Where in the cell is our cargo? Current methods to study intracellular cytosolic localization

NOTICE: This is the Accepted Version of the following article: Méndez Ardoy, A., Lostalé-Seijo, I., & Montenegro, J. (2018). Where in the cell is our cargo? Current methods to study intracellular cytosolic localization. Chembiochem. doi: 10.1002/cbic.201800390 © 2018 WILEY‐VCH Verlag GmbH & Co. KGaA, WeinheimThe internalization and delivery of active substances into cells is a field of growing interest for chemical biology and therapeutics. As we move from small‐molecule based drugs towards bigger cargos, such as antibodies, enzymes, nucleases or nucleic acids, the development of efficient delivery systems becomes critical for their practical application. Different strategies and synthetic carriers have been developed including cationic lipids, gold nanoparticles, polymers, cell‐penetrating peptides, protein surface modification, etc. However, all these methodologies still present limitations related to the precise targeting of the different intracellular compartments and, in particular, the difficult access to the cellular cytosol. Additionally, the precise quantification of the cellular uptake of a molecule is not enough to demonstrate delivery and/or functional activity. Therefore, methods to determine the cellular distribution of cargos and carriers are of critical importance to identify the barriers that are blocking the activity. In this mini‐review, we survey the different techniques that can be currently used to track and monitor the subcellular localization of the synthetic molecules that we deliver inside cellsWe acknowledge funding from the Spanish Agencia Estatal de Investigación (AEI) [CTQ2014-59646-R, SAF2017-89890-R], the Xunta de Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031) and the ERDF. A. M.-A. received a MCIF from the EC (GLYCONANOPEP-750248). J. M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Investigator Grant (DYNAP-677786) and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017)S

The emergence of order: a closer look on peptide assembly and its complexity

The self-assembly of peptides and proteins frequently shows structural plasticity depending on subtle alterations. The acquisition of structural data of these peptide assemblies with atomic level precision will be crucial to understand and predict assembled morphologies.We thank the AEI [PCI2019-103400, PID2020-117143RB-I00], the ERC (DYNAP-677786, TraffikGene-838002), the HFSP (RGY0066/2017)NO

pH-Triggered self-assembly and hydrogelation of cyclic peptide nanotubes confined in water micro-droplets

This is the Accepted Manuscript of the following article: Méndez-Ardoy, A., Granja, J., & Montenegro, J. (2018). pH-Triggered self-assembly and hydrogelation of cyclic peptide nanotubes confined in water micro-droplets. Nanoscale Horizons. http://dx.doi.org/10.1039/c8nh00009cThe controlled one-dimensional supramolecular polymerization of synthetic building blocks in confined spaces constitutes a key challenge to simplify the understanding of the fundamental physical principles behind the behavior of more complex encapsulated polymer networks. Cyclic peptide nanotubes constitute an optimal scaffold for the fabrication of hierarchical one-dimensional self-assembled architectures. Herein we report the pH-controlled nanotube formation and fibrillation of supramolecular cyclic peptides in confined aqueous droplets. The externally triggered self-assembly of these peptides gave rise to viscoelastic hydrogels in which the one-dimensional molecular arrangement was perfectly preserved from the nano- to the micro-scale. The cyclic peptide building blocks were confined inside water microdroplets and the base-triggered supramolecular polymerization was externally triggered and followed by confocal microscopy showing that the confined fibrillation spanned and affected the shape of the droplet micro containerThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [SAF2017-89890-R, CTQ2014-59646-R and CTQ2016-78423-R], the Xunta de Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031) and the ERDF. A. M.-A. received a MCIF from the EC (GLYCONANOPEP-750248). J. M. holds a Ramón y Cajal (RYC-2013-13784), an ERC-Stg (DYNAP-677786) and a Young Investigator Grant from the HFSP (RGY0066/2017)S

Synthesis and Supramolecular Functional Assemblies of Ratiometric pH Probes

Tracking the pH with spatiotemporal resolution is a critical challenge for synthetic chemistry, chemical biology and beyond. Over the last decade different small probes and supramolecular systems have emerged for in celluloor in vivo pH tracking. However, pH reporting still presents critical limitations such as background reduction, sensor improved stability, cell targeting, endosomal escape, near and far infrared ratiometric pH tracking, adaptation to the new imaging techniques (i.e. super‐resolution), etc. These challenges will demand the combined efforts of synthetic and supramolecular chemistry working together to develop a next generation of smart materials that will resolve the current limitations. In this review we describe the recent advances in the synthesis of small fluorescent probes together with new supramolecular functional systems employed for pH tracking with emphasis in ratiometric probes. The combination of organic synthesis and stimuli‐responsive supramolecular functional materials will be essential to solve future challenges of pH tracking such as the improved signal to noise ratio, on target activation and microenvironment reportingThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [SAF2017-89890-R], the Xunta de Galicia (ED431C 2017/25, 2016-AD031 and Centro Singular de Investigación de Galicia accreditation 2016–2019, ED431G/09), the ISCIII (RD16/0008/003), and the European Union (European Regional Development Fund –ERDF). A.M. received a Marie Curie fellowship (GLYCONANOPEP-750248). J.M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Investigator Grant (DYNAP-677786) and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017)S

