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In this study, we explored how chemical reactions of amphiphile compounds can be characterized and followed-up on model interfaces. A custom-made surfactant containing three alkyne sites was first adsorbed and characterized at a water/oil interface. These amphiphiles then underwent interfacial crosslinking by click chemistry upon the addition of a second reactive agent. The monolayer properties and dilatational elasticity, were compared before and after the polymerization. Using bulk phase exchange, the composition of the aqueous bulk phase was finely controlled and washed to specifically measure the interfacial effects of the entities adsorbed and trapped at the interface. In this study, we aim to emphasize an original experimental approach to follow complex phenomena occurring on model interfaces, and also show the potential of this method to characterize multifactorial processes.

In this work, we used an original experimental setup to examine the behavior of insoluble monolayers made with pH-sensitive lipids. Two kinds of unsaturated lipids were chosen: a cationic one (lipid 1) bearing an ammonium headgroup and an anionic one (lipid 2) terminated with an acidic phenol group. The lipids were deposited onto an air bubble interface maintained in an aqueous phase and, after stabilization, were subjected to a series of compressions performed at different pH values. These experiments disclosed a gradual increase in the specific area per molecule when lipids were neutralized. Imposing a pH variation at constant bubble volume also provided surface pressure profiles that confirmed this molecular behavior. As complementary characterization, dilatational rheology disclosed a phase transition from a purely elastic monophasic system to a viscoelastic two-phase system. We hypothesized that this unexpected increase in the specific area with lipid neutralization is related to the presence of unsaturations in each of the two branches of the hydrophobic tails that induce disorder, thereby increasing the molecular area at the interface. Application of the two-dimensional Volmer equation of state allowed the generation of quantitative values for the specific areas that showed variations with pH. It also allowed the determination of apparent pKa values, which are affected by both the electrostatic potential within the monolayer and the affinity of the lipid polar head for the aqueous phase.

Nano-emulsions are defined as stable oil droplets sizing below 300 nm. Their singular particularity lies in the loading capabilities of their oily core, much higher than other kinds of carrier. On the other hand, functionalizing the dynamic oil/water interface, to date, has remained a challenge. To ensure the best anchoring of the reactive functions onto the surface of the droplets, we have designed specific amphiphilic polymers (APs) based on poly(maleic anhydride-alt-1-octadecene), stabilizing the nano-emulsions instead of surfactants. Aliphatic C18 chains of the APs are anchored in the droplet core, while the hydrophilic parts of the APs are poly(ethylene glycol) (PEG) chains. In addition, PEG chains are terminated with reactive (i) azide functions in order to prove the concept of the droplet decoration with clickable rhodamine (Rh-DBCO, specifically synthesized for this study), or (ii) biotin functions to verify the potential droplet functionalization with fluorescent streptavidin (streptavidin-AF-488). This study describes AP synthesis, physico-chemical characterization of the functional droplets (electron microscopy), and finally fluorescence labeling and droplet decoration. To conclude, these APs constitute an interesting solution for the stable functionalization of nano-emulsion droplets, paving a new way for the applications of nano-emulsions in targeting drug delivery.

The eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) formulation with a ratio of 6:1 is a potent stimulator of the endothelial formation of nitric oxide (NO). The aim of the study was to investigate whether nanoencapsulation of EPA:DHA 6:1 followed by coating with gum increases its biological activity. Vascular reactivity was assessed using porcine coronary artery rings, the formation of NO in cultured endothelial cells (ECs) using DAF-FM and indirectly by platelet aggregation studies. Coated EPA:DHA 6:1 nanoparticles induced sustained relaxations of coronary artery rings that were greater in rings with than in those without endothelium, and more pronounced than with the native form. Treatment of ECs with coated EPA:DHA 6:1 nanoparticles caused greater and more sustained formation of NO and enhanced their anti-aggregatory effects. Thus, nanoencapsulation of EPA:DHA 6:1 is an attractive strategy to enhance the beneficial effect at the vascular endothelium.

Biphasic drug delivery systems are used for quick release of a specific amount of drug for immediate amelioration of a patient’s state, followed by sustained release, to avoid repeated administration. This type of delivery is often necessary for pain management and the treatment of many pathologies, such as migraines, hypertension, and insomnia. In this work, we propose a novel architecture of a biphasic release media that does not need the rapidly disintegrating layer and that allows for easily setting the sustained release rate. A drug-containing capsule is made by rolling up a thermally crosslinked gelatin strip on which drug reservoirs are formed by casting. The quick-release reservoir (QRR) is placed at the strip’s extremity, from which the rolling starts, while the sustained-release reservoir (SRR) is formed in the middle part of the strip. The strip is rolled around a cylinder that is a few millimeters wide, which is removed after rolling. The roll is stabilized by transglutaminase-catalyzed crosslinking of the consecutive shells. A biphasic release is successfully demonstrated with the use of model fluorescent drugs for single-dye and double-dye systems in phosphate-buffered saline (PBS) solution with pH = 7.4. In vitro, the drug from the QRR, placed at the walls of the cavity of the roll, is released immediately upon the capsule’s contact with the PBS solution. The drug from the SRR, embedded between the roll’s layers, diffuses steadily, with the lag time defined by the radial position of the reservoir.

