In Cellulo and In Vivo Comparison of Cholesterol, Beta-Sitosterol and Dioleylphosphatidylethanolamine for Lipid Nanoparticle Formulation of mRNA.

Lipid Nanoparticles (LNPs) are a leading class of mRNA delivery systems. LNPs are made of an ionizable lipid, a polyethyleneglycol (PEG)-lipid conjugate and helper lipids. The success of LNPs is due to proprietary ionizable lipids and appropriate helper lipids. Using a benchmark lipid (D-Lin-MC3) we compared the ability of three helper lipids to transfect dendritic cells in cellulo and in vivo. Studies revealed that the choice of helper lipid does not influence the transfection efficiency of immortalized cells but, LNPs prepared with DOPE (dioleylphosphatidylethanolamine) and ß-sitosterol were more efficient for mRNA transfection in murine dendritic cells than LNPs containing DSPC (distearoylphosphatidylcholine). This higher potency of DOPE and ß-sitosterol LNPs for mRNA expression was also evident in vivo but only at low mRNA doses. Overall, these data provide valuable insight for the design of novel mRNA LNP vaccines.

Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

Bambusuril Macrocycles as Mediators of Supramolecular Interactions: Application to the Europium Cage Helicate.

Herein, it is shown how bambusurils can be used for tuning and/or characterizing supramolecular systems. Indeed, the addition of bambusurils as anion scavengers to metal-mediated self-assemblies allows manipulation of the subtle equilibria in the given system. This is demonstrated for the case of the tetranuclear europium helical cage, which is well suited to different applications. Among the reported results, experimental evidence is provided showing that perchlorate and triflate anions act as a molecular template for the cage assembly. The complexation of inorganic anions with neutral bambusurils resulted in bulky non-coordinating counterions that may trigger the self-assembly process or stimulate specific interactions between components. Moreover, bambusuril was able to selectively remove coordinating nitrates from the mixture with non-coordinating anions, enabling the regeneration of the helical cage.

Kinetic theory of hyaluronan cleavage by bovine testicular hyaluronidase in standard and crowded environments.

In this paper, we introduce a comprehensive kinetic model describing the enzymatic cleavage of hyaluronan (HA) by bovine testicular hyaluronidase (BTH). Our theory focuses specifically on the late stage of the hydrolysis, where the concentrations of a limited number of oligomers may be determined experimentally with accuracy as functions of time. The present model was applied to fit different experimental sets of kinetic data collected by capillary electrophoresis at two HA concentrations and three concentrations of PEG crowder (0, 10, 17% w/w). Our theory seems to apply universally, irrespective of HA concentration and crowding conditions, reproducing to an excellent extent the time evolution of the individual molar fractions of oligomers. Remarkably, we found that the reaction mechanism in the late degradation stage essentially reduces to the cleavage or transfer of active dimers. While the recombination of dimers is the fastest reaction, the rate-limiting step turns out to be invariably the hydrolysis of hexamers. Crowding, HA itself or other inert, volume-excluding agents, clearly boosts recombination events and concomitantly slows down all fragmentation pathways. Overall, our results bring a novel and comprehensive quantitative insight into the complex reaction mechanism underlying enzymatic HA degradation. Importantly, rationalizing the effect of crowding not only brings the intricate conditions of in-vivo settings a little closer, but also emerges as a powerful tool to help pinpointing relevant kinetic pathways in complex systems.

Polyethylene glycol crowding effect on hyaluronidase activity monitored by capillary electrophoresis.