An Adhesive Peptide from the C-Terminal Domain of α-Synuclein for Single-Layer Adsorption of Nanoparticles onto Substrates

The two-dimensional (2D) homogeneous assembly of nanoparticle monolayer arrays onto a broad range of substrates constitutes an important challenge for chemistry, nanotechnology, and material science. α-Synuclein (αS) is an intrinsically disordered protein associated with neuronal protein complexes and has a high degree of structural plasticity and chaperone activity. The C-terminal domain of αS has been linked to the noncovalent interactions of this protein with biological targets and the activity of αS in presynaptic connections. Herein, we have systematically studied peptide fragments of the chaperone-active C-terminal sequence of αS and identified a 17-residue peptide that preserves the versatile binding nature of αS. Attachment of this short peptide to gold nanoparticles afforded colloidally stable nanoparticle suspensions that allowed the homogeneous 2D adhesion of the conjugates onto a wide variety of surfaces, including the formation of crystalline nanoparticle superlattices. The peptide sequence and the strategy reported here describe a new adhesive molecule for the controlled monolayer adhesion of metal nanoparticles and sets a stepping-stone toward the potential application of the adhesive properties of αSThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [BIO2015-70092-R, SAF2017-89890-R], the Xunta de Galicia (ED431C 2017/25, 2016-AD031, AGAUR (2017 SGR 324), and Centro Singular de Investigación de Galicia accreditation 2016–2019, ED431G/09), the ISCIII (COV20/00297), and the European Union (European Regional Development Fund – ERDF). X.S. acknowledges funding from the ERC (CONCERT-648201). IRB Barcelona is the recipient of a Severo Ochoa Award (Government of Spain). J.M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Grant (DYNAP-677786), and a Young Investigator Grant from the HFSP (RGY0066/2017)S

Harmonized tuning of nucleic acid and lectin binding properties with multivalent cyclodextrins for macrophage-selective gene delivery

Polycationic amphiphilic cyclodextrins (paCDs) have been shown to behave as efficient non-viral gene carriers paralleling the efficacy of commercial vectors towards a variety of cell lines. Their molecular framework and modular design allow the installation of saccharidic antennae to promote specific carbohydrate–protein interactions, thus potentially endowing them with selective targeting abilities. Yet, the presence of these additional functionalities onto the polycationic cluster may hamper paCD self-assembly and nucleic acid condensation. In this report we describe the influence of paCD mannosylation extent on paCD-pDNA nanocomplex stability as well as the consequences of varying glycotope density on mannose-specific lectin recognition and gene delivery capabilities. The work aims at exploring the potential of this approach to optimize both properties in order to modulate cell transfection selectivity.Ministerio de Economía y Competitividad SAF2013-44021-RJunta de Andalucía FQM-146

Cyclization and self-assembly of cyclic peptides

Part of the Methods in Molecular Biology book series (MIMB,volume 2371)Cyclic peptides are a fascinating class of molecules that can be programmed to fold or self-assemble into diverse mono- and multidimensional structures with potential applications in biomedicine, nanoelectronics, or catalysis. Herein we describe on-resin procedures to carry out head-to-tail peptide cyclization based on orthogonal protected linear structures. We also present essential characterization tools for obtaining dynamic and structural information, including the visualization cyclic peptide assembly into nanotubes (AFM, TEM) as well as the use of fluorescence microscopy2023-10-0

