Optimized Loading and Sustained Release of Hydrophilic Proteins

This two-step method ensures optimal loading of the particles with the proteins. .... containing different concentrations of polyglycerol ester (κ-va...
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Optimized Loading and Sustained Release of Hydrophilic Proteins from Internally Nanostructured Particles Angela Chemelli, Manuela Maurer, Roman Geier, and Otto Glatter* Institute of Chemistry, Karl-Franzens University of Graz, Heinrichstraße 28, A-8010 Graz, Austria S Supporting Information *

ABSTRACT: 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. substances.24−30 The pH dependent structural changes can be achieved by the introduction of fatty acids.31,32 The release rate from the discontinuous water compartments in the hexagonal, micellar cubic, and microemulsion phase is much slower compared to the release from continuous water channels, which are existent in the bicontinuous cubic structure.33−35 For application as drug delivery vehicles, the high viscosity of the liquid crystals is mostly undesirable. In order to create fluid formulations, these phases can be dispersed in water.24,25,29,36−39 The dispersed bicontinuous cubic and hexagonal phases, referred to as cubosomes and hexosomes, respectively, have been subjects of in vitro and in vivo investigations. In in vivo experiments, cubosomes have been studied as a drug delivery system for substances with low water solubility and it has been shown repeatedly that they have advantageous properties compared to common pharmaceutical formulations.8,36,40−51 Contrary to that, no prolonged release has been found in in vitro investigations. Hydrophilic and lipophilic substances show

1. INTRODUCTION Lipid based liquid crystals have been studied extensively as drug delivery systems for longer than a decade. The self-assembled bicontinuous cubic and hexagonal phases of monoglycerides, phytantriol and oleylglycerate were loaded successfully with substances of different size and solubility. In particular, pharmaceutically active substances have been loaded and their release investigated.1−8 Also high molecular weight substances such as proteins can be incorporated into the liquid crystalline phases.9−16 Besides the functionality as a delivery system, the self-assembled phase is also able to protect protein and peptide drugs from enzymatic, physical, and chemical degradation.1,11,17−20 The conformation and the bioactivity are not influenced despite of the inclusion in the self-assembled phases.11 It has been shown that the release rate of proteins and peptides from the inverse cubic phase is not dependent only on their size. It can be controlled by various factors such as the size of the water channels and the interaction of the load with the amphiphilic interface.21,22 Another feature that controls the release rate of the phases is the nanostructure. The structural transformation can be triggered by temperature changes,23 but it can also be induced by the addition of hydrophobic © 2012 American Chemical Society

Received: August 21, 2012 Revised: October 24, 2012 Published: October 26, 2012 16788

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about 60 °C. The amount of polyglycerol ester in the surfactant mixture is referred to as the κ-value.

a burst release under sink conditions.49,52,53 It was proposed that the load distributes between the particles and the continuous water phase according to its partition coefficient and its release is only limited by diffusion. Due to the high surface area and the small size of the particles, this process takes place within a split second. According to this, the particles are only suitable as delivery vehicles for substances having low water solubility and hence a high partition coefficient. Until now, only one contribution shows prolonged release of a watersoluble substance, the protein ovalbumin, from cubosome dispersions.15 Common dispersion processes use high shear forces and high temperatures or the addition of hydrotropes.54 Those processes are not suitable if sensitive substances such as proteins need to be incorporated. In the following study, we demonstrate that micellar cubosomes as well as emulsified microemulsions can be produced under mild conditions without the use of a hydrotrope. The loading of large proteins into the nanostructured material was optimized by increasing the size of the water compartments in order to achieve high loading efficiencies. The subsequent dispersing process was performed by using a Couette mixer.55 This two step preparation procedure, which is advantageous among common dispersion processes, makes it possible to load these particles with sensitive substances. The model proteins BSA and cytochrome c are released in a sustained manner. Accordingly, nanostructured particles are possible candidates as delivery vehicles for water-soluble proteins. In addition to the sustained release of proteins from the dispersed phase, we demonstrate the different release properties of the nondispersed nanostructured phases. With varying oil content, bicontinuous cubic, hexagonal, and micellar cubic structures and a water-in-oil microemulsion were prepared. The latter three discontinuous structures show significantly slower release of a hydrophilic dye than those of the bicontinuous cubic structure.

κ = ((mass of polyglycerol ester)/(mass of polyglycerol ester + mass of monoglyceride))100

(1)

The amount of tetradecane that was added in order to form the lipophilic mixture was given by the δ-value where the mass of monoglyceride was exchanged by the sum of monoglyceride and polyglycerol ester.

δ = ((mass of(monoglyceride + polyglycerol ester)) /(mass of(monoglyceride + polyglycerol ester) + mass of tetradecane))100

(2)

