Inverse ISAsomes in Bio-Compatible Oils-Exploring Formulations in Squalane, Triolein and Olive Oil.

In contrast to their more common counterparts in aqueous solutions, inverse ISAsomes (internally self-assembled somes/particles) are formulated as kinetically stabilised dispersions of hydrophilic, lyotropic liquid-crystalline (LC) phases in non-polar oils. This contribution reports on their formation in bio-compatible oils. We found that it is possible to create inverse hexosomes, inverse micellar cubosomes (Fd3m) and an inverse emulsified microemulsion (EME) in excess squalane with a polyethylene glycol alkyl ether as the primary surfactant forming the LC phase and to stabilise them with hydrophobised silica nanoparticles. Furthermore, an emulsified L1-phase and inverse hexosomes were formed in excess triolein with the triblock-copolymer Pluronic® P94 as the primary surfactant. Stabilisation was achieved with a molecular stabiliser of type polyethylene glycol (PEG)-dipolyhydroxystearate. For the inverse hexosomes in triolein, the possibility of a formulation without any additional stabiliser was explored. It was found that a sufficiently strong stabilisation effect was created by the primary surfactant alone. Finally, triolein was replaced with olive oil which also led to the successful formation of inverse hexosomes. As far as we know, there exists no previous contribution about inverse ISAsomes in complex oils such as triolein or plant oils, and the existence of stabiliser-free (i.e., self-stabilising) inverse hexosomes has also not been reported until now.

Structural Study of (Hydroxypropyl)Methyl Cellulose Microemulsion-Based Gels Used for Biocompatible Encapsulations.

(Hydroxypropyl)methyl cellulose (HPMC) can be used to form gels integrating a w/o microemulsion. The formulation in which a microemulsion is mixed with a hydrated HPMC matrix has been successfully used as a carrier of biocompatible ingredients. However, little is known about the structure of these systems. To elucidate this, scanning electron microscopy was used to examine the morphology and the bulk of the microemulsion-based gels (MBGs) and small-angle X-ray scattering to clarify the structure and detect any residual reverse micelles after microemulsion incorporation in the gel. Electron paramagnetic resonance spectroscopy was applied using spin probes to investigate the polar and non-polar areas of the gel. Furthermore, the enzyme-labelling technique was followed to investigate the location of an enzyme in the matrix. A structural model for HPMC matrix is proposed according to which, although a w/o microemulsion is essential to form the final gel, no microemulsion droplets can be detected after incorporation in the gel. Channels are formed by the organic solvent (oil), which are coated by surfactant molecules and a water layer in which the enzyme can be hosted.

Vancomycin Loaded Glycerol Monooleate Liquid Crystalline Phases Modified with Surfactants.

The influence of two tuning agents, polyglycerol ester (PE) and triblock copolymer (TC), on the properties of glycerol monooleate (MO) liquid crystalline phase (LCP) was investigated to achieve the therapeutic concentration of vancomycin hydrochloride (VHCl) into the eye, topically during 60 min (1 h) and intravitreally during 2880 min (48 h). Different techniques were used to elucidate the impact of surfactants on the structure of the LCP: polarized light microscopy (PLM), small-angle X-ray scattering (SAXS), and in vitro release tests I and II (simulating local and intravitreal application in the eye). The structure analysis by SAXS depicts that the inclusion of PE into the MO LCP provided partial transition of a hexagonal phase into a lamellar phase, and TC induced a partial transition of a hexagonal phase into an LCP which identification was difficult. The LCP modulated with PE and TC demonstrated different VHCl's release patterns and were evaluated by comparing our release data with the literature data. The comparison indicated that the LCP modulated with 30% w/w PE could be a promising VHCl delivery system intravitreally during 2880 min.

Preparation and Characterization of Stabilizer-Free Phytantriol-Based Water-in-Oil Internally Liquid Crystalline Emulsions.

