In vitro antileishmanial potentialities of essential oils from Citrus limon and Pistacia lentiscus harvested in Tunisia.

Leishmaniasis is a tropical parasitic disease that affects up to 12 million people worldwide. Current chemotherapies have limitations such as toxicity, high cost, and parasite resistance. This work aims to select an essential oil (EssOil) isolated from the Tunisian flora as a new antileishmanial candidate. Two plants were chosen for their antileishmanial potential: Citrus limon (Citrus) and Pistacia lentiscus (Pistacia). Each of these plants was harvested from two different sites (area 1 and area 2). Extracted EssOils were characterized using GC-MS. Their antiparasitic activity against axenic and intracellular Leishmania major amastigotes and their cytotoxicity were assessed. Citrus EssOil from area 1 displayed an interesting activity against L. major intramacrophage amastigotes with IC50 value at 4.2 ± 1.3 µg/mL. Interestingly, this activity was close to that of miltefosine. Moderate activities against intracellular amastigote were observed for Pistacia EssOil from area 1 and Citrus EssOil from area 2. However, low cytotoxicity with high selectivity index was proved only for Citrus EssOil from area 1, revealing its safety for macrophages. This study also demonstrated for the first time the antileishmanial activity of EssOil extracted from Citrus limon leaves. The EssOil interesting activity could be related to the lipophilic properties of terpenes that were shown in literature to contribute to the disruption of parasite intracellular metabolic pathways.

Bare and Sterically Stabilized PLGA Nanoparticles for the Stabilization of Pickering Emulsions.

Pickering emulsions were formulated using biodegradable and biocompatible poly(lactic- co-glycolic acid) (PLGA) nanoparticles (NPs) prepared without surfactants or any other polymer than PLGA. A pharmaceutical and cosmetic oil (Miglyol) was chosen as the oil phase at a ratio of 10% w/w. These emulsions were then compared with emulsions using the same oil but formulated with well-described PLGA-poly(vinyl alcohol) (PVA) NPs, i.e., with PVA as NP stabilizers. Strikingly, the emulsions demonstrated very different structures at macroscopic, microscopic, and interfacial scales, depending on the type of NPs used. Indeed, the emulsion layer was significantly thicker when using PLGA NPs rather than PLGA-PVA NPs. This was attributed to the formation and coexistence of multiple water-in-oil-in-water (W/O/W) and simple oil-in-water (O/W) droplets, using a single step of emulsification, whereas simple O/W emulsions were obtained with PLGA-PVA NPs. The latter NPs were more hydrophilic than bare PLGA NPs because of the presence of PVA at their surface. Moreover, PLGA NPs only slightly lowered the oil/water interfacial tension whereas the decrease was more pronounced with PLGA-PVA NPs. The PVA chains at the PLGA-PVA NP surface could probably partially desorb from the NPs and adsorb at the interface, inducing the interfacial tension decrease. Finally, independent of their composition, NPs were adsorbed at the oil/water interface without influencing its rheological behavior, possibly due to their mobility at their interface. This work has direct implications in the formulation of Pickering emulsions and stresses the paramount influence of the physicochemical nature of the NP surface into the stabilization of these systems.

Pickering emulsions stabilized with biodegradable nanoparticles for the co-encapsulation of two active pharmaceutical ingredients.

Innovative Pickering emulsions co-encapsulating two active pharmaceutical ingredients (API) were formulated for a topical use. An immunosuppressive agent, either cyclosporine A (CysA) or tacrolimus (TAC), was encapsulated at high drug loading in biodegradable and biocompatible poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP). These NP stabilized the oil droplets (Miglyol) containing an anti-inflammatory drug, calcitriol (CAL). The influence of the API on the physico-chemical properties of these emulsions were studied. Emulsions formulated with or without API had a similar macroscopic and microscopic structure, as well as interfacial properties, and they exhibited a good stability for at least 55 days. The emulsions did not alter the viability of human keratinocytes (HaCaT cell line) after 2 and 5 days of exposure to NP concentrations equivalent to efficient API dosages. Thus, these new Pickering emulsions appear as a promising multidrug delivery system for the treatment of chronical inflammatory skin diseases.

