This thesis project involves the comparison of two liposome preparation techniques in order to obtain stable and reproducible liposomes able to exploit their action as drug carriers for the perfusion of transplanted kidney. On this purpose, the liposomes have been loaded with myoglobin as model molecule for future loading of a growth factor. In vitro myoglobin release studies from liposome were conducted considering temperatures and timing of transplanted kidney perfusion (4 hours, 4 °C). More in details, two preparation technologies were compared, a traditional thin film hydration method followed by extrusion and an innovative one using the microfluidic technique (Nanoassemblr). The liposomes were formulated using different molar lipid concentration ratios of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol. The liposomes obtained with both preparation techniques were dimensionally characterized by analysis with DLS, NTA and SEM. In addition to the dimensional characterization, SEM was also used to conduct a morphological analysis on liposomes. The selected liposomes were subsequently frozen and lyophilized in order to improve their conservation over time and to evaluate their process yield. Several sugars have been investigated as possible cryoprotectants in order to maintain stable size and PDI after rehydration of lyophilized liposomes. The liposomes were loaded with myoglobin as a model protein. The encapsulation efficiency, in vitro release rate of myoglobin from liposomes at 4 ° C and 37 ° C were evaluated and the release kinetic fitting model was determined. Different concentrations of liposomes were used to conduct cell viability assays on human fibroblasts. In addition, fluorescent liposomes have been formulated to study cellular uptake on human fibroblasts and renal tubular cells through the use of fluorescence microscopy and confocal microscopy. Both manufacturing methods allowed to obtain DSPC: Chol 50:50 liposomes with dimensions below 300 nm. The process yields were higher than 80% for both manufacturing techniques. The encapsulation efficiency for myoglobin was found to be higher (71.20%) for liposomes prepared with thin film hydration method compared to liposomes prepared with microfluidic technique (33.50%). All liposomes tested guaranteed complete release of myoglobin after 4 hours at 37 ° C while at 4 ° C the complete release was reached after 5 hours. The release kinetics calculated for both formulations follows Peppas equation model with “n” between 0.45 and 0.89 typical of a non-Fickian release. Cell viability, with all tested concentrations, was always greater than 70% indicating good biocompatibility of the liposomes produced. The fluorescent liposomes showed the onset of cellular uptake already after 2 hours of incubation at both 4 ° C and 37 ° C. From the results obtained it can be concluded that liposomes were successfully obtained by both the manufacturing techniques tested. The liposomes showed good biocompatibility. Functional properties of the liposomes are affected by the manufacturing techniques in such a way the liposomes prepared by thin film hydration method resulted in higher encapsulation efficiency as copared to liposomes produced by Nanoassemblr.
Il progetto si basa sul confronto di due tecniche di preparazione di liposomi in modo da poter ottenere liposomi stabili e riproducibili, per poi essere utilizzati come veicoli di farmaci nella perfusione del rene espiantato. A tal fine i liposomi sono stati caricati con mioglobina, molecola modello per la futura veicolazione di un fattore di crescita. Gli studi di rilascio in vitro della mioglobina dai liposomi sono stati condotti rispettando le temperature e le tempistiche della perfusione di rene espiantato (4 ore, 4°C). Nello specifico sono state confrontate due tecnologie di preparazione, una tradizionale la thin film hydration method seguita da estrusione e una innovativa utilizzando la tecnica microfluidica (Nanoassemblr). Sono stati formulati liposomi utilizzando diversi rapporti di concentrazione lipidica molare di DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) e colesterolo. I liposomi ottenuti con entrambe le tecniche di preparazione sono stati caratterizzati dimensionalmente tramite analisi con DLS, NTA e SEM. Oltre alla caratterizzazione dimensionale, la SEM è stata anche utilizzata per condurre un’analisi morfologica sui liposomi. I liposomi selezionati sono stati successivamente congelati e liofilizzati al fine di migliorare la loro conservazione nel tempo e per valutarne la resa di processo. Diversi zuccheri sono stati studiati come possibili crioprotettori al fine di mantenere stabili le dimensioni e il PDI dopo la reidratazione dei liposomi liofilizzati. I liposomi sono stati caricati con mioglobina come proteina modello. Sono quindi state valutate l’efficienza di incapsulazione, la velocità di rilascio della mioglobina dai liposomi a 4°C e 37°C ed è stata determinata la cinetica di rilascio in vitro. Diverse concentrazioni di liposomi sono state utilizzate per condurre saggi di vitalità cellulare su fibroblasti umani. Inoltre, sono stati formulati