A nanomedicine approach for the treatment of long-lasting pain.

This study explores the potential of a nanomedicine approach, using Leu-enkephalin-squalene nanoparticles (LENK-SQ NPs) for managing long-lasting pain. It was observed that the nanomedicine significantly improved the pharmacological efficacy of the Leu-enkephalin, a fast metabolized neuropeptide, in a rat model of acute inflammatory pain, providing local analgesic effect, while minimizing potential systemic side effects by circumventing central nervous system. The LENK-SQ NPs were tested in a rat model of postoperative pain (Brennan's rodent plantar incision model) using continuous infusion via Alzet® pump, with an additional bolus injection. The analgesic activity was assessed through stimulus-evoked methods, such as the von Frey and Hargreaves tests. Both mechanical and thermal hyperalgesia were significantly reduced at days 2 and 3 post-incision. An additional pharmacokinetic study was conducted, showing that LENK-SQ NPs allowed a sustained circulation of the neuropeptide under its prodrug form. On the other hand, the biodistribution of fluorescently labelled LENK-SQ NPs revealed their selective accumulation in the incised paw within the first hour post administration, followed by a disassembly of the NPs, starting 24 h later. The study proposes the following multi-step mechanism for the anti-nociceptive pharmacological activity of LENK-SQ NPs: (i) protection of the neuropeptide from metabolization into the bloodstream, (ii) targeted accumulation of the nanoparticles within the incised painful tissue and (iii) gradual release of LENK at the onset of the inflammatory process, leading to the observed analgesic activity.

Multiscale organisation of lead carboxylates in artistic oil binders.

The supramolecular and mesoscopic architectures of lead-saponified linseed oil, used by painters since the Renaissance, have been characterised and linked to their rheological properties. The multi-scale organization of saponified oils has been demonstrated by SAXS (Small Angle X-ray Scattering), FF-TEM (Freeze-Fracture Transmission Electron Microscopy) and DIC (Differential Interference Contrast): some of the lead soaps (formed when the oil is heated in the presence of PbO) are organized into microscopic lamellar domains, distributed in a continuous matrix made up of unorganized species (partially saponified triglycerides, glycerol, remaining soaps, etc.). The concentration of lead soaps in the oil controls the average size and interaction between the lamellar domains. Linseed oil + PbO 17 mol% is viscous and consists of aggregates of lamellar domains isolated within the continuous unorganized matrix. In contrast, in linseed oil + PbO 50 mol%, the domains are homogeneously dispersed and form what can be described as a three-dimensional network, giving the system viscoelastic properties.

Development and Characterization of Innovative Multidrug Nanoformulation for Cardiac Therapy.

For several decades, various peptides have been under investigation to prevent ischemia/reperfusion (I/R) injury, including cyclosporin A (CsA) and Elamipretide. Therapeutic peptides are currently gaining momentum as they have many advantages over small molecules, such as better selectivity and lower toxicity. However, their rapid degradation in the bloodstream is a major drawback that limits their clinical use, due to their low concentration at the site of action. To overcome these limitations, we have developed new bioconjugates of Elamipretide by covalent coupling with polyisoprenoid lipids, such as squalenic acid or solanesol, embedding self-assembling ability. The resulting bioconjugates were co-nanoprecipitated with CsA squalene bioconjugate to form Elamipretide decorated nanoparticles (NPs). The subsequent composite NPs were characterized with respect to mean diameter, zeta potential, and surface composition by Dynamic Light Scattering (DLS), Cryogenic Transmission Electron Microscopy (CryoTEM) and X-ray Photoelectron Spectrometry (XPS). Further, these multidrug NPs were found to have less than 20% cytotoxicity on two cardiac cell lines even at high concentrations, while maintaining an antioxidant capacity. These multidrug NPs could be considered for further investigations as an approach to target two important pathways involved in the development of cardiac I/R lesions.

UV-Visible photo-reactivity of permanently polarized inorganic nanotubes coupled to gold nanoparticles.

