L’effet ouzo pour l’élaboration de nanocapsules hybrides (Hybridosomes®), l’encapsulation de composés hydrophobes par nanoprécipitation et la radiothérapie

The ouzo effect is a phenomenon of spontaneous emulsification in a "water/misciblesolvent/hydrophobic compound” system, leading to the formation of droplets of controlled size and low polydispersity. It has been used, for example, in the development of nanoparticles, encapsulation or for controlled drug delivery. For several years, our group has been using a process based on the ouzo effect to prepare hydrophilic nanocapsules composed of inorganic nanoparticles and polymer: Hybridosomes. Thus, this simple process leads to nanocapsules whose properties can be modulated according to the assembled nanoparticles (iron oxide, gold, quantum dots...). The objective of this work is to understand the physico-chemical mechanisms involved in the formation of hybridosomes as well as to explore their applications, in this case encapsulation and radiotherapy. The first part of this manuscript is devoted to the study of the ouzo system allowing the realization of hybridosomes. We seek to understand the parameters influencing the formation of the droplets, with a particular interest in the influence of the ligands present on the surface of nanoparticles. Besides, we investigate the use of nanoparticles of various nature and size. Then, we evaluate the use of our process for the encapsulation of hydrophobic compounds by nanoprecipitation. The very good encapsulation performances as well as the release (in vitro and in vivo) are exposed. Finally, we successfully use hybridosomes composed of gold and iron oxide nanoparticles for glioblastoma radiotherapy in a murine model.L’effet ouzo est un phénomène d’émulsification spontanée dans un système « eau/solvant miscible/composé hydrophobe », menant à la formation de gouttelettes de taille contrôlée et peu polydisperses. Il a par exemple été utilisé pour l’élaboration de nanoparticules, l’encapsulation ou encore la délivrance contrôlée de médicaments. Depuis plusieurs années, notre groupe a mis en place un procédé basé sur l’effet ouzo permettant la préparation de nanocapsules hydrophiles composées de nanoparticules inorganiques et de polymère : les Hybridosomes. Ainsi, ce procédé simple mène à des nanocapsules dont les propriétés sont modulables en fonction des nanoparticules assemblées (oxyde de fer, or, quantum dots…). L’objectif de ces travaux est de comprendre les mécanismes physico-chimiques impliqués dans la formation des hybridosomes ainsi que d’en explorer les applications, en l’occurrence l’encapsulation et la radiothérapie. La première partie de ce manuscrit est consacrée à l’étude du système ouzo permettant la réalisation des hybridosomes. Nous cherchons à comprendre les paramètres influençant l’établissement des gouttes, avec un intérêt tout particulier pour l’influence des ligands présents sur les nanoparticules. Par ailleurs, nous investiguons l’utilisation de nanoparticules de nature et de taille variées. Ensuite, nous évaluons l’utilisation de notre procédé pour l’encapsulation de composés hydrophobes par nanoprécipitation. Les très bonnes performances d’encapsulation ainsi que le relargage (in vitro et in vivo) sont exposés. Finalement, nous utilisons avec succès les hybridosomes composés de nanoparticules d’or et d’oxyde de fer pour la radiothérapie du glioblastome sur un modèle murin

The ouzo effect for the preparation of hybrid nanocapsules (Hybridosomes®), the encapsulation of hydrophobic compounds by nanoprecipitation and radiotherapy