Nanoemulsions (NEs) are biocompatible lipid nanoparticles composed of an oily core stabilized by a surfactant shell. It is acknowledged that the surface decoration with poly(ethylene glycol), through the use of nonionic surfactants, confers high stealth in biological medium with reduced nonspecific interactions. Tracking individual NE by fluorescence microscopy techniques would lead to a better understanding of their behavior in cells and thus require the development of bright single particles with enhanced photostability. However, the understanding of the relationship between the physicochemical properties and chemical composition of the NEs, on the one hand, and its fluorescence properties of encapsulated dyes, on the other hand, remains limited. Herein, we synthesized three new dioxaborine barbituryl styryl (DBS) dyes that displayed high molar extinction coefficients (up to 120 000 M

Attractive interest on double emulsions comes from their unique morphology, making them general multifunctional carriers able to encapsulate different hydrophilic and lipophilic molecules in the same particle. Over the past century, two different types of methods were followed to prepare double emulsions for pharmaceutics applications, so-called "one-step" and "two-step" processes. The two-step approach, consisting in two different emulsifications successively performed, allows the optimal and more efficient formulations due to simplicity of principle and controllability of the process. In this review, focused on the formulation of double emulsions by two-step process, we recount the historical development of this approach, along with the state-of-the-art, including a discussion on the role of the formulation parameters, surfactants, amphiphilic polymers, interface stabilization, volume fraction, and so forth, on the final formulation stability, morphology and properties as drug delivery system. Discussion was also extended to polymeric microparticles and nanoparticles made by solvent diffusion, on the basis of double emulsions made by two-step process, along with literature review on the impact of different formulation and processing parameters. In addition, the properties of the polymers used in the microparticles matrix (molecular weight, chemical nature) potentially impacting on the ones of the microparticles formed (drug release kinetics, stability, morphology), were also discussed. Finally, the future trends in double emulsions application were addressed, emphasizing some new advances made in the emulsifications method as potentially able to open the range of applications, for example to nanoscale with spontaneous emulsification or low energy microfluidic emulsification.

Since antiquity, the survival of human civilization has always been threatened by the microbial infections. An alarming surge in the resistant microbial strains against the conventional drugs is quite evident in the preceding years. Furthermore, failure of currently available regimens of antibiotics has been highlighted by the emerging threat of biofilms in the community and hospital settings. Biofilms are complex dynamic composites rich in extracellular polysaccharides and DNA, supporting plethora of symbiotic microbial life forms, that can grow on both living and non-living surfaces. These enforced structures are impervious to the drugs and lead to spread of recurrent and non-treatable infections. There is a strong realization among the scientists and healthcare providers to work out alternative strategies to combat the issue of drug resistance and biofilms. Plants are a traditional but rich source of effective antimicrobials with wider spectrum due to presence of multiple constituents in perfect synergy. Other than the biocompatibility and the safety profile, these phytochemicals have been repeatedly proven to overcome the non-responsiveness of resistant microbes and films via multiple pathways such as blocking the efflux pumps, better penetration across the cell membranes or biofilms, and anti-adhesive properties. However, the unfavorable physicochemical attributes and stability issues of these phytochemicals have hampered their commercialization. These issues of the phytochemicals can be solved by designing suitably constructed nanoscaled structures. Nanosized systems can not only improve the physicochemical features of the encapsulated payloads but can also enhance their pharmacokinetic and therapeutic profile. This review encompasses why and how various types of phytochemicals and their nanosized preparations counter the microbial resistance and the biofouling. We believe that phytochemical in tandem with nanotechnological innovations can be employed to defeat the microbial resistance and biofilms. This review will help in better understanding of the challenges associated with developing such platforms and their future prospects.

International audience; Endothelial senescence has been identified as an early event in the development of endothelial dysfunction, a hallmark of cardiovascular disease. This study developed theranostic nanocarriers (NC) decorated with VCAM-1 antibodies (NC-VCAM-1) in order to target cell surface VCAM-1, which is overexpressed in senescent endothelial cells (ECs) for diagnostic and therapeutic purposes. Incubation of Ang IIinduced premature senescent ECs or replicative senescent ECs with NC-VCAM-1 loaded with lipophilic fluorescent dyes showed higher fluorescence signals than healthy EC, which was dependent on the NC size and VCAM-1 antibodies concentration, and not observed following masking of VCAM-1. NC loaded with omega 3 polyunsaturated fatty acid (NC-EPA:DHA6:1) were more effective than native EPA:DHA 6:1 to prevent Ang II-induced VCAM-1 and p53 upregulation, and SA-β-galactosidase activity in coronary artery segments. These theranostic NC might be of interest to evaluate the extent and localization of endothelial senescence and to prevent pro-senescent endothelial responses.

International audience; Upconversion nanoparticles are a promising class of materials for bioimaging. Their attractive optical properties are influenced by their crystalline phase, size, and morphology resulting from the preparation method employed. Herein, we synthesized NaYF4:Yb,Er nanocrystals by the co-precipitation method followed by a heat treatment step. The influence of the treatment temperature and the type of reactor on the phase transition from the α-cubic to the β-hexagonal phase was investigated. We found that before the phase transition, the size and shape of α-NaYF4:Yb,Er nanocrystals evolved due to a coalescence mechanism with microtubular reactors. In contrast to the flask system, we also observed an anisotropic growth of β-NaYF4:Yb,Er nanorods in microtubes whose length can be controlled by adjusting the temperature. The diameter of microtubes plays a critical role in the shape evolution of the final products from spheres to nanorods. The type of reactor and thus the resulting temperature gradient could be considered as a promising parameter to control the phase, shape and size of UCNPs without changing the chemical composition.

International audience; Continuous progress of personalized medicine is insistently calling the pharmaceutical sector to increase product variability more than ever before. One of the challenges consists in personalization of the drug release rates and doses from drug-containing media. Herein is introduced an innovative methodology which resolves this problem by combining inkjet printing and rolling up procedure, allowing for the production of capsules with tailorable distributions of medical compounds. The prototype capsules consist of rolled-up polymeric films over which fluorescent probes have been deposited via inkjet printing prior to rolling. The position of the probes inside the capsules are determined by the printed pattern. The delay and the rate of the release of the probes in dissolution media can be shaped by the number of rolls and the position of the drug reservoir on the polymeric film before rolling. The innovative method proposed in the paper paves the way to a novel family of drug delivery devices with a broad range of therapeutic applications and it stands alongside other additive manufacturing techniques but allows to avoid their usual constraints.

