Milk sphingosomes as lipid carriers for tocopherols in aqueous foods: Thermotropic phase behaviour and morphology.

Foods containing polyunsaturated lipids are prone to oxidation. Designing food-grade hydrocolloidal encapsulation systems able to load lipophilic antioxidant molecules, such as tocopherols (vitamin E), is necessary to prevent oxidation and its deleterous consequences. In this study, we hypothesised that α-tocopherol molecules could incorporate in a host membrane composed of milk sphingomyelin (milk-SM) and performed a multi-scale biophysical study. The thermal properties of milk-SM bilayers with various molar proportions of α-tocopherol were characterised by differential scanning calorimetry (DSC), their structural properties were examined by X-ray diffraction (XRD). The miscibility between milk-SM and α-tocopherol was investigated in mixed Langmuir monolayers. The morphology of milk-SM sphingosomes was observed by confocal laser scanning microscopy (CLSM). We found that molecules of α-tocopherol inserted into the milk-SM bilayers and induced a physical desorganisation in the membrane packing, both in the ordered and fluid states. In the presence of α-tocopherol, the bilayers were no longer in a gel phase below the phase transition temperature Tm, but in the liquid ordered Lo phase. Furthermore, the sphingosomes formed elongated structures in presence of α-tocopherol as a result of membrane softening and changes in the bilayer curvature associated to membrane fusion. The findings of this work contribute in a better understanding of the capacity of milk-SM bilayers to incorporate guest molecules. Milk-SM sphingosomes loaded with tocopherols could be used to prevent oxidation in aqueous foods containing polyunsaturated lipids such as oil-in-water emulsions.

Breaking photoswitch activation depth limit using ionising radiation stimuli adapted to clinical application.

Electromagnetic radiation-triggered therapeutic effect has attracted a great interest over the last 50 years. However, translation to clinical applications of photoactive molecular systems developed to date is dramatically limited, mainly because their activation requires excitation by low-energy photons from the ultraviolet to near infra-red range, preventing any activation deeper than few millimetres under the skin. Herein we conceive a strategy for photosensitive-system activation potentially adapted to biological tissues without any restriction in depth. High-energy stimuli, such as those employed for radiotherapy, are used to carry energy while molecular activation is provided by local energy conversion. This concept is applied to azobenzene, one of the most established photoswitches, to build a radioswitch. The radiation-responsive molecular system developed is used to trigger cytotoxic effect on cancer cells upon gamma-ray irradiation. This breakthrough activation concept is expected to expand the scope of applications of photosensitive systems and paves the way towards the development of original therapeutic approaches.

Elongated self-assembled nanocarriers: From molecular organization to therapeutic applications.

Self-assembled cylindrical aggregates made of amphiphilic molecules emerged almost 40 years ago. Due to their length up to micrometers, those particles display original physico-chemical properties such as important flexibility and, for concentrated samples, a high viscoelasticity making them suitable for a wide range of industrial applications. However, a quarter of century was needed to successfully take advantage of those improvements towards therapeutic purposes. Since then, a wide diversity of biocompatible materials such as polymers, lipids or peptides, have been developed to design self-assembling elongated drug nanocarriers, suitable for therapeutic or diagnostic applications. More recently, the investigation of the main forces driving the unidirectional growth of these nanodevices allowed a translation toward the formation of pure nanodrugs to avoid the use of unnecessary side materials and the possible toxicity concerns associated.

Importance of Combining Advanced Particle Size Analysis Techniques To Characterize Cell-Penetrating Peptide-Ferrocifen Self-Assemblies.

The design of a simple platform to target the delivery of notably hydrophobic drugs into cancer cells is an ultimate goal. Here, three strategies were combined in the same nanovector, in limiting the use of excipients: cell-penetrating peptides, an amphiphilic prodrug, and self-assembly. Light scattering and cryogenic transmission electron microscopy revealed one size population of objects around 100 nm with a narrow size distribution. However, in-depth analysis of the suspension by nanoparticle tracking analysis, small-angle X-ray scattering, and nuclear magnetic resonance (NMR) diffusometry demonstrated the presence of another population of small objects (<2 nm). It has been shown that these small self-assemblies represented >99% of the matter! This presence was clearly and unambiguously demonstrated by NMR diffusometry experiments. The study highlights the importance and the complementary contribution of each characterization method to reflect the reality of the studied nanoassembly.

Deciphering the Peculiar Behavior of ß-Lapachone in Lipid Monolayers and Bilayers.

