostructured Materials with Intrinsic Proapoptotic Activities in CXCR4⁺ Solid Tumors

Intrinsic functional and architectonic heterogeneity of tumor-targeted protein nanoparticles

Improving Biomaterials Imaging for Nanotechnology: Rapid Methods for Protein Localization at Ultrastructural Level

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Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles

Cited by 28 publications

References 40 publications

Peptide‐Based Nanostructured Materials with Intrinsic Proapoptotic Activities in CXCR4+ Solid Tumors

Peptide‐Based Nanostructured Materials with Intrinsic Proapoptotic Activities in CXCR4+ Solid Tumors

Intrinsic functional and architectonic heterogeneity of tumor-targeted protein nanoparticles

Improving Biomaterials Imaging for Nanotechnology: Rapid Methods for Protein Localization at Ultrastructural Level

Contact Info

Product

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Peptide‐Based Nanostructured Materials with Intrinsic Proapoptotic Activities in CXCR4⁺ Solid Tumors

Peptide‐Based Nanostructured Materials with Intrinsic Proapoptotic Activities in CXCR4⁺ Solid Tumors