The experimental thesis project is based on the preparation of liposomes based on 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (Chol), having a net surface charge given by the addition of 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP) as cationic lipid, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) as anionic lipid and 1,2-dipalimitoyl-sn-glycero-3-phosphoethanolamine (PE) as zwitterionic lipid. Liposome batches DSPC: Chol: lipid-charged (47.5 : 47.5 :5.0) placebo and encapsulated with bovine serum albumin (BSA) as a model protein were prepared. Encapsulation was performed by two methods: direct microfluidic method and indirect method with Freeze-thawing (FT) cycling. To increase the loading of the protein (BSA 10 mg/mL) inside the liposomes, trehalose (20%w/w) was added in the aqueous phase. Liposome batches were characterized for their size and surface charge by Dynamic Light Scattering (DLS). The process yield in terms of particles/mL was evaluated by Nanoparticle tracking analysis (NTA). A transmission electron microscope (TEM) morphological characterization of placebo cationic DOTAP and anionic DOPS liposomes, and a scanning electron microscope (SEM) of previously freeze-dried placebo DOTAP liposomes was also performed. Different concentrations (particles/mL) of cationic, anionic and zwitterionic liposomes were employed to conduct the MTT assay as a cell viability assay on Normal Human Dermal Fibroblasts (NHDF). Finally, with the incorporation of N-(fluorescein-5-thiocarbamoyl)-1,2-diesadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (DHPE), fluorescent liposomes were created using the microfluidic technique. In particular were created DSPC:Chol:DOTAP:DHPE (47 : 47: 5: 1), DSPC:Chol:PE:DHPE (47 : 47: 5: 1) and DSPC:Chol:DOPS:DHPE (47 : 47: 5: 1) liposomes. The fluorescent batches were used to study, by fluorescence microscope, the cellular uptake on NHDF cells at 4°C and 37°C after 1h, 2h and 4h. As regards the characterization of the placebo and BSA encapsulating batches, all the batches were found to have dimensions of less than 200 nm and with concentrations of the order of 10^11 particles/mL. The best EE% (5.9 ± 0.8%) was obtained with DOTAP liposomes encapsulated with BSA with the indirect Freeze-thawing (FT) cycling method. With the MTT assay, good cell viability was observed with all concentrations tested. The concentration of 10^9 particles/mL was subsequently used to perform cellular uptake studies with fluorescent liposomes. From these studies, the best result was obtained with DSPC:Chol:DOTAP:DHPE liposomes placed in contact with the cells at 37°C for 4h.
Il progetto di tesi sperimentale si basa sulla preparazione di liposomi a base di 2-distearoil-sn-glicero-3-fosfocolina (DSPC) e colesterolo (Chol), aventi una carica superficiale netta data dall’aggiunta nella composizione di 1,2-dioleoil-3-trimetilammonio-propano (DOTAP) come lipide cationico, 1,2-dioleoil-sn-glicero-3-fosfo-L-serina (DOPS) come lipide anionico e 1,2-dipalimitoil-sn-glicero-3-fosfoetanolammina (PE) come lipide zwitterionico. Si sono preparati lotti DSPC: Chol: lipide carico (47.5: 47.5: 5.0) placebo e incapsulati con Albumina di siero bovino (BSA) come proteina modello. L’incapsulazione è stata effettuata mediante due metodi: metodo diretto microfluidico e metodo indiretto con Freeze-thawing (FT) cycling. Per incrementare il caricamento della proteina (BSA 10mg/mL) all’interno dei liposomi, è stato aggiunto trealosio (20%w/w) nella fase acquosa. I lotti di liposomi sono stati caratterizzati per le loro dimensioni e carica superficiale tramite Dynamic Light Scattering (DLS). La resa di processo in termini di particelle/mL è stata determinata tramite Nanoparticle tracking analysis (NTA). È stata condotta anche una caratterizzazione morfologica con microscopio elettronico a trasmissione (TEM) su liposomi cationici (DOTAP) e liposomi anionici (DOPS) placebo, e con microscopio elettronico a scansione (SEM) su liposomi DOTAP placebo precedentemente liofilizzati. Differenti concentrazioni (particles/mL) di liposomi cationici, anionici e zwitterionici