To mimic the activity of hyaluronidase in natural environment, the hydrolysis of hyaluronic acid (HA) by hyaluronidase was investigated for the first time in the presence of crowding agents using capillary electrophoresis (CE) as a simple and reliable technique for conducting enzymatic assay. Polyethylene glycol (PEG) 6000 was selected as a model crowder and the hyaluronic acid degradation catalyzed by bovine testes hyaluronidase (BTH) was carried out at different PEG concentrations (0%, 10%, and 17%). After optimization of the CE analytical method and enzymatic assay, the degradation products were monitored at different HA concentrations. At 10% of PEG and 0.3 mg mL-1 of HA, the activity of the enzyme was significantly reduced showing inconvenient interactions of PEG with the hyaluronidase blocking the release of hydrolysis products. A similar reduction of hyaluronidase activity was observed at 1 mg mL-1 of HA due to the presumable formation of the BTH-substrate complex. The experimental curves obtained by CE also evidence that the overall kinetics are governed by the hydrolysis of hexasaccharide intermediates. Finally, the effect of PEG on hyaluronidase activity was evaluated in the presence of natural or synthetic inhibitors. Our results show a significant difference of the inhibitors' affinity toward hyaluronidase in the presence of PEG. Surprisingly, the presence of the crowding agent results in a loss of the inhibition effect of small polycyclic inhibitors, while larger charged inhibitors were less affected. In this work, CE analyses confirm the importance of mimicking the cellular environment for the discovery and development of reliable inhibitors. Graphical abstract.

Crowding-Induced Uncompetitive Inhibition of Lactate Dehydrogenase: Role of Entropic Pushing.

The cell is an extremely complex environment, notably highly crowded, segmented, and confining. Overall, there is overwhelming and ever-growing evidence that to understand how biochemical reactions proceed in vivo, one cannot separate the biochemical actors from their environment. Effects such as excluded volume, obstructed diffusion, weak nonspecific interactions, and fluctuations all team up to steer biochemical reactions often very far from what is observed in ideal conditions. In this paper, we use Ficoll PM70 and PEG 6000 to build an artificial crowded milieu of controlled composition and density in order to assess how such environments influence the biocatalytic activity of lactate dehydrogenase (LDH). Our measurements show that the normalized apparent affinity and maximum velocity decrease in the same fashion, a behavior reminiscent of uncompetitive inhibition, with PEG resulting in the largest reduction. In line with previous studies on other enzymes of the same family, and in agreement with the known role of a surface loop involved in enzyme isomerization and regulation of access to the active site, we suggest that the crowding matrix interferes with the conformational ensemble of the enzyme. This likely results in both impaired enzyme-complex isomerization and thwarted product release. Molecular dynamics simulations confirm that excluded-volume effects lead to an entropic force that effectively tends to push the loop closed, thereby effectively shifting the conformational ensemble of the enzyme in favor of a more stable complex isoform. Overall, our study substantiates the idea that most biochemical kinetics cannot be fully explained without including the subtle action of the environment where they take place naturally, in particular accounting for important factors such as excluded-volume effects and also weak nonspecific interactions when present, confinement, and fluctuations.

Functionalized Triptycene-Derived Tripodal Ligands: Privileged Formation of Tetranuclear Cage Assemblies with Larger Ln(III).

In this Article, we report the self-assembly of lanthanide complexes formed with two new tripodal ligands, L2 and L3, where binding strands are connected to a rigid triptycene anchor. The pyridine moieties are functionalized with methoxy and PEG groups to enhance ligand solubility and to evaluate the effect of these substituents on lanthanide coordination. These ligands were successfully synthesized and characterized, and their coordination properties were examined along the lanthanide series through speciation studies with NMR and ESI-MS. Well-defined tetranuclear complexes are formed with both ligands, but their stabilities with heavier lanthanides are considerably reduced, especially for complexes with L3. This is attributed to a destabilizing effect of pending PEG arms in combination with increased steric hindrance between binding strands upon complexation with smaller cations. The sensitization of lanthanide luminescence in tetranuclear complexes occurs despite one water molecule being coordinated to a metal ion.

Designing artificial 3D helicates: unprecedented self-assembly of homo-octanuclear tetrapods with europium.

Herein, we report on the rational design, preparation and characterization of a novel homo-octanuclear helicate, which results from a spatial extension of the central tetranuclear platform. The 3D supramolecular assembly is obtained by complexing europium(III) with a new hexatopic tripodal ligand. The isolated octanuclear helicate is fully characterized by different methods clearly evidencing the structure predicted with molecular modelling. The ligand preorganization plays a crucial role in a successful self-assembly process and induces the formation of a well-defined triple-stranded helical structure. This prototypal octanuclear edifice accommodating functional lanthanides within a 3D scaffold offers attractive perspectives for further applications.