L’effet ouzo est un phénomène d’émulsification spontanée dans un système « eau/solvant miscible/composé hydrophobe », menant à la formation de gouttelettes de taille contrôlée et peu polydisperses. Il a par exemple été utilisé pour l’élaboration de nanoparticules, l’encapsulation ou encore la délivrance contrôlée de médicaments. Depuis plusieurs années, notre groupe a mis en place un procédé basé sur l’effet ouzo permettant la préparation de nanocapsules hydrophiles composées de nanoparticules inorganiques et de polymère : les Hybridosomes. Ainsi, ce procédé simple mène à des nanocapsules dont les propriétés sont modulables en fonction des nanoparticules assemblées (oxyde de fer, or, quantum dots…). L’objectif de ces travaux est de comprendre les mécanismes physico-chimiques impliqués dans la formation des hybridosomes ainsi que d’en explorer les applications, en l’occurrence l’encapsulation et la radiothérapie. La première partie de ce manuscrit est consacrée à l’étude du système ouzo permettant la réalisation des hybridosomes. Nous cherchons à comprendre les paramètres influençant l’établissement des gouttes, avec un intérêt tout particulier pour l’influence des ligands présents sur les nanoparticules. Par ailleurs, nous investiguons l’utilisation de nanoparticules de nature et de taille variées. Ensuite, nous évaluons l’utilisation de notre procédé pour l’encapsulation de composés hydrophobes par nanoprécipitation. Les très bonnes performances d’encapsulation ainsi que le relargage (in vitro et in vivo) sont exposés. Finalement, nous utilisons avec succès les hybridosomes composés de nanoparticules d’or et d’oxyde de fer pour la radiothérapie du glioblastome sur un modèle murin.The ouzo effect is a phenomenon of spontaneous emulsification in a "water/misciblesolvent/hydrophobic compound” system, leading to the formation of droplets of controlled size and low polydispersity. It has been used, for example, in the development of nanoparticles, encapsulation or for controlled drug delivery. For several years, our group has been using a process based on the ouzo effect to prepare hydrophilic nanocapsules composed of inorganic nanoparticles and polymer: Hybridosomes. Thus, this simple process leads to nanocapsules whose properties can be modulated according to the assembled nanoparticles (iron oxide, gold, quantum dots...). The objective of this work is to understand the physico-chemical mechanisms involved in the formation of hybridosomes as well as to explore their applications, in this case encapsulation and radiotherapy. The first part of this manuscript is devoted to the study of the ouzo system allowing the realization of hybridosomes. We seek to understand the parameters influencing the formation of the droplets, with a particular interest in the influence of the ligands present on the surface of nanoparticles. Besides, we investigate the use of nanoparticles of various nature and size. Then, we evaluate the use of our process for the encapsulation of hydrophobic compounds by nanoprecipitation. The very good encapsulation performances as well as the release (in vitro and in vivo) are exposed. Finally, we successfully use hybridosomes composed of gold and iron oxide nanoparticles for glioblastoma radiotherapy in a murine model

A Domain-Theoretic Approach to Statistical Programming Languages

We give a domain-theoretic semantics to a statistical programming language, using the plain old category of dcpos, in contrast to some more sophisticated recent proposals. Remarkably, our monad of minimal valuations is commutative, which allows for program transformations that permute the order of independent random draws, as one would expect. A similar property is not known for Jones and Plotkin’ s monad of continuous valuations. Instead of working with true real numbers, we work with exact real arithmetic, providing a bridge towards possible implementations. (Implementations by themselves are not addressed here.) Rather remarkably, we show that restricting ourselves to minimal valuations does not restrict us much: all measures on the real line can be modeled by minimal valuations on the domain $\mathbf {I}\mathbb {R}_\bot$ of exact real arithmetic. We give three operational semantics for our language, and we show that they are all adequate with respect to the denotational semantics. We also explore quite a few examples in order to demonstrate that our semantics computes exactly as one would expect, and in order to debunk the myth that a semantics based on continuous maps would not be expressive enough to encode measures with non-compact support using only measures with compact support, or to encode measures via non-continuous density functions, for instance. Our examples also include some useful, non-trivial cases of distributions on higher-order objects

Shedding light on the formation and stability of mesostructures in ternary "Ouzo" mixtures

International audienceHYPOTHESIS: Ternary systems made of water, a water-miscible solvent, and hydrophobic solutes spontaneously produce metastable particles by the "Ouzo effect" and thermodynamically stable "Surfactant-Free Micro Emulsions" (SFME). However, the use of different analyses has led to a variability in the criteria to determine the boundaries of the Ouzo domain. We hypothesized that this could be clarified by investigating the stability and the physical state of the particles. EXPERIMENTS: We investigate four systems using both solid and liquid solutes and two different solvents, and achieved a careful investigation of their phase diagrams, using DLS, Nanoparticle Tracking Analysis, NMR, Multiple Light Scattering, electrophoretic mobility, and fluorescence analysis. FINDINGS: Our results evidence that the transition from the monophasic to the Ouzo domains does not coincide with the cloudiness curve, and that compositions in the Ouzo domain can look fully transparent, in contrast to what is often considered. This transition is best determined by stability analysis. The cloudiness curve corresponds to the formation of particles with a large size dispersity. In the Ouzo domain, we observed an exchange of solute between the continuous phase and solute particles swollen with solvent. In addition, the particles are stabilized against coalescence by their high negative charge

