Bicelles as a carrier for bioactive compounds in beverages: application to nerolidol, an active sesquiterpene alcohol.

ABSTRACT: Nerolidol is a natural sesquiterpene alcohol with promising but limited application in food and pharmaceutical fields due to several factors including low photostability and low aqueous solubility. Recently, several carriers loading nerolidol were prepared and tested in fresh orange juice. Lipid vesicles loading nerolidol did not exhibit satisfactory organoleptic properties in this beverage. Hence, DMPC/DHPC bicelles were prepared as a new phospholipid-based carrier for nerolidol at different molar ratios. The bicelle suspensions were characterized in terms of homogeneity, particles size, and morphology. The optimal formulation (phospholipid:nerolidol molar ratio 100:1) was selected based on transparent appearance, homogeneity, and particle size (~ 45 nm). Besides, it showed a high encapsulation efficiency of nerolidol and a high incorporation rate of phospholipids. Transmission electron microscopy analysis demonstrated the formation of bicelles. The bicelles membrane fluidity was assessed by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and differential scanning calorimetry analysis. The membrane fluidity of bicelles appeared to increase in the presence of nerolidol in a concentration dependent manner. To our knowledge this is the first study dealing with the encapsulation of an essential oil component in bicelles.

Preparation of perfluorocarbon emulsions by premix membrane emulsification for Acoustic Droplet Vaporization (ADV) in biomedical applications.

Perfluorocarbon (PFC) droplets are used in acoustic droplet vaporization (ADV), a phenomenon where droplets vaporize into gas microbubbles under exposure to ultrasound. The size and the size distribution of a phase change contrast agent is an important factor in determining the ADV threshold and the biodistribution. Thus, high throughout manufacturing of uniform-sized droplets, required to maintain spatial control of the vaporization process, remains challenging. This work describes a parametric evaluation of a novel process using premix membrane emulsification (PME) to produce homogeneous PFC emulsions at high rate with moderate pressure using Shirasu Porous Glass (SPG) membranes. In this study, we investigated the effect of several process parameters on the resulting pressure and droplet size: membrane pore size, flow rate, and dispersed phase type. The functionality of the manufactured emulsions for ADV was also demonstrated. Vaporization of the PFC emulsions was obtained using an imaging ultrasound transducer at 7.813 MHz, and the ADV thresholds were determined. Here, the pressure threshold for ADV was determined to be 1.49 MPa for uniform-sized perfluorohexane (PFHex) droplets with a mean size of 1.51 µm and a sharp distribution (CV and span respectively of 20% and 0.6). Thus, a uniform-sized droplet showed a more homogeneous vaporization with a uniform response in the focal region of the transducer. Indeed, polydispersed droplets had a more diffuse response outside the focal region due to the presence of large droplets that vaporize at lower energies. The ADV threshold of uniform-sized PFC droplets was found to decrease with the droplet diameter and the bulk fluid temperature, and to increase with the boiling temperature of PFC and the presence of an oil layer surrounding the PFC core.

Interaction of Selected Phenylpropenes with Dipalmitoylphosphatidylcholine Membrane and Their Relevance to Antibacterial Activity.

The effect of structurally closely related phenylpropenes (PPs), estragole, anethole, eugenol, and isoeugenol, on the fluidity of dipalmitoyl phosphatidyl choline (DPPC) liposome membrane was investigated by DSC, Raman, and fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH). Liposomes were prepared by thin-film hydration method at various DPPC:PP molar ratios. The DPH anisotropy measurements of blank and PP-loaded liposomes were performed at 28, 41, and 50 °C, which correspond, respectively, to gel phase, main transition temperature of DPPC, and liquid phase. The Raman images showed the formation of nano- and micrometric spherical multi-lamellar vesicles. All studied PPs exhibited a membrane fluidizing effect which was reinforced by the presence of phenolic hydroxyl group in eugenol and isoeugenol. The PPs interacted with the choline head group and the alkyl chains of DPPC membrane, wherein isoeugenol and anethole possessing the same C7-C8 position of the double bond in the propenyl side chain, incorporated deeply in the bilayer. Additionally, the PPs were analyzed for antibacterial activity against E. coli by macrobroth dilution method. Anethole and estragole were more efficient in inhibiting the bacterial growth than eugenol and isoeugenol. We conclude that the fluidizing effect of PPs on the membrane is a common mechanism that is not related to the hydrophobicity of the PP molecule. Besides, other target sites may be involved in PP antibacterial activity against Gram-negative bacteria. The greater hydrophobicity of these PPs may contribute to their penetrability through the outer bacterial membrane.