Microemulsion phases, with a δ-value of 50, containing different concentrations of polyglycerol ester (κ-value from 0 to 60) were prepared in order to investigate the enlargement of the water compartments. The polyglycerol ester used for these experiments was DGMO. The phases were saturated with water. This was performed by dropwise addition of water. After each addition of water, the microemulsions were vortex mixed and allowed to equilibrate. For samples containing low polyglycerol ester portions, the equilibration time was a few minutes. This time increased with increasing polyglycerol ester content. Clear solutions were formed as long as the water content was below the saturation point. Upon further addition of water, the mixtures stayed turbid. This was determined by visual observation of the phases. For the following experiments, the polyglycerol ester used was Grindsted PGE O 80/D. It was the follow-up product of the no longer available DGMO and recommended by Danisco A/S, Denmark. Comparative studies on the water capacity of samples containing this polyglycerol ester did not show any differences from the samples containing DGMO. The microemulsion containing polyglycerol ester was loaded with BSA and cytochrome c. The lipophilic mixture was prepared as described above. At a κ-value of 33.3, the loading efficiency was remarkably increased in comparison to the microemulsion without polyglycerol ester. For all of the following experiments, a κ-value of 33.3 was chosen and kept constant. At a δ-value of 50, a microemulsion phase was formed. The proteins were loaded by using an aqueous protein solution instead of pure water. An aqueous solution of the protein (5% w/w) was added to the lipophilic mixture and quickly vortex mixed. Considering that the water content of the phases depends on their composition, the amount of protein was related to the lipophilic mixture, which was composed of the monoglyceride, polyglycerol ester, and tetradecane. One, two, and five mg protein per gram lipophilic mixture were used. Afterward, pure water was added, and the solution was vortex mixed again. The amount of water phase that was added was chosen to be the maximum that could be used without clouding the microemulsion. Mixtures containing different amounts of water between 10% (w/w) and 17% (w/w) were prepared. The turbidity of the microemulsions was visually observed. For the solutions containing BSA and cytochrome c 15% (w/w) and 13% (w/w) water phase were used, respectively. The mixing was performed as short as possible in order to minimize protein destruction. The microemulsions were allowed to equilibrate overnight. Before they were used, they were vortex mixed again for three seconds. At a δ-value of 57 and water saturation (21%), a micellar cubic structure is formed. The high viscosity of this structure was undesired for the loading with proteins as well as for the subsequent dispersion process. In order to overcome the mixing and dispersing problems that arise due to the high viscosity, a microemulsion phase was formed first. This could be achieved by using less of the aqueous phase. A mixture of monoglyceride, polyglycerol ester, and tetradecane (κ-value 33.3 and δ-value of 57) was prepared as described above. Before loading, this mixture as well as the protein solutions were heated up to 37 °C. A ratio of 1 and 2 mg of protein per gram of lipophilic mixture was

2. MATERIALS AND METHODS 2.1. Materials. Dimodan U/J a technical grade monoglyceride, mostly monolinoleate, and Grindsted PGE O 80/D, a mixture of polyglycerol esters mainly di-, tri-, and tetraglycerols, were a gift from Danisco A/S, Braband, Denmark. Diglycerol monooleate (DGMO), a customer-specified product was also provided from Danisco A/S, Denmark. Tetradecane (C14H30) puriss was purchased from Aldrich. Pluronic F127 (PEO99−PPO67−PEO99) was a gift from BASF SE (NJ, U.S.). Albumin, bovine serum (BSA) fraction V (∼99%) and bovine serum albumin fluorescein isothiocyanate conjugate (BSA-FITC) were acquired from SIGMA. Cytochrome c from equine heart was purchased from Sigma. Phosphate buffered Saline (PBS buffer) was prepared from disodium hydrogen phosphate dodecahydrate (10 mM), potassium dihydrogen phosphate (1.8 mM), potassium chloride (2.7 mM) (all p.a. from Merck), and sodium chloride (137 mM) (>99.8% Riedel-deHaën), and the pH was adjusted to 7.4 using hydrochloric acid (1 M diluted from 35% VWR). Milli-Q water was used for all the experiments. 2.2. Optimized Loading of the Self-Assembled Bulk Phases with Proteins. The addition of oil causes a decrease of the water capacity of the self-assembled bulk phases.28 The water compartments in the water-in-oil microemulsion are small, which complicates the loading of large water-soluble molecules such as proteins. In order to increase the loading capacity of the microemulsion, the water droplets needed to be enlarged, which was realized by the addition of polyglycerol ester.56 For the preparation of the bulk phases, the polyglycerol ester was molten and mixed with the monoglycerol at 16789

