Nucleoside-Derived Low-Molecular-Weight Gelators as a Synthetic Microenvironment for 3D Cell Culture.

For the last few decades, many efforts have been made in developing cell culture methods in order to overcome the biological limitations of the conventional two-dimensional culture. This paradigm shift is driven by a large amount of new hydrogel-based systems for three-dimensional culture, among other systems, since they are known to mimic some living tissue properties. One class of hydrogel precursors has received interest in the field of biomaterials, low-molecular-weight gelators (LMWGs). In comparison to polymer gels, LMWG gels are formed by weak interactions upon an external trigger between the molecular subunits, giving them the ability to reverse the gelation, thus showing potential for many applications of practical interest. This study presents the use of the nucleoside derivative subclass of LMWGs, which are glyco-nucleo-bola-amphiphiles, as a proof of concept of a 3D cell culture scaffold. Physicochemical characterization was performed in order to reach the optimal features to fulfill the requirements of the cell culture microenvironment, in terms of the mechanical properties, architecture, molecular diffusion, porosity, and experimental practicality. The retained conditions were tested by culturing glioblastoma cells for over a month. The cell viability, proliferation, and spatial organization showed during the experiments demonstrate the proof of concept of nucleoside-derived LMWGs as a soft 3D cell culture scaffold. One of the hydrogels tested permits cell proliferation and spheroidal organization over the entire culture time. These systems offer many advantages as they consume very few matters within the optimal range of viscoelasticity for cell culture, and the thermoreversibility of these hydrogels permits their use with few instruments. The LMWG-based scaffold for the 3D cell culture presented in this study unlocked the ability to grow spheroids from patient cells to reach personalized therapies by dramatically reducing the variability of the lattice used.

Biomaterials for Three-Dimensional Cell Culture: From Applications in Oncology to Nanotechnology.

Three-dimensional cell culture has revolutionized cellular biology research and opened the door to novel discoveries in terms of cellular behavior and response to microenvironment stimuli. Different types of 3D culture exist today, including hydrogel scaffold-based models, which possess a complex structure mimicking the extracellular matrix. These hydrogels can be made of polymers (natural or synthetic) or low-molecular weight gelators that, via the supramolecular assembly of molecules, allow the production of a reproducible hydrogel with tunable mechanical properties. When cancer cells are grown in this type of hydrogel, they develop into multicellular tumor spheroids (MCTS). Three-dimensional (3D) cancer culture combined with a complex microenvironment that consists of a platform to study tumor development and also to assess the toxicity of physico-chemical entities such as ions, molecules or particles. With the emergence of nanoparticles of different origins and natures, implementing a reproducible in vitro model that consists of a bio-indicator for nano-toxicity assays is inevitable. However, the maneuver process of such a bio-indicator requires the implementation of a repeatable system that undergoes an exhaustive follow-up. Hence, the biggest challenge in this matter is the reproducibility of the MCTS and the associated full-scale characterization of this system's components.

Self-Assembly of Nucleoside-Derived Low-Molecular-Weight Gelators: A Thermodynamics and Kinetics Study on Different Length Scales.

Biocompatible materials are of paramount importance in numerous fields. Unlike chemically bridge polymer-based hydrogels, low-molecular-weight gelators can form a reversible hydrogel as their structures rely on noncovalent interaction. Although many applications with this type of hydrogel can be envisioned, we still lack their understanding due to the complexity of their self-assembly process and the difficulty in predicting their behaviors (transition temperature, gelation kinetics, the impact of solvent, etc.). In this study, we extend the investigations of a series of nucleoside-derived gelators, which only differ by subtle chemical modifications. Using a multitechnique approach, we determined their thermodynamic and kinetic features on various scale (molecular to macro) in different conditions. Monitored at the supramolecular level by circular dichroism as well as macroscopic scales by rheology and turbidimetry, we found out that the sol-gel and gel-sol transitions are greatly dependent on the concentration and on the mechanisms that are probed. Self-assembly kinetics depends on hydrogel molecules and is modulated by temperature and solvent. This fundamental study provides insight on the impact of some parameters on the gelation process, such as concentration, cooling rate, and the nature of the solvent.

Nucleoside-lipid-based nanocarriers for methylene blue delivery: potential application as anti-malarial drug.