In vitro efficacy of essential oils against Sarcoptes scabiei.

The mite Sarcoptes scabiei is responsible for scabies, a pruritic and contagious skin disease in humans. S. scabiei is also responsible for mange in a wide range of animal species. The treatment of S. scabiei infection is hampered by an under-effectiveness of the few available drugs. The objective of this work was to evaluate the in vitro acaricide activity of a large number of plant essential oils (EOs) against S. scabiei. EOs were selected mainly on the basis of traditional treatments for dermatological infections in Madagascar. The sarcoptes originating from a porcine animal model were tested at concentrations ranging from 10 to 0.1%. The viability of sarcoptes was assessed by stereomicroscopic observation at 5 min, 15 min, 30 min, 45 min and then every hour until 6 h after treatment. Estimates of lethal time and lethal concentration producing 50% mortality were generated using a probit analysis. The survival curves were estimated using the Kaplan Meier method. A total of 31 EOs from different plants were tested. Cinnamomum zeylanicum (cinnamom) and Ocimum sanctum (tulsi) oils were the most active for all concentrations tested. They may be included in in vivo studies, in order to further assess their potential interest as topical treatments.

Cochleate formulations of Amphotericin b designed for oral administration using a naturally occurring phospholipid.

The purpose of this work was to formulate the poor soluble antifungal and antiparasitic agent Amphotericin B (AmB) in cost-effective lipid-based formulations suitable for oral use in developing countries, overcoming the limitations of poor water solubility, nephrotoxicity and low oral bioavailability. The antifungal agent was formulated, at different molar proportions, in cochleate nanocarriers prepared using an accessible naturally occurring phospholipid rich in phosphatidylserine (Lipoid PSP70). These nanoassemblies were prepared by condensation of negatively charged phospholipid membrane vesicles with divalent cations (Ca2+). Small-angle X-ray scattering studies revealed the Ca2+-triggered condensation of loosely packed multilamellar vesicles into tightly packed bilayers of strongly dehydrated multilamellar organization characterized by narrow Bragg peaks. Transmission electron microscopy and quasi-elastic light scattering studies demonstrated the formation of nanosized particles. AmB drug loading was above 55% in all formulations. Circular dichroism demonstrated the prevalence of monomeric and complexed forms of AmB over toxic aggregates. The stability of AmB in gastric medium was improved by loading in cochleates and its release in gastrointestinal media was retarded. Confocal microscopy studies revealed the in-vitro interactions of Lipoid PSP70-based cochleates with Caco2 intestinal cell monolayers. The results suggest that the low-cost AmB-loaded cochleates may increase the therapeutic range of this drug.

Effect of high pressure homogenization on the structure and the interfacial and emulsifying properties of ß-lactoglobulin.

The effect of high pressure homogenization (HPH) on the structure of ß-lactoglobulin (ß-lg) was studied by combining spectroscopic, chromatographic, and electrophoretic methods. The consequences of the resulting structure modifications on oil/water (O/W) interfacial properties were also assessed. Moderated HPH treatment (100 MPa/4 cycles) showed no significant modification of protein structure and interfacial properties. However, a harsher HPH treatment (300 MPa/5 cycles) induced structural transformation, mainly from ß-sheets to random coils, wide loss in lipocalin core, and protein aggregation via intermolecular disulfide bridges. HPH-modified ß-lg displayed higher surface hydrophobicity leading to a faster adsorption rate at the interface and an earlier formation of an elastic interfacial film at Cß-lg = 0.1 wt%. However, no modification of the interfacial properties was observed at Cß-lg = 1 wt%. At this protein concentration, the prior denaturation of ß-lg by HPH did not modify the droplet size of nanoemulsions prepared with these ß-lg solutions as the aqueous phases. A slightly increased creaming rate was however observed. The effects of HPH and heat denaturations appeared qualitatively similar, but with differences in their extent.

Complex coacervation between beta-lactoglobulin and Acacia gum: a nucleation and growth mechanism.