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Figure 1. Schematic drawing of protein loaded nanostructured particles. used. The solutions were then vortex mixed for 3 s. Subsequently, they were allowed to cool to room temperature and equilibrate overnight. At an aqueous phase content of 17% for BSA and 10% for cytochrome c, clear water-in-oil microemulsions were formed. These microemulsions can later be transformed into micellar cubic phases by the subsequent addition of water. 2.3. Phase Diagram. In order to establish the phase diagram, nanostructured dispersions containing monoglyceride and polyglycerol ester with a κ-value of 33.3 were prepared. Samples were prepared having δ-values from 50 up to 100. One gram of lipophilic phase was dispersed in 9 g of a 1.1% Pluronic F127 solution. For the dispersion process, ultrasonication was used. The ultrasonic processor (SY-LAB G.m.b.H., Purkersdorf, Austria) was used at 30% of its maximum power at a pulse mode where the ultrasound was turned on for 0.5 s and off for 0.5 s. The ultrasonication was performed for 10 min. The dispersion was allowed to cool to room temperature before the SAXS measurements were performed. The sample with δ 57 was prepared as described in the following. 2.4. Preparation of Protein Loaded Nanostructured Particles. After the microemulsion phases were loaded with the proteins, a subsequent dispersion process was performed at 25 °C (Figure 1). Two phases were filled separately in a Couette mixer. In the first compartment, an aqueous solution of Pluronic F127 (7.5% w/w) was filled. The second chamber contained lipophilic phase consisting of the protein loaded microemulsion phase. This phase could be either the water-saturated phase with a δ-value of 50 or the phase with a δ-value of 57, which was not saturated with water. For the release experiments, these phases were loaded with BSA (2 mg/g lipophilic mixture) and cytochrome c (1 mg or 2 mg per gram lipophilic mixture). Equal volumes of the two solutions were then pressed simultaneously into a premixing chamber. In there, the solutions were premixed by a stirrer with approximately 800 rpm. The crude mixture formed was directly transferred into a Couette mixer in order to give the final nanostructured dispersion with a small droplet size. The Couette mixer consisted of a fixed outer and a rotating inner cylinder. The inner cylinder had a diameter of 60 mm and its height was 20 mm. The gap width in between the outer and the inner cylinder was 100 μm. A shear rate of 31 300 s−1 was used. This process was performed from the bottom up so that the flow rate through the mixer could be controlled. A flow rate of approximately 1.4 mL/sec was used in order to form homogeneous dispersions. Using this procedure, 10 mL of a highly concentrated dispersion could be prepared within a few seconds. This concentrate was further diluted with water or buffer in order to obtain the desired final concentration. The formation of the micellar cubic structure after the dispersion process was monitored by time dependent small-angle X-ray scattering measurements. In order to ensure that the protein was in the dispersion, a semiquantitative determination was performed. 200 μL of the protein loaded dispersion was mixed with 800 μL of acetone. The solution was cooled to −18 °C overnight. After centrifugation, the upper phase was condemned. The precipitate was then dissolved in 400 μL buffer and

the protein concentration was measured by reversed phase high performance liquid chromatography (RP-HPLC). 2.5. Release of Proteins from Nanostructured Particles. The concentrated nanostructured dispersions which were prepared by a Couette mixer were further diluted with water or buffer in order to get a final concentration of 2.5% lipophilic phase (monoglyceride, polyglycerol ester and tetradecane). The release of the proteins BSA and cytochrome c in the nanostructured dispersions was monitored by separating the particles from the continuous outer aqueous phase. For this purpose, the diluted sample was filled into a Millipore stirred ultrafiltration cell 8050, fitted with a Biomax-300 polyether sulfone membrane MWCO 300 kDa both purchased from Millipore, and stirred at 100 rpm throughout the whole experiment. At certain times, samples were withdrawn by applying a pressure of 0.5 atm to the cell. Before a sample was taken, the first 1.5 mL of the filtrate were collected. The following 1 mL was withdrawn for the RP-HPLC analysis. The first 1.5 mL filtrate was poured back into the sample and the 1 mL sample withdrawn was replaced with water or buffer, respectively, in order to keep the volume of the release medium constant. All experiments were carried out in duplicate. Possible losses of the protein due to the filtering step were evaluated by filtering pure protein solutions. The concentration of the proteins in the solutions and the filtrates were also measured by RP-HPLC analysis. 2.6. Small Angle X-ray Scattering. Small angle X-ray scattering (SAXS) measurements were carried out in order to determine the selfassembled structure in the nanostructured dispersions or in the bulk material. The SAXS equipment that was used, consisted of a SAXSess camera (Anton-Paar, Graz, Austria), connected to an X-ray generator (Philips,PW1730/10) operating at 40 kV and 50 mA with a sealedtube Cu anode. The divergent polychromatic X-ray beam was focused into a line shaped beam of Cu Kα radiation (λ = 0.154 nm) with a Goebel mirror. A PI-SCX fused fiber optic taper CCD camera from Princeton Instruments, which is a division of Roper Scientific, Inc. (Trenton, NJ, U.S.) was used to record the two-dimensional scattering patterns. It has a 2084 × 2084 array with 24 × 24 μm pixel size (chip size: 50 ×50 mm). The CCD detector was used at −30 °C (10 °C water assisted cooling) for the purpose of reducing the thermally generated charge. The scattering patterns were edited by subtraction of the background and correcting the cosmic X-ray impacts. The twodimensional scattering patterns were integrated into a one-dimensional scattering function. The samples were filled into a capillary for the measurement. The temperature in the sample holder was controlled by a Peltier element. All of the samples were measured at 25 °C. The samples were placed into the SAXS camera at least 10 min before the measurement to ensure temperature equilibration. For measuring the phase diagram, the temperature was allowed to equilibrate for 20 min. The scattering curves were recorded for 5 min three times and averaged. The formation of the micellar cubic phase after the dispersion process was followed by recording scattering curves every 3 min. The dispersion was placed in the SAXS camera right after preparation, and 16790

