http://elea.unisa.it/xmlui/handle/10556/925 2026-04-20T09:41:31Z http://elea.unisa.it/xmlui/handle/10556/2025 Supercritical assisted processes for the production of biopolymeric micro and nanocarriers Campardelli, Roberta This work provides an innovative point of view on obtaining nanoparticles by supercritical fluids. Supercritical fluids have already been exploited in the production and processing of micro or sub-micro particles, but the production of nanoparticles is still more ambitious, requiring a deep understanding of the thermodynamic of multiphase systems involved and of the fluid dynamics and mass transfer of the process. This thesis focused on the development of supercritical assisted process for the production of biopolymeric micro and nanocarrier for pharmaceutical and biomedical applications. After a wide study of the state of the, two different processes have been focused on the production of different kind of devices: 1) Supercrititical Emulsion Extraction process (SEE) for the production of multifunctional nanodevices; 2) Supercritical Assisted Injection in Liquid Antisolvent process (SAILA) for the production of stabilized nanoparticle water suspensions. Supercritical Emulsion Extraction (SEE) has been recently proposed in the literature for the production of drug/polymer microspheres with controlled size and distribution, starting from oil-in-water (o-w) and water-in-oil-in-water (w-o-w) emulsions. This process uses supercritical carbon dioxide (SC-CO) to extract the “oil” phase of emulsions, leading to near solvent-free microparticles. SEE offers the advantage of being a one-step process and is superior to other conventional techniques for the better particle size control, higher product purity and shorter processing times; the SEE process has been proposed in the continuous layout, using a high pressure packed tower. However, monodisperse sub-micro, nanoparticles of biopolymer suitable for pharmaceutical formulations have not been produced until now by SEE. Therefore, this part of the work was performed on the production of monodisperse biopolymer nanoparticles. Particularly, emulsion formulation parameters have been tested, such as, different surfactant concentrations and biopolymer percentages in the oily phase, and several emulsification techniques (ultrasound or high speed emulsification) and their interactions with SEE processing have been tested to obtain small droplets dimensions from the micro size to the nano size range. Poly-lactic-co-glycolic acid (PLGA), poly-lactic acid (PLA) and poly-caprolactone (PCL) were the polymers selected to produce micro and nano devices, relative results are shown in Chapter 7. Then, after the optimization of the process conditions for the production of monodisperse sub-micro and nanoparticles encapsulation of drugs, proteins, peptides and metals has been carried out. For the encapsulation study the SEE technique starting from single or double emulsions was used for the production of nanospheres. The SEE technology has a great potential regarding the field of protein encapsulation thanks to the mild extraction condition and the short process time, and mainly thanks to the possibility to use multiple emulsions. For this reason, the SEE has been applied to the production of biopolymeric micro and nanoparticles of PLA and PLGA encapsulating peptides and proteins starting from double emulsion. Results are shown in Chapter 8. Bovine serum albumin (BSA) has been selected as protecting protein for growth factor, such as Vascular Endothelial Growth Factor (VEGF) and Bone Morphogenetic Proteins (BMP). Both micro and nanoparticles were produced; the effect of size of particles on protein loading and release has been evaluated. PLGA microspheres loaded with GFs were charged inside a bioactive alginate scaffold to monitor the effect of the local release of these biosignals on cells differentiation. Human Mesenchimal Steam Cells (h-MSC), where used in view of the fact that they are a promising cell source for bone tissue engineering. Good cells differentiation indicates that the SEE process was successful in the encapsulation of proteins and peptides, preserving the functional structure of the proteins, thanks to the mild operating conditions used. Another important challenge that has been managed is also the production of nanocomposite biopolymeric particles encapsulating metal nanoparticles for the production of light sensitive drug delivery devices. The encapsulation of TiO2 and Au nanoparticles has been performed for different biomedical applications (Chapter 9 and 10). In this work PLA/nano-TiO2 microparticles have been produced using SEE for photodynamic therapy of cells and bacteria. Anatase type nano-TiO2 ethanol stabilized suspension has been synthesized by precipitation from solutions of titanium alcoxides and directly used as the water internal phase a of double emulsion water in oil in water and also TiO2 fine powder was produced and used for encapsulation experiments using a solid in oil in water emulsion. Both micro and sub-microparticles have been produced. [edited by Author] 2011 - 2012 2015-04-20T00:00:00Z http://elea.unisa.it/xmlui/handle/10556/2026 In silico and in vitro models in pharmacokinetic studies Cascone, Sara One of the aims of the thesis was to design and realize an in vitro device able to reproduce the gastrointestinal