Relationship between structural features and in vitro release of doxorubicin from biocompatible anionic microemulsion

In this work structural features of anionic microemulsions, containing the pharmaceutical biocompatible components soya phosphatidylcholine (SPC), eumulgin HRE 40 (EU) and sodium oleate (SO) as surfactant, cholesterol (CHO) as oil phase and aqueous buffer were studied. Microemulsions were formulated with and without the antitumor drug doxorubicin (DOX). The various microstructures characterized in the pseudo-ternary phase diagram were analyzed by polarized light microscopy, small-angle X-ray scattering (SAXS) and X-ray diffraction (XRD) as well as by their ability to incorporate and release DOX. The experimental results demonstrated a correlation between the composition, the structural features and drug delivery. It was found that at higher cholesterol contents, the crystallization of CHO polymorph phases changed the mobility of DOX molecules. Droplets were formed with short-range spatial correlation from a microemulsion (ME) with a low surfactant:oil ratio. More ordered structures with lamellar arrangements formed by the increasing of the CHO proportions in the formulation may be due to CHO crystallization. The in vitro release of DOX showed that the presence of a high content of crystalline CHO prolongs the release of DOX from ME. The retention of DOX in the internal oil phase of the ME may modulate the drug release for a prolonged time. These results clearly demonstrate the potential of ME as a drug-delivery system.     

Structural changes of biocompatible neutral microemulsions stabilized by mixed surfactant containing soya phosphatidylcholine and their relationship with doxorubicin release

Depending on the composition, the mixture of surfactant, oil and water, may form supramolecular aggregates with different structures which can significantly influence the drug release. In this work several microemulsion (ME) systems containing soya phosphatidylcholine (SPC) and eumulgin HRE40™ (EU) as surfactant, cholesterol (O) as oil phase, and ultra-pure water as an aqueous phase were studied. MEs with and without the antitumoral drug doxorubicin (DOX) were prepared. The microstructures of the systems were characterized by photon correlation spectroscopy, rheological behavior, polarized light microscopy, small-angle X-ray scattering (SAXS) and X-ray diffraction (XRD). The results reveal that the diameter of the oil droplets was dependent on the surfactant (S) amount added to formulations. The apparent viscosity was dependent on the O/S ratio. High O/S ratio leads to the crystallization of cholesterol polymorphs phases which restricts the mobility of the DOX molecules into the ME structure. Droplets with short-range spatial correlation were formed from the ME with the low O/S ratio. The increase of the cholesterol fraction in the O/S mixture leads to the formation of ordered structures with lamellar arrangements. These different structural organizations directly influenced the drug release profiles. The in vitro release assay showed that the increase of the O/S ratio in the formulations inhibited the constant rate of DOX release. Since the DOX release ratio was directly dependent on the ratio of O/S following an exponential decay profile, this feature can be used to control the DOX release from the ME formulations.     

On the incorporation of the non-steroidal anti-inflammatory naproxen into cationic O/W microemulsions

Microemulsions (ME) containing hexadecyltrimethylammonium bromide (HTAB)/ethanol as surfactant, isopropylmyristate (IM) or butylstearate (BS) as oil phase and aqueous buffer were studied. Pseudo-ternary phase diagrams of the investigated systems were obtained at constant surfactant/cosurfactant molar ratio (1:5) by titration in order to characterize the proportions between the components to obtain clear systems. Oil in water microemulsions were prepared in a wide range of phase volume (phi). UV-vis absorption spectra of naproxen at pH 5.5 showed that the solubility of Np increases significantly in the presence of O/W ME in high phase volumes. For both, IM and BS microemulsions, the dynamic light scattering experiments showed that the size of the oil droplets remains constant in low values of phi, increasing abruptly in high phi values. Phase solubility study revealed that for both IM and BS microemulsions, the drug incorporation followed a straight-line profile in all range of phi. The data could be analyzed through the phase-separation model and the association constants (K) calculated varied from 27 to 90 M(-1), depending on the pH and on the microemulsion oil phase.     