Tetra- and Penta-Cyclic Triterpenes Interaction with Lipid Bilayer Membrane: A Structural Comparative Study.

The effect of tetracyclic (cortisol, prednisolone, and 9-fluorocortisol acetate) and pentacyclic (uvaol and erythrodiol) triterpenes (TTPs) on the fluidity of dipalmitoyl phosphatidyl choline (DPPC) liposome membrane was investigated by differential scanning calorimetry, Raman spectroscopy, and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Liposomes were prepared in the absence and presence of TTPs at molar ratios DPPC:TTP 100:1, 100:2.5, and 100:10. All the studied TTPs abolished the pre-transition and modified the intensity of the Raman peak at 715 cm(-1) proving the interaction of TTP molecules with the choline head group of phospholipids. An increase in the Raman height intensity ratios of the peaks I 2935/2880, I 2844/2880, and I 1090/1130, giving information about the ratio disorder/order of the alkyl chains, and a decrease of the main transition temperature demonstrated the interaction of TTPs with the alkyl chains. The tetracyclic TTPs produced broadening of the phase transition profile. Besides, a scarcely splitting of the main transition peak was obtained with prednisolone and 9-fluorocortisol acetate. The results of fluorescence depolarization of DPH showed that the studied molecules fluidized the liposomal membrane at 25, 41, and 50 °C. Pentacyclic TTPs, being more hydrophobic than tetracyclic ones, demonstrated higher fluidizing effect than tetracyclic TTPs in the liquid crystalline phase suggesting a deeper incorporation in the lipid bilayer. The presence of a free polar head group at the ring D seems to control the TTP incorporation in the bilayer and consequently its effect on the membrane fluidity.

Large-scale preparation of clove essential oil and eugenol-loaded liposomes using a membrane contactor and a pilot plant.

Based on our previous study where optimal conditions were defined to encapsulate clove essential oil (CEO) into liposomes at laboratory scale, we scaled-up the preparation of CEO and eugenol (Eug)-loaded liposomes using a membrane contactor (600 mL) and a pilot plant (3 L) based on the principle of ethanol injection method, both equipped with a Shirasu Porous Glass membrane for injection of the organic phase into the aqueous phase. Homogenous, stable, nanometric-sized and multilamellar liposomes with high phospholipid, Eug loading rates and encapsulation efficiency of CEO components were obtained. Saturation of phospholipids and drug concentration in the organic phase may control the liposome stability. Liposomes loaded with other hydrophobic volatile compounds could be prepared at large scale using the ethanol injection method and a membrane for injection.

Effect of Progesterone, Its Hydroxylated and Methylated Derivatives, and Dydrogesterone on Lipid Bilayer Membranes.

The interaction of progesterone (PG), 17-hydroxyprogesterone (17-OHPG), 21-hydroxyprogesterone (21-OHPG), medroxyprogesterone (MP), medroxyprogesterone acetate (MPA), and dydrogesterone (DYG), with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposome, was investigated as a function of drug concentration using Fourier transform infrared spectroscopy and differential scanning calorimetry. The results reveal that progesterone and its derivatives changed the physical properties of the DPPC bilayers by decreasing the main phase-transition temperature (T m) and enthalpy (ΔH m), abolishing the pre-transition and disordering the membrane. From the thermodynamic parameters analysis, we concluded that PG, 21-OHPG, and MPA are localized inside the membrane. Whereas, the insertion of 17-OHPG in the lipid bilayers cannot be excluded in view of the significant decrease in the transition enthalpy at two molar ratios. MP and DYG are rather localized near the polar heads of phospholipids at the interface water-lipid bilayer. PG derivatives increase the membrane fluidity in the order: PG ≈ 21-OHPG ≈ MPA > 17-OHPG > MP ≈ DYG. The distinct effects produced by steroids are discussed in terms of hydrophobicity and chemical structure.

Effect of Erythrodiol, A Natural Pentacyclic Triterpene from Olive Oil, on the Lipid Membrane Properties.