ß-Lapachone (ß-Lap) is a promising anticancer drug whose applications have been limited so far because of its poor solubility and stability. Its encapsulation in liposomes has been proposed to overcome these issues. However, surface pressure measurements show that ß-Lap exhibits atypical interfacial behavior when mixed with lipids. Although the drug does not seem to be retained in lipid monolayers as deduced from the π-A isotherms, small changes in compressibility moduli suggest that ß-Lap actually interacts with lipids, either disorganizing or rigidifying their monolayers. Thermal and structural analyses of lipid bilayers confirm the existence of ß-Lap/lipid interactions and show that the drug inserts between hydrophobic chains, close to the polar headgroup in DPPC bilayers and deeper in the acyl chains in POPC bilayers. Molecular dynamics simulations allow a comprehensive description of the drug position and orientation in DOPC and POPC bilayers in the presence or absence of cholesterol.

Stacking as a Key Property for Creating Nanoparticles with Tunable Shape: The Case of Squalenoyl-Doxorubicin.

The development of elongated nanoparticles for drug delivery is of growing interest in recent years, due to longer blood circulation and improved efficacy compared to spherical counterparts. Squalenoyl-doxorubicin (SQ-Dox) conjugate was previously shown to form elongated nanoparticles with improved therapeutic efficacy and decreased toxicity compared to free doxorubicin. By using experimental and computational techniques, we demonstrate here that the specific physical properties of SQ-Dox, which include stacking and electrostatic interactions of doxorubicin as well as hydrophobic interactions of squalene, are involved in the formation of nanoassemblies with diverse elongated structures. We show that SQ-Dox bioconjugate concentration, ionic strength, and anion nature can be used to modulate the shape and stiffness of SQ-Dox nanoparticles. As those parameters are involved in nanoparticle behavior in biological media, these findings could bring interesting opportunities for drug delivery and serve as an example for the design of original nanodrugs with stacking properties tuned for particular clinical purposes.

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.

The Critical Role of Thioacetamide Concentration in the Formation of ZnO/ZnS Heterostructures by Sol-Gel Process.

ZnO/ZnS heterostructures have emerged as an attractive approach for tailoring the properties of particles comprising these semiconductors. They can be synthesized using low temperature sol-gel routes. The present work yields insight into the mechanisms involved in the formation of ZnO/ZnS nanostructures. ZnO colloidal suspensions, prepared by hydrolysis and condensation of a Zn acetate precursor solution, were allowed to react with an ethanolic thioacetamide solution (TAA) as sulfur source. The reactions were monitored in situ by Small Angle X-ray Scattering (SAXS) and UV-vis spectroscopy, and the final colloidal suspensions were characterized by High Resolution Transmission Electron Microscopy (HRTEM). The powders extracted at the end of the reactions were analyzed by X-ray Absorption spectroscopy (XAS) and X-ray diffraction (XRD). Depending on TAA concentration, different nanostructures were revealed. ZnO and ZnS phases were mainly obtained at low and high TAA concentrations, respectively. At intermediate TAA concentrations, we evidenced the formation of ZnO/ZnS heterostructures. ZnS formation could take place via direct crystal growth involving Zn ions remaining in solution and S ions provided by TAA and/or chemical conversion of ZnO to ZnS. The combination of all the characterization techniques was crucial to elucidate the reaction steps and the nature of the final products.

Supramolecular Organization and siRNA Binding of Hyaluronic Acid-Coated Lipoplexes for Targeted Delivery to the CD44 Receptor.

The dynamics of the formation of siRNA-lipoplexes coated with hyaluronic acid (HA) and the parameters influencing their supramolecular organization were studied. The insertion of a HA-dioleylphosphatidylethanolamine (DOPE) conjugate in the liposome structure as well as subsequent complexation with siRNA increased the liposome size. Lipoplexes were around 110 nm at high ± charge ratios with a zeta potential around +50 mV and around 230 nm at low ± ratios, with a zeta potential that decreased to negative values, reaching -45 mV. The addition of the conjugate did not compromise siRNA binding to liposomes, although these nucleic acids induced a displacement of part of the HA-DOPE conjugate upon lipoplex formation, as confirmed by capillary electrophoresis. Isothermal titration calorimetry, X-ray diffraction studies, and cryo-TEM microscopy demonstrated that in addition to electrostatic interactions with siRNA a rearrangement of the lipid bilayers takes place, resulting in condensed oligolamellar vesicles. This phenomenon is dependent on the number of siRNA molecules and the degree of modification with HA. Finally, the suitable positioning of HA on the lipoplex surface and its ability to bind specifically to the CD44 receptors in a concentration-dependent manner was demonstrated by surface plasmon resonance analysis.