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the measurement was started as soon as possible. The first curve was measured 3 min after the dispersion process. 2.7. Dynamic Light Scattering. Dynamic light scattering (DLS) was performed in order to measure the sizes of the nanostructured particles. The DLS equipment used consisted of a diode laser (Coherent Verdi V5, λ = 532 nm, Pmax = 5 W) and a goniometer. The signal was detected with a single mode fiber detection optics (OZ from GMP, Zürich, Switzerland), an ALV/SO-SIPD/DUAL photomultiplier with pseudo cross correlation and ALV 5000/E correlator with fast expansion (ALV, Langen, Germany). The light scattering was measured at a scattering angle of 90°. The diffusion coefficient and the mean hydrodynamic radius of the particles were calculated from the resulting correlation function by means of the cumulant method.57 The samples were diluted in order to give a final concentration of the particles of about 10 ppm. This dilution was sufficient to avoid multiple scattering and particle interaction. During the dilution process, only slight shaking by hand was performed so that the particle size did not decrease. All of the measurements were performed at 25 °C. The samples were placed into the goniometer at least 5 min before the measurement in order to ensure the right temperature. 2.8. Reversed-Phase High Performance Liquid Chromatography. Reversed-phase high performance liquid chromatography (RPHPLC) was used to separate and quantify components in the aqueous phase of the nanostructured dispersions. Samples collected during the release experiments of the proteins were analyzed. The filtrates from the ultrafiltration were clear so they could be used without further treatment. The separation of the components was performed by a gradient RPHPLC. Mobile phase A consisted of 0.1% (v/v) trifluor acetic acid in Milli-Q water and mobile phase B of 0.1% (v/v) trifluor acetic acid in acetonitrile. The column was heated up to 40 °C and the absorbance was measured at 220 nm and additionally at 408 nm for cytochrome c. Before each series, the system was rinsed with pure mobile phase A and B for a few minutes. Afterward, the initial A/B ratio of 25/75 was pumped through the system until a flat baseline was observed. This took approximately 1 h. A full loop of 100 μL of the sample was injected. After injection, the initial A/B ratio of 25/75 was maintained for 3 min. It was gradually changed to 70/30 within the following 4 min and was kept constant for 4 min. The composition was changed back to the initial condition within the following 2 min and maintained for 7 min before the next sample was measured. Between samples, a gradient was run as a blank in order to prevent carryover of the analytes. After each series, the system was rinsed with acetonitrile: Milli-Q water 66:34 until the baseline was flat again. The RP-HPLC equipment used was from Merck Hitachi. The system consisted of a D-6000A Interface which allowed computer control. The mobile phases were pumped through the system by an L6200A pump. The temperature of the column was controlled by a T6300 column oven. The samples were injected into the system by an AS-4000 autosampler and analyzed by an L-4250 UV−vis-Detector. The column used for the separation was a Jupiter C4 from Phenomenex with dimensions of 150 × 4.6 mm, particle size 5 μm, and pore size 300 Å.

that is safe for the desired application. In this case, the structural changes would have to be measured beforehand. This would have expanded the study enormously and so, for simplicity, tetradecane was used. 3.1. Optimized Loading of the Self-Assembled Bulk Phases with Proteins. Contrary to loading with small hydrophilic molecules such as the dye Erioglaucine (see Supporting Information), the loading of the phases with proteins could not be performed as easily. Especially heating should be avoided. Water-in-oil microemulsions could be prepared at room temperature and thus are suitable for loading of proteins. The disadvantage of the microemulsion is that the water compartments and consequently the water capacity are small compared to the other phases. When the protein solution was added to the lipid mixture, a slightly turbid liquid was formed. The turbidity of the solution indicated that phase separation occurred. The upper phases consisted of a water-in-oil microemulsion, as observed by SAXS measurements (results not shown). Additionally, a lower aqueous phase was formed. In order to locate the protein, a colored BSA, namely BSA-FITC, was used. Through visual observation, it could be seen that only small portions of the protein were in the microemulsion phase and that most of it was located in the lower aqueous phase. In order to be able to load larger proteins such as BSA, the water pools needed to be enlarged. This could be achieved by partial substitution of the monoglyceride with polyglycerol ester. The size of the microemulsion droplets at different polyglycerol ester concentrations was followed by SAXS (Figure 2a). It should be mentioned that in such systems, only the hydrophilic headgroups and the water molecules contribute (nonzero contrast). During the preparation, an increase of water solubility had already been observed. By substitution of half of the monoglyceride with polyglycerol ester (κ = 50) the water content could be increased from 12% to 19%. In order to get a better understanding in which way the polyglycerol ester altered the structure of the microemulsion droplets the SAXS measurements were evaluated by generalized indirect Fourier transformation (GIFT) using a structure factor for polydisperse hard spheres. The resulting pair distance distribution functions (PDDFs) show an enlargement of the water droplet size with increasing polyglycerol ester concentration (Figure 2b). The maximum of the PDDF is related to the mean radius of the droplets. The polydispersity of the microemulsion droplets was analyzed by GIFT as well. The maxima of the asymmetric size distributions (not shown) were compared to the mean interaction radii (half center-to-center distance for hard spheres) which were calculated from the structure factor (Figure 2c). For all of the microemulsions, the mean radius was smaller than the interaction radius. The latter is not only controlled by the hydrophilic core but also, at least partially, by the alkyl chains of the monoglycerides. The increase of the size of the water compartments enabled the incorporation of large water-soluble molecules such as proteins. As a model protein bovine serum albumin was chosen. This globular protein has good water solubility. It has a molecular weight of 66 kDa and an approximate diameter of 8 nm (measured by SAXS). Assuming that the sizes of the droplets do not change upon incorporation of the protein, a polyglycerol ester content of at least 20% was necessary to create droplets with sufficient size for BSA. If no polyglycerol ester was used, then the solution separated already at protein concentrations as low as 100 μg/g.