sono state impiegate per condurre il test MTT come saggio di vitalità cellulare su fibroblasti umani dermici. Infine, tramite l’incorporazione di N-(fluoresceina-5-tiocarbamoil)-1,2-diesadecanoil-sn-glicero-3-fosfoetanolammina, sale di trietilammonio (DHPE), si sono creati con tecnica microfluidica liposomi fluorescenti DSPC:Chol:DOTAP:DHPE (47 : 47: 5: 1), DSPC:Chol:PE:DHPE (47 : 47: 5: 1) e DSPC:Chol: DOPS:DHPE (47 : 47: 5: 1) . Sui lotti fluorescenti è stato condotto uno studio di uptake cellulare su fibroblasti umani dermici a 4°C e 37°C per 1h, 2h e 4h; le analisi sono state condotte con microscopia a fluorescenza. Per quanto riguarda la caratterizzazione dei lotti placebo e incapsulanti BSA, tutti i lotti sono risultati avere dimensioni inferiori ai 200 nm e con concentrazioni dell’ordine 10^11 particle/mL. L’EE% migliore (5,9 ± 0,8%) si è ottenuta con i liposomi DOTAP incapsulanti BSA con metodo indiretto Freeze-thawing (FT) cycling. Con il test MTT si è osservata una buona vitalità cellulare con tutte le concentrazioni testate. La concentrazione 10^9 particle/mL è stata impiegata successivamente per eseguire gli studi di uptake cellulari con liposomi fluorescenti. Da questi studi, il miglior risultato si è ottenuto con i liposomi DSPC:Chol:DOTAP:DHPE messi a contatto con le cellule a 37°C per 4h.
Valutazione comparativa di formulazioni liposomiali
IEMMA, LISA LUCREZIA
2021/2022
Abstract
The experimental thesis project is based on the preparation of liposomes based on 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (Chol), having a net surface charge given by the addition of 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP) as cationic lipid, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) as anionic lipid and 1,2-dipalimitoyl-sn-glycero-3-phosphoethanolamine (PE) as zwitterionic lipid. Liposome batches DSPC: Chol: lipid-charged (47.5 : 47.5 :5.0) placebo and encapsulated with bovine serum albumin (BSA) as a model protein were prepared. Encapsulation was performed by two methods: direct microfluidic method and indirect method with Freeze-thawing (FT) cycling. To increase the loading of the protein (BSA 10 mg/mL) inside the liposomes, trehalose (20%w/w) was added in the aqueous phase. Liposome batches were characterized for their size and surface charge by Dynamic Light Scattering (DLS). The process yield in terms of particles/mL was evaluated by Nanoparticle tracking analysis (NTA). A transmission electron microscope (TEM) morphological characterization of placebo cationic DOTAP and anionic DOPS liposomes, and a scanning electron microscope (SEM) of previously freeze-dried placebo DOTAP liposomes was also performed. Different concentrations (particles/mL) of cationic, anionic and zwitterionic liposomes were employed to conduct the MTT assay as a cell viability assay on Normal Human Dermal Fibroblasts (NHDF). Finally, with the incorporation of N-(fluorescein-5-thiocarbamoyl)-1,2-diesadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (DHPE), fluorescent liposomes were created using the microfluidic technique. In particular were created DSPC:Chol:DOTAP:DHPE (47 : 47: 5: 1), DSPC:Chol:PE:DHPE (47 : 47: 5: 1) and DSPC:Chol:DOPS:DHPE (47 : 47: 5: 1) liposomes. The fluorescent batches were used to study, by fluorescence microscope, the cellular uptake on NHDF cells at 4°C and 37°C after 1h, 2h and 4h. As regards the characterization of the placebo and BSA encapsulating batches, all the batches were found to have dimensions of less than 200 nm and with concentrations of the order of 10^11 particles/mL. The best EE% (5.9 ± 0.8%) was obtained with DOTAP liposomes encapsulated with BSA with the indirect Freeze-thawing (FT) cycling method. With the MTT assay, good cell viability was observed with all concentrations tested. The concentration of 10^9 particles/mL was subsequently used to perform cellular uptake studies with fluorescent liposomes. From these studies, the best result was obtained with DSPC:Chol:DOTAP:DHPE liposomes placed in contact with the cells at 37°C for 4h.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/20.500.14239/15686