Hybrid aluminosilicate nanotubes (Imo-CH3) have the ability to trap small organic molecules inside their hydrophobic internal cavity while being dispersed in water owing to their hydrophilic external surface. They also display a curvature-induced polarization of their wall, which favors reduction outside the nanotubes and oxidation inside. Here, we coupled bare plasmonic gold nanoparticles (GNPs) with Imo-CH3 and analyzed for the first time the redox reactivity of these hybrid nano-reactors upon UV illumination. We show that the coupling between GNPs and Imo-CH3 significantly enhances the nanotube photocatalytic activity, with a large part of water reduction occurring directly on the gold surface. The coupling mechanism strongly influences the initial H2 production rate, which can go from ×10 to more than ×90 as compared to bare Imo-CH3 depending on the synthesis route of the GNPs. The present results show that this hybrid photocatalytic nano-reactor benefits from a synergy of polarization and confinement effects that facilitate efficient H2 production.

Rheo-SAXS characterization of lead-treated oils: Understanding the influence of lead driers on artistic oil paint's flow properties.

From the 15th century onwards, painters began to treat their oils with lead compounds before grinding them with pigments. Such a treatment induces the partial hydrolysis of the oil triglycerides and the formation of lead soaps, which significantly modify the rheological properties of the oil paint. Organization at the supramolecular scale is thus expected to explain these macroscopic changes. Synchrotron Rheo-SAXS (Small Angle X-ray Scattering) measurements were carried out on lead-treated oils, with different lead contents. We can now propose a full picture of the relationship between structure and rheological properties of historical saponified oils. At rest, lead soaps in oil are organized as lamellar phases with a characteristic period of 50 Å. Under shear, the loss of viscoelastic properties can be linked to the modification of this organization. Continuous shear resulted in a preferential and reversible orientation of the lamellar domains which increased with the concentration of lead soaps. The parallel orientation predominates over the entire shear range (0-1000 s-1). Conversely, oscillatory shear coiled the lamellae into cylinders that oriented themselves vertically in the rheometer cell. This is the first report of such a vertical cylindrical structure obtained under shear from lamellae.

Coexistence of Transient Liquid Droplets and Amorphous Solid Particles in Nonclassical Crystallization of Cerium Oxalate.

Crystallization from solution often occurs via "nonclassical" routes; that is, it involves transient, non-crystalline states like reactant-rich liquid droplets and amorphous particles. However, in mineral crystals, the well-defined thermodynamic character of liquid droplets and whether they convert─or not─into amorphous phases have remained unassessed. Here, by combining cryo-transmission electron microscopy and X-ray scattering down to a 250 ms reaction time, we unveil that crystallization of cerium oxalate involves a metastable chemical equilibrium between transient liquid droplets and solid amorphous particles: contrary to the usual expectation, reactant-rich droplets do not evolve into amorphous solids. Instead, at concentrations above 2.5 to 10 mmol L-1, both amorphous and reactant-rich liquid phases coexist for several tens of seconds and their molar fractions remain constant and follow the lever rule in a multicomponent phase diagram. Such a metastable chemical equilibrium between solid and liquid precursors has been so far overlooked in multistep nucleation theories and highlights the interest of rationalizing phase transformations using multicomponent phase diagrams not only when designing and recycling rare earths materials but also more generally when describing nonclassical crystallization.

Atomic structure of Lanreotide nanotubes revealed by cryo-EM.

Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different from the molecular model, largely based on X-ray fiber diffraction, proposed 20 y ago. Comparison of the nanotube with a crystal structure at 0.83-Å resolution of a Lanreotide derivative highlights the polymorphism for this peptide family. This work shows once again that higher-order assemblies formed by even well-characterized small peptides are very difficult to predict.

Supramolecular organization and biological interaction of squalenoyl siRNA nanoparticles.