The effect of erythrodiol, a natural pentacyclic triterpene to which humans are exposed through nutrients, on the lipid membranes is studied using liposomes as a membrane model. Empty and erythrodiol-loaded liposomes were prepared by the reverse phase evaporation method followed by the extrusion and by the thin film hydration method. Liposomes were characterized in terms of size and zeta potential and were imaged by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The effect of erythrodiol on thermotropic behavior of DPPC bilayers is also examined by differential scanning calorimetry (DSC). The DSC thermograms suggested that erythrodiol interacted with the polar head groups of phospholipids and may produce a disruption of the ordering of the alkyl chains. The diffraction light scattering analysis showed that erythrodiol-loaded liposomes presented a decrease in the vesicle size when compared to blank liposomes. Images obtained by TEM confirmed the formation of unilamellar and spherical liposomes. AFM images showed spherical vesicles and single lipid bilayers. The latter were more abundant in the preparations containing erythrodiol than in the blank ones. Moreover, erythrodiol-loaded liposomes tended to rupture into single lipid bilayers during scanning. The study may provide a better understanding of pentacyclic triterpenes-membrane interaction.

Optimisation of rosemary oil encapsulation in polycaprolactone and scale-up of the process.

Rosemary essential oil (REO) has many biological activities, such as antioxidant, anticarcinogenic, cognition-enhancing, analgesic and antimicrobial activities. The aim of this study was to prepare, at laboratory scale and larger scale, nanoencapsulating REO in order to reduce its volatilisation, light sensitivity and to enhance its water solubility. The nanoprecipitation method was applied to prepare polycaprolactone (PCL)-based nanocapsules loaded with REO at laboratory scale and then the optimal formulation obtained was scaled-up (×6) using the membrane contactor technique. The effect of several parameters, such as the evaporation method, the type of emulsifiers and the amount of the formulation products (PCL, REO, emulsifiers, etc.) on the REO-loaded nanocapsules properties (mean size, polydispersity index (PdI), zeta potential and REO loss) was evaluated at laboratory scale in order to obtain the optimal formulation. REO-loaded nanocapsules obtained from nanoprecipitation presented a nanometric mean size (220 ± 10 nm) with a PdI below 0.25, indicating an adequate homogeneity of the system, a negative zeta potential (-19.9 ± 4.6 mV) and a high encapsulation efficiency (∼99% for the major components). In addition, the membrane contactor technique gave similar results using an adequate pressure of the organic phase (0.8-1.2 bar). It is then suggested that the nanoprecipitation method can be suitable for the preparation of essential oil-loaded nanocapsules.

Essential oils encapsulated in liposomes: a review.

In the recent years there has been an increased interest toward the biological activities of essential oils. However, essential oils are unstable and susceptible to degradation in the presence of oxygen, light and temperature. So, attempts have been made to preserve them through encapsulation in various colloidal systems such as microcapsules, microspheres, nanoemulsions and liposomes. This review focuses specifically on encapsulation of essential oils into liposomes. First, we present the techniques used to prepare liposomes encapsulating essential oils. The effects of essential oils and other factors on liposome characteristics such as size, encapsulation efficiency and thermal behavior of lipid bilayers are then discussed. The composition of lipid vesicles membrane, especially the type of phospholipids, cholesterol content, the molar ratio of essential oils to lipids, the preparation method and the kind of essential oil may affect the liposome size and the encapsulation efficiency. Several essential oils can decrease the size of liposomes, homogenize the liposomal dispersions, increase the fluidity and reduce the oxidation of the lipid bilayer. Moreover, liposomes can protect the fluidity of essential oils and are stable at 4-5 °C for 6 months at least. The applications of liposomes incorporating essential oils are also summarized in this review. Liposomes encapsulating essential oils are promising agents that can be used to increase the anti-microbial activity of the essential oils, to study the effect of essential oils on cell membranes, and to provide alternative therapeutic agents to treat several diseases.

Classical and Recent Developments of Membrane Processes for Desalination and Natural Water Treatment.