Influence of the nanoprecipitation conditions on the supramolecular structure of squalenoyled nanoparticles.

Hydrophobic organic compounds dissolved in a polar solvent can self-assemble into nanoparticles (NPs) upon nanoprecipitation into water. In the present study, we have investigated the structure of squalenacetyl-adenosine (SQAc-Ad) nanoparticles which were previously found to exhibit impressive neuroprotective activity. When obtained by nanoprecipitation of a SQAc-Ad ethanolic solution into water, two different supramolecular organizations of SQAc-Ad NPs were evidenced, depending on the water-to-ethanol volume ratio. It has been shown that a fraction of the solvent remained associated with the NPs, despite prolonged evaporation under reduced pressure after nanoprecipitation, and that this residual solvent dramatically affected their structure. This study points to the importance of being in the "Ouzo" region to minimize the amount and effect of residual solvent and to control the structure of NPs.

Novel self assembling nanoparticles for the oral administration of fondaparinux: synthesis, characterization and in vivo evaluation.

Fondaparinux (Fpx) is the anticoagulant of choice in the treatment of short- and medium-term thromboembolic disease. To overcome the low oral bioavailability of Fpx, a new nanoparticulate carrier has been developed. The nanoparticles (NPs) contain squalenyl derivatives, known for their excellent oral bioavailability. They spontaneously self-assemble upon both electrostatic and hydrophobic interactions between the polyanionic Fpx and cationic squalenyl (CSq) derivatives. The preparation conditions were optimized to obtain monodisperse, stable NPs with a mean diameter in the range of 150-200 nm. The encapsulation efficiencies were around 80%. Fpx loadings reached 39 wt.%. According to structural and morphological analysis, Fpx and CSq organized in spherical multilamellar ("onion-type") nanoparticles. Furthermore, in vivo studies in rats suggested that Fpx was well absorbed from the orally administered NPs, which totally dissociated when reaching the blood stream, leading to the release of free Fpx. The Fpx:CSq NPs improved the plasmatic concentration of Fpx in a dose-dependent manner. However, the oral bioavailability of these new NPs remained low (around 0.3%) but of note, the Cmax obtained after oral administration of 50mg/kg NPs was close to the prophylactic plasma concentration needed to treat venous thromboembolism. Moreover, the oral bioavailability of Fpx could be dramatically increased up to 9% by including the nanoparticles into gastroresistant capsules. This study opens up new perspectives for the oral administration of Fpx and paves the way towards elaborating squalene-based NPs which self assemble without the need of covalently grafting the drug to Sq.

Self-assembly of polyisoprenoyl gemcitabine conjugates: influence of supramolecular organization on their biological activity.

An amphiphilic prodrug of gemcitabine, a cytidine analogue used clinically against various tumors, had been previously synthesized by covalent coupling to squalene, a natural isoprenoid chain. The resulting bioconjugate self-assembled spontaneously in water as nanoparticles, displaying an impressive activity both in vitro and in vivo. The aim of the present study was to determine the influence of the length of the isoprene moiety on the structure of the nanoparticles, in an attempt to establish a relationship between the chemical structure of the prodrug, its supramolecular organization, and its pharmacological activity. Remarkably, gemcitabine-squalene and gemcitabine-5-isoprenes, which differ only in the position of two methyl groups on the hydrophobic chain, displayed different supramolecular organizations and different anticancer activities on some cell lines. This difference in activity was related to the ability of nanoparticles to be internalized by cells.

Interactions of anticancer drugs with biomembranes: what can we learn from model membranes?

The interactions of anticancer drugs with cell membranes are of primary importance for drug transport, accumulation and activity. However, these interactions are very difficult to investigate because of the complexity of biological membranes. Lipid model membranes have therefore been built to gain insight into the collective role of lipids in drug-membrane interactions. Membranes can act as a barrier for drug molecules, sequester them or conversely may allow them to freely diffuse, thereby modulating the accumulation of drugs into cells. Lipid membranes also affect the ability of the efflux pump Pgp to bind and efflux anticancer drugs from cells. On the other hand, anticancer drugs can alter the structure and properties of lipid membranes, which are expected to influence the functioning of embedded proteins. The relevance of lipid model membranes to assess interactions between anticancer drugs and biomembranes is evidenced.

A unique squalenoylated and nonpegylated doxorubicin nanomedicine with systemic long-circulating properties and anticancer activity.