Nano-emulsion consists of a dispersion of oil droplets sizing below 200 nm, in aqueous continuous phase, and generally stabilized by low-molecular-weight surfactants. These stable nano-carriers are able to encapsulate and transport lipophilic molecules poorly soluble in water. However, the question on the leakage and release mechanisms of an active pharmaceutical ingredient, from oil nano-droplets to an acceptor medium has not been clearly addressed. Herein, we developed a simple fluorescence approach based on self-quenching of lipophilic fluorophore-based on Nile Red (NR668) to monitor cargo transfer from lipid nano-droplets to the acceptor medium. In this method, the fluorophore release can be monitored by the increase in its fluorescence quantum yield and the blue shift in its emission spectrum. The studies of the release process allow emphasizing an important role of the bulk aqueous medium in controlling the droplet to droplet fluorophore transfer and the attained equilibrium. The developed methodology could be applied to monitor release of other lipophilic dyes and it could help to better understand the cargo release from nanocarriers.

Statins have been proposed as potential adjuvant to periodontal treatment due to their pleiotropic properties. A new thermosensitive chitosan hydrogel loaded with statins (atorvastatin and lovastatin) nanoemulsions was synthesized to allow a spatially controlled local administration of active compounds at lesion site. Spontaneous nano-emulsification method was used to synthesize statins loaded nanoemulsions. In vitro, atorvastatin and lovastatin loaded nanoemulsions were cytocompatible and were able to be uptake by oral epithelial cells. Treatment of Porphyromonas gingivalis infected oral epithelial cells and gingival fibroblasts with atorvastatin and lovastatin loaded nanoemulsions decreased significantly pro-inflammatory markers expression (TNF-α and IL-1β) and pro-osteoclastic RANKL. Nevertheless, such treatment induced the expression of Bone sialoprotein 2 (BSP2) in osteoblast emphasizing the pro-healing properties of atorvastatin and lovastatin nanoemulsions. In vivo, in a calvarial bone defect model (2 mm), treatment with the hydrogel loaded with atorvastatin and lovastatin nanoemulsions induced a significant increase of the neobone formation in comparison with systemic administration of statins. This study demonstrates the potential of this statins loaded hydrogel to improve bone regeneration and to decrease soft tissue inflammation. Its use in the specific context of periodontitis management could be considered in the future with a reduced risk of side effects.

International audience; Efficient and safe delivery of anticancer drugs such as 5-fluorouracil (5-FU) is still a challenge in chemotherapy. The combination of polymers with a luminescent probe offers a promising venue to develop nanoobjects for theranostics. Herein, upconversion nanoparticles (UCNPs) decorated with polymers based on N-(2-hydroxypropyl) methacrylamide at their surface were investigated as potential carriers of 5-FU. The hyperbranched topology of the polymer offered a higher loading capacity of the polymer shell as compared to this linear analog even though the loading in 5-FU remained low and its release was fast (few hours). To overcome these limitations, 5-FU was conjugated to the polymer through esterification leading to a four-fold increase of the loading and a more sustained release over few days. The introduction of branching points with a disulfide bridge on the polymer afforded nanohybrids susceptible to degradation in the reductive environment of cancer cells. The degradation of the polymer was confirmed by incubating the polymer with dithiothreitol. These results pave the way to theranostics through the use of luminescent properties of UCNPs and the tuning of the polymer architecture.

International audience; Hyperbranched poly(N-(2-hydroxypropyl) methacrylamide)s grafted on upconversion nanoparticles (UCNPs) were prepared by surface-initiated self-condensing vinyl RAFT copolymerization from the surface of UCNPs modified with a RAFT chain transfer agent. The effect of the grafting density in RAFT chain transfer agent on UCNPs and the concentration in transmer were investigated. The propagation of the polymerization was strongly affected by termination and transfer reactions associated to the high local concentration in propagating chains due to the immobilization of the polymer chains at the surface of UCNPs and increased number of active centers during the polymerization, but also to the hopping and rolling mechanisms related to the surface-initiated RAFT polymerization. The development of such nanohybrids opens the way to novel nano-objects for biomedical imaging and theranostics.

Nanosized gel particles, so-called nanogels, have attracted substantial interest in different application fields, thanks to their controllable and three-dimensional physical structure, good mechanical properties and potential biocompatibility. Literature reports many technologies for their preparation and design, however a recurrent limitation remains in their broad size distributions as well as in the poor size control. Therefore, the monodisperse and size-controlled nanogels preparation by simple process –like emulsification– is a real challenge still in abeyance to date. In this study we propose an original low energy emulsification approach for the production of monodisperse nanogels, for which the size can be finely controlled in the range 30 to 200 nm. The principle lies in the fabrication of a direct nano-emulsion containing both oil (medium chain triglycerides) and a bi-functional acrylate monomer. The nanogels are thus formed in situ upon UV irradiation of the droplet suspension. Advantage of such modification of the oil nano-carriers are the potential modulation of the release of encapsulated drugs, as a function of the density and/or properties of the polymer chain network entrapped in the oil nano-droplets. This hypothesis was confirmed using a model of hydrophobic drug –ketoprofen– entrapped into the nanogels particles, along with the study of the release profile, carried out in function of the nature of the monomers, density of polymer chains, and different formulation parameters.

International audience; Currently, drug delivery strategies using nanocarriers (NCs) deal with encapsulation of cargo or its covalently modified prodrug. Here, we propose a concept of reversible pH-controlled capture and delivery of active cargo based on dynamic covalent chemistry inside lipid nano-droplets (nanoemulsions), coined as "drug sponge". We designed a highly lipophilic hydrazide (LipoHD) capable to react with a free cargo-ketone (fluorescent dye and doxorubicin drug) directly inside lipid NCs, yielding lipophilic hydrazone prodrug efficiently captured in the oil core. LipoHD-loaded NCs spontaneously accumulated cargo-ketones, yielding formulations stable against cargo leakage at pH 7.4, and further released their dye/drug cargo at low pH range (5.0-6.8) in solution and live cells. Doxorubicin-loaded drug-sponge NCs showed cytotoxicity in four cancer cell lines and capacity to inhibit tumor growth in subcutaneous xenografts of mice. Finally, unprecedented extraction of dye/drug cargos directly from cells and tissues (i.e. detoxification) was realized by the drug-sponge NCs.