3. RESULTS First of all, it has to be stated that this study was carried out in order to demonstrate the possiblility to use monoglyceridebased formulations as delivery vehicles. It should also give an insight into how the internal structure and additional components can affect the release properties. The components used were model substances. Tetradecane was used as an oil because its effect on the self-assembled phase has been extensively studied.25,26 It can be purchased at high purity and thus allows high reproducibility. Also natural oils, like triglycerides58 and R-(+)-limonene,25,28 can create similar structural changes in the monoglyceride system. For drug delivery formulations, tetradecane has to be exchanged by an oil 16791

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measured by SAXS. The structural changes were followed in the heating direction (Figure 3).

Figure 3. Phase diagram, measured in the heating direction of monoglyceride and polyglycerol ester based nanostructured particles (κ = 33.3). Lines are only a guide to the eyes. Schematic drawings of the structures are added.

In a previous study, the phase behavior of tetradecane loaded glycerol monolinoleate based nanostructured dispersions has been investigated.25 Depending on the composition and the temperature, the particles could have bicontinuous cubic, hexagonal, micellar cubic internal structure or they were emulsified microemulsions. Also in the polyglycerol ester containing system all these structures were present but the symmetry of the bicontinuous cubic phase was Im3m instead of Pn3m. Additionally, the formation of vesicles was observed. In the nondispersed polyglycerol ester containing system, a bicontinuous cubic phase with Pn3m symmetry was formed under water saturation when no oil was added. The formation of the Im3m phase and the vesicles is most probably due to the stabilizer Pluronic F127.59 The substitution of the monoglyceride with polyglycerol ester not only increased the sizes of the water pools, but also had an influence on the phase behavior. polyglycerol ester itself forms a lamellar phase with water. The addition of this surfactant to the nanostructured particles decreased the negative curvature of the interfacial layer. In the previous study,25 the SAXS patterns were recorded only during cooling. Therefore, the phase diagrams are compared in the cooling direction (for our corresponding diagram, see the Supporting Information). Generally, the substitution of monoglyceride with polyglycerol ester caused a shift of the phase boundaries to higher oil content. For the formation of the emulsified microemulsion 35% of tetradecane in the lipophilic phase were needed. The addition of polyglycerol ester increased the oil content necessary for the formation of emulsified microemulsions by 10%. For the development of the other structures, a 5% to 10% higher oil content was required. However, only tiny amounts of oil could be incorporated into the bicontinuous cubic structure in the PGE-free system before a change to hexagonal was caused. In the system containing polyglycerol ester, more than 7% of oil could be loaded without disturbing the bicontinuous cubic structure.

Figure 2. Structural transformation of the microemulsion due to the substitution of monoglyceride with polyglycerol ester. (a) SAXS curves. The curves were shifted vertically for better visibility. (b) Normalized PDDFs showing the enlargement of the microemulsion droplets. (c) Comparison of the mean radius (■) and the mean interaction radius (▲) of the microemulsion droplets.

Replacement of one-third of the monoglyceride by polyglycerol ester (κ = 33.3) was sufficient to enable a loading efficiency higher than 5 mg/g without causing phase separation, i.e., the loading efficiency was increased by a factor higher than 50. 3.2. Phase Diagram. For the preparation of nanostructured dispersions using the Couette mixer, it was necessary to know at which temperature the liquid crystalline phase transformed into a water in oil microemulsion.55 The internal structure of nanostructured particles having different amounts of oil was 16792

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The temperatures at which the liquid crystalline phases transformed to a water in oil microemulsion was also affected by the inclusion of the second surfactant. The Fd3m phase for example “melted” into a microemulsion at approximately 50 °C. This temperature increased 5 to 15 °C with polyglycerol ester. In the case of the hexagonal phase, an elevation of 15 °C was observed as well. 3.3. Protein Loaded Nanostructured Dispersions. The dispersion process presented here was based on the use of a Couette mixer in order to produce emulsified microemulsions and micellar cubosomes. The process could be performed at ambient temperature without the addition of a hydrotrope. Water-in-oil microemulsions, which already contained the protein, were prepared before the dispersion step was performed. Especially for water-soluble substances, it was essential that loading and dispersing were performed in two successive steps. This ensured that the entire load was inside the self-assembled phase before the particles were formed. Due to the high viscosity of the micellar cubic phase some problems arose. Mixing during the loading process is difficult and the dispersion of this gel-like phase with the Couette mixer is not possible. Usually for dispersing highly viscous phases they need to be transformed to a fluid water-in-oil microemulsion by heating, which should be avoided when proteins are loaded. At a δ-value of 57, heating to 45 °C would be necessary (Figure 3). In order to overcome those problems, a different preparation procedure was used. In comparison to the microemulsion with a δ-value of 50, the water compartments in the micellar cubic phase at a δ-value of 57 are larger under water-saturated conditions. This allowed for decreasing the water content during the loading process. As the water content of the phase decreased a water-in-oil microemulsion was developed. A clear protein containing microemulsion was formed at a water content of 17% for BSA and 10% for cytochrome c. This fluid phase enabled proper mixing and consequently homogeneous distribution of the protein could be ensured. Subsequently, this microemulsion could be dispersed at room temperature using the Couette mixer. After the dispersion process, the particles take up water and the equilibrium micellar cubic structure was formed. This development was slow enough not to hinder the fast dispersion process and to be followed by SAXS experiments. Already in the first scattering curve, which was recorded 3 min after preparation, a micellar cubic structure could be observed (Figure 4). The small height of the peaks in the scattering curve indicated that after 3 min, the structure was not yet fully developed. During the experiment, the peaks of the curves got sharper and higher, which reflected the continued transformation into the micellar cubic structure. Most of the structural arrangements were completed after 120 min. Only slight changes could be observed up to 450 min. This slow formation of the structure could be utilized during the dispersion process, which took only a few seconds, so no heating was required. The size of the nanostructured particles was determined by measuring the mean diffusion coefficient using DLS. The average hydrodynamic radius of the particles was 310 ± 33 nm and the width of the size distribution is 77 ± 6%. The size remained constant up to at least three months. The internal structure was measured by means of SAXS. No structural changes could be observed due to the incorporation of the protein. The presence of BSA in the nanostructured dispersions was confirmed by a semiquantitative determination. By the addition of acetone to the dispersion, the particles were