Tripodal europium complex with triangulenium dye: a model bifunctional metallo-organic system.

A new tripodal ligand has been designed by coupling pyridyldicarbonyl binding strands with a triazatriangulenium platform (TATA). The complexation reaction with europium provides a C(3)-symmetrical mononuclear compound that is characterized with NMR, ESMS and qualitatively with single-crystal X-ray crystallography. In addition, photophysical studies of this dual emissive system have been performed, since the combination of the TATA fluorophore with trivalent lanthanides is of potential interest for the further development of imaging applications.

Lanthanide-mediated triangular cationic assemblies: structural and physico-chemical properties.

This contribution investigates Ln(III) complexes formed with a small ditopic ligand, L1, and their structural, thermodynamic and photophysical properties. The spectrophotometric and NMR titrations evidence the triangular assemblies [Ln(3)(L1-H)(3)](6+) at stoichiometric conditions and their properties are discussed in relation to L2-containing analogues. In addition, the dinuclear species, [Ln(2)(L1-H)](5+), is observed with an excess of metal.

Building large supramolecular nanocapsules with europium cations.

A new tripodal ligand has been designed by connecting pyridine-based coordination units to a rigid triptycene moiety. Its reaction with europium(III) provides three-dimensional tetranuclear edifices, whose structural and photophysical characteristics as well as host-guest interactions are discussed in this contribution.

Multistage complexation of fluoride ions by a fluorescent triphenylamine bearing three dimesitylboryl groups: controlling intramolecular charge transfer.

A propeller-shaped boron-nitrogen compound (NB(3)) with three binding sites for fluoride anions was synthesized and investigated by optical absorption, luminescence, and ((1)H, (11)B, (13)C, (19)F) NMR spectroscopy. Binding of fluoride in dichloromethane solution occurs in three clearly identifiable steps and leads to stepwise blocking of the three initially present nitrogen-to-boron charge transfer pathways. As a consequence, the initially bright blue charge transfer emission is red-shifted and decreases in intensity, until it is quenched completely in presence of large fluoride excess. Fluoride binding constants were determined from global fits to optical absorption and luminescence titration data and were found to be K(a1) = 4 × 10(7) M(-1), K(a2) = 2.5 × 10(6) M(-1), and K(a3) = 3.2 × 10(4) M(-1) in room temperature dichloromethane solution. Complexation of fluoride to a given dimesitylboryl site increases the electron density at the central nitrogen atom of NB(3), and this leads to red shifts of the remaining nitrogen-to-boron charge transfer transitions involving yet unfluorinated dimesitylboryl groups.

Tris(6-carb-oxy-pyridine-2-carboxyl-ato)terbium(III) 2.75-hydrate.

In the title compound, [Tb(C(7)H(4)NO(4))(3)]·2.75H(2)O, the Tb(3+) atom is coordinated by three tridentate 6-carb-oxy-pyridine-2-carboxyl-ate ligands and lies on a crystallographic threefold rotation axis. The coordination polyhedron around Tb(III) adopts a distorted tricapped trigonal-prismatic geometry. Disordered water mol-ecules with partial occupancy are also present in the crystal, one of which is associated with each of the carboxyl-ate O atoms of the complex unit.

Lanthanide-mediated supramolecular cages and host-guest interactions.

The structure and thermodynamic properties of lanthanide complexes with a new tripodal ligand L2 have been elucidated using different physicochemical methods. At stoichiometric ratios, the tetrahedral three-dimensional complexes with lanthanide cations are formed in acetonitrile with good stabilities. Despite minor structural changes comparing to previously investigated tripodal ligands, the resulting assembly exhibits different features revealed with the crystal structure of [Eu(4)L2(4)](OH)(ClO(4))(11) (orthorhombic, Pbcn). Interestingly, the highly charged edifice contains an inner cage encapsulating a perchlorate anion. Such lanthanide mediated cage-like assemblies are rare, and may be of interest for different sensing applications. Indeed, the anionic guest can be exchanged with different anions. The related host-guest equilibria were investigated with NMR techniques. Various aspects of these reactions are qualitatively discussed.

Rational design of a ternary supramolecular system: self-assembly of pentanuclear lanthanide helicates.