The prevalence of nosocomial infections due to multidrug resistant (MDR) bacterial strains is associated with high morbidity and mortality. Folk medicine and ethnopharmacological data can provide a broad range of plants with promising antimicrobial activity. Triphala, an Ayurvedic formula composed of three different plants: Terminalia chebula Retz., Terminalia bellirica (Gaertn.) Roxb. (Combretaceae), and Phyllanthus emblica L. (Phyllanthaceae), is used widely for various microbial infections. Various extraction techniques were applied in the extraction of the biologically active constituents of Triphala in order to compare their efficiency. Microwave-assisted extraction (MAE) was shown to be the most efficient method based on yield, extraction time, and selectivity. The Triphala hydroalcoholic extract (TAE) has been chemically characterized with spectroscopic and chromatographic techniques. Triphala hydroalcoholic extract was evaluated alone or with carvacrol. Different drug formulations including cream and nanoemulsion hydrogel were prepared to assess the antimicrobial activity against selected microorganism strains including Gram-positive and Gram-negative bacteria and fungi. We used a lipophilic oil of carvacrol (5 mg/mL) and a hydrophilic TAE (5 mg/mL) ingredient in a dosage form. Two solutions were created: hydrogel containing nanoemulsion as a lipophilic vector dispersed in the gel as a hydrophilic vehicle and a cream formulation, an oil-in-water emulsion. In both cases, the concentration was 250 mg of active ingredient in 50 mL of final formulation. The formulas developed were stable from a physical and chemical perspective. In the nanoemulsion hydrogel, the oil droplet size ranged from 124 to 129 nm, with low polydispersity index (PdI) 0.132 ± 0.013 and negative zeta potential −46.4 ± 4.3 mV. For the cream, the consistency factor (cetyl alcohol and white wax) induced immobilization of the matrix structure and the stability. Triphala hydroalcoholic extract in drug nanoformulation illustrated might be an adjuvant antimicrobial agent for treating various microbial infections.

This chapter presents the fundamental and application of lipid nanocarriers, dealing with the classification, preparative methods, and recent applications. It covers applications in various areas such as the drug delivery, cosmetics, food industry and fluorecence imaging application.

International audience; The purpose of this study was to develop Pickering water-in-oil nano-emulsions only stabilized by Eudragit RL100 nanoparticles (NPs), in order to increase the nano-emulsion stability and create a barrier to improve the drug encapsulation and better control the drug release. The first part of this study was dedicated to investigating the nano-emulsion formulation by ultrasonication and understanding the interfacial behavior and role of NPs in the stabilization of the water/oil interface. The focus was on the surface coverage in the function of the formulation parameters (volume fractions) to disclose the extents and limitations of the process. The main physicochemical analysis of the Pickering nano-emulsions was performed by dynamic light scattering and transmission electron microscopy. On the other hand, the second experimental approach was dedicated to understanding the interfacial behavior of the Eudragit RL100 NPs toward a model water/oil interface, using a dynamic tensiometer with axisymmetric drop shape analysis. The study investigated the NPs’ adsorption, as well as their rheological behavior. The aim of this part was to reveal the main phenomena that govern the interactions between NPs and the interface in order to understand the origin of Pickering nano-emulsions’ stability. The last part of the study was concerned with the stability and in vitro release of a model encapsulated drug (ketoprofen) in a gastric and simulated intestinal environment. The results showed that Pickering nano-emulsions significantly improved the resistance to gastric pH, inducing a significantly slower drug release compared to classical nano-emulsions’ stabilized surfactants. These Pickering nano-emulsions appear as a promising technology to modify the delivery of a therapeutic agent, in the function of the pH, and can be, for instance, applied to the oral drug delivery of poorly soluble drugs.

International audience; This study investigates the formulation of surfactant-free Pickering nano-emulsions able to release a drug at specific pH, in order to enhance its oral bioavailability. The stabilizing nanoparticles composed of magnesium hydroxide, were obtained by nano-precipitation method. The oil-in-water Pickering nano-emulsions stabilized with Mg(OH)2 nanoparticles, and encapsulating a model of hydrophobic drug (ibuprofen) were formulated following a high-energy process, using a sonication probe. The experimental approach explored the impact of all formulation parameters, composition and size of Mg(OH)2 nanoparticles, on the physico-chemical properties of the Pickering nano-emulsions. The system was characterized by DLS and transmission electron microscopy. In addition, Mg(OH)2 has the advantage of being solubilized in an acid medium leading to the destabilization of the nano-emulsion and the release of the active ingredient orally. The acid release study (pH = 1.2) showed cumulative release as a function of initial nanodroplet loading and saturation concentration. In basic media (pH = 6.8), we found a significant release of ibuprofen from the nano-emulsions that already had saturation in an acid medium. These nano-emulsions can not only protect patients from the side effects of acid medicines through the basic properties of hydroxides but also can contribute to the increase of the bioavailability of these drugs. In addition, once in the stomach pH is increased by hydroxides and promotes the release of active ingredients such as ibuprofen whose solubility is strongly influenced by pH.

