Fernandez-Megia Lab

16/11/2022

In a recent paper published in Pharmaceutics, we report the synthesis of multivalent aptamers using a dendritic scaffold; a work in collaboration with the group of Carla Cruz (Universidade da Beira Interior, Portugal). These conjugates, named Aptadendrimers, incorporate up to 34 copies of the G-quadruplex AT11 aptamer on a dendrimer. Aptadendrimers are able to complex an acridine orange ligand (anticancer drug and stabilizer of the G-quadruplex structure), which is internalized in cells via endocytosis. The rapid cell internalization makes these conjugates attractive for the development of drug delivery nanocarriers.

Herein, we describe the synthesis of an aptadendrimer by covalent bioconjugation of a gallic acid–triethylene glycol (GATG) dendrimer with the G-quadruplex (G4) AT11 aptamer (a modified version of AS1411) at the surface. We evaluated the loading and interaction of an acridine orange ligand, te...

01/08/2022

In communication published last week in Analytical Chemistry, our group describes the use of Cu2+ (paramagnetic) for the selective NMR-filtering of mixtures.
The paramagnetic spin relaxation (PSR) filter allows the selective NMR signal suppression of components in mixtures according to their complexation ability to a paramagnetic ion. It relies on the faster relaxation of nuclei in paramagnetic environments and thus is complementary to classical diffusion and relaxation filters. So far, the PSR filter has established Gd3+ as the sole PSR agent, restricting the paramagnetic filtering repertoire. Herein, we present Cu2+ as a robust, alternative PSR agent with characteristic filtering properties. While Gd3+ depends on unspecific ion-pair interactions with anionic components, Cu2+ stands out for filtering species via ordered coordination complexes. An evaluation of the paramagnetic effect of Cu2+ over more than 50 small molecules and polymers has unveiled different sensitivities to Cu2+ (especially high for pyridines, diamines, polyamines, and amino alcohols) and precise filtering conditions for mixtures (1H, COSY, and HMQC) that were challenged with a test bed of commercial drugs. The advantage of integrating Cu2+ and Gd3+ for the stepwise PSR filtering of complex mixtures is also shown.

The paramagnetic spin relaxation (PSR) filter allows the selective NMR signal suppression of components in mixtures according to their complexation ability to a paramagnetic ion. It relies on the faster relaxation of nuclei in paramagnetic environments and thus is complementary to classical diffusio...

03/06/2022

Dendrimers are tree-like macromolecules with precise structure and molecular weight. Their globular architecture and the possibility of controlling the number of terminal groups entitle dendrimers with properties and applications unattainable for classical linear polymers. However, the synthesis of dendrimers is so tedious and slow (compared to the one-step synthesis of linear polymers), that unique dendrimer applications might be at risk for the high costs associated. For this reason, the development of accelerated syntheses of dendrimers is a hot topic.
In a recent paper published in Green Chemistry, our group describes an accelerated synthesis of dendrimers combining the thermal azide–alkyne cycloaddition and azide substitution reactions. In addition to highly flexible and modular, the strategy is so user-friendly and accelerated that allows the preparation of a G5 dendrimer with 96 terminal groups in less than 12 h.
https://doi.org/10.1039/D2GC00473A

05/01/2022

In a paper recently published in ACS Appl. Mater. Interfaces, our group in collaboration with that of Ricardo A. Pires (University of Minho, Portugal) have described dendrimers with a precise number of gallic acids on the periphery that are able to interact with monomeric/oligomeric amyloid-β (Aβ) peptides, promoting their remodeling into noncytotoxic aggregates in a process controlled by the multivalent presentation of gallic acids. As the self-assembled Aβ oligomers are linked to the onset and progression of Alzheimer’s disease, we believe our results set a strong background for testing the ability of these dendrimers to cross the blood−brain barrier in vivo and reach the affected region of the brain.

The self-assembly of amyloid-β (Aβ) generates cytotoxic oligomers linked to the onset and progression of Alzheimer’s disease (AD). As many fundamental molecular pathways that control Aβ aggregation are yet to be unraveled, an important strategy to control Aβ cytotoxicity is the development of ...

19/11/2021

A new paper from the group published in ACS Macro Lett describes the Saturation Transfer Difference (STD) experiment to quickly identify the existence of an invisible fraction of nuclei (IF) in the 1H NMR of polymers in solution.
It is well known that the observation of signals in solution NMR requires nuclei with sufficiently large transverse relaxation times (T2). Otherwise, broad signals embedded in the baseline afford an IF. IF-STD is presented as a quick tool to unveil IF in the 1H NMR spectra of polymers. Analysis of a wide collection of polymers by IF-STD reveals IF more common than previously thought, with relevant IF figures when STD > 0.4% at 750 MHz. Interestingly, since nuclei observed (edited) by IF-STD at the visible–invisible interphase are in close spatial proximity to the IF, they emerge as a privileged platform from which gaining an insight into the IF itself.