Figure 4. SAXS curves showing the BSA loaded microemulsion bulk phase and the formation of the micellar cubic structure after dispersing a in water. The curves were shifted vertically for better visibility.

dissolved and simultaneously the protein precipitated. More than 44% of the protein could be found. By this method, the protein could not be recovered quantitatively. 3.4. Release of Proteins from Nanostructured Particles. The continuous water phase was separated from the nanostructured particles by ultrafiltration at low pressure. The suitability of the filter membrane was investigated before the experiments were carried out. This was done by filtering aqueous protein solutions with concentrations between 1 μg/ mL up to 100 μg/mL. Less than 3% of BSA was held back during the filtration. The amount of cytochrome c that got lost due to the filtering was higher. The concentration measured was decreased by approximately 30%. The concentration of released protein in the outer water phase was measured by RP-HPLC. The release of the model proteins BSA and cytochrome c from the nanostructured particles into the continuous phosphate buffered saline phase was very slow. The amount of BSA that was released into the outer aqueous phase increased with time within the first 2 to 3 h (Figure 5). After this time, the release rate seems to decrease for the following 5 h. A following acceleration of the release could be found. In this study, the reason for this intermediate slowing down of the delivery could not be clarified. A significant difference of the

Figure 5. Release of BSA from nanostructured particles with different internal structures in PBS; water-in-oil microemulsion (d50, ■) and micellar cubic phase (d57, ▲). 16793

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release of BSA from particles with microemulsion and micellar cubic internal structure could not be measured. In comparison to that the release of cytochrome c was influenced by the nanostructure (Figure 6). The concentration of this protein in

Figure 6. Release of cytochrome c from nanostructured particles with different internal structures in PBS; water-in-oil microemulsion (d50, ■) and micellar cubic phase (d57, ▲).

the outer aqueous phase increased with time in case of the micellar cubosomes. The slower release from the emulsified microemulsion could not be measured accurately because the protein concentration was close to the quantification limit. Before the first measurement was carried out, a small amount of the protein was already released. It is possible that this release occurred during the dispersion process. The release of the proteins was also measured in pure water. Within the first 2 to 3 h of the experiments, a similar release behavior of BSA in water and PBS was observed (Figure 7). However, the release of cytochrome c was much faster in pure water especially in case of the micellar cubosomes (Figure 8). At longer times, the concentration of the proteins in the outer aqueous phase seemed to decrease. Again, the reason for this could not be clarified during this study. In comparison to the results shown above, the amount of cytochrome c in the experiments with micellar cubosomes (Figure 8b) was 2 mg per gram lipophilic phase, whereas only 1 mg per gram was used for all of the other experiments. The amount of protein that was released into the PBS phase before the first sample was withdrawn was higher compared to the experiment where less protein was loaded (Figure 6). Only a small increase of released cytochrome c was measured within the first 4 h, whereas the particles loaded with less protein show a sustained release over the whole time of the experiment. Due to the low amounts of BSA and cytochrome c that were released during the experimental time, the accuracy of the measurements was not sufficient to fit the data with a linear dependence. However, a sustained release from nanostructured particles could be found for both proteins.

Figure 7. Release of BSA in PBS (■) and pure water (▲) from emulsified microemulsion (δ50) (a) and micellar cubosomes (δ57) (b).

called hydrotrope method has been invented.54 Although it is used for the preparation of protein-containing cubosomes, one has to keep in mind that proteins may be denatured irreversibly due to the high alcohol content in the precursor formulation. The first step of the preparation demonstrated in this study included the optimized loading of emulsified microemulsions with proteins. The water capacity of the water-in-oil microemulsion (δ = 50) is only 12% which is very low compared to the bicontinuous cubic structured phase (δ = 100) which can take up 32.2% of water.28 In order to be able to load watersoluble substances with high molecular weight the size of the water droplets needed to be enlarged. This could be achieved by decreasing the curvature of the monoglyceride film, which enclosed the water compartments. Polyglycerol ester arranged in this film and caused the desired growth of the microemulsion droplets. By the inclusion of polyglycerol ester, the loading efficiency of BSA could be increased by a factor higher than 50. In order to load the highly viscous micellar cubic phase and disperse it in water, it is usually transformed into a microemulsion phase by heating.55 This was circumvented by decreasing the initial water content. In this case, the selfassembled phase was not water saturated so that a water-in-oil microemulsion was formed at room temperature. The loading of this microemulsion was then optimized analogously to the water saturated microemulsion described above. The composition of the microemulsions was optimized for every protein that was loaded.