We identified that the chemical linkage of the anticancer drug doxorubicin onto squalene, a natural lipid precursor of the cholesterol's biosynthesis, led to the formation of squalenoyl doxorubicin (SQ-Dox) nanoassemblies of 130-nm mean diameter, with an original "loop-train" structure. This unique nanomedicine demonstrates: (i) high drug payload, (ii) decreased toxicity of the coupled anticancer compound, (iii) improved therapeutic response, (iv) use of biocompatible transporter material, and (v) ease of preparation, all criteria that are not combined in the currently available nanodrugs. Cell culture viability tests and apoptosis assays showed that SQ-Dox nanoassemblies displayed comparable antiproliferative and cytotoxic effects than the native doxorubicin because of the high activity of apoptotic mediators, such as caspase-3 and poly(ADP-ribose) polymerase. In vivo experiments have shown that the SQ-Dox nanomedicine dramatically improved the anticancer efficacy, compared with free doxorubicin. Particularly, the M109 lung tumors that did not respond to doxorubicin treatment were found inhibited by 90% when treated with SQ-Dox nanoassemblies. SQ-Dox nanoassembly-treated MiaPaCa-2 pancreatic tumor xenografts in mice decreased by 95% compared with the tumors in the saline-treated mice, which was significantly higher than the 29% reduction achieved by native doxorubicin. Concerning toxicity, SQ-Dox nanoassemblies showed a fivefold higher maximum-tolerated dose than the free drug, and moreover, the cardiotoxicity study has evidenced that SQ-Dox nanoassemblies did not cause any myocardial lesions, such as those induced by the free doxorubicin treatment. Taken together, these findings demonstrate that SQ-Dox nanoassemblies make tumor cells more sensitive to doxorubicin and reduce the cardiac toxicity, thus providing a remarkable improvement in the drug's therapeutic index.

Nanoprecipitation and the "Ouzo effect": Application to drug delivery devices.

Biodegradable nanocarriers such as lipid- or polymer-based nanoparticles can be designed to improve the efficacy and reduce the toxic side effects of drugs. Under appropriate conditions, nanoprecipitation of a hydrophobic compound solution in a non-solvent can generate a dispersion of nanoparticles with a narrow distribution of sizes without the use of surfactant ("Ouzo" effect). The aim of this review is to present the main parameters controlling the nucleation and growth of aggregates in a supersaturated solution and the characteristics of the obtained nanoparticles. The importance of the kinetics of mixing of the solution containing the hydrophobic compound and the non-solvent is highlighted. Illustrative examples of polymeric nanoparticles for drug delivery or terpenoid-based nanoprodrugs obtained by nanoprecipitation are reported.

Self-assembly of squalene-based nucleolipids: relating the chemical structure of the bioconjugates to the architecture of the nanoparticles.

Squalene-based nucleolipids, including anticancer or antiviral prodrugs, gave rise to nanoparticles displaying a diversity of structures upon nanoprecipitation in water. Synchrotron small-angle X-ray scattering and cryo-TEM imaging revealed that both the nature of the nucleoside and the position of the squalene moiety relative to the nucleobase determined the self-assembly of the corresponding bioconjugates. It was found that small chemical differences resulted in major differences in the self-organization of nucleolipids when squalene was grafted onto the nucleobase whereas only lamellar phases were observed when squalene was linked to the sugar moiety. The key role of hydrogen bonds between nucleobases in the formation of the lamellar phases was suggested, in agreement with molecular simulations. These findings provide a way to fine tune the supramolecular organization of squalene-based prodrugs, with the aim of improving their pharmacological activity.

Nanostructured fluids from pluronic® mixtures.

Micellization and gelation of binary mixtures of EO99PO69EO99 (pluronic(®) F127) and EO80PO27EO80 (pluronic(®) F68) in aqueous solutions were investigated by means of micro-differential scanning calorimetry and rheology and for a total copolymer concentration fixed at 20 wt%. The aim of this investigation is to determine the interplay between micellization and macroscopic gelation of the mixed solutions. Micro-DSC reveals the formation of two distinct populations in F127/F68 mixtures during heating and subsequent cooling of the solutions. The enthalpies of micellization of each copolymer and the respective onset temperatures remained constant after mixing indicating the predominance of two independent processes of micellization in the mixtures. The F127 exhibits a crystallization transition, at a distinct temperature which persists, but increases in the mixtures with concentrations higher than 10 wt%. Rheological measurements were performed during heating ramps or after maturation periods versus frequency. They showed two types of gelation transitions: either a steep increase of the storage and the loss moduli, which corresponds to the crystallization temperature of the F127 micelles or a progressive jamming transition when no crystal can form. Maturation process has a major effect on the rheological properties of the mixed gels, possibly related to local rearrangements of the two micellar phases. This investigation highlights the unique features of the binary pluronic(®) mixtures, compared to dilution effects of single component aqueous solutions.