Nano-emulsions consist of stable suspensions of nano-scaled droplets that have huge loading capacities and are formulated with safe compounds. For these reasons, a large number of studies have described the potential uses of nano-emulsions, focusing on various aspects such as formulation processes, loading capabilities, and surface modifications. These studies typically concern direct nano-emulsions (i.e., oil-in-water), whereas studies on reverse nano-emulsions (i.e., water-in-oil) remain anecdotal. However, reverse nano-emulsion technology is very promising (e.g., as an alternative to liposome technology) for the development of drug delivery systems that encapsulate hydrophilic compounds within double droplets. The spontaneous emulsification process has the added advantages of optimization of the energetic yield, potential for industrial scale-up, improved loading capabilities, and preservation of fragile compounds targeted for encapsulation. In this study, we propose a detailed investigation of the processes and formulation parameters involved in the spontaneous nano-emulsification that produces water-in-oil nano-emulsions. The following details were addressed: (i) the order of mixing of the different compounds (method A and method B), (ii) mixing rates, (iii) amount of surfactants, (iv) type and mixture of surfactants, (v) amount of dispersed phase, and (vi) influence of the nature of the oil. The results emphasized the effects of the formulation parameters (e.g., the volume fraction of the dispersed phase, nature or concentration of surfactant, or nature of the oil) on the nature and properties of the nano-emulsions formed.

Purpose: Multidrug resistance (MDR) of tumors to chemotherapeutics often leads to failure of cancer treatment. The aim of the study was to prepare novel MDR-overcoming chemotherapeutics based on doxorubicin (DOX) derivatives and to evaluate their efficacy in 2D and 3D in vitro models. Methods: To overcome MDR, we synthesized five DOX derivatives, and then obtained non-covalent complexes with human serum albumin (HSA). Drug efficacy was evaluated for two tumor cell lines, namely human breast adenocarcinoma MCF-7 cells and DOX resistant MCF-7/ADR cells. Additionally, MCF-7 cells were entrapped in alginate-oligochitosan microcapsules, and generated tumor spheroids were used as a 3D in vitro model to study cytotoxicity of the DOX derivatives. Results: Due to 3D structure, the tumor spheroids were more resistant to chemotherapy compared to monolayer culture. DOX covalently attached to palmitic acid through hydrazone linkage (DOX-N2H-Palm conjugate) was found to be the most promising derivative. Its accumulation levels within MCF-7/ADR cells was 4- and 10-fold higher than those of native DOX when the conjugate was added to cultivation medium without serum and to medium supplemented with 10% fetal bovine serum, respectively. Non-covalent complex of the conjugate with HSA was found to reduce the IC50 value from 32.9 µM (for free DOX-N2H-Palm) to 16.8 µM (for HSA-DOX-N2H-Palm) after 72 h incubation with MCF-7/ADR cells. Conclusion: Palm-N2H-DOX conjugate was found to be the most promising DOX derivative in this research. The formation of non-covalent complex of Palm-N2H-DOX conjugate with HSA allowed improving its anti-proliferative activity against both MCF-7 and MCF-7/ADR cells.

Double emulsions are very attractive systems for many reasons; the most important of these are their capacity to encapsulate hydrophilic and lipophilic molecules simultaneously in a single particle and their potentiality to protect fragile hydrophilic molecules from the continuous phase. Double emulsions represent a technology that is widely present down to the micrometer scale; however, double nanoemulsions, with their new potential applications as nanomedicines or diagnosis agents, currently present a significant challenge. In this study, we propose an original two-step approach for the fabrication of double nanoemulsions with a final size below 200 nm. The process consists of the formulation of a primary water-in-oil (w1/O) nanoemulsion by high-pressure homogenization, followed by the re-emulsification of this primary emulsion by a low-energy method to preserve the double nanostructure. Various characterization techniques were undertaken to confirm the double structure and to evaluate the encapsulation efficiency of a small hydrophilic probe in the inner aqueous droplets. Complementary fluorescence confocal and cryo-TEM microscopy experiments were conducted to characterize and confirm the double structure of the double nanoemulsion.

The development of biomaterials with low environmental impact has seen increased interest in recent years. In this field, lipid nanoparticles have found a privileged place in research and industry. The purpose of this study was to develop Pickering O/W nano-emulsions only stabilized by solid lipid nanoparticles (SLNs), as a new generation of safe, non-toxic, biocompatible, and temperature-sensitive lipid nano-carriers. The first part is dedicated to understanding the interfacial behavior of SLNs and their related stabilization mechanisms onto nano-emulsions formulated by ultrasonication. Investigations were focused on the surface coverage as a function of the SLN size and volume fraction of dispersed oil, in order to prove that the droplet stabilization is effectively performed by the nanoparticles, and to disclose the limitations of this formulation. Characterization is performed by dynamic light scattering and transmission electron microscopy. The second part of the study investigated SLN adsorption on a model oil/water interface (surface tension and rheology) through an axisymmetrical drop shape analysis (drop tensiometer), following the interfacial tension and the rheological behavior. The objective of this part is to characterize the phenomenon governing the droplet/interface interactions, and disclose the rheological behavior of the interfacial SLN monolayer. The effect of temperature was also investigated, proving a real destabilization of the nano-suspension when the sample is heated above a temperature threshold, impacting on the integrity of the SLNs, which partially melt, and strongly enhancing the release of a model drug (ketoprofen) encapsulated in the nano-emulsion oil core. To conclude, Pickering nano-emulsions only stabilized by SLNs appear to be a very efficient innovative drug nano-carrier, opening new doors as a potential temperature-sensitive drug delivery system.

International audience; Through this study, we propose to specifically focus on a particular stage of the fabrication of polymeric nanoparticles intended to be used as contrast agent for biomedical X-ray imaging. These nanoparticles, made from nanoprecipitation of preformed polymer, poly(MAOTIB) (poly(2-methacryloyloxyethyl(2,3,5-triiodobenzoate))) follow a solvent displacement process. This method, widely used in literature, is sensitive to the formulation and process parameters such as nature and concentrations of surfactant and polymer, solvent / non-solvent ratio, rate of addition of one phase in the other one, respective volumes of the phase, and homogenization shearing rate. On the other hand, in function of the aimed administration route, the final suspension should obey to specific constraints on final product, e.g. size range and polydispersity, final particle concentration (i.e. iodine concentration) and surfactant concentration. In the present work, we report a specific investigation on the nanoprecipiation of poly(MAOTIB) in tetrahydrofuran, dropped in water or ethanol (as non-solvent) and stabilized by nonionic surfactant. The objective is to show and explain the potentials and limitations of such the process, but also to provide a guidance on the way to optimize it.

