Dr. Gyorgy Levai defended past Monday 28 th November his PhD thesis at the University of Valladolid. The thesis was directed by Dr. Ángel Martín and Dr. Soraya Rodriguez Rojo from the High Pressure Processes Group and it has the mention of International PhD because of his secondment in the Department of Experimental Thermodynamics in Ruhr-University of Bochum, under the supervision of Prof. Tobias M. Fieback.
Polyphenols are widely applied in pharmaceutic and cosmetic areas due to their beneficial effects on health, such as strong antioxidant-, antiviral- and antihistaminic capability. Quercetin is a member of flavonoids, which is the major group of the polyphenols, and is highly available in various fruits, vegetables and oils. It has an anti-proliferative effect in a wide range of human cancer cell lines, and it is a highly promising active compound against a wide variety of diseases. A major limitation for the clinical application of quercetin is its low bioavailability (lower than 1% in humans), due to its low water solubility, which makes necessary to administrate it in doses, as high as 50 mg/kg.
Application of supercritical fluid technologies are promising alternative in the processing of natural bioactive compounds, because upon applying an adequate supercritical medium, such as supercritical carbon-dioxide (scCO2), they allow carrying out the encapsulation process at near ambient temperatures and in an inert atmosphere, thus avoiding the thermal degradation or oxidation of the product, and reducing its contamination with organic solvents.
The aim of this thesis is to increase water solubility and hence bioavailability of quercetin, by increasing its specific surface area, precipitating it in sub-micrometric scale, and encapsulating it in biocompatible polymers, such as soy-bean lecithin and a poloxamer (Pluronic L64®). For this, Supercritical Fluid Extraction of Emulsions (SFEE) technology was used to obtain aqueous suspension product, and Particles from Gas Saturated Solutions (PGSS)-drying technology was used, to obtain dry quercetin loaded particles in controlled micrometric range.
SFEE technology (Fig 1), (presented in Chapter I), can be considered as an evolution of SAS technology, which is especially suitable to encapsulate poorly water soluble drugs in an aqueous suspension. The process consists of forming an oil in water (o/w) emulsion, containing the quercetin in the dispersed organic phase. The organic solvent is extracted from this emulsion using scCO2, which has high affinity to the organic solvent, meanwhile low affinity to the active compound of interest. Due to the solubility differences, the supercritical solvent extracts quickly the organic solvent from the emulsion, leading to the rapid super-saturation of the active compound in the aqueous phase, and promoting its fast precipitation in nanometric scale. Quercetin is encapsulated in the aqueous suspension product by a surfactant material, which was originally added to the o/w emulsion in order to increase its stability, which is a crucial issue in SFEE process.
In Chapter I two different biopolymers (Pluronic L64® poloxamer and soy-bean lecithin) were used as carriers and surfactant materials in the formation of o/w emulsions. Emulsions were treated by a batch SFEE equipment (Fig 2), at operating conditions around 100 bar and 40°C, and a part of scCO2, contained by the system was renewed several times during the process, without any kind of pressure and temperature change. Optimal number of extraction cycles and duration of each cycle was determined in these experiments, to decrease residual organic content of aqueous suspensions under the restrictions of FDA, without degradation or agglomeration of encapsulated quercetin particles. Applying Pluronic L64® as surfactant, needle quercetin particles were obtained, with an average particle sizes around 1 µm and poor encapsulation efficiency. In case of soy-bean lecithin, quercetin loaded multivesicular liposomes were obtained, with a mean particle size around 100 nm and around 70% of encapsulation efficiency, without the presence of segregated quercetin crystals. Moreover, antioxidant activity of quercetin was enhanced by encapsulation in lecithin, in agreement with previous reports, that describe a synergistic effect of these two compounds.