Oil-in-water lecithin-based microemulsions as a potential delivery system for amphotericin B

In this work the structural features of microemulsions (MEs) containing the pharmaceutical biocompatible Soya phosphatidylcholine/Tween 20 (1:1) as surfactant (S), Captextrade mark 200 as oil phase (O), and phosphate buffer 10mM, pH 7.2 as aqueous phase (W) were studied. Systems obtained with different proportions of the components were described by pseudo-ternary phase diagrams in order to characterize the microemulsions studied here. MEs were prepared with and without the polyene antifungal drug amphotericin B (AmB). The maximum AmB incorporation into the ME system was dependent on both the oil phase and surfactant proportions with 6.80 and 5.7mg/mL in high contents, respectively. The incorporation of AmB into the ME systems significantly increased the profile of the droplet size of the ME for all ranges of surfactant proportions used in the formulations. The microstructures of the system were characterized by dynamic light scattering (DLS) and rheological behavior. The DLS results showed that the size of the oil droplets increases 4.6-fold when AmB is incorporated into the ME system. In all cases the increase in the proportion of the oil phase of the ME leads to a slight increase in the diameter of the oil droplets of the system. Furthermore, for both the AmB-loaded and AmB-unloaded MEs, the size of the oil droplets decrease significantly with the increase of the S proportion in the formulations, demonstrating the efficiency of the surfactant in stabilizing the ME. Depending on the ME composition, an anti-thixotropic behavior was found. The maximum increases of the consistency index caused by the increase of the oil phase of the ME were of 17- and 25-times for the drug-loaded and drug-unloaded MEs, respectively. However, the observed effect for the drug-loaded ME was about 4.6 times higher than that for the drug-unloaded one, demonstrating the strong effect of the drug on the rheological characteristics of the ME system. Therefore, it is possible to conclude that the investigated ME can be used as a very promising vehicle for AmB.     

Recent advances in microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) is an electrodriven separation technique. Separations are typically achieved using oil-in-water microemulsions, which are composed of nanometre-sized droplets of oil suspended in aqueous buffer. The oil droplets are coated in surfactant molecules and the system is stabilised by the addition of a short-chain alcohol cosurfactant. The novel use of water-in-oil microemulsions for MEEKC separations has also been investigated recently. This report summarises the different microemulsion types and compositions used to-date and their applications with a focus on recent papers (2002-2004). The effects of key operating variables (pH, surfactant, cosurfactant, oil phase, buffer, additives, temperature, organic modifier) and methodology techniques are described.     

Recent developments in the methodology and application of MEEKC

MEEKC is an electrodriven separation technique that utilises the unique properties of a microemulsion (ME) as a background electrolyte to achieve separation of a diverse range of solutes. MEs are composed of nanometre-sized oil droplets suspended in aqueous buffer, which is commonly referred to as oil-in-water ME. The droplets are stabilised by the presence of both a surfactant and co-surfactant. The use of water-in-oil MEs in MEEKC has also been investigated. This review details the advances in MEEKC-based separations from the period June 2008 - June 2010. Areas covered include online sample concentration, suppressed electroosmosis MEEKC, chiral separations, MEEKC-MS, MEEKC-ICP-MS and ME structure characterisation. The review also includes a fundamental introduction to MEEKC, along with a review of recent applications.     

Phase behavior of the orange essential oil/sodium bis(2-ethylhexyl)sulfosuccinate/water system

The ternary phase diagram for the orange essential oil (OEO)/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water system was constructed at 25 °C. It indicates a large single phase region, comprising an isotropic water-in-oil (W/O) microemulsion (ME) phase (L₂), a liquid crystal (LC) (lamellar or hexagonal) and a large unstable emulsion phase that separates in two phases of normal and reverse micelles (L₁ and L₂). In this communication the properties of the ME are investigated by viscosity, electric conductivity and small angle X-ray scattering (SAXS) indicating that the isotropic ME phase exhibits different behaviors depending on composition. At low water content low viscous “dry” surfactant structures are formed, whereas at higher water content higher viscous water droplets are formed. The experimental data allow the determination of the transition from “dry” to the water droplet structures within the L₂ phase. SAXS analyses have also been performed for selected LC samples.     

Steric repulsion between internal aqueous droplets and the external aqueous phase in double emulsions

A theoretical model for analyzing the steric repulsion energy between internal aqueous droplets and the external aqueous phase in double emulsions, which results from the steric interaction between the surfactant molecules adsorbed at the two interfaces, has been established. The steric interaction is dependent on the separation distance between the internal aqueous droplets and the external aqueous phase, the thicknesses of the two adsorbed surfactant layers, and the size of the internal aqueous droplets and the oil globules, all of which determine the extent of the compression of the adsorbed surfactant molecules. The thickness of each of the two surfactant layers have the same effect on the steric repulsion, and stronger steric interaction can be achieved with thicker adsorbed layers, which can effectively prevent coalescence between the internal aqueous droplets and the external aqueous phase. Increasing the internal aqueous droplet size can produce stronger steric repulsion; however, larger oil globules will weaken the steric repulsion, indicating that a more stable double-emulsion system can be achieved by preparing the system with smaller oil globules and larger internal aqueous droplets.     