Water supply and water treatment are of major concern all around the world. In this respect, membrane processes are increasingly used and reported for a large range of applications. Desalination processes by membranes are well-established technologies with many desalination plants implemented in coastal areas. Natural water treatment is also well implemented to provide purified water for growing population. This review covers various aspects of desalination: membranes and modules, plants, fouling (scaling, biofouling, algal blooms), cleaning, pretreatment (conventional and membrane treatments), energy and environmental issues, renewable energies, boron removal and brine disposal. Treatment of natural water focuses on removal of natural organic matter, arsenic, iron, nitrate, fluoride, pesticides and herbicides, pharmaceutical and personal care products. This review underlines that desalination and natural water treatment require identical knowledge of membrane fouling, construction of large plants, cleaning procedures, energy and environmental issues, and that these two different fields can learn from each other.

Encapsulation of curcumin within oil-in-water emulsions prepared by premix membrane emulsification: Impact of droplet size and carrier oil type on physicochemical stability and in vitro bioaccessibility.

Oil-in-water emulsions containing curcumin with different droplet size (small ≈ 0.5 µm, medium ≈ 0.8 µm, large ≈ 3.7 µm and premix ≈ 60 µm) were prepared through premix membrane emulsification using different carrier oils: tributyrin (short chain triglycerides, SCT), medium chain triglycerides (MCT) and corn oil (long chain triglycerides, LCT). An in vitro gastrointestinal model was used to evaluate the impact of oil and droplet size on lipid digestion and curcumin bioaccessibility. Lipid digestion and bioaccessibility decreased with the increase of droplet size for LCT-based emulsions, whereas there was no significant difference for small, medium and large emulsions in SCT and MCT-based emulsions. In addition, encapsulation efficiency played an important role in determining bioaccessibility. Bioaccessibility in MCT premix was significantly lower than that in other size MCT-based emulsions because of its low encapsulation efficiency. The bioaccessibility decreased in the order MCT > SCT > LCT in each size of emulsions..

Encapsulation of curcumin within oil-in-water emulsions prepared by premix membrane emulsification: Impact of droplet size and carrier oil on the chemical stability of curcumin.

Oil-in-water emulsions containing curcumin with different droplet sizes were produced by premix membrane emulsification with different carrier oils: tributyrin (short chain triglycerides, SCT), medium chain triglycerides (MCT) and corn oil (long chain triglycerides, LCT). The influence of carrier oil type and droplet size on the physical stability, chemical stability of curcumin and lipid oxidation stability of emulsions were investigated. Turbiscan results indicated that the physical stability of emulsions was related to both carrier oils and emulsion droplet sizes. The oil type and droplet size of emulsions stored at 25 °C showed limited effect on the stability of curcumin, but significantly affected the stability of curcumin at 55 °C. Chemical stability of curcumin decreased with the decrease of emulsion droplet sizes. For each droplet size emulsions, the stability of curcumin decreased in the order SCT > MCT > LCT. Moreover, the lipid oxidation in LCT-based emulsions resulted in lower zeta potential of droplets, which was independent of emulsion droplet sizes. The presence of curcumin improved the oxidative stability of emulsions.

Effect of cysteamine hydrochloride-loaded liposomes on skin depigmenting and penetration.

Skin hyperpigmentation is caused by an excessive production of melanin. Cysteamine, an aminothiol compound physiologically synthetized in human body cells, is known as depigmenting agent. The aim of this study was to evaluate the depigmenting activity and skin penetration of liposome formulations encapsulating cysteamine hydrochloride. First, cysteamine hydrochloride-loaded liposomes were prepared and characterized for their size, polydispersity index, zeta potential and the encapsulation efficiency of the active molecule. The stability of cysteamine hydrochloride in the prepared liposome formulations in suspension and freeze-dried forms was then assessed. The in vitro cytotoxicity of cysteamine and cysteamine-loaded liposome suspensions (either original or freeze-dried) was evaluated in B16 murine melanoma cells. The measurement of melanin and tyrosinase activities was assessed after cells treatment with free and encapsulated cysteamine. The antioxidant activity of the free and encapsulated cysteamine was evaluated by the measurement of ROS formation in treated cells. The ex vivo human skin penetration study was also performed using Franz diffusion cell. The stability of cysteamine hydrochloride was improved after encapsulation in liposomal suspension. In addition, for the liposome re-suspended after freeze-drying, a significant increase of vesicle stability was observed. The free and the encapsulated cysteamine in suspension (either original or freeze-dried) did not show any cytotoxic effect, inhibited the melanin synthesis as well as the tyrosinase activity. An antioxidant activity was observed for the free and the encapsulated cysteamine hydrochloride. The encapsulation enhanced the skin penetration of cysteamine hydrochloride. The penetration of this molecule was better for the re-suspended freeze-dried form than the original liposomal suspension where the drug was found retained in the epidermis layer of the skin.