The effect of the main SFEE parameters, such as pressure and temperature, initial concentration of surfactants and quercetin, organic to water ratio, and kinetics of the elimination of the organic solvent from the emulsion, has key importance for the optimization of the SFEE process. For that purpose, in Chapter II ethyl acetate and dichloromethane based o/w emulsions were prepared, using soy-bean lecithin and/or Pluronic L64® as surfactants, and they were contacted with supercritical carbon-dioxide (scCO2), and mass transport properties of these systems were measured by Magnetic Suspension Balances (MSB), which provide an on-line, contactless sample weight monitoring method in a closed system. Static and dynamic measurements (applying a continuous flow of scCO2 through the emulsion) were performed, and the effect on mass transport properties of the initial organic/water proportion, the concentration of surfactant materials (soy-bean lecithin and additional Pluronic L64®), and the density of scCO2 atmosphere were studied. In the static system the dissolution of scCO2 by the sample, and the extraction of organic phase was observed simultaneously, dominating the process in different moments as presented on Fig 3.
A five-parameter mass transfer model was developed for the measured results, and two parameters of them were fixed, indicating the significant process steps of SFEE: dissolution of the scCO2 and the organic solvent in the water phase, and the extraction of the organic phase by the scCO2 through the water phase boundary. Experimental and modelling results proved that DCM/w emulsions are not adequate for SFEE processing, as phase separation occurs immediately upon pressurization, due to the high interfacial tension between the aqueous and organic phase. In the case of EtAc/w emulsion – scCO2 system, only the density of scCO2 was significantly influencing the speed of the mass transfer of compounds. According to dynamic measurements, the higher proportion of the organic phase is extracted with the pressurization of the system by scCO2, due to the Marangoni effect, or additional mixing effect between phases, upon pressurizing by CO2 the system.
In Chapter III the combination of surfactant materials (soy-bean lecithin and Pluronic L64®) was used, in order to increase the encapsulation efficiency of quercetin, without significant morphological change of the obtained aqueous suspension product. Based on mass transport measurement results detailed in Chapter II, a successful scale-up of SFEE process is done, and a semi-continuous SFEE device is designed (Fig 4) and presented in Chapter III, and with a significantly shorter processing time a significantly higher amount of o/w emulsion is treated. By comparing the results of batch and scaled-up systems, robustness of scaled-up, semi-continuous SFEE process is proved.
Aqueous suspensions produced by SFEE were further treated by Particles from Gas Saturated Solutions (PGSS)-drying technology (Fig 5), to produce micronized, quercetin loaded dry particles, and to increase long-term stability of product. An experimental plan with PGSS-drying is done, in order to choose the applicable settings of significantly influencing process parameters, such as Gas to Liquid ratio (GLR), pre-expansion pressure and temperature, and to obtain less possible quercetin degradation and residual water content of product.
A free flowing powder in micrometric scale (Fig 6) with a residual moisture content of less than 10 w/w%, and is produced, with a maximum quercetin encapsulation efficiency of 86.4%, which is corresponding only 0.6% of quercetin loss, comparing to the aqueous suspension from which PGSS-drying experiment was performed. Lyophilization (assumed as a quercetin-degradation-free process) of two SFEE produced aqueous suspension mixtures is parallelly done with PGSS-drying, and encapsulation efficiency, and antioxidant activity of with PGSS drying and with lyophilization prepared dried products are measured and compared with each other. According to transdermal diffusion measurement, quercetin permeability of SFEE and PGSS-drying micronized dry-product through transdermal membrane into simulated intestinal fluid is increased significantly, comparing to lyophilized dry-product, which did not proved an adequate methodology, as no quercetin permeability increase was obtained – comparing to physical mixture -, as micronization and encapsulation of quercetin did not take place.
Thesis supervised by:
- Dr. Ángel Martín Martínez, Universidad de Valladolid – Spain
- Dr. Soraya Rodríguez Rojo, Universidad de Valladolid – Spain
- Prof. Tobias M. Fieback, Ruhr-University of Bochum – Germany
Members of the Committee:
- Dr. Ing. Marcus Peterman, Ruhr-University Bochum – Germany
- Dra Sagrario Beltran Calvo, Universidad de Burgos – Spain
- Dra Lourdes Calvo Garrido, Universidad Complutense de Madrid – Spain
- Dra Ana Matias, iBET – Instituto de Biologia Experimental e Tecnológica – Portugal
- Dr. Ing. Salima Varona Iglesias, Fraunhofer Institute for Interfacial Engineering and Biotechnology – Germany