Nucleolipid supramolecular assemblies are promising Drug Delivery Systems (DDS), particularly for nucleic acids. Studies based on negatively and positively charged nucleolipids (diC16dT and DOTAU, respectively) demonstrated appropriate stability, safety, and purity profile to be used as DDS. Methylene Blue (MB) remains a good antimalarial drug candidate, and could be considered for the treatment of uncomplicated or severe malaria. However, the development of MB as an antimalarial drug has been hampered by a high dose regimen required to obtain a proper effect, and a short plasmatic half life. We demonstrated that nanoparticles formed by nucleolipid encapsulation of MB using diC16dT and DOTAU (MB-NPs) is an interesting approach to improve drug stability and delivery. MB-NPs displayed sizes, PDI, zeta values, and colloidal stability allowing a possible use in intravenous formulations. Nanoparticles partially protected MB from oxido-reduction reactions, thus preventing early degradation during storage, and allowing prolongated pharmacokinetic in plasma. MB-NPs' efficacy, tested in vitro on sensitive or multidrug resistant strains of Plasmodium falciparum, was statistically similar to MB alone, with a slightly lower IC50. This nucleolipid-based approach to protect drugs against degradation represents a new alternative tool to be considered for malaria treatment.

Silver Ions Detection via Nucleolipids Self-Assembly.

A novel hybrid bioinspired amphiphile featuring a cytosine moiety, which self-assembles into liposomes can be used to detect silver ions in aqueous media. The coordination of Ag+ ions by the nucleotide moiety increases membrane rigidity, which enhances the fluorescence of a common reporter, Thioflavin T. Ag+ can be sensed even at trace concentrations (3 ppb) with great specificity over other metals ions. These nucleotide based supramolecular structures can be used to detect silver ions in drinking water, demonstrating the robustness of this approach.

Cu and Zn coordination to amyloid peptides: From fascinating chemistry to debated pathological relevance.

Several diseases share misfolding of different peptides and proteins as a key feature for their development. This is the case of important neurodegenerative diseases such as Alzheimer's and Parkinson's diseases and type II diabetes mellitus. Even more, metal ions such as copper and zinc might play an important role upon interaction with amyloidogenic peptides and proteins, which could impact their aggregation and toxicity abilities. In this review, the different coordination modes proposed for copper and zinc with amyloid-ß, α-synuclein and IAPP will be reviewed as well as their impact on the aggregation, and ROS production in the case of copper. In addition, a special focus will be given to the mutations that affect metal binding and lead to familial cases of the diseases. Different modifications of the peptides that have been observed in vivo and could be relevant for the coordination of metal ions are also described.

Cu(II) binding to various forms of amyloid-ß peptides. Are they friends or foes?

In the present micro-review, we describe the Cu(II) binding to several forms of amyloid-ß peptides, the peptides involved in Alzheimer's disease. It has indeed been shown that in addition to the "full-length" peptide originating from the precursor protein after cleavage at position 1, several other shorter peptides do exist in large proportion and may be involved in the disease as well. Cu(II) binding to amyloid-ß peptides is one of the key interactions that impact both the aggregating properties of the amyloid peptides and the Reactive Oxygen Species (ROS) production, two events linked to the etiology of the disease. Binding sites and affinity are described in correlation with Cu(II) induced ROS formation and Cu(II) altered aggregation, for amyloid peptides starting at position 1, 3, 4, 11 and for the corresponding pyroglutamate forms when they could be obtained (i.e. for peptides cleaved at positions 3 and 11). It appears that the current paradigm which points out a toxic role of the Cu(II) - amyloid-ß interaction might well be shifted towards a possible protective role when the peptides considered are the N-terminally truncated ones.

Mutations of Histidine†13 to Arginine and Arginine 5 to Glycine Are Responsible for Different Coordination Sites of Zinc(II) to Human and Murine Peptides.

Because mice and rats do not naturally develop Alzheimer's disease, genetically modified animals are required to study this pathology. This striking difference in terms of disease onset could be due to three alterations in the murine sequence (R5G, Y10F and H13R) of the amyloid-ß peptide with respect to the human counterpart. Whether the metal-ion binding properties of the murine peptide are at the origin of such different amyloidogenicity of the two peptides is still an open question. Herein, the main zinc binding site to the murine amyloid-ß at physiological pH has been determined through the combination of several spectroscopic and analytical methods applied to a series of six peptides with one or two of the key mutations. These results have been compared with the zinc binding site encountered in the human peptide. A coordination mechanism that demonstrates the importance of the H13R and R5G mutations in the different zinc environments present in the murine and human peptides is proposed. The nature of the minor zinc species present at physiological pH is also suggested for both peptides. Finally, the biological relevance and fallouts of the differences determined in zinc binding to human versus murine amyloid-ß are also discussed.

Cytidine- and guanosine-based nucleotide-lipids.

Hybrid nucleotide-lipids composed of a lipid covalently attached to purine and pyrimidine nucleobases exhibit supramolecular properties. The novel cytidine and guanosine derivatives are promising bioinspired materials, which can act as supramolecular gelators depending on both the nucleobase and the presence of salts. These supramolecular properties are of broad interest for biomedical applications.

Nucleoside-Lipid-Based Nanocarriers for Sorafenib Delivery.