A new method for liposome preparation using a membrane contactor.

In this article, we present a novel, scalable liposomal preparation technique suitable for the entrapment of pharmaceutical agents into liposomes. This new method is based on the ethanol-injection technique and uses a membrane contactor module, specifically designed for colloidal system preparation. In order to investigate the process, the influence of key parameters on liposome characteristics was studied. It has been established that vesicle-size distribution decreased with a decrease of the organic-phase pressure, an increase of the aqueous-phase flow rate, and a decrease of the phospholipid concentration. Additionally, special attention was paid on reproducibility and long-term stability of lipid vesicles, confirming the robustness of the membrane contactor-based technique. On the other hand, drug-loaded liposomes were prepared and filled with two hydrophobic drug models. High entrapment-efficiency values were successfully achieved for indomethacin (63%) and beclomethasone dipropionate (98%). Transmission electron microscopy images revealed nanometric quasispherical-shaped multilamellar vesicles (size ranging from 50 to 160 nm).

Pentacyclic triterpenes modulate liposome membrane fluidity and permeability depending on membrane cholesterol content.

Since the membrane-related processes represent an integral part of the biological activities of drugs, their effect on the membrane dynamics is actually considered. In this study, we investigated the effect of pentacyclic triterpenes (TTPs), oleanolic acid (OA) and erythrodiol (ER), on the fluidity and permeability of liposomes membranes differing by their cholesterol content. All liposomes were prepared by reverse phase evaporation technique (REV). Spin-labeled liposomes exposed or not to TTPs were used for fluidity studies by using 5- and 16-doxyl stearic acids (DSA). TTPs-loaded liposomes (phospholipid:cholesterol of 1:1), and preformed vesicles exposed to TTPs were used for permeability studies by monitoring the release of sulforhodamine B (SRB) at 37 °C. The apparent release constants of SRB were determined by Higuchi model based on a biphasic curve shape (0-10 h; 10-48 h). TTPs-loaded liposomes were characterized for their size and homogeneity. Results showed that ER increased the membrane fluidity at the upper region of the membrane while the both TTPs produced a condensing effect at the deeper region of the membrane. The membrane composition was a critical parameter modulating the effect of TTPs on the membrane permeability. Also, this study consolidated the fact that a fluidizing membrane agent is not necessarily a permeabilizing-membrane compound.

Challenges for cysteamine stabilization, quantification, and biological effects improvement.

The aminothiol cysteamine, derived from coenzyme A degradation in mammalian cells, presents several biological applications. However, the bitter taste and sickening odor, chemical instability, hygroscopicity, and poor pharmacokinetic profile of cysteamine limit its efficacy. The use of encapsulation systems is a good methodology to overcome these undesirable properties and improve the pharmacokinetic behavior of cysteamine. Besides, the conjugation of cysteamine to the surface of nanoparticles is generally proposed to improve the intra-oral delivery of cyclodextrin-drug inclusion complexes, as well as to enhance the colorimetric detection of compounds by a gold nanoparticle aggregation method. On the other hand, the detection and quantification of cysteamine is a challenging mission due to the lack of a chromophore in its structure and its susceptibility to oxidation before or during the analysis. Derivatization agents are therefore applied for the quantification of this molecule. To our knowledge, the derivatization techniques and the encapsulation systems used for cysteamine delivery were not reviewed previously. Thus, this review aims to compile all the data on these methods as well as to provide an overview of the various biological applications of cysteamine focusing on its skin application.

Development of cysteamine loaded liposomes in liquid and dried forms for improvement of cysteamine stability.