Despite the widespread use of surfactants, there are known issues such as allergic reactions and formulation complications in their use as emulsion stabilizers. In this study, stabilizer-free water-in-oil (W/O) emulsions containing water, phytantriol, and almond oil were prepared by an ultra-turrax homogenizer, a standard laboratory equipment, and a high specialized high-shear device. Parameters such as mixing time, stirring rate, composition, order of addition of phases, and temperature were investigated to systematically optimize the preparation of the formulations through evaluating their accelerated physical stability by a centrifugal sedimentation technique. The liquid crystalline structure of the continuous phase was studied by small-angle X-ray scattering indicating a reverse hexagonal phase (H2). Microscopy images showed the emulsions prepared via high-shear method had smaller water droplets with more uniform shape and better dispersion as confirmed by Fourier-transform infrared-attenuated total reflection spectroscopy. Rheology studies showed a larger yield stress value for emulsions with higher content of phytantriol. Our results indicated that emulsions prepared by the high-shear device with higher amount of phytantriol were the most stable formulations. Applying the correct variables in the preparation of the stabilizer-free emulsions using ultra-turrax homogenizer, one could obtain similarly stable emulsions lacking the uniformity of the droplets.

Vancomycin ocular delivery systems based on glycerol monooleate reversed hexagonal and reversed cubic liquid crystalline phases.

Lyotropic bulk reversed hexagonal and reversed cubic liquid crystalline phases (hexagonal and cubic phases) composed of glycerol monooleate (GM) were used to design the vancomycin hydrochloride's (VHCl) delivery systems aiming to maintain VHCl's therapeutic concentration during 24 h in the eye, locally (as an insert) and/or intravitreally (as a bulk phase injection). Bulk VHCl's hexagonal and cubic phases were successfully prepared by melted homogenization and solvent evaporation method, and then an insert was prepared. The structural characteristics of liquid crystalline phases were studied using cross polarized light microscopy and small angle X-ray scattering technique. The presence of VHCl (1-9.5% w/w VHCl solution) did not exhibit any change in the liquid crystalline phase's structure to another liquid crystalline phase, and showed little effect on the lattice parameter of the existing liquid crystalline phase structure. In order to relate the liquid crystalline phase structure to VHCl's release rate locally into the eye, in-vitro release test of an implant has been done using a simulated tear fluid. VHCl's release in the simulated tear fluid from the cubic phase obeyed Higuchi kinetics, with linear VHCl's release versus the square root of time. The hexagonal phase released VHCl in simulated tear fluid significantly slower than the cubic phase. In order to relate the liquid phase structure to VHCl's diffusion intravitreally, in vitro release test by the Sirius' Subcutaneous Injection Site Simulator (Scissor) has been performed. Comparing the release properties by a Scissor, the VHCl's cubic phase demonstrated slower permeation through extra-cellular matrix than the VHCl solution. To evaluate the efficacy of the system investigated, the release properties of VHCl's cubic phase were compared with literature data indicating that the cubic phase could be a potential matrix system in the delivery of VHCl intravitreally during 24 h after intravitreal injection. The release data in the simulated tear fluid indicated that the cubic phase should be further optimized to achieve a therapeutic VHCl concentration locally in the eye during 24 h.

Microstructure of ionic liquid (EAN)-rich and oil-rich microemulsions studied by SANS.

In a previous study we investigated the phase behavior of microemulsions consisting of the ionic liquid ethylammonium nitrate (EAN), an n-alkane and a nonionic alkyl polyglycolether (CiEj). We found the same general trends as for the aqueous counterparts, i.e. a transition from an oil-in-EAN microemulsion via a bicontinuous microemulsion to an EAN-in-oil microemulsion with increasing temperature. However, unlike what happens in the corresponding aqueous systems, in EAN-in-oil microemulsions only a very small amount of EAN was detected by NMR-measurements. This is why we investigated the phase behavior and microstructure of EAN-rich n-dodecane-in-EAN microemulsions and oil-rich EAN-in-n-octane microemulsions. We found that the ionic liquid emulsification failure boundary has an extraordinarily small slope, which suggests that the amphiphilic film loses its ability to solubilize EAN with an increase in temperature by only a few degrees. The analysis of the small angle neutron scattering (SANS) curves unambiguously shows that this behavior is due to the fact that the EAN molecules form a substructure with a characteristic length scale of Λ ≈ 8 Å inside the EAN-in-oil droplets. In more detail, the analysis of the SANS data with the GIFT method revealed a transition from spherical to cylindrical structures approaching the respective critical endpoint temperatures. By using the respective form factors and combining them with a Gaussian spatial intensity distribution to account for the EAN sub-structure we were able to describe the scattering curves nearly quantitatively.