Complex coacervation between proteins and polysaccharides is a demixing process mainly driven by electrostatic interactions. During this process many structural transitions occur, involving the formation of soluble complexes, aggregated complexes, and coacervates. The dynamic mechanism of complexation/coacervation was studied on beta-lactoglobulin (BLG)/Acacia gum (AG) mixed dispersions (0.1 wt% total concentration; BLG:AG ratio of 2:1) using small angle static light scattering (SALS). Acidification of BLG/AG dispersions was induced by dissolution of 0.11 wt% glucono-delta-lactone, allowing in situ SALS measurements. Time evolution of turbidity, scattered light intensity at 46 degrees scattering angle (I46) or slope of scattering functions at high q range revealed the existence of six pH-induced structural transitions. During BLG/AG complexation and before coacervation took place, scattering profiles displayed a monotonic decrease of I(q) as a function of q. A correlation peak in the scattering functions was only observed when coacervates appeared in the system. The wave vector q(max) corresponding to the maximum in scattered intensity first shifted toward larger q values, indicating an increasing number of coacervates, then shifted toward smaller q values, as a consequence of the system coarsening. The power laws q(max) approximately t(-alpha) and I(max) approximately t(-beta) gave values of 1.9 and 9.2, respectively, values much larger than those expected for intermediate and late stages of spinodal decomposition. From these results, it was concluded that complex coacervation between BLG and AG was a nucleation and growth type process. In addition, the temporal evolution of I46 followed power laws with two different exponents. First exponent corresponding to BLG/AG complexation was 3.0+/-0.3 and indicated a diffusion-controlled growth mechanism. Second exponent corresponding to the initiation of phase separation to the coacervation process was 6.5+/-0.3 and revealed an interfacially-controlled growth mechanism.

ß-lactoglobulin stabilized nanemulsions--Formulation and process factors affecting droplet size and nanoemulsion stability.

To avoid the toxicological concerns associated to synthetic surfactants, proteins might be an alternative for the stabilization of pharmaceutical nanoemulsions. The present study investigates the use of ß-lactoglobulin (ß-lg) to stabilize oil in water biocompatible nanoemulsions intended for a pharmaceutical use and prepared by high pressure homogenization (HPH). The effects of composition (nature and weight fraction of oil, ß-lg concentration) and of process parameters (pressure and number of cycles) on the droplet size and on the stability of nanoemulsions were thoroughly assessed. The nanoemulsions prepared with ß-lg at 1 wt% and with 5 wt% Miglyol 812 (the oil with the lowest viscosity) displayed a relatively small particle size (about 200 nm) and a low polydispersity when a homogenization pressure of 100 MPa was applied for 4 cycles. These nanoemulsions were the most stable formulations over 30 days at least. Emulsification efficiency of ß-lg was reduced at higher homogenization pressures (200 MPa and 300 MPa). The effect of HPH process on the interfacial properties of ß-lg was evaluated by drop shape analysis. This treatment had an effect neither on the interfacial tension nor on the interfacial dilatational rheology of ß-lg at the Miglyol 812/water interface.

Calixarene cleansing formulation for uranium skin contamination.

An oil-in-water cleansing emulsion containing calixarene molecule, an actinide specific chelating agent, was formulated in order to improve the decontamination of uranium from the skin. Commonly commercialized cosmetic ingredients such as surfactants, mineral oil, or viscosifying agents were used in preparing the calixarene emulsion. The formulation was characterized in terms of size and apparent viscosity measurements and then was tested for its ability to limit uranyl ion permeation through excoriated pig-ear skin explants in 24-h penetration studies. Calixarene emulsion effectiveness was compared with two other reference treatments consisting of DTPA and EHBP solutions. Application of calixarene emulsion induced the highest decontamination effect with an 87% decrease in uranium diffusion flux. By contrast, EHBP and DTPA solutions only allowed a 50% and 55% reduction of uranium permeation, respectively, and had the same effect as a simple dilution of the contamination by pure water. Uranium diffusion decrease was attributed to uranyl ion-specific chelation by calixarene within the formulation, since no significant effect was obtained after application of the same emulsion without calixarene. Thus, calixarene cleansing emulsion could be considered as a promising treatment in case of accidental contamination of the skin by highly diffusible uranium compounds.

Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: alternatives to synthetic surfactants in the pharmaceutical field?