Despite the high aqueous solubility of cysteamine, its unpleasant organoleptic properties, hygroscopicity, instability in solutions, and poor pharmacokinetic profile are the main drawbacks that limit its use for medical and cosmetic purposes. In this study, cysteamine-loaded liposomes were prepared using the ethanol injection method. Liposomes were characterized for their size, homogeneity, surface charge, and morphology. The incorporation ratios of cholesterol and phospholipids, the encapsulation efficiency and the loading ratio of cysteamine in liposomes were determined. Moreover, the stability of free and encapsulated cysteamine was assessed at different temperatures (4, 25, and 37 °C) in the presence and absence of light. Cysteamine-loaded liposomes were freeze-dried and reconstituted liposomes were characterized. Finally, the storage stability of the freeze-dried cysteamine-loaded liposomes was studied. Liposomes were nanometric, oligolamellar, and spherical. The encapsulation efficiency and the loading ratio of cysteamine varied between 12 and 40% in the different formulations. The encapsulation improved the stability of cysteamine in the various storage conditions. The dried form of cysteamine-loaded liposomes conserved the size of the vesicles and retained 33% of cysteamine present in the liposomal suspension before lyophilization. The freeze-dried liposomes formulations were stable after four months of storage at 4 °C.

Recent advances in encapsulation of curcumin in nanoemulsions: A review of encapsulation technologies, bioaccessibility and applications.

Curcumin is widely acknowledged for its beneficial activities. However, its application has remained challenging due to its low aqueous solubility, biochemical/structural degradation and poor bioavailability. For these reasons, many researches are aimed at overcoming these limitations using lipid-based nanosystems to encapsulate curcumin, especially nanoemulsions. This review highlights the theoretical aspects and recent advances of preparation technologies (phase inversion temperature, phase inversion composition, ultrasonication, high pressure homogenization and microfluidization) for encapsulation of curcumin in nanoemulsions. Additionally, the specific factors in designing nanoemulsions systems that affect the chemical stability and in vitro bioaccessibility of the encapsulated curcumin are discussed. Also, the importance of nanoemulsions in improving antioxidant, anti-inflammatory and anticancer activities of curcumin is underlined. Curcumin-loaded nanoemulsions preparation technologies have been proposed to provide efficient, systematic, and practical protocols for improved applications of curcumin. Additionally, key factors that influence curcumin delivery include the nature of emulsifier, the type and the amount of carrier oil and emulsifier-curcumin interactions. The pharmacological activities of curcumin including antioxidant, anti-inflammatory and anticancer activities can be improved by nanoemulsions.

Use of free and encapsulated nerolidol to inhibit the survival of Lactobacillus fermentum in fresh orange juice.

Lactobacillus fermentum is commonly responsible for fruit juice fermentation and spoilage. The aim of this study was to investigate the potential use of nerolidol to control the spoilage of fresh orange juice by L. fermentum. Nerolidol was incorporated into hydroxypropyl-ß-cyclodextrin inclusion complex, conventional liposome, and drug-in-cyclodextrin-in liposome systems. The systems were lyophilized and characterized with respect to their nerolidol content, size, and morphology. The effects of the acidity and cold storage of orange juice on the survival of L. fermentum were evaluated. Subsequently, the antibacterial activity of nerolidol in refrigerated orange juice was assessed at pH 3.3. Nerolidol showed a faster antibacterial activity at 4 000 µM (5 days) compared to 2 000 µM (8 days). Under the same conditions, the inclusion complex completely killed bacteria within 6 days of incubation at 4 000 µM, suggesting its potential application in fruit juices. Nerolidol-loaded liposomes did not exhibit an antibacterial activity and altered the appearance of juice.

Comparison of Three Processes for Parenteral Nanoemulsion Production: Ultrasounds, Microfluidizer, and Premix Membrane Emulsification.

Nanoemulsions are of great interest for pharmaceutical applications, including parenteral dosage forms. However, their production is still limited and requires more efficient and adaptive technologies. The more common systems are high-shear homogenization such as microfludizers at industrial scale and ultrasounds at research scale, both based on high energy, limiting their application for sensitive drugs. Recently a process based on premix membrane emulsification (PME) was developed to produce nanoemulsions. These 3 processes have been compared for the production of a model parenteral nanoemulsion containing all-trans retinoic acid, a thermolabile molecule that is used in the treatment of acute promyelocytic leukemia in a parenteral form. Droplet size and active integrity were studied because of their major interest for efficacy and safety assessment. Regarding droplet size, PME produced monodispersed droplets of 335 nm compared with the other processes that produced nanoemulsions of around 150 nm but with the presence of micron-size droplets detected by laser diffraction and optical microscopy. No real difference between the 3 processes was observed on active degradation during emulsifcation. However regarding stability, especially at 40°C, nanoemulsions obtained with the microfluidizer showed a greater molecule degradation and unstable nanoemulsion with a 4-times droplet size increase under stress conditions.