Reverse Hexosome Dispersions in Alkanes-The Challenge of Inverting Structures.

Monoglycerides form lipophilic liquid-crystalline (LC) phases when mixed with water. The corresponding LC nanostructures coexist with excess water, which is a necessary condition for the formation of internally nanostructured dispersed particles. These nanostructures comprise bicontinuous cubic phases, inverted hexagonal phases, and inverted micellar cubic phases. The dispersed particles are therefore named cubosomes, hexosomes, or micellar cubosomes. Such dispersions are usually stabilized by hydrophilic high-molecular-weight triblock (TB) copolymers. Another way to stabilize such dispersions is by forming the so-called Pickering or Ramsden emulsions using nanoparticles as stabilizers. In this contribution, we explore the possibility of forming and stabilizing inverted or reverse systems, that is, dispersions of hydrophilic LC phases in an excess oil phase like tetradecane. Our aim was to change from oil-in-water emulsions to water-in-oil emulsions, where the water phase is a LC phase in equilibrium with excess oil and where the oil is nonpolar, for example, an alkane. This work consists of three parts: (1) to find a hexagonal hydrophilic LC phase that can not only incorporate a certain amount of tetradecane but can also coexist with excess tetradecane in the case of higher oil concentration, (2) to find a suitable stabilizer-either polymeric or nanoparticle type-that can stabilize the emulsion without destroying the hexagonal LC phase, and finally (3) to check the stability of this reverse hexosome emulsion. We discovered that it is possible to create a hexagonal hydrophilic LC phase with short-chain nonionic surfactants such as polyethylene glycol alkyl ethers or with high-molecular-weight TB copolymers of type A-B-A. Furthermore, it is possible to successfully stabilize the reverse hexosomes with low hydrophilic-lipophilic balance TB copolymers-either synthesized in our laboratory or commercially available ones-as well as with hydrophobized, commercially available silica nanoparticles.

Uniform Distance Scaling Behavior of Planet-Satellite Nanostructures Made by Star Polymers.

Planet-satellite nanostructures from RAFT star polymers and larger (planet) as well as smaller (satellite) gold nanoparticles are analyzed in experiments and computer simulations regarding the influence of arm number of star polymers. A uniform scaling behavior of planet-satellite distances as a function of arm length was found both in the dried state (via transmission electron microscopy) after casting the nanostructures on surfaces and in the colloidally dispersed state (via simulations and small-angle X-ray scattering) when 2-, 3-, and 6-arm star polymers were employed. This indicates that the planet-satellite distances are mainly determined by the arm length of star polymers. The observed discrepancy between TEM and simulated distances can be attributed to the difference of polymer configurations in dried and dispersed state. Our results also show that these distances are controlled by the density of star polymers end groups, and the number of grabbed satellite particles is determined by the magnitude of the corresponding density. These findings demonstrate the feasibility to precisely control the planet-satellite structures at the nanoscale.

The Structure of Gold-Nanoparticle Networks Cross-Linked by Di- and Multifunctional RAFT Oligomers.