Radiosensitizing Fe-Au nanocapsules (hybridosomes®) increase survival of GL261 brain tumor-bearing mice treated by radiotherapy

International audienceGlioblastoma remains a cancer for which the effectiveness of treatments has shown little improvement over the last decades. For this pathology, multiple therapies combining resection, chemotherapy and radiotherapy remain the norm. In this context, the use of high-Z nanoparticles such as gold or hafnium to potentiate radiotherapy is attracting more and more attention. Here, we evaluate the potentiating effect of hollow shells made of gold and iron oxide nanoparticles (hybridosomes®) on the radiotherapy of glioblastoma, using murine GL261-Luc+ brain tumor model. While iron oxide seems to have no beneficial effect for radiotherapy, we observe a real effect of gold nanoparticles —despite their low amount— with a median survival increase of almost 20% compared to radiotherapy only and even 33% compared to the control group. Cellular and in vivo studies show that a molecule of interest nano-precipitated in the core of the hybridosomes® is released and internalized by the surrounding brain cells. Finally, in vivo studies show that hybridosomes® injected intra-tumorally are still present in the vicinity of the brain tumor more than 5 days after injection (duration of the Stupp protocol’s radiation treatment). Interestingly, one mouse treated with radiotherapy in the presence of gold-containing hybridosomes® survived 78 days. Monitoring of the tumoral growth of this long-term survivor using both MRI and bioluminescence revealed a decrease of the tumor size after treatment. These very encouraging results are a proof-of-concept that hybridosomes®, are really effective tools for combined therapies development (chemo-radiotherapy)

Radiosensitizing Fe-Au nanocapsules (hybridosomes®) increase survival of GL261 brain tumor-bearing mice treated by radiotherapy

International audienceGlioblastoma remains a cancer for which the effectiveness of treatments has shown little improvement over the last decades. For this pathology, multiple therapies combining resection, chemotherapy and radiotherapy remain the norm. In this context, the use of high-Z nanoparticles such as gold or hafnium to potentiate radiotherapy is attracting more and more attention. Here, we evaluate the potentiating effect of hollow shells made of gold and iron oxide nanoparticles (hybridosomes®) on the radiotherapy of glioblastoma, using murine GL261-Luc+ brain tumor model. While iron oxide seems to have no beneficial effect for radiotherapy, we observe a real effect of gold nanoparticles —despite their low amount— with a median survival increase of almost 20% compared to radiotherapy only and even 33% compared to the control group. Cellular and in vivo studies show that a molecule of interest nano-precipitated in the core of the hybridosomes® is released and internalized by the surrounding brain cells. Finally, in vivo studies show that hybridosomes® injected intra-tumorally are still present in the vicinity of the brain tumor more than 5 days after injection (duration of the Stupp protocol’s radiation treatment). Interestingly, one mouse treated with radiotherapy in the presence of gold-containing hybridosomes® survived 78 days. Monitoring of the tumoral growth of this long-term survivor using both MRI and bioluminescence revealed a decrease of the tumor size after treatment. These very encouraging results are a proof-of-concept that hybridosomes®, are really effective tools for combined therapies development (chemo-radiotherapy)

Simple elaboration of drug-SPION nanocapsules (hybridosomes®) by solvent shifting: effect of the drug molecular structure and concentration

International audienceDrug nanocapsules coated with iron oxide nanoparticles (SPION) were elaborated by the simultaneous nanoprecipitation of the drug and the nanoparticles, through solvent shifting. We examined four drugs: sorafenib, sorafenib tosylate, α-tocopherol and paclitaxel, to cover the cases of molecular solids, ionic solids, and molecular liquids. We first investigated the formation of the drug core in the final mixture of solvents at different concentrations. A Surfactant Free Micro-Emulsion domain (SFME, thermodynamically stable) was observed at low drug concentration and an Ouzo domain (metastable) at high drug concentration, except for the case of paclitaxel which crystallizes at high concentration without forming an Ouzo domain. When co-nanoprecipitated with the molecular drugs in the Ouzo domain (sorafenib or α-tocopherol), the SPION limited the coalescence of the drug particles to less than 100 nm, forming capsules with a drug encapsulation efficiency of ca 80 %. In contrast, larger capsules were formed from the SFME or when using the ionic form (sorafenib tosylate). Finally, the sorafenib-SPION capsules exhibit a similar chemotherapeutic effect as the free drug on the hepatocellular carcinoma in vitro