Although the application of sorafenib, a small inhibitor of tyrosine protein kinases, to cancer treatments remains a worldwide option in chemotherapy, novel strategies are needed to address the low water solubility (< 5 µM), toxicity, and side effects issues of this drug. In this context, the use of nanocarriers is currently investigated in order to overcome these drawbacks. In this contribution, we report a new type of sorafenib-based nanoparticles stabilized by hybrid nucleoside-lipids. The solid lipid nanoparticles (SLNs) showed negative or positive zeta potential values depending on the nucleoside-lipid charge. Transmission electron microscopy of sorafenib-loaded SLNs revealed parallelepiped nanoparticles of about 200 nm. Biological studies achieved on four different cell lines, including liver and breast cancers, revealed enhanced anticancer activities of Sorafenib-based SLNs compared to the free drug. Importantly, contrast phase microscopy images recorded after incubation of cancer cells in the presence of SLNs at high concentration in sorafenib (> 80 µM) revealed a total cancer cell death in all cases. These results highlight the potential of nucleoside-lipid-based SLNs as drug delivery systems.

Chemical and functional properties of metal chelators that mobilize copper to elicit fungal killing of Cryptococcus neoformans.

A panel of iron (Fe) and copper (Cu) chelators was screened for growth inhibitory activity against the fungal pathogen Cryptococcus neoformans. Select bidentate metal-binding ligands containing mixed O,S or O,N donor atoms were identified as agents that induce cell killing in a Cu-dependent manner. Conversely, structurally similar ligands with O,O donor atoms did not inhibit C. neoformans growth regardless of Cu status. Studies of Cu(ii) and Cu(i) binding affinity, lipophilicity, and growth recovery assays of Cu-import deficient cells identified lipophilicity of thermodynamically stable CuIIL2 complexes as the best predictor of antifungal activity. These same complexes induce cellular hyperaccumulation of Zn and Fe in addition to Cu. The results described here present the utility of appropriate metal-binding ligands as potential antifungal agents that manipulate cellular metal balance as an antimicrobial strategy.

Zinc(II) Binding Site to the Amyloid-ß Peptide: Insights from Spectroscopic Studies with a Wide Series of Modified Peptides.

The Zn(II) ion has been linked to Alzheimer's disease (AD) due to its ability to modulate the aggregating properties of the amyloid-ß (Aß) peptide, where Aß aggregation is a central event in the etiology of the disease. Delineating Zn(II) binding properties to Aß is thus a prerequisite to better grasp its potential role in AD. Because of (i) the flexibility of the Aß peptide, (ii) the multiplicity of anchoring sites, and (iii) the silent nature of the Zn(II) ion in most classical spectroscopies, this is a difficult task. To overcome these difficulties, we have investigated the impact of peptide alterations (mutations, N-terminal acetylation) on the Zn(Aß) X-ray absorption spectroscopy fingerprint and on the Zn(II)-induced modifications of the Aß peptides' NMR signatures. We propose a tetrahedrally bound Zn(II) ion, in which the coordination sphere is made by two His residues and two carboxylate side chains. Equilibria between equivalent ligands for one Zn(II) binding position have also been observed, the predominant site being made by the side chains of His6, His13 or His14, Glu11, and Asp1 or Glu3 or Asp7, with a slight preference for Asp1.

Sequence proximity between Cu(II) and Cu(I) binding sites of human copper transporter 1 model peptides defines reactivity with ascorbate and O2.

The critical nature of the copper transporter 1 (Ctr1) in human health has spurred investigation of Ctr1 structure and function. Ctr1 specifically transports Cu(I), the reduced form of copper, across the plasma membrane. Thus, extracellular Cu(II) must be reduced prior to transport. Unlike yeast Ctr1, mammalian Ctr1 does not rely on any known mammalian reductase. Previous spectroscopic studies of model peptides indicate that human Ctr1 could serve as both copper reductase and transporter. Ctr1 peptides bind Cu(II) at an amino terminal high-affinity Cu(II), Ni(II) ATCUN site. Ascorbate-dependent reduction of the Cu(II)-ATCUN complex is possible by virtue of an adjacent HH (bis-His), as this bis-His motif and one methionine ligand constitute a high affinity Ctr1 Cu(I) binding site. Here, we synthetically varied the distance between the ATCUN and bis-His motifs in a series of peptides based on the human Ctr1 amino terminal, with the general sequence MDHAnHHMGMSYMDS, where n=0-4. We tested the ability of each peptide to reduce Cu(II) with ascorbate and stabilize Cu(I) under ambient conditions (20% O2). This study reveals that significant differences in coordination structure and chemical behavior with ascorbate and O2 result from changes in the sequence proximity of ATCUN and bis-His. Peptides that deviate from the native Ctr1 pattern were less effective at forming stable Cu(I)-peptide complexes and/or resulted in O2-dependent oxidative damage to the peptide.