Small interfering RNAs (siRNA) are attractive and powerful tools to inhibit the expression of a targeted gene. However, their extreme hydrophilicities combined with a negative charge and short plasma half-life counteract their use as therapeutics. Previously, we chemically linked siRNA to squalene (SQ) which self-assembled as nanoparticles (NPs) with pharmacological efficiency in cancers and recently in a hereditary neuropathy. In order to understand the siRNA-SQ NP assembly and fate once intravenously injected, the present study detailed characterization of siRNA-SQ NP structure and its interaction with serum components. From SAXS and SANS analysis, we propose that the siRNA-SQ bioconjugate self-assembled as 11-nm diameter supramolecular assemblies, which are connected one to another to form spherical nanoparticles of around 130-nm diameter. The siRNA-SQ NPs were stable in biological media and interacted with serum components, notably with albumin and LDL. The high specificity of siRNA to decrease or normalize gene expression and the high colloidal stability when encapsulated into squalene nanoparticles offer promising targeted therapy with wide applications for pathologies with gene expression dysregulation.

Catalytically active peptides affected by self-assembly and residues order.

Amphiphilic peptides that induce catalysis are interesting alternatives to natural enzymes thanks to robustness of their synthesis and the ability to induce certain types of conformations by specific motifs of amino acid sequences. Various studies aimed at mimicking the activity of serine proteases by designed peptides. Here we demonstrate that the order by which the catalytic triad residues are positioned along amphiphilic ß-strands influences both assembly structures and catalytic activity. A set of three ß-sheet amphiphilic peptides, decorated with different orders of the catalytic triad amino acids, Glu, His and Ser along the strands were evaluated for their catalytic hydrolysis efficiency of p-nitrophenyl acetate (pNPA) substrate. Among the three peptides, Ac-Cys-Phe-Glu-Phe-Ser-Phe-His-Phe-Pro-NH2 (ESH) achieved the greatest catalytic efficiency with a value of 0.19 M-1 s-1, at peptide concentration of 250 µM. This study sheds light on an overlooked factor in designing catalytic amphiphilic assemblies whereby charged residues that make up the active sites, are in fact engaged in intermolecular stabilizing interactions that in turn may hamper their catalytic action.

New Nanoparticle Formulation for Cyclosporin A: In Vitro Assessment.

Cyclosporin A (CsA) is a molecule with well-known immunosuppressive properties. As it also acts on the opening of mitochondrial permeability transition pore (mPTP), CsA has been evaluated for ischemic heart diseases (IHD). However, its distribution throughout the body and its physicochemical characteristics strongly limit the use of CsA for intravenous administration. In this context, nanoparticles (NPs) have emerged as an opportunity to circumvent the above-mentioned limitations. We have developed in our laboratory an innovative nanoformulation based on the covalent bond between squalene (Sq) and cyclosporin A to avoid burst release phenomena and increase drug loading. After a thorough characterization of the bioconjugate, we proceeded with a nanoprecipitation in aqueous medium in order to obtain SqCsA NPs of well-defined size. The SqCsA NPs were further characterized using dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryoTEM), and high-performance liquid chromatography (HPLC), and their cytotoxicity was evaluated. As the goal is to employ them for IHD, we evaluated the cardioprotective capacity on two cardiac cell lines. A strong cardioprotective effect was observed on cardiomyoblasts subjected to experimental hypoxia/reoxygenation. Further research is needed in order to understand the mechanisms of action of SqCsA NPs in cells. This new formulation of CsA could pave the way for possible medical application.

Albumin-driven disassembly of lipidic nanoparticles: the specific case of the squalene-adenosine nanodrug.

In the field of nanomedicine, nanostructured nanoparticles (NPs) made of self-assembling prodrugs emerged in the recent years with promising properties. In particular, squalene-based drug nanoparticles have already shown their efficiency through in vivo experiments. However, a complete pattern of their stability and interactions in the blood stream is still lacking. In this work we assess the behavior of squalene-adenosine (SQAd) nanoparticles - whose neuroprotective effect has already been demonstrated in murine models - in the presence of fetal bovine serum (FBS) and of bovine serum albumin (BSA), the main protein of blood plasma. Extensive physicochemical characterizations were performed using Small Angle Neutron Scattering (SANS), cryogenic transmission electron microscopy (Cryo-TEM), circular dichroism (CD), steady-state fluorescence spectroscopy (SSFS) and isothermal titration calorimetry (ITC) as well as in silico by means of ensemble docking simulations with human serum albumin (HSA). Significant changes in the colloidal stability of the nanoparticles in the presence of serum albumin were observed. SANS, CD and SSFS analyses demonstrated an interaction between SQAd and BSA, with a partial disassembly of the nanoparticles in the presence of BSA and the formation of a complex between SQAd and BSA. The interaction free energy of SQAd nanoparticles with BSA derived from ITC experiments, is about -8 kcal mol-1 which is further supported in silico by ensemble docking simulations. Overall, our results show that serum albumin partially disassembles SQAd nanoparticles by extracting individual SQAd monomers from them. As a consequence, the SQAd nanoparticles would act as a circulating reservoir in the blood stream. The approach developed in this study could be extended to other soft organic nanoparticles.

