The present work is part of a range of studies related to the encapsulation of molecular probe inside of the erythrocytes that are able to induce the temporary shaping of pores on the cell membrane by exploiting the mechanical stress, in particular way the shear stress. Thereby the purpose of this study is to develop a new technique of encapsulation that uses the application of shear stress on Red Blood Cells causing them to flow within micro channels. To manage this, first I studied the fluid dynamics of a suspension of erythrocytes and saline solution inside a rectangular capillary of a few tens of microns (60x40μm), for different values of hematocrit, 1% and 5%, and at different values of flow rate, 0,25μl/min e 0,50μl/min. This analysis was performed using a micro-PIV system, which previously allowed me to determine the velocity profile in a experimental way and secondly the relative profile of shear stress, calculated by using the Casson model. Subsequently, I carried out with tests of encapsulation of fluorescent probes in the fluid condition considered to be more favorable for the my purpose, in particular for the solution at the hematocrit of 5% and the flow rate of 0,50μl/min. I chose FITC-dextrans as probative molecules which have Stokes’ radius of 45Å and atomic whight equal to 40000Da. After a careful centrifugation of the treated samples, I observed them through the fluorescence microscope, and found out the presence of these molecules inside Red Blood Cells but the following cell counting in the Burker chamber was not enough for an evaluation of their number because they were insufficient. In the end, I carried out with an analysis through the spectrofluorimeter which allowed me to quantify the concentration of encapsulated dextrans. The results of my study show the possibility of encapsulating molecules within the erythrocyte as a result of mechanical stress using micro capillaries as well, showing the way forward to the realization of a device consisting of several channels in parallel, aimed to the direct therapeutic application for the patient.
Il presente lavoro di tesi si colloca nell’ambito degli studi relativi all’incapsulamento di molecole probanti all’interno degli eritrociti sfruttando la sollecitazione meccanica, in particolar modo lo shear stress, in grado di indurre la formazione temporanea di pori sulla membrana cellulare. Tale studio è quindi volto a realizzare una tecnica innovativa d’incapsulamento che prevede la sollecitazione degli eritrociti tramite scorrimento in canali di dimensioni micrometriche. Per fare ciò, è stata dapprima studiata la fluidodinamica di una sospensione di eritrociti e soluzione fisiologica all’interno di un capillare rettangolare di qualche decina di micron (60x40μm), a differenti valori di ematocrito, 1% e 5%, e a differenti valori di portata, 0,25μl/min e 0,50μl/min. Tale analisi è stata effettuata tramite micro-PIV ed ha permesso di determinare in modo sperimentale i profili di velocità ed in seconda analisi i relativi profili di sforzo tangenziale, calcolati secondo il modello proposto da Casson. In seguito, sono state eseguite prove d’incapsulamento di molecole fluorescenti nella condizione fluidodinamica ritenuta più favorevole allo scopo, in particolare per la soluzione ad ematocrito del 5% e alla portata di 0,50μl/min. Come molecole probanti sono stati scelti FITC-destrani, aventi raggio di Stokes di 45Å e peso atomico pari a 40000Da. L’osservazione al microscopio in fluorescenza, dopo un’accurata centrifugazione dei campioni trattati, ha dimostrato la presenza di dette molecole all’interno degli eritrociti ma la successiva conta cellulare in camera di Burker non ha permesso una stima del loro numero in quanto insufficiente. È stata infine eseguita un’analisi allo spettrofluorimetro che ha permesso di quantificare la concentrazione di destrani incapsulati. I risultati ottenuti dimostrano quindi la possibilità di incapsulare molecole all’interno degli eritrociti per effetto della sollecitazione meccanica anche utilizzando microcapillari, aprendo la strada alla realizzazione di un dispositivo costituito da numerosi condotti in parallelo, volto all’applicazione terapeutica in linea col paziente.
Studio reologico del sangue in microcapillari : lo sforzo di taglio, fattore incapsulante di molecole in eritrociti
D'URSO, DAVIDE GIUSEPPE
2010/2011
Abstract
The present work is part of a range of studies related to the encapsulation of molecular probe inside of the erythrocytes that are able to induce the temporary shaping of pores on the cell membrane by exploiting the mechanical stress, in particular way the shear stress. Thereby the purpose of this study is to develop a new technique of encapsulation that uses the application of shear stress on Red Blood Cells causing them to flow within micro channels. To manage this, first I studied the fluid dynamics of a suspension of erythrocytes and saline solution inside a rectangular capillary of a few tens of microns (60x40μm), for different values of hematocrit, 1% and 5%, and at different values of flow rate, 0,25μl/min e 0,50μl/min. This analysis was performed using a micro-PIV system, which previously allowed me to determine the velocity profile in a experimental way and secondly the relative profile of shear stress, calculated by using the Casson model. Subsequently, I carried out with tests of encapsulation of fluorescent probes in the fluid condition considered to be more favorable for the my purpose, in particular for the solution at the hematocrit of 5% and the flow rate of 0,50μl/min. I chose FITC-dextrans as probative molecules which have Stokes’ radius of 45Å and atomic whight equal to 40000Da. After a careful centrifugation of the treated samples, I observed them through the fluorescence microscope, and found out the presence of these molecules inside Red Blood Cells but the following cell counting in the Burker chamber was not enough for an evaluation of their number because they were insufficient. In the end, I carried out with an analysis through the spectrofluorimeter which allowed me to quantify the concentration of encapsulated dextrans. The results of my study show the possibility of encapsulating molecules within the erythrocyte as a result of mechanical stress using micro capillaries as well, showing the way forward to the realization of a device consisting of several channels in parallel, aimed to the direct therapeutic application for the patient.| File | Dimensione | Formato | |
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2012_04_D'Urso.pdf
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https://hdl.handle.net/10589/49141