International audience; Delivery systems able to coencapsulate both hydrophilic and hydrophobic species are of great interest in both fundamental research and industrial applications. Water-inoil-in-water (w 1 /O/W 2) emulsions are interesting systems for this purpose, but they suffer from limited stability. In this study, we propose an innovative approach to stabilize double emulsions by the synthesis of a silica membrane at the water/oil interface of the primary emulsion (i.e., inner w 1 /O emulsion). This approach allows the formulation of stable double emulsions through a twostep process, enabling high encapsulation efficiencies of model hydrophilic dyes encapsulated in the internal droplets. This approach also decreases the scale of the double droplets up to the nanoscale, which is not possible without silica stabilization. Different formulation and processing parameters were explored in order to optimize the methodology. Physicochemical characterization was performed by dynamic light scattering, encapsulation efficiency measurements, release profiles, and optical and transmission electron microscopies.

International audience; In general, nano-emulsions are submicron droplets composed of liquid oil phase dispersed in liquid aqueous bulk phase. They are stable and very powerful systems when it regards the encapsulation of lipophilic compounds and their dispersion in aqueous medium. On the other hand, when the properties of the nano-emulsions aim to be modified, e.g. for changing their surface properties, decorating the droplets with targeting ligands, or modifying the surface charge, the dynamic liquid / liquid interfaces make it relatively challenging. In this study, we have explored the development of nano-emulsions which were not anymore stabilized with a classical low-molecular weight surfactant, but instead, with an amphiphilic polymer based on poly(maleic anhydride-alt-1-octadecene) (PMAO) and Jeffamine®, a hydrophilic amino-terminated PPG/PEG copolymer. Using a polymer as stabilizer is a potential solution for the nano-emulsion functionalization, ensuring the droplet stabilization as well as being a platform for the droplet decoration with ligands (for instance after addition of function groups in the terminations of the chains). The main idea of the present work was to understand if the spontaneous emulsification –commonly performed with nonionic surfactants– can be transposed with amphiphilic polymers, and a secondary objective was to identify the main parameters impacting on the process. PMAO was modified with two different Jeffamine®, additionally differentoils and different formulation conditions were evaluated. As a control, the parent monomer, octadecyl succinic anhydride (OSA) was also modified and studied in the similar way as that of polymer. The generated nano-emulsions were mainly studied by dynamic light scattering and electron microscopy, that allows discriminating the crucial parameters in the spontaneous process, originally conducted with polymers as only stabilizer.

Lipid nanocarriers are considered as promising candidates for drug delivery and cancer targeting because of their low toxicity, biodegradability and capacity to encapsulate drugs and/or contrasting agents. However, their biomedical applications are currently limited because of a poor understanding of their integrity in vivo. To address this problem, we report on fluorescent nano-emulsion droplets of 100 nm size encapsulating lipophilic near-infrared cyanine 5.5 and 7.5 dyes with a help of bulky hydrophobic counterion tetraphenylborate. Excellent brightness and efficient Förster Resonance Energy Transfer (FRET) inside lipid NCs enabled for the first time quantitative fluorescence ratiometric imaging of NCs integrity directly in the blood circulation, liver and tumor xenografts of living mice using a whole-animal imaging set-up. This unique methodology revealed that the integrity of our FRET NCs in the blood circulation of healthy mice is preserved at 93% at 6 h of post-administration, while it drops to 66% in the liver (half-life is 8.2 h). Moreover, these NCs show fast and efficient accumulation in tumors, where they enter in nearly intact form (77% integrity at 2 h) before losing their integrity to 40% at 6 h (half-life is 4.4 h). Thus, we propose a simple and robust methodology based on ratiometric FRET imaging in vivo to evaluate quantitatively nanocarrier integrity in small animals. We also demonstrate that nano-emulsion droplets are remarkably stable nano-objects that remain nearly intact in the blood circulation and release their content mainly after entering tumors., Graphical abstract Image 1

Introduction The omega 3 polyunsaturated fatty acid (PUFAs) formulation containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) with a 6:1 ratio is a potent inducer of endothelium-dependent nitric oxide (NO)-mediated vasorelaxation that also improves ageing-related and angiotensin II-induced endothelial dysfunction in rats. Purpose Since PUFAs are unstable, the possibility that nanoencapsulation of EPA:DHA 6:1 followed by coating potentiates the bioactivity was evaluated. Methods EPA:DHA 6:1 was emulsified in water phase and coated with proteins and gum derivatives or used in native form. Changes in isometric tension of porcine coronary artery rings were determined using organ chambers. The role of NO was assessed using Nw-nitro-L-arginine (L-NA, NO synthase inhibitor), prostanoids using indomethacin (Indo, COX inhibitor) and endothelium-derived hyperpolarization (EDH) using TRAM-34 and UCL-1684 (Ca2 + -dependent potassium channel inhibitors). Results Coated EPA:DHA 6:1 nanoparticles (NP) caused in a concentration-dependent manner endothelium-dependent relaxations that were more sustained than those of the native form. At 0.3%, coated and native forms induced 56.6 ± 2.8% and 51.1 ± 1.5%relaxations at 10 min, and 93.8 ± 2.1% and 35.1 ± 1.7% at 60 min, respectively. The response to the coated EPA:DHA 6:1 NP was inhibited by L-NA and not affected by Indo, TRAM-34 plus UCL-1684, and by the previous incubation of rings with another oil (corn oil 0.3%). In contrast, L-NA, Indo, and TRAM-34 plus UCL-1684 inhibited the relaxation to EPA:DHA 6:1. Conclusion Nanoencapsulation of EPA:DHA 6:1 followed by coating perpetuated their ability to cause endothelium-dependent relaxation of coronary artery rings that is exclusively mediated by NO. In contrast, the relaxing activity of the native form involved NO, EDH and vasorelaxant prostanoids. Therefore, coated EPA:DHA 6:1 NP appear to be an attractive approach to enhance the vasoprotective effect of omega 3 PUFAs.