Translation of nanomedicines from lab to industrial scale synthesis: The case of squalene-adenosine nanoparticles.

A large variety of nanoparticle-based delivery systems have become increasingly important for diagnostic and/or therapeutic applications. Yet, the numerous physical and chemical parameters that influence both the biological and colloidal properties of nanoparticles remain poorly understood. This complicates the ability to reliably produce and deliver well-defined nanocarriers which often leads to inconsistencies, conflicts in the published literature and, ultimately, poor translation to the clinics. A critical issue lies in the challenge of scaling-up nanomaterial synthesis and formulation from the lab to industrial scale while maintaining control over their diverse properties. Studying these phenomena early on in the development of a therapeutic agent often requires partnerships between the public and private sectors which are hard to establish. In this study, through the particular case of squalene-adenosine nanoparticles, we reported on the challenges encountered in the process of scaling-up nanomedicines synthesis. Here, squalene (the carrier) was functionalized and conjugated to adenosine (the active drug moiety) at an industrial scale in order to obtain large quantities of biocompatible and biodegradable nanoparticles. After assessing nanoparticle batch-to-batch consistency, we demonstrated that the presence of squalene analogs resulting from industrial scale-up may influence several features such as size, surface charge, protein adsorption, cytotoxicity and crystal structure. These analogs were isolated, characterized by multiple stage mass spectrometry, and their influence on nanoparticle properties further evaluated. We showed that slight variations in the chemical profile of the nanocarrier's constitutive material can have a tremendous impact on the reproducibility of nanoparticle properties. In a context where several generics of approved nanoformulated drugs are set to enter the market in the coming years, characterizing and solving these issues is an important step in the pharmaceutical development of nanomedicines.

Towards a clinical application of freeze-dried squalene-based nanomedicines.

Squalene-adenosine (SQAd) nanoparticles (NPs) were found to display promising pharmacological activity similar to many other nanomedicines, but their long-term stability was still limited, and their preparation required specific know-how and material. These drawbacks represented important restrictions for their potential use in the clinic. Freeze-drying nanoparticles is commonly presented as a solution to allow colloidal stability, but this process needs to be adapted to each nanoformulation. Hence, we aimed at developing a specific protocol for freeze-drying SQAd NPs while preserving their structural features. NPs were lyophilised, resuspended and analysed by dynamic light scattering, atomic force microscopy and small-angle scattering. Among four different cryoprotectants, trehalose was found to be the most efficient in preserving NPs physico-chemical characteristics. Interestingly, we identified residual ethanol in NP suspensions as a key parameter which could severely affect the freeze-drying outcome, leading to NPs aggregation. Long-term stability was also assessed. No significant change in size distribution or zeta potential could be detected after three-month storage at 4 °C. Finally, freeze-dried NPs innocuity was checked in vitro on cultured hepatocytes and in vivo on mice. In conclusion, optimisation of freeze-drying conditions resulted in safe lyophilised SQAd NPs that can be easily stored, shipped and simply reconstituted into an injectable form.

Partial Transformation of Imogolite by Decylphosphonic Acid Yields an Interface Active Composite Material.

The phosphonic acid moiety is commonly used as an anchoring group for the surface modification of imogolite. However, the impact of the reaction on its structure has never been clearly analyzed before. We study the reaction of imogolite and decylphosphonic acid by combining infrared spectroscopy, X-ray scattering, scanning electron microscopy, transmission electron microscopy, and solid-state nuclear magnetic resonance spectroscopy. Instead of a surface functionalization, we observe the formation of a lamellar phase interconnected with imogolite bundles. Although we find no evidence for grafted imogolite tubes, we observe the expected dispersion characteristics and stabilization of water in toluene emulsions described in the literature. Based on the surface chemistry of imogolite, we propose an explanation for the observed reactivity and link the structural features of the obtained composite material to its dispersibility in toluene and its observed properties at the toluene-water interface.