Emulsions are widely used in pharmaceutics for the encapsulation, solubilization, entrapment, and controlled delivery of active ingredients. In order to answer the increasing demand for clean label excipients, natural polymers can replace the potentially irritative synthetic surfactants used in emulsion formulation. Indeed, biopolymers are currently used in the food industry to stabilize emulsions, and they appear as promising candidates in the pharmaceutical field too. All proteins and some polysaccharides are able to adsorb at a globule surface, thus decreasing the interfacial tension and enhancing the interfacial elasticity. However, most polysaccharides stabilize emulsions simply by increasing the viscosity of the continuous phase. Proteins and polysaccharides may also be associated either through covalent bonding or electrostatic interactions. The combination of the properties of these biopolymers under appropriate conditions leads to increased emulsion stability. Alternative layers of oppositely charged biopolymers can also be formed around the globules to obtain multi-layered "membranes". These layers can provide electrostatic and steric stabilization thus improving thermal stability and resistance to external treatment. The novel biopolymer-stabilized emulsions have a great potential in the pharmaceutical field for encapsulation, controlled digestion, and targeted release although several challenging issues such as storage and bacteriological concerns still need to be addressed.

Stabilization mechanism of oil-in-water emulsions by ß-lactoglobulin and gum arabic.

Natural biopolymer stabilized oil-in-water emulsions were formulated using ß-lactoglobulin (ß-lg), gum arabic (GA), and ß-lg:GA solutions as an alternative to synthetic surfactants. Emulsions using these biopolymers and their complexes were formulated varying the biopolymer total concentration, the protein-to-polysaccharide ratio, and the emulsification protocol. This work showed that whereas ß-lg enabled the formulation of emulsions at concentration as low as 0.5 (w/w)%, GA allowed to obtain emulsions at concentrations equal to or higher than 2.5 (w/w)%. In order to improve emulsion stability, ß-lg and GA were complexed through strong attractive electrostatic interactions. GA solution had to be added to previously prepared ß-lg emulsions in order to obtain stable emulsions. Interfacial tension and interfacial rheological measurements allowed a better understanding of the possible stabilizing mechanism. ß-lg and GA both induced a very effective decrease in interfacial tension and showed interfacial elastic behaviour. In the mixed system, ß-lg adsorbed at the interface and GA electrostatically bound to it, leading to the formation of a bi-layer stabilized emulsion. However, emulsion stability was not improved compared to ß-lg stabilized emulsion, probably due to depletion or bridging flocculation.

pH-Induced structural transitions during complexation and coacervation of beta-lactoglobulin and acacia gum.

pH-Induced structural changes during complex coacervation between beta-lactoglobulin (BLG) and Acacia gum (AG) in aqueous solutions were determined by coupling slow in situ acidification of BLG/AG mixed dispersions and different experimental methods. The combined signal evolution of dynamic light scattering at 90 degrees scattering angle (I(90)), electrophoretic mobility, turbidimetry (tau), circular dichroism, and phase contrast microscopy allowed the distinction of critical structural transitions and the definition of their corresponding pH. The formation of soluble BLG/AG complexes was initiated at pH(sc) (4.90), since I(90) and tau significantly increased from the baseline. In parallel or just following complexation, a conformational change of BLG was detected at pH(pct) (4.8). An increase in positive charge density of BLG induced complex aggregation at pH(ca) (4.7). More efficient charge neutralization of aggregated complexes, especially through the lowering of the number of AG negative charges, promoted initiation of phase separation at pH(psi) (4.4). Mixed dispersions became unstable and phase separation occurred at pH(ps) (4.2). The phase separation of mixed dispersions was suggested by the maximum value of scattered light, by an important acceleration of the dispersion turbidity, by a strong increase of hydrodynamic radii, and by the first appearance of light fluctuations as observed by phase contrast microscopy. At the microscopic level, the first coacervates were observed at pH(coa) (4.0), near the pH of the maximum of turbidity. It was also noticed that, from the onset of interactions between biopolymers, the pH decrease led to (i) a gradual homogenization of particle size in the mixed dispersion as suggested by the decrease of dispersion polydispersity and (ii) conformational transitions of the protein (a loss of alpha-helix structure at pH(pct) and a gain in protein secondary structure near pH(coa), probably involving beta-sheet components).