Effect of alpha-lactalbumin on the phase behavior of AOT-brine-isooctane mixtures: role of charge interactions

We have found that both electrostatic and hydrophobic interactions are involved in the ability of the protein alpha-lactalbumin (alpha-LA) to affect the self-assembly of the anionic surfactant sodium bis(ethylhexyl) sulfosuccinate (AOT, 3.5 wt %) in equivolume mixtures of organic and aqueous solutions. The composition and size of AOT phase structures that form in the presence of 0.35 wt % protein were evaluated as a function of pH and ionic strength. In the absence of protein, AOT forms water-in-oil microemulsion droplets for all pH and salt concentrations studied here. The presence of the protein in the water-in-oil microemulsion phase boosts water solubilization and droplet size, as the spontaneous curvature of the surfactant interface becomes less negative. Aggregates of protein, surfactant, and oil also form in the water-continuous phase. The size and composition of structures in both phases can be tuned in the presence of protein by varying the pH and ionic strength. alpha-LA induces the appearance of an anisotropic surfactant phase at pH <5.8. At intermediate salt concentrations, a third isotropic, viscous aqueous phase appears that contains 55-60% of the protein, 10-14% of the surfactant, and significant amounts of oil. Circular dichroism and fluorescence spectroscopy indicate that the protein contains enhanced alpha-helical secondary structure when self-assembling with surfactant, and has a loosened tertiary structure. The protein does not interact with the surfactant as an unfolded random coil. Although the conformation of alpha-LA in aqueous salt solutions is known to depend on pH, when self-assembling with AOT the protein adopts a structure whose features are quite pH insensitive, and likely reflect an intrinsic interaction with the interface.     

Effect of alpha-lactalbumin on aerosol-OT phase structures in oil/water mixtures

The ability of water-soluble, globular proteins to tune surfactant/oil/water self-assemblies has potential for the formation of biocompatible microemulsions and also plays a role in protein function at biological interfaces. In this work, we examined the effect of the protein alpha-lactalbumin on Aerosol-OT (AOT) phase structures in equivolume mixtures of oil and 0.1 M brine. In this pseudo-ternary system, surfactants are free to move to either oil or water phase to adopt phase structures close to the spontaneous curvature of the surfactants. Using small-angle X-ray scattering, we observed that addition of this protein changed the spontaneous curvature of the surfactant monolayer substantially. In the absence of protein, AOT adopted a negative spontaneous curvature to form spherical w/o microemulsion droplets. When less than 1 wt % of alpha-lactalbumin was added into the system, the w/o droplets became nonspherical and larger in volume, corresponding to an increase in water uptake into the droplets. As the protein-to-surfactant ratio increased, protein, surfactant, and oil increasingly partitioned toward the aqueous phase. There the protein triggered the formation of o/w microemulsions with a positive spontaneous curvature. These protein-containing structures exhibited significant interparticle attraction. We also compared the influence of two oil types, isooctane and cyclohexane, on the protein/surfactant interactions. We propose that the more negative natural curvature of the AOT/cyclohexane monolayer in the absence of protein prevented protein incorporation within organic phase structures and consequently pushed the system self-assembly toward aqueous aggregate formation.     

On the interaction of doxorubicin with oleate ions: fluorescence spectroscopy and liquid-liquid extraction study

Increase of lipophilicity of cationic doxorubicin (DOX) by its association with a fatty acid ion is of interest for pharmaceutical formulations and could have an impact on the drug delivery into cancer cells. On the basis of spectroscopic analysis of intrinsic DOX fluorescence, this study provides an experimental evidence of DOX-oleate interactions as function of ion/drug molar ratio (R) and pH. An electrostatic attraction to oleates is dominant for the cationic form of DOX (pH 6.5) and a hydrophobic interaction is characteristic of the molecular form of DOX (pH 8.6). A high content of sodium oleate vesicles ([oleate]>/=0.2 mM, R>/=20) limits the electrostatic and hydrophobic interactions at pH 6.5 while favoring the hydrophobic interactions at pH 8.6. The influence of these interactions on the lipophilicity of the cationic form of DOX is analyzed by measuring the apparent partition coefficient (aqueous buffer pH 6.5/methylene chloride). The results show a lipophilicity gain for the cationic form of DOX in presence of 10 : 1 ion/drug molar ratio, while no lipophilicity increase is observed at 50 : 1 molar ratio.     