behavior. To reproduce the temperature and pH history an USP apparatus II coupled with a control system was used. The temperature was kept constant using the USP apparatus, a pH probe was inserted in the dissolution medium to measure the pH. The measured pH was compared (by a software) with a set point. Proportionally at the mean error, a quantity of an acidic or basic solution was inserted, by pumps, in the dissolution medium adjusting the pH at the desired value. Using the real pH history of the gastrointestinal tract, which provide a decrease in the pH value from 4.8 to about 2.0 during the first two hours of dissolution, and then an increase to 6.8, the release pattern from tablets was evaluated. The release patterns of these tablets obtained with the new device were compared with those obtained using the conventional method (which provides a pH 1 during the first two hours of dissolution, and then the neutralization at pH 6.8) and it was found that the drug released during the first two hours was higher in the case in which the real pH history was reproduced. This is due to the fact that the higher pH in the first stage damages the coating of the tablet. Once the chemical and thermal conditions were reproduced, the reproduction of the transport across the intestinal membrane was faced. An high throughput device which is able to reproduce continuously the exchange between the compartments has been necessary. The USP apparatus was equipped with a device composed by an hollow filter (which simulate the intestinal wall) and two pumps for the fluids simulating the intestinal content and the circulatory system surrounding the gastrointestinal tract content. The fluids enter in contact in the filter and the fluid rich in drug content (that simulates the intestinal content) gives the drug to the fluid poor in drug (simulating the blood content). The release patterns obtained by the use of this device were studied and compared with those obtained following the conventional dissolution method. Moreover these release patterns obtained using the real pH evolution were coupled with the effect of mass exchange and compared with those obtained using the conventional methods. The results showed that the effect of the real history of pH is higher in the first stage of dissolution, than the effect of the mass exchange is dominant. The reproduction of the mechanical history of the stomach is than faced. The peristaltic waves were reproduced using a lattice bag (elastic and compressible) connected to a camshaft which, with its rotation ensured the contraction of the bag. The bag was shrunk by connectors and the right position was ensured by guides. Changing the rotation speed of the shaft, the frequency of the contractions could be adjusted. The release pattern of a commercial tablet in the new device was evaluated and compared with the conventional one. The results showed that the non-perfect mixing of the stomach was satisfactory reproduced and this lead to a release pattern completely different. Moreover, the effect of the frequency of the contractions on the release pattern was evaluated. Second, but not secondary, aim of the thesis was to develop an in silico model (physiologically based) which is able to simulate the plasma concentration of drugs. The model is composed by seven compartments, which simulate the human organ, tissue, or a group of them. The compartments are interconnected between them and seven differential equations (with their initial conditions) describe their behavior. Once the parameter are obtained (by fitting or in literature), using an in vitro release pattern, the model is able to simulate the concentrations in all the compartments, including the plasma compartment. The plasma concentration are simulated both in the case in which the new release pattern (with the real pH history) is used as input, and the case in which the conventional one is used. The results show that in the real case the plasma concentration is very different both in value and in shape than the expected. The model then was used to simulate the fate of several molecules simultaneously in the human body (i.e. if a racemic mixture is administered or if the drug is metabolized to another molecule). The system of differential equations is expanded to describe the fate of each molecule. Then, the physiological parameters, such as gender and age, were integrated in the model; in this way, the dependence of the model parameter on the physiological parameter was evaluated. Finally, the gastrointestinal concentration simulated with the in silico model was successfully compared with the drug concentration measured with the in vitro model. It could be concluded that the combined approach which uses the in vitro and the in silico models is a powerful tool in the pharmacokinetic studies. [edited by Author] 2011 - 2012 2015-04-21T00:00:00Z http://elea.unisa.it/xmlui/handle/10556/2030 Novel technologies and process intensification in the production of micro-systems with pharmacological/nutraceutical activity Dalmoro, Annalisa Purpose of the PhD thesis was to develop a novel microencapsulation process, designing and building a single-pot semi-continuous bench scale apparatus. The novel process is based on the coupling of two emerging techniques, involving ultrasound and microwave, used in atomization and heating operations, respectively. The