liposomi fluorescenti per studiare l’uptake cellulare su fibroblasti umani e cellule tubulari renali tramite l’utilizzo della microscopia a fluorescenza e della microscopia confocale. Entrambi i metodi di produzione hanno permesso di ottenere liposomi DSPC:Chol 50:50 con dimensioni inferiori ai 300nm. Le rese di processo ottenute sono state superiori all’80% per entrambe le tecniche. L’efficienza di incapsulazione per la mioglobina è risultata essere maggiore (71.20%) per i liposomi prodotti con la tecnica di thin film hydration method rispetto ai liposomi prodotti con Nanoassemblr (33.50%). I liposomi ottenuti con entrambe le tecniche preparative, hanno garantito un rilascio completo di mioglobina dopo 4 ore a 37°C mentre a 4°C il rilascio completo è stato raggiunto dopo 5 ore. La cinetica di rilascio calcolata per entrambe le formulazioni segue il modello di Peppas con “n” compreso tra 0.45 e 0.89 tipico di un rilascio non-Fickiano. La vitalità cellulare con tutte le concentrazioni testate è stata sempre superiore al 70% indicando la buona biocompatibilità dei liposomi prodotti. I liposomi fluorescenti hanno evidenziato l’inizio di un uptake cellulare già dopo le 2 ore di incubazione sia a 4°C che 37°C. Dai risultati ottenuti si può concludere che i liposomi sono stati prodotti con successo con entrambe le tecniche di produzione testate. I liposomi hanno mostrato una buona biocompatibilità. Le proprietà funzionali dei liposomi sono influenzate dalle tecniche di produzione, in quanto i liposomi preparati con la tecnica del thin film hydration method mostrano una maggiore efficienza di incapsulazione rispetto ai liposomi preparati con Nanoassembler.
“Sviluppo e caratterizzazione in vitro di liposomi come sistemi di rilascio di proteine per applicazioni nella perfusione del rene espiantato”
CERIANA, FRANCESCA
2020/2021
Abstract
This thesis project involves the comparison of two liposome preparation techniques in order to obtain stable and reproducible liposomes able to exploit their action as drug carriers for the perfusion of transplanted kidney. On this purpose, the liposomes have been loaded with myoglobin as model molecule for future loading of a growth factor. In vitro myoglobin release studies from liposome were conducted considering temperatures and timing of transplanted kidney perfusion (4 hours, 4 °C). More in details, two preparation technologies were compared, a traditional thin film hydration method followed by extrusion and an innovative one using the microfluidic technique (Nanoassemblr). The liposomes were formulated using different molar lipid concentration ratios of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol. The liposomes obtained with both preparation techniques were dimensionally characterized by analysis with DLS, NTA and SEM. In addition to the dimensional characterization, SEM was also used to conduct a morphological analysis on liposomes. The selected liposomes were subsequently frozen and lyophilized in order to improve their conservation over time and to evaluate their process yield. Several sugars have been investigated as possible cryoprotectants in order to maintain stable size and PDI after rehydration of lyophilized liposomes. The liposomes were loaded with myoglobin as a model protein. The encapsulation efficiency, in vitro release rate of myoglobin from liposomes at 4 ° C and 37 ° C were evaluated and the release kinetic fitting model was determined. Different concentrations of liposomes were used to conduct cell viability assays on human fibroblasts. In addition, fluorescent liposomes have been formulated to study cellular uptake on human fibroblasts and renal tubular cells through the use of fluorescence microscopy and confocal microscopy. Both manufacturing methods allowed to obtain DSPC: Chol 50:50 liposomes with dimensions below 300 nm. The process yields were higher than 80% for both manufacturing techniques. The encapsulation efficiency for myoglobin was found to be higher (71.20%) for liposomes prepared with thin film hydration method compared to liposomes prepared with microfluidic technique (33.50%). All liposomes tested guaranteed complete release of myoglobin after 4 hours at 37 ° C while at 4 ° C the complete release was reached after 5 hours. The release kinetics calculated for both formulations follows Peppas equation model with “n” between 0.45 and 0.89 typical of a non-Fickian release. Cell viability, with all tested concentrations, was always greater than 70% indicating good biocompatibility of the liposomes produced. The fluorescent liposomes showed the onset of cellular uptake already after 2 hours of incubation at both 4 ° C and 37 ° C. From the results obtained it can be concluded that liposomes were successfully obtained by both the manufacturing techniques tested. The liposomes showed good biocompatibility. Functional properties of the liposomes are affected by the manufacturing techniques in such a way the liposomes prepared by thin film hydration method resulted in higher encapsulation efficiency as copared to liposomes produced by Nanoassemblr.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/14472