Introduction Endothelial senescence has been identified as an early event promoting the development of endothelial dysfunction (ED), a hallmark of vascular ageing and major cardiovascular diseases. In vivo, senescent endothelial cells (EC) appear initially at arterial sites at risk characterized by disturbed flow and low shear stress (i.e., bifurcations and curvatures) compared to those at low risk characterized by laminar flow and a high level of shear stress (i.e., aorta). Objective The aim is to develop fluorescent nano-carriers (NC) to target a cell surface protein up-regulated in senescent ECs, for diagnostic purposes. Method NC with a lipid core containing a fluorescent probe (NR668 or Cy5.5 derivative) stabilized by a polymer scaffold and decorated with VCAM-1 antibodies (NC-VCAM-1) were applied to different models of EC senescence in cultured porcine coronary artery (premature senescence induced by angiotensin II and replicative senescence observed at passage P3). The fluorescence signal was visualized by confocal microscopy. Results Both models of EC senescence demonstrated higher levels of senescence-associated beta-galactosidase activity, senescence markers and VCAM-1 than healthy EC at P1. Incubation of either P3 EC or Ang II-treated P1 EC with fluorescent NC-VCAM-1 showed a higher level of fluorescence than P1 EC. The EC-related NC-VCAM-1 fluorescent signal is abolished by the previous incubation of EC with an antibody directed against VCAM-1, demonstrating the ability to target cell surface VCAM-1. The highest level of NC-VCAM-1 fluorescence was observed with NC with an average diameter below 100 nm and at 37 °C. Conclusion The present findings indicate that fluorescent NC-VCAM-1 allow the detection of senescent EC. Thus, using fluorescent functionalized NC targeting a senescence-associated cell surface protein appears to be an attractive strategy to evaluate the burden of EC senescence, which promotes ED and, ultimately, cardiovascular events.

The purpose of this study was to develop a pH-thermosensitive oral chitosan-based hydrogels, able to release phenobarbital in the small intestine of the newborn. Phenobarbital is an active drug used in neonatal treatment of epilepsy. pH-thermosensitive hydrogels will improve its bioavailability and therapeutic efficiency with less side effects. This study allowed us to understand the free energy variation at the interface between chitosan chains themselves and the surrounding available molecules with its interactions behavour. Indeed, inverted tube method was used to prepare hydrogels containing 2.45 and 2.55% of chitosan, eudragitE100 and phenobarbital at 37+/-1°C via sol-gel transition. The characterization of their morphology was done by using XL SIRION 200 FEG SEM. In addition, conductivity, refractive index and density’s values were determinated. The phenobarbital release mechanism from hydrogels at different pH values, simulating the gastrointestinal tract of the newborn was also studed. UV / visible spectrophotometer SHIMADZU from the UV-2400PC series was used to determine the phenobarbital released amount as a function of time. The results showed that sol-gel transition time decreases when the chitosan concentration increases. In addition, it showed that the hydrogels structure was heterogeneous and the phenobarbital released amount were more important at pH simulating the small intestine at 2.45% of chitosan, final solution’s pH6.85 and with the presence of eudragitE100. These results were confirmed by conductivity’s values. The sols and hydrogels had a comparable refractive index and density’s values. The Korsmeyer-Peppas model was used to fit the phenobarbital release profiles. In short, the hydrogels formulated lend themselves to a phenobarbital pulsatile release usable in the newborn. The phenobarbital release profiles fitting makes possible to predict the phenobarbital amounts, which will be released at the action sites according to the need.

Noninvasive diagnostic by imaging combined with a contrast agent (CA) is by now the most used technique to get insight into human bodies. X-ray and magnetic resonance imaging (MRI) are widely used technologies providing complementary results. Nowadays, it seems clear that bimodal CAs could be an emerging approach to increase the patient compliance, accessing different imaging modalities with a single CA injection. Owing to versatile designs, targeting properties, and high payload capacity, nanocarriers are considered as a viable solution to reach this goal. In this study, we investigated efficient superparamagnetic iron oxide nanoparticle (SPION)-loaded iodinated nano-emulsions (NEs) as dual modal injectable CAs for X-ray imaging and MRI. The strength of this new CA lies not only in its dual modal contrasting properties and biocompatibility, but also in the simplicity of the nanoparticulate assembling: iodinated oily core was synthesized by the triiodo-benzene group grafting on vitamin E (41.7% of iodine) via esterification, and SPIONs were produced by thermal decomposition during 2, 4, and 6 h to generate SPIONs with different morphologies and magnetic properties. SPIONs with most anisotropic shape and characterized by the highest r2/ r1 ratio once encapsulated into iodinated NE were used for animal experimentation. The in vivo investigation showed an excellent contrast modification because of the presence of the selected NEs, for both imaging techniques explored, that is, MRI and X-ray imaging. This work provides the description and in vivo application of a simple and efficient nanoparticulate system capable of enhancing contrast for both preclinical imaging modalities, MRI, and computed tomography.

