The aim of my thesis project is to combine electroporation with nanotecnology as an innovative treatment for cancers treatment. Electroporation is a well-established technique for the treatment of various tumors, as it involves the application of electric pulses at specific voltages to create transient pores in the cell membrane, facilitating the passage of numerous molecules such as drugs (Bleomycin), enzymes and peptides. The innovation lies in the use of this therapeutic approach in conjunction with nanotechnology, where liposomes are created to deliver the chemotherapy agent of interest with the final goal to selectively target cancer cells with the anti-cancer drug. As proof of concept, was studied what the best formulation of liposomes would be, in terms of chemical composition with the selection of anionic or cationic lipids (DSPC: Chol: DOPS or DSPC: chol: DOTAP), considering the molar ratio between them (47.5:47.5:5 or 67.5:27.5:5). