Nanostructure Empowers Active Tumor Targeting in Ligand‐Based Molecular Delivery

López‐Laguna, Hèctor; Sala, Rita; Sánchez, Julieta M.; Álamo, Patricia; Unzueta, Ugutz; Sánchez‐Chardi, Alejandro; Serna, Naroa; Sánchez‐García, Laura; Voltà‐Durán, Eric; Mangues, Ramón; Villaverde, Antonio; Vázquez, Esther

doi:10.1002/ppsc.201900304

Cited by 10 publications

(17 citation statements)

References 45 publications

(39 reference statements)

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“…Flanking sequences (three residues each end) are also indicated together with the original amino acid numbering of the protein. b) Hydrodynamic diameters of the resulting nanoparticles were previously determined (Table 1in[48]) and confirmed here by DLS.nal and plasma membranes with CellMask TM Deep Red (ThermoFisher) at 2.5 µg mL −1 displaying red signal, both incubated for 10 min at room temperature. The cells were then washed in phosphate saline buffer (Sigma-Aldrich) and the images collected on an inverted TCS SP5 Spectral confocal microscope (Leica Microsystems) using 63× (1.4 NA) oil immersion objective lens.…”

supporting

confidence: 74%

“…To explore the impact of Hn in selfassembling, the H6 end-terminal peptide in T22-GFP-H6 had been replaced by the alternative histidine-rich peptides of similar length, H3A, H5T and H5E, corresponding to HAAHAH, HTHTHTHTH and HEHEHEHEH amino acid sequences respectively. These peptides show at least 90% of homology to sequences of human origin (Table 1), and as in the case of T22-GFP-H6, they also promote the spontaneous formation of nanoparticles ranging from 10 to 18 nm [48]. In this context, we have comparatively characterized the Hn variant nanoparticles, namely T22-GFP-H3A, T22-GFP-H5T, T22-GFP-H5E and T22-GFP-H6 ( Fig.…”

Section: Resultsmentioning

confidence: 95%

“…1), and the histidine-rich (Hn) sequences used here have a human origin (see Table 1). Further details of these proteins can be found elsewhere [48]. E. coli Origami B (BL21, OmpT − , Lon − , TrxB, Gor − ; Novagen) transformed cells were then grown at 37°C overnight in Lysogeny Broth (LB) and the encoded proteins were produced at 20°C overnight by the induction of gene expression with 0.1 mmol L −1 of isopropyl-β-D-thiogalactopyronaside (IPTG) at an optical density (OD) 550 of 0.5-0.7.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

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

et al. 2019

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Poly-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 H6tagged 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 fulllength 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.

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confidence: 74%

Section: Resultsmentioning

confidence: 95%

Section: Protein Production and Purificationmentioning

confidence: 99%

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

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“…More details about the modified His tags used here can be found elsewhere. [36] www.afm-journal.de www.advancedsciencenews.com…”

Section: (4 Of 12)mentioning

confidence: 99%

In Vitro Fabrication of Microscale Secretory Granules

López‐Laguna

Parladé

Álamo

et al. 2021

Adv Funct Materials

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Advanced medical treatments involving drug delivery require fully biocompatible materials with the ability to release functional drugs in a time-prolonged way. Ideally, the delivered molecules should be self-contained as chemically homogenous entities to prevent the use of potentially toxic scaffolds or hold matrices. In nature, peptidic hormones are self-stored in protein-only secretory granules formed by the reversible coordination of Zn 2+ and histidine residues. Inspired by this concept, an in vitro transversal procedure is developed, analyzed, and comparatively applied for the fabrication of protein-only secretory granules at the microscale. These materials can be produced from any polyhistidine-tagged protein using physiological concentrations of Zn 2+ as a potent and versatile glue-like agent. The screening of granules formed by 12 engineered and nonengineered proteins at different Zn 2+ concentrations revealed optimal fabrication conditions and the consequent release profiles. Moreover, the functional and structural properties of the delivered protein are fully validated using a drug-targeting protein platform in a mouse model of human colorectal cancer. In summary, short histidine tags allow the packaging of structurally and functionally dissimilar polypeptides, which supports the proposed fabrication method as a powerful protocol extensible to diverse clinical scenarios in which slow protein drug delivery is required.

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“…[ 31 ] Apparently, this combination of factors ensures reaching the proper biodistribution intrinsically defined by the homing peptide that is necessary but not sufficient for tumor accumulation. [ 32 ] The nanoformulation, dimerization and other particular presentations of venom peptides have been described as positive influencers of their therapeutic values. [ 33 ] However, the targeting effect here provided by T22, namely CXCR4 binding, but also its multiple surface display on oligomeric nanoparticles, are expected to offer additional values over the mere nanoscale size, such as for instance the mimicking of the cooperative attachment of viral particles to target cells.…”

Section: Figurementioning

confidence: 99%

Engineering Protein Venoms as Self‐Assembling CXCR4‐Targeted Cytotoxic Nanoparticles

Serna

Cano‐Garrido

Sánchez‐García

et al. 2020

Part & Part Syst Charact

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as self-assembling at the nanoscale, cell targeting and fluorescent emission, which might be useful for imaging purposes. This can be achieved by the construction of modular proteins that combine protein segments from different origins and with different biological activities. [3] Among such functions, the oligomerization within nanoscale dimensions and the multivalent display of cell surface peptidic ligands [4] are highly desirable to favor the enhanced retention and permeability (EPR) effect and a proper tumor biodistribution, and to estimulate the intracellular delivery in target tissues through cooperative cell binding. The incorporation of C-terminal polyhistidine peptides to modular proteins represents a simple strategy to promote robust self-assembling, as divalent metal and non-metal cations in the media, acting as molecular cross-linkers, induce protein clustering [5] and regular oligomerization in form of stable nanoparticles. [6,7] An N-terminal cationic region in the building blocks is also required for oligomerization. [6,7] Among cytotoxic proteins, venoms developed for animal predation and defense are particularly appealing as cytotoxic drugs, [8,9,10] and the possibility to engineer venoms as Protein venoms are effective cytotoxic molecules that when conveniently targeted to tumoral markers can be exploited as promising anticancer drugs. Here, it is explored whether the structurally unrelated melittin, gomesin, and CLIP71 could be functionally active when engineered, in form of GFP fusions, as self-assembling multimeric nanoparticles. Incorporated in modular constructs including a C-terminal polyhistidine tag and an N-terminal peptidic ligand of the cytokine receptor CXCR4 (overexpressed in more than 20 human neoplasias), these venoms are well produced in recombinant bacteria as proteolytically stable regular nanoparticles ranging between 12 and 35 nm. Being highly fluorescent, these materials selectively penetrate, label, and kill CXCR4 + tumor cells in a CXCR4-dependent fashion. The obtained data support the concept of recombinant venoms as promising drugs, through the precise formulation as tumor-targeted nanomaterials for selective theragnostic applications in CXCR4 + cancers.

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Nanostructure Empowers Active Tumor Targeting in Ligand‐Based Molecular Delivery

Cited by 10 publications

References 45 publications

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

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

In Vitro Fabrication of Microscale Secretory Granules

Engineering Protein Venoms as Self‐Assembling CXCR4‐Targeted Cytotoxic Nanoparticles

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