4. DISCUSSION The two-step preparation procedure presented here is advantageous among common methods like ultrasonication, hot homogenization, and the use of hydrotropes.37,39,54,60 The first two processes use high temperatures and high shear forces in order to form homogeneous nanostructured dispersions which make them inapplicable if sensitive substances have to be incorporated. In order to avoid those harsh conditions, the so16794

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micelles of the microemulsion enabled faster release compared to the two discontinuous liquid crystals. The water compartments of the rod-like micelles in the hexagonal phase were larger than in the two other structures. Small hydrophilic molecules could diffuse comparable long distances inside the micelles. This might be the reason for the slightly faster diffusion inside this phase compared to the micellar cubic phase. The diffusion of the load was more hindered in the spherical micelles that might lead to the slowest release rate. The addition of polyglycerol ester increased the release rate from the microemulsion phase. The reason for this acceleration could be that the water cores of the microemulsion droplets got closer as the polyglycerol ester content increased. The release of hydrophilic and lipophilic substances from nanostructured particles, mainly with bicontinuous cubic internal structure, has been studied previously by other groups. A sudden release of the load was found and within a split second an equilibrium concentration in the outer water phase was reached.49,52,53 Contrary to the release of small molecules, the release of proteins was not governed by their partition coefficient. The delivery of the proteins from the internal water compartments to the outer water phase was sustained and not a burst release. Only small portions of the entrapped proteins were released during 24 h. This was in accordance with one investigation that showed sustained release of ovalbumin from cubosomes.15 Cytochrome c, which has a lower molecular weight than BSA, was released faster from the nanostructured particles. However, the differences in the release rate could not be attributed to only the sizes of the proteins. Proteins can have lipophilic domains even if they are water-soluble. There are three different ways in which hydrophilic proteins can reside in an inverse micelle.61 It can be located in the water core, partly in the interfacial region, and parts of it can even be arranged in the lipophilic compartment. Serum albumins have binding sites for monoglycerides.62 Cytochrome c is part of the electron transport chain and is naturally located in the intermembrane space of the mitochondrion. Thus, it is most likely that BSA and cytochrome c were located close to the interfacial region of the micelles. The dependence of the release rate on the nanostructure could not be found for BSA. A possible explanation could be that the interaction with the interface of the micelles had more influence on the release rate than the nanostructure. The release of cytochrome c was faster from the micellar cubosomes than from the emulsified microemulsion, which was in contrast to the release from small hydrophilic molecules from the nondispersed phases. It is possible that the nanostructure was locally altered due to the arrangement of the protein in the interfacial film which may have led to the different release rates. The release rate of proteins was also affected by the composition of the release medium. PBS affected the protein and its interaction with the self-assembled phase in various ways. The buffer and salt in it resulted in an increased ionic strength and a slight increase in pH. This could affect the intramolecular electrostatic interaction of the protein which might induce changes in its structure. In the presence of PBS, the delivery of cytochrome c was decelerated, whereas the release of BSA within the first 3 h of the experiment was not significantly affected by the presence of the buffer. Nevertheless, there are several factors that contribute to the release of proteins from nanostructured particles. More studies

Figure 8. Release of cytochrome c in PBS (■) and pure water (▲) from emulsified microemulsion (δ50) (a) and micellar cubosomes (δ57) (b).

In the second step, the microemulsions were dispersed in water at room temperature using a Couette mixer. In order to prepare micellar cubosomes, the same procedure was used. In this case, the self-assembled phase was not saturated with water and consequently a water-in-oil microemulsion was formed. The dispersion process could be performed at ambient temperature so heating, which was usually necessary, could be avoided. After the dispersion, the particles took up water and the internal structure transformed to the equilibrium micellar cubic structure. The preparation method used here showed that heating was not required because the transformation to a micellar cubic structure was slow enough so that the dispersion process was not disturbed. Previous studies showed repeatedly that the release from the self-assembled bulk phases is controlled by the diffusion of the load inside the gel and it is not controlled by their macroscopic viscosity.33−35 This behavior was also found for the hydrophilic dye Erioglaucine (see the Supporting Information). Even though the bicontinuous structure had the highest viscosity, the release rate was much higher compared to the microemulsion that was fluid. The bicontinuous cubic structure was built up of two interwoven three-dimensional networks of percolating water channels that were surrounded by the lipids. The diameter of the water channels was sufficient so that the dye could diffuse through them. In the microemulsion, cubic micellar, and hexagonal phase, the water compartments were closed. The discontinuous water pools in those structures caused the slowing down of the release. The relatively mobile 16795

