Supercritical carbon dioxide spray drying for the production of stable dried protein formulations

The main goals of this thesis are to understand the supercritical carbon dioxide (scCO2) spray drying mechanisms and parameters that influence the stability of proteins, to evaluate the excipients to stabilize protein formulations during scCO2 spray drying, and to study the scalability of the scCO2 spray drying process. For this study, lysozyme and myoglobin were used as model proteins. More specifically, the detailed aims are as follows: 1) to study the scCO2 spray drying parameters (i.e., pressure, protein solution and CO2 flow rate, feed volume) without the use of organic solvents, in order to produce dried protein formulations with minimal residual water content in a single drying step, 2) to evaluate the scalability of the scCO2 spray drying process, 3) To gain fundamental insight into the effect of the CO2 spray drying parameters at sub- and supercritical conditions (65-130bar and 25-50°C) on the stability of myoglobin, 4) to understand the effect of the CO2/water interface and pH shift on heme destabilization and aggregation in myoglobin solutions using a gas bubbling method at atmospheric conditions, 5) to evaluate the influence of pharmaceutical excipients on the stability of myoglobin in terms of heme binding and aggregation during scCO2 spray drying.The Royal Thai Government ScholarshipDrug Delivery Technolog

Critical processing parameters of carbon dioxide spray drying for the production of dried protein formulations: A study with myoglobin.

The aim of this study was to gain fundamental insight into protein destabilization induced by supercritical CO2 spray drying processing parameters. Myoglobin was used as a model protein (5mg/ml with 50mg/ml trehalose in 10mM phosphate buffer, pH 6.2). The solution was exposed to sub- and supercritical CO2 conditions (65-130bar and 25-50°C), and CO2 spray drying under those conditions. The heme binding of myoglobin was determined by UV/Vis, fluorescence, and circular dichroism spectroscopy, while myoglobin aggregation was studied by using size-exclusion chromatography and flow imaging microscopy. It was found that pressure and temperature alone did not influence myoglobin's integrity. However, when pressurized CO2 was introduced into myoglobin solutions at any condition, the pH of the myoglobin formulation shifted to about 5 (measured after depressurization), resulting in heme binding destabilization and aggregation of myoglobin. When exposed to CO2, these degradation processes were enhanced by increasing temperature. Heme binding destabilization and myoglobin aggregation were also seen after CO2 spray drying, and to a greater extent. Moreover, the CO2 spray drying induced the partial loss of heme. In conclusion, pressurized CO2 destabilizes the myoglobin, leading to heme loss and protein aggregation upon spray drying

Characterization of drug delivery particles, produced by supercritical carbon dioxide technologies

Drug Delivery Technolog

In Vitro Characterization and Mosquito (Aedes aegypti) Repellent Activity of Essential-Oils-Loaded Nanoemulsions

The nanoemulsions composed of citronella oil, hairy basil oil, and vetiver oil with mean droplet sizes ranging from 150 to 220 nm were prepared and investigated both in vitro and in vivo. Larger emulsion droplets (195–220 nm) shifted toward a smaller size (150–160 nm) after high-pressure homogenization and resulted in higher release rate. We proposed that thin films obtained from the nanoemulsions with smaller droplet size would have higher integrity, thus increasing the vaporization of essential oils and subsequently prolonging the mosquito repellant activity. The release rates were fitted with Avrami’s equations and n values were in the same range of 0.6 to 1.0, implying that the release of encapsulated limonene was controlled by the diffusion mechanism from the emulsion droplet. By using high-pressure homogenization together with optimum concentrations of 5% (w/w) hairy basil oil, 5% (w/w) vetiver oil (5%), and 10% (w/w) citronella oil could improve physical stability and prolong mosquito protection time to 4.7 h due to the combination of these three essential oils as well as small droplet size of nanoemulsion

Citotoxic activity evaluation of essential oils and nanoemulsions of Drimys angustifolia and D. brasiliensis on human glioblastoma (U-138 MG) and human bladder carcinoma (T24) cell lines in vitro

The species Drimys angustifolia Miers and D. brasiliensis Miers, commonly known as "casca-de-anta", have in their leaves essential oils that can confer cytotoxic effects. In this study, we evaluated the citotoxic effects of the volatile oils from these two species. We also proposed a nanoemulsion formulation for each of the species and assessed the in vitro cytotoxicity on U-138 MG (human glioblastoma) and T24 (human bladder carcinoma) cell lines. The plant chemical composition was evaluated by gas chromatography coupled to mass spectrometer. Furthermore, the nanoemulsions were prepared and characterized. Our results showed that; bicyclogermacrene (19.6%) and cyclocolorenone (18.2%) were the most abundant for the D angustifolia oil and D brasiliensis oil, respectively. Both nanoemulsions, D angustifolia and D brasiliensis appeared macroscopically homogeneous and opalescent bluish liquids, with nanometric mean diameters of 168 nm for D brasiliensis and 181 nm for D angustifolia. The polydispersity indices were below 0.10, with an acid pH of 4.7-6.3, and negative zeta potentials about -34 mV. The results of transmission electron microscopy showed that droplets are present in the nanometer range. Only the D brasiliensis oil was efficient in reducing the cell viability of both U-138 MG (42.5%±7.0 and 67.8%±7.8) and T24 (33.2%±2.8, 60.3%±1.6 and 80.5%±8.8) cell lines, as assessed by MTT assay. Noteworthy, similar results were obtained with cell counting. Finally, D brasiliensis oil incubation caused an increase of annexin-V and propidium iodite population, according to evaluation by cytometry analysis, what is characteristic of late apoptosis. The results presented herein lead us to consider the potential therapeutic effects of the essential oils and nanoformulations as novel strategies to inhibit tumor growth