Exploring Hybrid Imogolite Nanotube Formation via Si/Al Stoichiometry Control.

Hybrid imogolite aluminosilicate nanotubes with methylated internal surface can be obtained by introduction of the corresponding organosilane during their synthesis. However, similarly to pristine imogolite, a number of side products, including proto-imogolite (open-imoLS), allophanes, and aluminum hydroxides, are formed, which ultimately impact on the properties of the dispersions. In order to minimize the proportion of these side products, we have here systematically explored the impact of the initial Si/Al ratio on the content of hybrid imogolite dispersions before and after dialysis. By combining cryo-transmission electron microscopy, inductively coupled plasma mass spectrometry, infrared spectroscopy, and small-angle X-ray scattering, we evidenced that the Si/Al ratio has a large impact on the formation of aluminum hydroxides that can be minimized with a slight excess of Si precursor. However, a large excess of Si is detrimental to the reaction yield leading to an important proportion of proto-imogolite. We propose that the optimal Si/Al ratio of ca. 0.6 can both minimize the proportion of aluminum hydroxides and proto-imogolite. These results suggest that the dynamic and therefore reactive character of imogolite dispersions may have been so far underlooked.

Contribution to Accurate Spherical Gold Nanoparticle Size Determination by Single-Particle Inductively Coupled Mass Spectrometry: A Comparison with Small-Angle X-ray Scattering.

Small-angle X-ray scattering spectroscopy (SAXS) is the method of choice for nanoparticle diameter and concentration determination. On the one hand, it is metrologically traceable for spherical nanoparticle mean diameter determination and does not require any sample preparation or calibration. On the other hand, single-particle inductively coupled mass spectrometry (SPICPMS) is still under development and requires involved process clarification and accuracy improvement. The strategy of this study is the comparison of the two techniques to study comprehensively SPICPMS performance and observe phenomena otherwise hidden. Six spherical gold nanoparticle suspensions distributed over a large size range (30, 50, 60, 80,100, and 150 nm) are studied as calibration points. Potential matrix effects are eliminated by stabilizing nanoparticles with chitosan in HCl. Chitosan encapsulates nanoparticles, stabilizes their dispersion, and protects them from dissolution. Detection counting/analogue threshold and timeout appear as the relevant parameters for transient signals. They show an influence not only on mean signal but also on signal distribution. The detection tuning proposed allows to linearly calibrate the nanoparticle distribution signal to cubed diameter over the entire range studied with no sensitivity diminution. Comparing the three classical transport efficiency methods, size transport efficiency is shown as the most accurate. The new procedure is validated analyzing three gold nanoparticle suspensions (135, 40, and 50 nm). The results are consistent with SAXS measurements.

Reversible Assembly of a Drug Peptide into Amyloid Fibrils: A Dynamic Circular Dichroism Study.

The common view on the amyloid fibril formation is that it is a multistep process that involves many oligomeric intermediate species, which leads to a high degree of polymorphism. This view derives from numerous kinetic studies whose vast majority was carried out with amyloid ß fragments or other pathological amyloidogenic sequences. Yet, it is not clear whether the mechanisms inferred from these studies are universal and also apply to functional amyloids, in particular to peptide hormones which form reversible amyloid structures. In the present work, we study the self-assembly properties of atosiban, a nonapeptide drug, whose sequence is very close to those of the oxytocin and vasopressin hormones. We show that this very soluble peptide consistently self-assembles into 7 nm wide amyloid fibrils above a critical aggregation concentration (2-10 w/w % depending on the buffer conditions). The peptide system is characterized in details, from the monomeric to the assembled form, with osmotic concentration measurements, transmission electron microscopy, small-angle X-ray scattering, infrared and fluorescence spectroscopy, and circular dichroism (CD). We have followed in situ the fibril assembly with fluorescence and synchrotron radiation CD and noticed that the peptide undergoes conformational changes during the process. However, several lines of evidence point toward the association of monomers and dimers into fibrils without passing through oligomeric intermediate species contrary to what is usually reported for pathogenic amyloids. The native ß-hairpin conformation of the monomer could explain the straightforward assembly. The tyrosine stacking is also shown to play an important role.