Polyisoprenoyl gemcitabine conjugates self assemble as nanoparticles, useful for cancer therapy.

A series of new polyisoprenoyl prodrugs of gemcitabine, which can be formulated as nanoassemblies are described. These prodrugs were designed to improve gemcitabine efficacy and to overcome the limitations due to the systemic toxicity of this anticancer compound. In vitro biological assessment showed that these polyisoprenoyl gemcitabine nanoassemblies displayed notable cytotoxicity on several cancer cell lines, including murine melanoma cell line B16F10, human pancreatic carcinoma cell line MiaPaCa-2, human lung carcinoma cell line A549 and human breast adenocarcinoma cell line MCF7. Interestingly, it was observed that the anticancer efficacy of these nanoassemblies was dependant on the size of polyisoprenoyl moiety. The polyisoprenoyl prodrug of gemcitabine containing three isoprene units (2d) was the more active on all the cancer cell lines tested. The antitumor efficacy of the nanoassemblies (NAs) constructed with the most active prodrug 2d was further evaluated on a human pancreatic (MiaPaCa-2) carcinoma xenograft model in mice. The prodrug 2d NAs showed an increased antitumor efficacy as compared to free gemcitabine or to squalene-gemcitabine (SQ-gem, 2a) nanoassemblies. Interestingly, MiaPaCa-2 tumors that did not respond to gemcitabine were inhibited by 76% after treatment with prodrug 2d NAs, whereas SQ-gem-treated MiaPaCa-2 tumor xenografts decreased only by 41% compared to saline or to gemcitabine-treated mice. Together, these findings demonstrated that the modulation of the length of nanoassemblies polyisoprenoyl moiety made tumor cells more sensitive to gemcitabine treatment without flagrant toxicity, thus providing a significant improvement in the drug therapeutic index.

Improving the antitumor activity of squalenoyl-paclitaxel conjugate nanoassemblies by manipulating the linker between paclitaxel and squalene.

A series of new lipid prodrugs of paclitaxel, which can be formulated as nanoassemblies, are described. These prodrugs which are designed to overcome the limitations due to the systemic toxicity and low water solubility of paclitaxel consist of a squalene chain bound to the 2'-OH of paclitaxel through a 1,4-cis,cis-dienic linker. This design allows the squalene-conjugates to self-assemble as nanoparticular systems while preserving an efficient release of the free drug, thanks to the dienic spacer. The size, steric hindrance, and functional groups of the spacer have been modulated. All these prodrugs self-assemble into nanosized aggregates in aqueous solution as characterized by dynamic light scattering and transmission electron microscopy and appear stable in water for several days as determined by particle size measurement. In vitro biological assessment shows that these squalenoyl-paclitaxel nanoparticles display notable cytotoxicity on several tumor cell lines including A549 lung cell line, colon cell line HT-29, or KB 3.1 nasopharyngeal epidermoid cell line. The cis,cis-squalenyl-deca-5,8-dienoate prodrug show improved activity over simple 2'-squalenoyl-paclitaxel prodrug highlighting the favourable effect of the dienic linker. The antitumor efficacy of the nanoassemblies constructed with the more active prodrugs has been investigated on human lung (A549) carcinoma xenograft model in mice. The prodrug bearing the cis,cis-deca-5,8-dienoyl linker shows comparable antitumor efficacy to the parent drug, but reveals a much lower subacute toxicity as seen in body weight loss. Thus, nanoparticles with the incorporated squalenoyl paclitaxel prodrug may prove useful for replacement of the toxic Cremophor EL.

Self-assembled nucleolipids: from supramolecular structure to soft nucleic acid and drug delivery devices.

This short review aims at presenting some recent illustrative examples of spontaneous nucleolipids self-assembly. High-resolution structural investigations reveal the diversity and complexity of assemblies formed by these bioinspired amphiphiles, resulting from the interplay between aggregation of the lipid chains and base-base interactions. Nucleolipids supramolecular assemblies are promising soft drug delivery systems, particularly for nucleic acids. Regarding prodrugs, squalenoylation is an innovative concept for improving efficacy and delivery of nucleosidic drugs.