The self-assembly of the first pentanuclear helicate was predicted on the structural basis obtained for linear and tetranuclear parent supramolecular compounds. Accordingly, the designed ternary supramolecular system requires appropriate polytopic organic receptors, which were successfully synthesized. Indeed, the formation of pentanuclear complexes was experimentally evidenced with NMR and ESMS spectra that perfectly reflect the expected pattern. The structural features in the europium pentanuclear complex are highlighted with semiempirical molecular modeling. The present work validates the combinatorial approach leading to the thermodynamically driven formation of tower-like pentanuclear edifices.

Thermodynamics, structure and properties of polynuclear lanthanide complexes with a tripodal ligand: insight into their self-assembly.

Self-assembly processes between a tripodal ligand and Ln(III) cations have been investigated by means of supramolecular analytical methods. At an equimolar ratio of components, tetranuclear tetrahedral complexes are readily formed in acetonitrile. The structural analysis of the crystallographic data shows a helical wrapping of binding strands around metallic cations. The properties of this series of highly charged 3D compounds were examined by using NMR spectroscopy and optical methods in solution and in the solid state. In the presence of excess metal, a new trinuclear complex was identified. The X-ray crystal structure elucidated the coordination of metallic cations with two ligands of different conformations. By varying the metal/ligand ratio, a global speciation of this supramolecular system has been evidenced with different spectroscopic methods. In addition, these rather complicated equilibria were successfully characterised with the thermodynamic stability constants. A rational analysis of the self-assembly processes was attempted by using the thermodynamic free energy model and the impact of the ligand structure on the effective concentration is discussed.

Structure, stability and relaxivity of trinuclear triangular complexes.

The self-assembly of a carbonylpyridine-based heptadentate ligand with Ln(III) results in the formation of triangular trinuclear europium complexes, which exhibit interesting luminescent properties in the solid state and in solution. With a view to developing multimodal responsive systems, we report here the preparation and characterisation of analogous complexes with Gd(III). The X-ray crystal structure of Gd(3)L2(3) indeed reveals the isostructurality with the Eu(III) complexes. A combination of (1)H NMRD and variable temperature studies yields the parameters elucidating the exchange of coordinated water and relaxivity properties. Conveniently, the competitive spectrophotometric titrations with EDTA and NTA are used to determine the thermodynamic stability constants of the europium complexes in aqueous media. In addition, the exchange reaction with EDTA is monitored with NMR and fluorimetry. The interactions of the Eu(III) trinuclear complex with some potentially interfering ligands are qualitatively investigated by means of luminescence titrations.

Unsymmetrical tripodal ligand for lanthanide complexation: structural, thermodynamic, and photophysical studies.

Two tridentate and one bidentate binding strands have been anchored on a carbon atom to provide a new unsymmetrical tripodal ligand L for Ln(III) coordination. The ligand itself adopts a single conformation in solution stabilized by intramolecular hydrogen bonds evidenced in the solid state. The reaction of L with trivalent lanthanides provides different coordination complexes depending on the metal/ligand ratio. The speciation studies with selected lanthanides were performed in solution by means of NMR, ESMS, and spectrophotometric titrations. Differences in coordination properties along the lanthanide series were evidenced and may be associated with the changes in the ionic size. However, thermodynamic stability constants for the species of the same stoichiometry do not significantly vary. In addition, the structure of the dinuclear complex [Eu(2)L(2)](6+) has been elucidated in the solid state, where the complex crystallizes predominantly as an M-isomer. The crystal structure shows the coordination of two different ligands to each europium cation through tridentate strands, and the europium nine-coordinate sphere is completed with three solvent molecules. Finally, the results of photophysical investigations of [Eu(2)L(2)](6+) are in close agreement with the structural parameters determined by crystallography.

Self-assembly of a trinuclear luminescent europium complex.

Triangular luminescent box: Self-assembly of a new multidentate receptor with europium cations results in the formation of trinuclear discrete complexes. X-ray crystallography shows that nine-coordinate cations are linked by ligands to provide a triangular complex in the solid state and in solution. Despite the coordinated solvent molecules, this topologically unusual complex exhibits remarkable luminescent properties.