The observation of signals in solution NMR requires nuclei with sufficiently large transverse relaxation times (T2). Otherwise, broad signals embedded in the baseline afford an invisible fraction of nuclei (IF). Based on the STD (saturation transfer difference) sequence, IF-STD is presented as a qui...

16/10/2021

Review on dendrimer-containing LbL nanoassemblies
A few days ago a review on the use of dendrimer-containing layer-by-layer (LbL) nanoassemblies for bioapplications has been published in Polymer Chemistry. The review written in collaboration with Cristiana F. V. Sousa, Dr. João Borges and Prof. João F. Mano (Univ. Aveiro, Portugal) covers the multitude of intermolecular interactions behind the build-up of supramolecular dendrimer-containing multifunctional LbL with improved properties for bioapplications in controlled drug/therapeutics/nucleic acid delivery, gene therapy, biosensing, bioimaging, and tissue engineering and regenerative medicine.

Dendrimers are powerful synthetic macromolecular architectures for a wide variety of bioapplications owing to their unique and superior features, including monodispersity, well-defined and highly branched architecture, multivalency, tunable size and shape, good water solubility, bioavailability, and...

15/03/2021

A dendritic polymer therapeutic reduces amyloid-beta (Aβ) oligomers
The progressive accumulation of Aβ in specific areas of the brain is a common prelude to late-onset of Alzheimer’s disease (AD). Although activation of liver X receptors (LXR) with agonists decreases Aβ levels, concomitant hypercholesterolemia/hypertriglyceridemia limits their clinical application.
A new paper in collaboration with the group of Prof. Martin A. Bruno (Universidad Católica de Cuyo, Argentina) and other groups from Argentina, Canada and USA has been recently published in ACS Nano. In this paper we describe a dendritic polymer therapeutic for the delivery of DMHCA, an LXR partial agonist that, despite inducing the expression of apolipoprotein E (main responsible of Aβ drainage from the brain), shows nil activity in vivo because of a low solubility and inability to cross the blood brain barrier.
The covalent incorporation of DMHCA into a PEG-dendritic scaffold via carboxylate esters produces an amphiphilic copolymer that efficiently self-assembles into nanometric micelles that exert a biological effect in primary cultures of the central nervous system (CNS) and experimental animals using the intranasal route. After CNS biodistribution and an effective dose were determined in nontransgenic mice, a transgenic AD-like mouse model of cerebral amyloidosis was treated with the micelles for 21 days. The benefits of the treatment included prevention of memory deterioration and a significant reduction of hippocampal Aβ oligomers without affecting plasma lipid levels. These results represent a proof of principle for further clinical developments of DMHCA delivery systems.

The progressive accumulation of amyloid-beta (Aβ) in specific areas of the brain is a common prelude to late-onset of Alzheimer’s disease (AD). Although activation of liver X receptors (LXR) with agonists decreases Aβ levels and ameliorates contextual memory deficit, concomitant hypercholesterol...

09/03/2021

Dendrimers for food smart packaging
In a new paper recently published in ACS Appl Polym Mater, we describe an anionic dendrimer as host for the color stabilization of the short-living flavylium cation of pyranoanthocyanins. By using NMR (chemical shift, diffusion, relaxation) and UV−vis titrations, it was revealed that the structure and concentration of the dye modulate the host−guest interaction mechanisms. This research in collaboration with the group of Luis Cruz (University of Porto) aims at developing pH-sensitive biocompatible dyes for food smart packaging.
Stay tuned! Completely redesigned host-guest dendritic systems in the oven are able to stabilize the flavylium cation hundred times more efficiently.
https://pubs.acs.org/doi/10.1021/acsapm.0c01321

28/11/2020

The COVID-19 pandemic is disturbing our lives with an unexpected intensity. Only the efforts by the research community worldwide put some light at the end of the tunnel. We are proud to announced financial aid to our group from Axencia Galega de Innovación (GAIN), Xunta de Galicia and FEDER to develop Affidendrons as multivalent nanotools for the early diagnosis and antiviral treatment of COVID-19.
Affidendrons are multivalent conjugates incorporating small affitiny proteins named Affitins, develop at the CNRS in Nantes, around a dendritic scaffold. As a result of the multivalent presentation, their affinity increases exponentially as recently described with a prototype against Staphylococcus aureus (https://doi.org/10.1021/acsami.9b05702), a bacteria causing numerous hospital- and community-acquired infections. Affidendrons recognizing the SARS-CoV-2 virus will be used to develop antigen tests for the early diagnosis of COVID-19. These tests are rapid (15-20 min) and benefit from a reduced price and easy handling, which makes them an excellent complement to PCR for the quick and periodic analysis of large groups of population.