Reversible Morphological Control of Cholecystokinin Tetrapeptide Amyloid Assemblies as a Function of pH.

Most amyloid assemblies are seen as irreversible and exhibit polymorphism because their assembly is kinetically controlled and different structures are trapped during the aggregation process. However, in the specific case of peptide hormones, formation of amyloid assemblies for storage purposes has been reported. This suggests a strict control of assembly and the ability to disassemble upon hormone secretion. In the present work, we have sought to test these assertions with a short peptide, the cholecystokinin (or gastrin) tetrapeptide (CCK-4), that has been found in both gastrointestinal tract and central nervous system, and whose sequence is shared by a large number of hormones. We have thus studied in vitro this peptide's self-assembling properties in dense phases at different pH levels, thus mimicking in vivo storage conditions. The solubility and morphology of the supramolecular assemblies have been shown to vary with the pH. At low pH, the tetrapeptide exhibits a low solubility and forms microcrystals. At higher pH levels, peptide solubility increases and above a high enough concentration, peptide monomers self-assemble into typical amyloid fibrils of 10-20 nm diameter. The physical network formed by these fibrils results in a birefringent hydrogel phase. Despite the different morphological features exhibited at different pH, structural analysis shows strong similarities. Both supramolecular assemblies-microcrystals and fibrils-are structured by ß-sheets. We also show that all these morphologies are reversible and can be either dissolved or changed into one another by switching the pH. In addition, we demonstrate that a modification in the charge sequence of the peptide by amino acid mutation modifies its self-assembly properties. In conclusion, just as the CCK-4 sequence is the minimal sequence required for a complete biological activity at CCKB receptors in the brain, it is also sufficient to form amyloid fibers whose properties can be related to hormone storage and release purposes in vivo.

Biocompatible Stimuli-Responsive W/O/W Multiple Emulsions Prepared by One-Step Mixing with a Single Diblock Copolymer Emulsifier.

Multiple water-in-oil-in-water (W/O/W) emulsions are promising materials in designing carriers of hydrophilic molecules or drug delivery systems, provided stability issues are solved and biocompatible chemicals can be used. In this work, we designed a biocompatible amphiphilic copolymer, poly(dimethylsiloxane)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMS-b-PDMAEMA), that can stabilize emulsions made with various biocompatible oils. The hydrophilic/hydrophobic properties of the copolymer can be adjusted using both pH and ionic strength stimuli. Consequently, the making of O/W (oil in water), W/O (water in oil), and W/O/W emulsions can be achieved by sweeping the pH and ionic strength. Of importance, W/O/W emulsions are formulated over a large pH and ionic strength domain in a one-step emulsification process via transitional phase inversion and are stable for several months. Cryo-TEM and interfacial tension studies show that the formation of these W/O/W emulsions is likely to be correlated to the interfacial film curvature and microemulsion morphology.

Directing peptide crystallization through curvature control of nanotubes.

In the absence of efficient crystallization methods, the molecular structures of fibrous assemblies have so far remained rather elusive. In this paper, we present a rational method to crystallize the lanreotide octapeptide by modification of a residue involved in a close contact. Indeed, we show that it is possible to modify the curvature of the lanreotide nanotubes and hence their diameter. This fine tuning leads to crystallization because the radius of curvature of the initially bidimensional peptide wall can be increased up to a point where the wall is essentially flat and a crystal is allowed to grow along a third dimension. By comparing X-ray diffraction data and Fourier transform Raman spectra, we show that the nanotubes and the crystals share similar cell parameters and molecular conformations, proving that there is indeed a structural continuum between these two morphologies. These results illustrate a novel approach to crystallization and represent the first step towards the acquisition of an Å-resolution structure of the lanreotide nanotubes ß-sheet assembly.