This study investigates the impact of the chemical nature of lipids and additive on the formulation and properties of pH sensitive liposomes. The objective is to understand the respective role of the formulation parameters on the liposome properties in order to optimize the conditions for efficient encapsulation of doxorubicin (DOX). These liposomes should be stable at physiological pH, and disrupt in slightly acidic media such as the tumor microenvironment to release their DOX load. The major challenge for encapsulating DOX in pH sensitive liposomes lies in the fact that this drug is soluble at low pH (when the pH-sensitive liposomes are not stable), but the DOX aqueous solubility decreases in the pH conditions corresponding to the stability of the pH-sensitive liposomes. The study of pH-sensitivity of liposomes was conducted using carboxyfluorescein (CF) encapsulated in high concentration, i.e. quenched, and following the dye dequenching as sensor of the liposome integrity. We studied the impact of (i) the chemical nature of lipids (dioleoyl phosphatidyl ethanolamine (DOPE), palmitoyl-oleoyl phosphatidyl ethanolamine (POPE) and dimyristoyl phosphatidyl ethanolamine (DMPE)) and (ii) the lipid/stabilizing agent ratio (alpha-tocopheryl succinate), on the pH sensitivity of the liposomes. Optimized liposome formulations were then selected for the encapsulation of DOX by an active loading procedure, i.e. driven by a difference in pH inside and outside the liposomes. Numerous experimental conditions were explored, in function of the pH gradient and liposome composition, which allowed identifying critical parameters for the efficient DOX encapsulation in pH-sensitive liposomes.

This review article presents the state-of-the-art in the major imaging modalities supplying relevant information on patient health by real-time monitoring to establish an accurate diagnosis and potential treatment plan. We draw a comprehensive comparison between all imagers and ultimately end with our focus on two main types of scanners: X-ray CT and MRI scanners. Numerous types of imaging probes for both imaging techniques are described, as well as reviewing their strengths and limitations, thereby showing the current need for the development of new diagnostic contrast agents (CAs). The role of nanoparticles in the design of CAs is then extensively detailed, reviewed and discussed. We show how nanoparticulate agents should be promising alternatives to molecular ones and how they are already paving new routes in the field of nanomedicine.

In the last two decades, nanoparticle contrast agents have emerged as an essential tool for preclinical imaging and diagnosis. Besides their main advantage, related to their size range inhibiting glomerular filtration, they exhibit excellent X-ray attenuation when fabricated with heavy metal and thus high contrast in tomodensitometry. Another strength of inorganic nanoparticles, making them very adaptable to preclinical imaging applications, is the modularity of their surface chemistry, which is compatible with decoration by ligands and biomolecules. The present review draws a state-of-the art picture of the different inorganic nanoparticles synthesized as X-ray contrast agents. We present the panel of heavy metals and materials used, their X-ray attenuation properties, related applications, potential surface modifications, and in vitro and in vivo behaviors. An important aspect of this review is that the majority of inorganic nanoparticles are based on gold. We summarize the latest technologies for targeting nanoparticles designed to improve imaging techniques and advanced diagnostic methods.

ObjectivesThis review highlights both the physicochemical characteristics of the nanocarriers (NCs) and the physiological features of tumour microenvironment (TME) to outline what strategies undertaken to deliver the molecules of interest specifically to certain lesions. This review discusses these properties describing the convenient choice between passive and active targeting mechanisms with details, illustrated with examples of targeting agents up to preclinical research or clinical advances.Key findingsTargeted delivery approaches for anticancers have shown a steep rise over the past few decades. Though many successful preclinical trials, only few passive targeted nanocarriers are approved for clinical use and none of the active targeted nanoparticles. Herein, we review the principles and for both processes and the correlation with the tumour microenvironment. We also focus on the limitation and advantages of each systems regarding laboratory and industrial scale.SummaryThe current literature discusses how the NCs and the enhanced permeation and retention effect impact the passive targeting. Whereas the active targeting relies on the ligand-receptor binding, which improves selective accumulation to targeted sites and thus discriminates between the diseased and healthy tissues. The latter could be achieved by targeting the endothelial cells, tumour cells, the acidic environment of cancers and nucleus.

We present a two-step process to produce dry-state Ketoprofen-loaded poly(methyl methacrylate) nanoparticles (NPs) with controllable size and tunable drug release profile. A colloidal suspension of drug-loaded nanoparticles was first obtained from a nanoprecipitation process and then transferred into a commercial spray dryer. Three micromixers of different designs and mixing principles (molecular diffusion and impact mixing) were tested. After the first step, highly monomodal NPs in the size range 100 to 210 nm were obtained as seen by the low polydispersity index value (ca. PDI ∼ 0.2) returned by a dynamic light scattering detector. Physicochemical properties, encapsulation efficiency/ratio and drug release kinetics of NPs before and after drying were determined. For similar operating conditions, the best micromixer tested (impact mixing) allowed concluding that the NPs size was not significantly affected by the spray drying while encapsulation parameters and drug release rate were slightly decreased compared to the non spray-dried NPs. A sustained drug release was observed over 6 h and the drug release rate (up to 70%) was found to vary with the size of the NPs which in turn was a function of the flow rate ratio between the polymer solution and the non-solvent solution.

In this paper, superparamagnetic iron oxide nano particles (SPIONs, around 6 nm) encapsulated in poly(methyl methacrylate) nanoparticles (PMMA NPs) with controlled sizes ranging from 100 to 200 nm have been successfully produced. The hybrid polymeric NPs were prepared following two different methods: (1) nanoprecipitation and (2) nanoemulsification evaporation. These two methods were implemented in two different microprocesses based on the use of an impact jet micromixer and an elongational-flow microemulsifier. SPIONs-loaded PMMA NPs synthesized by the two methods presented completely different physicochemical properties. The polymeric NPs prepared with the micromixer-assisted nanoprecipitation method showed a heterogeneous dispersion of SPIONs inside the polymer matrix, an encapsulation efficiency close to 100 wt %, and an irregular shape. In contrast, the polymeric NPs prepared with the microfluidic-assisted nanoemulsification evaporation method showed a homogeneous dispersion, an almost complete encapsulation, and a spherical shape. The properties of the polymeric NPs have been characterized by dynamic light scattering, thermogravimetric analysis, and transmission electron microscope. In vitro cytotoxicity assays were also performed on the nanohybrids and pure PMMA NPs.