Gold nanoparticle (AuNP) network structures featuring particles from the two-phase Brust-Schiffrin synthesis and linear RAFT oligomers of styrene with two and multiple trithiocarbonate (TTC) groups along their backbone have been investigated in detail. Insights into the internal structures of these particle networks could be obtained from small-angle X-ray scattering experiments, showing that primary AuNPs are cross-linked by the employed molecular linker. The extent of AuNP network formation was investigated by means of dynamic light scattering and UV/visible extinction spectroscopy, showing an abrupt attenuation of network formation after a critical degree of polymerization of the cross-linker is exceeded. Analysis of transmission electron micrographs indicated a three-dimensional shape of the particle superstructures, which is evenly filled with the primary AuNPs. From the results obtained in this study, guidelines for the fabrication of nanoparticle networks from the self-assembly with macromolecular cross-linkers are suggested.

Self-assembled nanostructured aqueous dispersions as dermal delivery systems.

Due to their high interfacial area and capability of loading hydrophobic, hydrophilic and amphiphilic drugs, self-assembled nanoparticles are the subject of much attention in view of an application of these dispersions as carrier systems for a variety of different active ingredients. Therefore, the effect of the internal nanostructure of oil-loaded monoglyceride-based nanoparticles on the dermal delivery of diclofenac sodium was investigated. The different self-assembled phases of the nanostructured aqueous dispersions were characterized by small angle X-ray scattering (SAXS). The influence of the different phases ranging from cubic-bicontinuous, over hexagonal and cubic-micellar phases to emulsified microemulsions on the dermal delivery of the incorporated active was examined by Franz-type diffusion cell and in vitro tape stripping experiments on porcine skin. These studies revealed a dependency of the skin permeation of diclofenac sodium on the formulation's internal structure, which could be modified by varying the amount of R-(+)-limonene in the oil phase. A superiority of the emulsified microemulsion, possessing the highest amount of R-(+)-limonene, over cubic or hexagonal phases was evidenced in terms of dermal drug delivery.

Amino Acid Induced Modification of Self-Assembled Monoglyceride-Based Nanostructures.

Self-assembled phases based on monoglycerides are promising candidates for drug delivery systems. Alterations of these phases need to be performed by addition of substances which are biocompatible. Inverse bicontinuous cubic phases are altered by the addition of five amino acids, namely, glycine, phenylalanine, alanine, glutamine, and tryptophan. These natural molecules have a diversity of side chains which predicts their polarity and subsequently their interaction with the interfacial region. Whereas polar amino acids cause a slight shrinking of the fully hydrated phase, amino acids with a nonpolar side chain expand it. Tryptophan is also able to provoke a growth of inverse hexagonal, micellar cubic, and micellar structures. Amino acid concentrations in the aqueous phase, even above the amino acid's solubility, further affect all aforementioned structures and cause a significant enlargement of up to 26%. Besides the amino acids' impact on the structural sizes, they also affect the phase transition temperatures.

Structural analysis and adsorbability onto the corneal epithelial cells-model interface of vitamin nano-emulsions.

O/W nano-emulsions can be used as effective drug carriers of hydrophobic active ingredients in an aqueous solution, because nano-emulsions are comparatively stable and their structure can be controlled by changing the compositions and the preparation methods. In this paper, we focused on vitamin A and its derivatives (VA), which are among the widely-used lipophilic active ingredients, and tried to develop the nano-emulsions, which can bring out the efficiency of VA for the healing of injured corneas, with the detailed structural analysis of them using the small-angle X-ray scattering (SAXS) method. As a result, we elucidated that the nano-emulsions bearing the hydrophobic oil/water interface can be prepared by decreasing the surfactant concentration against vitamins. Moreover, we clarified that the nano-emulsions composed of lower surfactant concentration tend to adsorb VA onto the corneal epithelial cells-model interface. Therefore it is necessary to prepare the nano-emulsions, which have the hydrophobic oil/water interface for improving the adsorbability onto cell membranes.

Lipid transfer between submicrometer sized Pickering ISAsome emulsions and the influence of added hydrogel.