Free Superoxide is an Intermediate in the Production of H2O2 by Copper(I)-Aß Peptide and O2.

Oxidative stress is considered as an important factor and an early event in the etiology of Alzheimer's disease (AD). Cu bound to the peptide amyloid-ß (Aß) is found in AD brains, and Cu-Aß could contribute to this oxidative stress, as it is able to produce in vitro H2O2 and HO˙ in the presence of oxygen and biological reducing agents such as ascorbate. The mechanism of Cu-Aß-catalyzed H2O2 production is however not known, although it was proposed that H2O2 is directly formed from O2 via a 2-electron process. Here, we implement an electrochemical setup and use the specificity of superoxide dismutase-1 (SOD1) to show, for the first time, that H2O2 production by Cu-Aß in the presence of ascorbate occurs mainly via a free O2˙(-) intermediate. This finding radically changes the view on the catalytic mechanism of H2O2 production by Cu-Aß, and opens the possibility that Cu-Aß-catalyzed O2˙(-) contributes to oxidative stress in AD, and hence may be of interest.

A prochelator peptide designed to use heterometallic cooperativity to enhance metal ion affinity.

A peptide has been designed so that its chelating affinity for one type of metal ion regulates its affinity for a second, different type of metal ion. The prochelator peptide (PCP), which is a fusion of motifs evocative of calcium loops and zinc fingers, forms a 1 : 2 Zn : peptide complex at pH 7.4 that increases its affinity for Zn2+ ∼3-fold in the presence of Tb3+ (log ß2 from 13.8 to 14.3), while the 1 : 1 luminescent complex with Tb3+ is brighter, longer lived, and 20-fold tighter in the presence of Zn2+ (log K from 6.2 to 7.5). This unique example of cooperative, heterometallic allostery in a biologically compatible construct suggests the possibility of designing conditionally active metal-binding agents that could respond to dynamic changes in cellular metal status.

Concept for simultaneous and specific in situ monitoring of amyloid oligomers and fibrils via Förster resonance energy transfer.

Oligomeric species of amyloidogenic peptides or proteins are often considered as the most toxic species in several amyloid disorders, like Alzheimer or Parkinson's diseases, and hence came into the focus of research interest and as a therapeutic target. An easy and specific monitoring of oligomeric species would be of high utility in the field, as it is the case for thioflavin T fluorescence for the fibrillar aggregates. Here, we show proof of concept for a new sensitive method to increase specific detection of oligomers by two extrinsic fluorophores. This is achieved by exploiting a Förster resonance energy transfer (FRET) between the two fluorophores. Thus, a mixture of two extrinsic fluorophores, bis-ANS and a styrylquinoxalin derivative, enabled one to monitor simultaneously and in situ the presence of oligomers and fibrils of amyloidogenic peptides. Thereby, the formation of oligomers and their transformation into fibrils can be followed.

The role of metal ions in amyloid formation: general principles from model peptides.

The mechanistic understanding of peptide self-assembly mediated by metal ions into amyloids or other structures has gained a lot of interest, mainly due to their importance in several neurodegenerative diseases. The use of short, easy-to-handle peptides has contributed to a better knowledge of structures and mechanisms that are also relevant for the native and longer peptides involved in the neurodegenerative diseases. Here, we review the results obtained with such "model systems" with the aim to identify and discuss fundamental parameters determining the self-aggregation with a special focus on the role of metal ions in this process.

Zn impacts Cu coordination to amyloid-ß, the Alzheimer's peptide, but not the ROS production and the associated cell toxicity.

Combined coordination of Zn(II) and Cu(I) or Cu(II) to the amyloid-ß peptide has been investigated using XANES, EPR and NMR spectroscopies. While Zn(II) does alter Cu(II) binding to Aß, this has no effect on (Aß)Cu induced ROS production and associated cell toxicity.

Cu(II) affinity for the Alzheimer's peptide: tyrosine fluorescence studies revisited.

Copper(II) binding to the amyloid-ß peptide has been proposed to be a key event in the cascade leading to Alzheimer's disease. As a direct consequence, the strength of the Cu(II) to Aß interaction, that is, the Cu(II) affinity of Aß, is a very important parameter to determine. Because Aß peptide contain one Tyr fluorophore in its sequence and because Cu(II) does quench Tyr fluorescence, fluorescence measurements appear to be a straightforward way to obtain this parameter. However, this proved to be wrong, mainly because of data misinterpretation in some previous studies that leads to a conflicting situation. In the present paper, we have investigated in details a large set of fluorescence data that were analyzed with a new method taking into account the presence of two Cu(II) sites and the inner-filter effect. This leads to reinterpretation of the published data and to the determination of a unified affinity value in the 10(10) M(-1) range.