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desmopressin on the properties of reverse hexagonal mesophase. J. Phys. Chem. B 2009, 113, 6336−6346. (7) Libster, D.; Ishai, P. B.; Aserin, A.; Shoham, G.; Garti, N. Molecular interactions in reverse hexagonal mesophase in the presence of Cyclosporin A. Int. J. Pharm. 2009, 367, 115−126. (8) Lopes, L. B. Enhancement of skin penetration of vitamin K using monoolein-based liquid crystalline systems. Eur. J. Pharm. Sci. 2007, 32, 209−215. (9) Angelova, A.; Angelov, B.; Mutafchieva, R.; Lesieur, S.; Couvreur, P. Self-assembled multicompartment liquid crystalline lipid carriers for protein, peptide, and nucleic acid drug delivery. Acc. Chem. Res. 2011, 44, 147−156. (10) Ericsson, B.; Larsson, K.; Fontell, K. A cubic protein-monooleinwater phase. Biochim. Biophys. Acta, Biomembr. 1983, 729, 23−27. (11) Leslie, S. B.; Puvvada, S.; Ratna, B. R.; Rudolph, A. S. Encapsulation of hemoglobin in a bicontinuous cubic phase lipid. Biochim. Biophys. Acta 1996, 1285, 246−254. (12) Libster, D.; Aserin, A.; Garti, N. Interactions of biomacromolecules with reverse hexagonal liquid crystals: Drug delivery and crystallization applications. J. Colloid Interface Sci. 2011, 356, 375−386. (13) Mishraki, T.; Libster, D.; Aserin, A.; Garti, N. Temperaturedependent behavior of lysozyme within the reverse hexagonal mesophases (HII). Colloids Surf., B 2010, 75, 391−397. (14) Mishraki, T.; Libster, D.; Aserin, A.; Garti, N. Lysozyme entrapped within reverse hexagonal mesophases: Physical properties and structural behavior. Colloids Surf., B 2010, 75, 47−56. (15) Rizwan, S. B.; Assmus, D.; Boehnke, A.; Hanley, T.; Boyd, B. J.; Rades, T.; Hook, S. Preparation of phytantriol cubosomes by solvent precursor dilution for the delivery of protein vaccines. Eur. J. Pharm. Biopharm. 2011, 79, 15−22. (16) Zabara, A.; Mezzenga, R. Plenty of room to crystallize: Swollen lipidic mesophases for improved and controlled in-meso protein crystallization. Soft Matter 2012, 8, 6535−6541. (17) Barauskas, J.; Razumas, V.; Nylander, T. Entrapment of glucose oxidase into the cubic Q230 and Q224 phases of aqueous monoolein. Prog. Colloid Polym. Sci. 2000, 116, 16−20. (18) Sadhale, Y.; Shah, J. C. Glyceryl monooleate cubic phase gel as chemical stability enhancer of cefazolin and cefuroxime. Pharm. Dev. Technol. 1998, 3, 549−56. (19) Sadhale, Y.; Shah, J. C. Stabilization of insulin against agitationinduced aggregation by the GMO cubic phase gel. Int. J. Pharm. 1999, 191, 51−64. (20) Sadhale, Y.; Shah, J. C. Biological activity of insulin in GMO gels and the effect of agitation. Int. J. Pharm. 1999, 191, 65−74. (21) Chang, C.-M.; Bodmeier, R. Effect of dissolution media and additives on the drug release from cubic phase delivery systems. J. Control. Release 1997, 46, 215−222. (22) Clogston, J.; Caffrey, M. Controlling release from the lipidic cubic phase. Amino acids, peptides, proteins and nucleic acids. J. Control. Release 2005, 107, 97−111. (23) Mezzenga, R.; Meyer, C.; Servais, C.; Romoscanu, A. I.; Sagalowicz, L.; Hayward, R. C. Shear rheology of lyotropic liquid crystals: A case study. Langmuir 2005, 21, 3322−3333. (24) de Campo, L.; Yaghmur, A.; Sagalowicz, L.; Leser, M. E.; Watzke, H.; Glatter, O. Reversible phase transitions in emulsified nanostructured lipid systems. Langmuir 2004, 20, 5254−5261. (25) Guillot, S.; Moitzi, C.; Salentinig, S.; Sagalowicz, L.; Leser, M. E.; Glatter, O. Direct and indirect thermal transitions from hexosomes to emulsified micro-emulsions in oil-loaded monoglyceride-based particles. Colloids Surf., A 2006, 291, 78−84. (26) Moitzi, C.; Guillot, S.; Fritz, G.; Salentinig, S.; Glatter, O. Phase reorganisation in self-assembled systems through interparticle material transfer. Adv. Mater. 2007, 19, 1352−1358. (27) Sagalowicz, L.; Michel, M.; Adrian, M.; Frossard, P.; Rouvet, M.; Watzke, H. J.; Yaghmur, A.; Campo, L. d.; Glatter, O.; Leser, M. E. Crystallography of dispersed liquid crystalline phases studied by cryotransmission electron microscopy. J. Microsc. 2006, 221, 110−121.

on the release of several proteins are required in order to get a better insight into the release mechanisms.

5. CONCLUSIONS In a two step preparation method, the nanostructured material can be loaded with sensitive substances under optimized conditions and in the following it can be dispersed at low temperature. Both proteins in this study are releases in a sustained manner from the particles. The release of the protein cytochrome c is affected by the internal structure of the particles. In prospective investigations, these differences may be used in order to adjust the release rate by changing the nanostructure. Accordingly, nanostructured particles are promising candidates as controlled delivery systems for proteins.

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ASSOCIATED CONTENT

S Supporting Information *

The phase diagram in cooling direction as well as the study of the release of the hydrophilic dye Erioglaucine from nanostructured materials with four different structures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

This manuscript was written through contributions of all authors. All authors have given their approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the Department of Pharmaceutical Technology, Karl-Franzens University Graz, especially Bettina Bauer, Stephan Bertl, Martin Griesbacher, and Andreas Zimmer, for providing the HPLC equipment and their support during the HPLC analysis.

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REFERENCES

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NOTE ADDED AFTER ASAP PUBLICATION This article was published ASAP on November 16, 2012. A change has been made in equation 2. The correct version was published on November 27, 2012.

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