Fatty acid chemistry at the oil-water interface: self-propelled oil droplets

Fatty acids have been investigated as boundary structures to construct artificial cells due to their dynamic properties and phase transitions. Here we have explored the possibility that fatty acid systems also demonstrate movement. An oil phase was loaded with a fatty acid anhydride precursor and introduced to an aqueous fatty acid micelle solution. The oil droplets showed autonomous, sustained movement through the aqueous media. Internal convection created a positive feedback loop, and the movement of the oil droplet was sustained as convection drove fresh precursor to the surface to become hydrolyzed. As the system progressed, more surfactant was produced and some of the oil droplets transformed into supramolecular aggregates resembling multilamellar vesicles. The oil droplets also moved directionally within chemical gradients and exhibited a type of chemotaxis.     

Formulation and characterization of a biocompatible microemulsion composed of mixed surfactants: lecithin and Triton X-100

We report on the formation and characterization of a biocompatible microemulsion (ME) system composed of lecithin (L), Triton X-100 (T) as the surfactant(s), butyl lactate (BL) as the cosurfactant, and isopropyl myristate (IPM) as the oil phase and water. Detailed phase construction reveals that mixing of surfactants (L and T) produces larger single-phase ME region compared to L. In the mixed surfactant systems, a three-phase body appears which is otherwise not obtained in the single surfactant counterparts signifying the synergistic solubilization behaviour upon mixing. The maximum solubilization capacity decreases as the content of T increases in the mixture. Viscosity, conductance and adiabatic compressibility measurements of the single-phase ME systems at a constant amphiphile concentration (80 % w/w) show a linear trend with increasing water content revealing a droplet-type structure of all the studied formulations. FTIR studies in the water-in-oil (w/o) region identify the presence of three distinct types of water molecules in these systems and their relative content changes with the interfacial composition as well as the total water content in the system. Our study offers a biocompatible mixed ME system in which the physical properties do not differ much from those of the lecithin-based systems with the additional advantage of having higher solubilization capacity, low pH dependency and low viscosity, which renders its potential to be used for specific pharmaceutical applications.     

Release of nortriptyline hydrochloride from oil-water microemulsions

The release of nortritptyline hydrochloride from oil-in-water (o/w) microemulsions (isopropyl myristate as oil, propylene glycol as cosurfactant, polysorbate 80 as surfactant and phosphate buffer, pH 7.4, as the continuous phase) containing increasing concentrations of polyethylene glycol 400, used to facilitate the diffusion of a drug from the inner oily phase of the microemulsion to the outer aqueous phase of such a dispersion system, was studied by determining the permeability constants of the drug through hydrophilic and lipophilic membranes separating the o/w microemulsions from the receiving aqueous phase (phosphate buffer pH 7.4). The permeability of nortriptyline hydrochloride from microemulsions through the lipophilic membrane increased as the concentration of polyethylene glycol 400 in the disperse system increased. The apparent permeability constant for nortriptyline hydrochloride, from the microemulsion without polyethylene glycol, was 1.36 x 10(-3) cm x h(-1), it increased up to 7.80 x 10(-3) cm x h(-1) in the presence of polyethylene glycol at a concentration of 50% (v/v) of the initial volume of the aqueous phase.     

Migration mechanism of bases and nucleosides in oil-in-water microemulsion capillary electrophoresis.

The electrophoretic behaviors of five bases and corresponding nucleosides in the oil in water (o/w) microemulsion capillary electrophoresis, microemulsion electrokinetic chromatography (MEEKC), were examined in comparison with those in normal capillary zone electrophoresis (CZE). The microemulsion systems were composed of heptane, sodium dodecyl sulfate (SDS), 1-butanol and 10 mM phosphate buffer (pH 7.0) or toluene, SDS, 1-butanol and 5 mM carbonate buffer (pH 10.0). CZE was carried out in the range of pH 9.7-10.9, and the dissociation constants, pKa, of the bases and nucleosides and the electrophoretic mobilities of the anionic forms were determined. The electrophoretic behaviors of the solutes in the microemulsion systems were analyzed from their pKa, the electrophoretic mobilities of the anions determined by CZE, and the distribution constants, K(D), of the neutral forms between the microemulsion droplets and the outer aqueous phase. The importance of adsorption mechanism in MEEKC system was suggested from the correlation between log K(D) and log P.     