process has been designed to respond to the needs for process intensification, i.e. improvement of process efficiency and cutting down of energy consumption. With this aim, a review of the main processes used for microencapsulation was first performed: conventional processes showed a number of drawbacks, such as high energy consumption, batch configuration, use of solvents and long times of production. On the basis of the state of the art, the idea of an intensified apparatus for particles production, exploiting alternative resources, such as ultrasound and microwave, was formulated. The apparatus was composed of three main sections: feeding, atomization, separation/stabilization. The feeding and atomization sections were built connecting a double channel ultrasonic atomizer to a system for feeding solutions in a purposely designed separation/stabilization section, thus realizing a semi-continuous apparatus. Separation section consisted of a wet-collector, i.e. a sort of hydrocyclone, which allowed a uniform distribution of the hardening solution and the consequent contact with the atomized drops, a filtering device, and a microwave oven. The wet-collector was placed into the microwave oven to obtain an “on-line” drying. Recirculation of the hardening solution, to renew contact surface between droplets and cross-linker, was guaranteed by a system of centrifugal pumps. In this configuration, when atomization occurred, drops were harvested in the wet-collector. After atomization, the obtained suspension was collected in the cross-linker tank, then the filtering device was inserted in the lower part of the wet-collector, so that hardening solution was recovered and particles settled on the filter, when the suspension was brought again to the wet-collector and after its complete emptying. An eventual following washing step can be done in a similar way to the previous hardening step. Finally, particles were stabilized by microwave drying, and then recovered. The steps for building the microencapsulation apparatus were accurately shown. Then, criteria used for components selection, in order to obtain the best performances from the plant, were highlighted. After building the plant, the process parameters were defined. First, the research for the best combination of feeding parameters, such as type of materials, composition, concentration and feed rate, that assure the encapsulation of the core material in the shell, was carried out. Then, the parameters of the ultrasonic atomizer (atomization section), essentially power, were tuned. Finally, for stabilization/separation section, fundamental was the relevant stabilization step, where microwave power was set to avoid too high temperatures that could degrade molecules... [edited by Author] 2011 - 2012 2015-04-22T00:00:00Z http://elea.unisa.it/xmlui/handle/10556/1986 Development of selective materials and methods for analytical techniques combined with mass spectrometry Euterpio, Maria Anna Mass spectrometry (MS) is a powerful detection technique that has become very important in several chemical disciplines for detection of both small molecules (environmental pollutants, small metabolites) and large biomolecules (proteins, peptides). The "heart" of the mass spectrometer is the analyzer, that uses electrical or magnetic fields, or combination of both, from the region where they are produced, to a detector, where they produce a signal which is amplified. This element separates the gas phase ions according to their m/z (mass to charge ratio) value. MS is mostly used coupled to high performance liquid chromatography (HPLC) or ultra- high performance liquid chromatography (UHPLC) with an atmospheric pressure ionization (API) interface between the LC and MS. The most important API technique is electrospray ionization (ESI), with a wide range of application from small molecules to large molecules such as proteins or polymers. Complex matrix, as environmental samples, biological fluids, food or tissue extracts, are not usually compatible with MS detection without extensive sample preparation. In facts, complex matrix could contains substances that can cause the detector signal to decrease or increase for a selected analyte compared to the signal from the same compound in a standard solution. ESI is the ionization technique in which this phenomenon is most common because in the ESI process ions compete for ionization/desorption. Matrix effect is observed in ESI when compounds co-elute with the analyte of interest: this affects sensitivity, linearity, accuracy, precision and the limit of detection (LOD). Furthermore, the target compound(s) are often present at concentrations lower than their detection limits and require a preliminary concentration step. Therefore, the first step of an analysis is usually some kind of sample pretreatment to improve both the selectivity and sensitivity of the subsequent detection. Many techniques are available for this purpose, the suitability of which depends primarily on the physical state of the sample, e.g. solid-phase extraction (SPE) for liquid and pressurized-liquid extraction for solid samples. Proper sample preparation is critical for MS analysis, because the quality and reproducibility of sample extraction and preparation significantly impact results from MS instruments... [edited by Author] 2011 - 2012 2015-04-23T00:00:00Z