Transfer of lipids between droplets in Pickering emulsions has been studied by time-resolved small-angle X-ray scattering (SAXS). The special features of self-assembled liquid-crystalline phases have been applied to examine the kinetics of internal phase reorganization imposed by lipid release and uptake by the droplets. The findings reveal faster transfer kinetics in Pickering emulsions than in emulsions stabilized with Pluronic F127. It is shown that the transfer kinetics can be accelerated by adding free surfactant to the dispersions and that this acceleration becomes more dominant when micelles are formed. The effect of immobilization of the droplets has been studied by incorporating them into the appropriate hydrogel network. The droplets are arrested, and the transfer slows down significantly at high enough concentrations of the hydrogel where nonergodic systems are obtained.

Location of cholesterol in liposomes by using small-angle X-ray scattering (SAXS) data and the generalized indirect Fourier transformation (GIFT) method.

We investigated the location of cholesterol (Chol) in liposomes and its interaction with phospholipids using small-angle x-ray scattering (SAXS) data and applying the generalized indirect Fourier transformation (GIFT) method. The GIFT method has been applied to lamellar liquid crystal systems and it gives quantitative data on bilayer thickness, electron density profile, and membrane flexibility (Caillé parameter). When the GIFT method is applied to the SAXS data of dipalmitoylphosphatidylcholine (DPPC) alone (Chol [-]) or a DPPC/Chol = 7/3 mixed system (Chol [+], molar ratio), change in the bilayer thickness was insignificant in both systems. However, the electron density for the Chol (+) system was higher than that for the Chol (-) system at the location of hydrophilic groups of phospholipids, and whereas Caillé parameter value increased with temperature for the Chol (-) system, no significant change with temperature was observed in the Caillé parameter for the Chol (+) system. These results indicated that Chol is located in the vicinity of the hydrophilic group of the phospholipids and constricts the packing of the acyl chain of phospholipids in the bilayer.

Lipid transfer in oil-in-water isasome emulsions: influence of arrested dynamics of the emulsion droplets entrapped in a hydrogel.

The transfer kinetics of lipids between internally self-assembled droplets of O/W emulsions is studied. The droplets (isasomes) consist of various liquid-crystalline phases or W/O microemulsions stabilized by a polymeric stabilizer F127. The various internal phases were identified by the relative peak positions in the small-angle X-ray scattering (SAXS) curves. An arrested system composed of isasomes embedded in a gel matrix actually provides an additional possibility to control these systems in terms of the release of various host molecules. These experiments have been applied to examine the kinetics of the internal phase reorganization imposed by the lipids' release and uptake by the droplets embedded in a κ-carrageenan (KC) hydrogel network. Increasing the concentration of the gelling agent slows down the transfer from one droplet to the other through the aqueous phase. We examined the region where the free diffusion is stopped. i.e., the point where the system changes from the ergodic to the nonergodic state and the kinetics is essentially slowed down. This effect can be balanced by the addition of small amounts of free polymeric stabilizer, which speeds up the kinetics. This is even possible in the case of highly arrested dynamics of the emulsion droplets, as found for the highest KC hydrogel concentrations forming nonergodic systems.

Continuous-flow synthesis of CdSe quantum dots: a size-tunable and scalable approach.

In recent years, continuous-flow/microreactor processing for the preparation of colloidal nanocrystals has received considerable attention. The intrinsic advantages of microfluidic reactors have opened new opportunities for the size-controlled synthesis of nanocrystals either in the laboratory or on a large scale. Herein, an experimentally simple protocol for the size-tunable continuous-flow synthesis of rather monodisperse CdSe quantum dots (QDs) is presented. CdSe QDs are manufactured by using cadmium oleate as cadmium source, selenium dioxide as selenium precursor, and 1-octadecene as solvent. Exploiting selenium dioxide as selenium source and 1-octadecene as solvent allows execution of the complete process in open air without any requirement for air-free manipulations using a glove box or Schlenk line. Continuous-flow processing is performed with a stainless steel coil of 1.0 mm inner diameter pumping the combined precursor solution through the reactor by applying a standard HPLC pump. The effect of different reaction parameters, such as temperature, residence time, and flow rate, on the properties of the resulting CdSe QDs was investigated. A temperature increase from 240 to 260 °C or an extension of the residence time from 2 to 20 min affords larger nanocrystals (range 3-6 nm) whereas the size distribution does not change significantly. Longer reaction times and higher temperatures result in QDs with lower quantum yields (range 11-28 %). The quality of the synthesized CdSe QDs was confirmed by UV/Vis and photoluminescence spectroscopy, small-angle X-ray scattering, and high-resolution transmission electron microscopy. Finally, the potential of this protocol for large-scale manufacturing was evaluated and by operating the continuous-flow process for 87 min it was possible to produce 167 mg of CdSe QDs (with a mean diameter of 4 nm) with a quantum yield of 28 %.