Radiosensitizing Fe-Au Nanocapsules (Hybridosomes®) increase survival of GL261 brain tumor-bearing mice treated by radiotherapy

International audienceGlioblastoma remains a cancer for which the effectiveness of treatments has shown little improvement over the last decades. For this pathology, multiple therapies combining resection, chemotherapy and radiotherapy remain the norm. In this context, the use of high-Z nanoparticles such as gold or hafnium to potentiate radiotherapy is attracting more and more attention. Here, we evaluate the potentiating effect of hollow shells made of gold and iron oxide nanoparticles (hybridosomes®) on the radiotherapy of glioblastoma, using murine GL261-Luc+brain tumor model. While iron oxide seems to have no beneficial effect for radiotherapy, we observe a real effect of gold nanoparticles -despite their low amount- with a median survival increase of almost 20% compared to radiotherapy only and even 33% compared to the control group. Cellular and in vivo studies show that a molecule of interest nano-precipitated in the core of the hybridosomes® is released and internalized by the surrounding brain cells. Finally, in vivo studies show that hybridosomes® injected intra-tumorally are still present in the vicinity of the brain tumor more than 5days after injection (duration of the Stupp protocol’s radiation treatment). Interestingly, one mouse treated with radiotherapy in the presence of gold-containing hybridosomes® survived 78days. Monitoring of the tumoral growth of this long-term survivor using both MRI and bioluminescence revealed a decrease of the tumor size after treatment. These very encouraging results are a proof-of-concept that hybridosomes®, are really effective tools for combined therapies development (chemo-radiotherapy)

Effect of nanoparticles on spontaneous Ouzo emulsification

International audienceParticles stabilize fluid interfaces. In particular, oil/water Pickering emulsions undergo limited coalescence, yielding droplets of smaller size as the amount of particles is increased. Herein, we studied the effect of hydrophobic nanoparticles (<10 nm, alkyl-coated) on submicronic droplets (ca 100 nm) formed in an Ouzo system. We investigated thoroughly the water/tetrahydrofuran (THF)/butylated hydroxytoluene (BHT) reference diagram, in the absence and in the presence of nanoparticles, using the Nanoparticle Tracking Analysis (NTA) technique. This allowed us to characterize the size distributions in a much finer way than what is usually obtained using conventional Dynamic Light Scattering (DLS). Both a Surfactant-Free Microemulsion (SFME, thermodynamically stable) and an Ouzo (metastable spontaneous emulsion) domains were identified and the transition from one to the other could be characterized by specific features of the droplet size distributions. We found that the presence of the nanoparticles limits coalescence in the metastable domain. We also show that the alkyl-coated nanoparticles are irreversibly attached to the liquid-liquid interface

The Ouzo effect: A tool to elaborate high-payload nanocapsules

International audienceWe investigate the encapsulation in hybridosomes®, a type of capsules unique regarding their structure and method of elaboration. Hybridosomes® are made of a single shell of inorganic nanoparticles (~5 nm) crosslinked with a polymer and are easily obtained via spontaneous emulsification in a ternary mixture THF/water/butylated hydroxytoluene (BHT). Our main finding is that an exceptionally high concentration of a hydrophobic model dye can be loaded in the hybridosomes®, up to 0.35 mol.L or equivalently 170 g.L or 450,000 molecules/capsule. The detailed investigation of the encapsulation mechanism shows that the dye concentrates in the droplets during the emulsification step simultaneously with capsule formation. Then it precipitates inside the capsules during the course of solvent evaporation. In vitro fluorescence measurements show that the nano-precipitated cargo can be transferred from the core of the hybridosomes® to the membrane of liposomes. In vivo studies suggest that the dye diffuses through the body during several days. The released dye tends to accumulate in body-fat, while the inorganic nanoparticles remain trapped into the liver and the spleen macrophages