Bottom-up supramolecular assembly in two dimensions

This article is part of the themed collections: Most popular 2022 supramolecular chemistry articles and 2022 Chemical Science Perspective & Review CollectionThe self-assembly of molecules in two dimensions (2D) is gathering attention from all disciplines across the chemical sciences. Attracted by the interesting properties of two-dimensional inorganic analogues, monomers of different chemical natures are being explored for the assembly of dynamic 2D systems. Although many important discoveries have been already achieved, great challenges are still to be addressed in this field. Hierarchical multicomponent assembly, directional non-covalent growth and internal structural control are a just a few of the examples that will be discussed in this perspective about the exciting present and the bright future of two-dimensional supramolecular assembliesThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) (SAF2017-89890-R, PCI2019-103400, and PID2020-117143RB-I00), Xunta de Galicia (ED431C 2017/25, Centro singular de investigación de Galicia accreditation 2019–2022, ED431G 2019/03) and the European Commission (ERDF). I. I. thanks the European Commission (EC) and AEI for MSCA-IF (2018-843332) and Juan de la Cierva (FJCI-2017-31795) fellowships, respectively. J. M. thanks the ERC-STG (DYNAP, 677786), Instituto de Salud Carlos III (COV20/00297), ERC-POC (TraffikGene, 838002), Xunta de Galicia (Oportunius Program) and Human Frontier Science Programme Young Investigator Grant (RGY0066/2017) for fundingS

Targeted gene delivery by new folate-polycationic amphiphilic cyclodextrin-DNA nanocomplexes in vitro and in vivo

Aim Development and evaluation of a new targeted gene delivery system by first preforming self-assembled nanocomplexes from a polycationic amphiphilic cyclodextrin (paCD) and pDNA and then decorating the surface of the nanoparticles with folic acid (FA). Experimental section The cyclodextrin derivative (T2) is a tetradecacationic structure incorporating 14 primary amino groups and 7 thioureido groups at the primary face of a cyclomaltoheptaose (β-CD) core and 14 hexanoyl chains at the secondary face. Results and conclusions T2 complexed and protected pDNA (luciferase-encoding plasmid DNA, pCMVLuc) and efficiently mediated transfection in vitro and in vivo with no associated toxicity. The combination of folic acid with CDplexes afforded ternary nanocomplexes (Fol-CDplexes) that enhanced significantly the transfection activity of pCMVLuc in human cervix adenocarcinoma HeLa cells, especially when formulated with 1 μg FA/μg DNA. The observed transfection enhancement was associated to specific folate receptor (FR)-mediated internalization of Fol-CDplexes, as corroborated by employing a receptor-deficient cell line (HepG2) and an excess of free folic acid. The in vivo studies, including luciferase reporter gene expression and biodistribution, indicated that 24 h after intravenous administration of the T2-pDNA nanocomplexes, transfection takes part mainly in the liver and partially in the lung. Interestingly, the corresponding Fol-CDplexes lead to an increase in the transfection activity in the lung and the liver compared to non-targeted CDplexes. Folate-CDplexes developed in this study have improved transfection efficiency and although various methods have been used for the preparation of ligand–DNA-complexes, covalent binding is usually needed and insoluble aggregates are formed unless the concentration of the components is minimized. However, the complexes developed by first time in this work were prepared by simple mixing. The synthetic nature of this formulation provides the potential of flexibility in terms of composition and the capability of inexpensive and large-scale production of the complexes. These nanovectors may be an adequate alternative to viral vectors for gene therapy in the future.Ministerio de Economía y Competitividad, CTQ2010 - 15848, SAF2010 - 1567

Spatially Controlled Supramolecular Polymerization of Peptide Nanotubes by Microfluidics

The recent advances in the supramolecular polymerization of synthetic building blocks in aqueous conditions has given rise to new artificial and biocompatible functional materials. However, despite the importance of spatially resolved self‐assembly for natural and artificial molecular machines, the spatial control of supramolecular polymerization with synthetic monomers has not been experimentally established yet. Here we describe a microfluidic‐regulated tandem process of supramolecular polymerization and droplet encapsulation to control the position of self‐assembled microfibrillar bundles of cyclic peptide nanotubes in water droplets. This method allowed the precise preferential localization of the fibres either at the interface or into the core of the droplets. UV absorbance, circular dichroism and fluorescence microscopy indicated that the microfluidic control of the stimuli (changes in pH or ionic strength) can be employed to adjust the packing degree and the spatial position of microfibrillar bundles of cyclic peptide nanotubes. Additionally, this spatially organized supramolecular polymerization of peptide nanotubes was applied in the assembly of highly ordered two‐dimensional droplet networksThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [SAF2017-89890-R, CTQ2016-78423-R], the Xunta de Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031) and the ERDF. A. M.-A. received a MCIF from the EC (GLYCONANOPEP-750248). J. M. holds a Ramón y Cajal (RYC-2013-13784), an ERC-Stg (DYNAP-677786) and a Young Investigator Grant from the HFSP (RGY0066/2017). J. R. G. thanks to the mobility program (PRX17/00147)S