Submicrometer-sized Pickering emulsions stabilized by silica nanoparticles with adsorbed oleic acid.

Oil-water Pickering emulsions of about 200 nm were stabilized by nanosized hydrophilic silica after a simple surface treatment method. We have modified the aqueous silica nanoparticle dispersions by simple adsorption of oleic acid to their surfaces, improving the hydrophobicity of the particles while maintaining their charge and stability. The adsorption was monitored by small-angle X-ray scattering and electrophoretic measurements to estimate the interparticle interactions and surface charges. The effect of various parameters, such as nanoparticle concentration, amount of oleic acid, ionic strength, and pH, on the droplets' size and stability was investigated by dynamic light scattering. Furthermore, the ability of these modified silica nanoparticles to stabilize long-chain alkanes, liquid paraffin, and liquid-crystalline phases was examined.

Optimized loading and sustained release of hydrophilic proteins from internally nanostructured particles.

In this study, we demonstrate that emulsified microemulsions and micellar cubosomes are suitable as sustained delivery vehicles for water-soluble proteins. Through structural modifications, the loading efficiency of two model proteins, namely bovine serum albumin (BSA) and cytochrome c could be remarkably increased. A procedure for preparing these particles loaded with optimized amounts of sensitive substances is presented. Loading and dispersion at low temperatures is performed in two successive steps. First, a water-in-oil microemulsion is loaded with the proteins. Subsequently, this phase is dispersed in water resulting in particles with microemulsion and micellar cubic internal structure and a size of approximately 620 nm. This two-step method ensures optimal loading of the particles with the proteins. These nanostructured particles are able to sustain the release of the water-soluble BSA and cytochrome c. Within one day, less than 10% of BSA and 15% of cytochrome c are released. The release rate of cytochrome c is influenced by the nanostructure of the particles.

Highly concentrated emulsified microemulsions as solvent-free plant protection formulations.

Effective plant protection agents are readily available and well implemented in industry. However, delivery to the plant and application on the leaf are processes that still need to be optimized. Up to now plant protection formulations represent either emulsion or suspension concentrates that often contain environmentally harmful organic solvents and/or adjuvants. Emulsified microemulsions are hierarchically organized systems comprising emulsion droplets that confine a water-in-oil microemulsion. In the present contribution we show that emulsified microemulsions prepared from environmentally friendly components can be loaded with the plant-protection agent Fenpropimorph® up to 48 wt.% without organic solvent. The emulsion itself is highly concentrated, containing 60 wt.% of dispersed phase, and can be readily diluted with water for spraying in farming applications. Small-angle X-ray measurements reveal the existence of a water-in-Fenpropimorph® microemulsion confined inside the emulsion droplets. Dynamic light scattering shows that the emulsions prepared are monomodal, comprising droplet radii in the hundred nanometer range.

Internally self-assembled submicrometer emulsions stabilized with a charged polymer or with silica particles.

Internally self-assembled submicrometer emulsions were stabilized by F127, by the charged diblock copolymer K151, by L300 particles, and by sodium dodecyl sulfate (SDS). The stabilization of all investigated internal phases and the impact of the stabilizer on them are discussed. The use of charged stabilizers results in a highly negative zeta potential of the emulsion droplets, which can be exploited as a means to control their adsorption onto charged surfaces. Small-angle X-ray scattering and dynamic light scattering were used to determine the internal structure and size of the emulsion droplets, respectively.