One-step inversion process to a Janus emulsion with two mutually insoluble oils

High internal phase ratio (HIPR) aqueous Janus emulsions of two immiscible oils, silicone oil (SO) and a vegetable oil (VO), were prepared using a vibration mixer. The simple HIPR Janus emulsions, (VO + SO)/W, were found at weight fractions of the aqueous phase in excess of 0.3, while at a corresponding fraction of 0.1, a triple emulsion was obtained with the Janus emulsion forming a drop inside the vegetable oil to give a double Janus emulsion, (VO + SO)/W/VO, which in turn formed drops in the silicone oil resulting in a triple Janus emulsion (VO + SO)/W/VO/SO. Increasing the aqueous-phase fraction from 0.1 to 0.3 consequently meant an inversion, of which one intermediate stage was observed: a more complex configuration, e.g., one in which large SO drops with highly distorted VO drops attached were dispersed in a regular aqueous emulsion with spherical Janus (VO + SO) drops. A preliminary investigation was made into the destabilization process of the triple emulsions.     

Effect of added alpha-lactalbumin protein on the phase behavior of AOT-brine-isooctane systems

We have found that the presence of <1 wt% of the globular protein alpha-lactalbumin has a significant impact on the equilibrium phase behavior of dilute sodium bis(ethylhexyl) sulfosuccinate (AOT)/brine/isooctane systems. Nuclear magnetic resonance (NMR), Karl Fischer titration, and ultraviolet spectroscopy were used to determine the surfactant, oil, water, and protein content of the organic and aqueous phases as a function of the total surfactant and protein present. As a small amount of alpha-lactalbumin is added to the mixture, there is a substantial increase (up to 80%) in the maximum water solubility in the water-in-oil microemulsion phase. Dynamic light scattering measurements indicate that this increase is due to a decrease in the magnitude of the (negative) spontaneous curvature of the surfactant monolayer, as droplets swell in size. As the molar ratio of alpha-lactalbumin to AOT surpasses approximately 1:300, the partitioning of water, protein, and surfactant shifts to the excess aqueous phase, where soluble assemblies with positive curvature are detected by dynamic light scattering. Significant amounts of isooctane are solubilized in these aggregates, consistent with the formation of oil-in-water microemulsion droplets. Circular dichroism studies showed that the tertiary structure of the protein in the microemulsion is disrupted while the secondary structure is increased. In light of these findings, the protein most likely expands to a molten-globule type conformation in the AOT interfacial environment, but does not substantially unfold to become an extended chain.     

Background and operating parameters in microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) is an electrodriven separation technique. Separations are generally achieved using microemulsions consisting of surfactant-coated nanometer-sized oil droplets suspended in aqueous buffer. A cosurfactant such as a short-chain alcohol is generally used to stabilize the microemulsion. This review summarizes the various microemulsion types and compositions that have been used in MEEKC. The effects of key-operating variables such as surfactant type and concentration, cosurfactant type and concentration, buffer pH and type, oil type and concentration, use of organic solvent and cyclodextrin additions, and temperature are described. Specific examples of water-in-oil microemulsions and chirally selective separations are also covered.     

Mechanism of oil-in-water emulsification using a water-soluble amphiphilic polymer and lipophilic surfactant

A new O/W (oil-in-water) emulsification system was developed using the amphiphilic polymer HHM-HEC (hydrophobically-hydrophilically modified hydroxyethylcellulose) and a lipophilic surfactant. HHM-HEC was used as a thickener and polymeric surfactant, and the addition of small quantities of various types of nonionic lipophilic surfactant (hydrophilic-lipophilic balance <5) decreased the droplet size of several types of oil due to a lowering of the tension at the water/oil interface. The oil droplets were held by the strong network structure of the aqueous HHM-HEC solution, preserving the O/W phase without inversion. These stable O/W emulsions were prepared without the addition of hydrophilic surfactants and thus show improved water repellency.     

二-(2-乙基己基)磷酸/正辛醇反胶束萃取L-辛弗林

利用二-(2-乙基己基)磷酸(P204)/正辛醇反胶束从枳实粗提物中萃取L-辛弗林。 研究了萃取机理和水相pH值、含水量W0、阳离子浓度、萃取时间以及P204浓度对L-辛弗林萃取率的影响。 通过原子力显微镜观察了P204/正辛醇反胶束;萃取L-辛弗林的最佳条件为：萃取原料液水相pH=6.5,含水量W0=15、P204浓度为0.08 mol/L,在此条件下萃取15 min,对L-辛弗林的单次萃取率为68.0%。 基于萃取率建立的数学模型可反映pH值、P204浓度与萃取率的关系,在一定pH值及P204浓度区间可预测萃取率的变化趋势。