02/09/2019

Polysaccharides Meet Dendrimers to Fine-tune the Stability and Release Properties of Polyion Complex Micelles:
Despite the extensive application of charged polysaccharides (chitosan, alginate, hyaluronic acid) in the preparation of charged polyelectrolyte complexes for drug delivery, their application to neutral polyion complex (PIC) micelles is surprisingly very scarce, with reports describing unfeasible micelle formation or complete dissociation at physiological pH/ionic strength.
In a recent paper appeared in Polymer Chemistry, our lab has described that polysaccharide-based PIC micelles can be readily obtained at physiological pH/ionic strength bringing into play PEG-dendritic block copolymers. Indeed, the intrinsic rigidity and globular nature of the dendritic block results in micelles with stabilities that largely exceed those from linear copolymers; with sizes and polydispersities unaltered even after days at 37 ºC in the presence of 150 mM NaCl. Remarkably, prolonged micelle stability was revealed on reducing the polysaccharide molecular weight (MW), an effect not previously observed for PIC and associated to the high stiffness of charged polysaccharides. The polysaccharide MW-dependent properties of these micelles were analyzed via encapsulation of the anticancer drug doxorubicin, which confirmed a higher compactness and slower intracellular drug release of micelles prepared from shorter polysaccharides. Overall, dendritic-polysaccharide PIC micelles represent promising delivery systems where dendritic rigidity and polysaccharide stiffness synchronize to determine the stability, release properties, and cytotoxicity of the system.
https://pubs.rsc.org/en/content/articlelanding/2019/py/c9py00727j/unauth #!divAbstract

09/07/2019

Dendrimers as Color-Stabilizing Agents
Anthocyanins are glycosylated flavonoids responsible for a pallet of colors found in fruits and flowers which rely on complex, pH-dependent equilibria. While colored species are observed at acidic pH, color fading results under moderated acidic and neutral conditions. To circumvent this shortcoming, many plants have developed red and blue color-stabilizing mechanisms that via non-covalent interactions protect at high pH a key flavylium cation intermediate responsible of color.
In a paper recently accepted in Chemistry, A European Journal in collaboration with the group of Luís Cruz from the University of Porto (Portugal), we describe that anionic dendrimers are able to protect that cationic species via electrostatic ion pair recognition. Using UV-Vis, stopped-flow and NMR techniques, we have demonstrated that a G4 dendrimer with 162 terminal sulfate groups is able to interact with two flavylium cations per sulfate (association constant ca 700 M-1), which ultimately results in a red color stabilization.
Applications of this dendrimer to the development of sensors for biomedical devices and smart packaging solutions are envisaged. You can read the paper at

True colors: The interaction of two anthocyanins, cyanidin‐3‐glucoside (cy3glc) and malvidin‐3‐glucoside (mv3glc), with a water‐soluble polyanionic dendrimer was studied through UV/Vis, stopped‐flow,...

25/06/2019

New paper on dendrimers interacting with intrinsically disordered proteins (IDP)
Protein-protein interactions (PPI) are fundamental processes for programmed cell death, cell division, transcription and signal transduction. While in well-folded proteins, drugs mimicking the interacting surfaces can be used as a first step for the design of PPI inhibitors, when targeting an IDP , the design of drugs is a mounting task.
NUPR1 is an 82-residue-long (8 kDa), monomeric, basic IDP over-expressed during the acute phase of pancreatitis. It plays key roles in apoptosis by interacting with prothymosin alpha, another IDP, and it is also implicated in DNA binding and repair, through contacts with the male specific lethal protein 1 (MSL1).

In this work, a joint effort coordinated by J. L. Neira (Universidad Miguel Hernández) and our group, which has also involved groups from Universidad de Zaragoza, University of Calabria, and Aix-Marseille Universite, we hypothesize that since dendrimers can alter the quaternary interactions of well-folded proteins, they could also have the potential to destabilize the PPI of IDP. With this aim we have selected NUPR1 as a proof-of-concept. Our results, obtained with several generations of different GATG dendrimers (anionic, cationic, neutral) indicate that indeed dendrimers are able to bind NUPR1. The functionality at the dendrimer periphery modulates the affinity for NUPR1, and in contrast to well-folded proteins, affinity increases with generation. Several techniques have been used in this study namely, fluorescence, CD, NMR, ITC, and in silico techniques consisting of molecular dynamics and docking simulations.

The affinities of most of the dendrimers were in the range 4-40 x 103 M-1, and for [Gn]-PhCO2Na were similar to those of NUPR1 for its natural partners (0.1-1 x 106 M-1). In all dendrimers, the residues of NUPR1 firstly affected upon binding were located around Ala33, indicating that NUPR1 employs the same hot-spot to recognize any natural or synthetic molecule.

Because of the inherently modular and adaptable nature of dendrimers, and the complexity and variety of interactions that can be stablished with proteins, it is likely that dendrimers can be developed with increased affinity and specificity toward IDP. We suggest that the chemical space of dendrimers may include molecular combinations that are capable of competing with the biological binders of an IDP, ultimately resulting in the possibility to modulate or inhibit its function.

NUPR1 is a protumoral multifunctional intrinsically disordered protein, which is activated during the acute phases of pancreatitis, interacting with several biomolecules through residues around Ala33 and Thr68. Because of the large size of this hot-spot, designed small molecules could be insufficien...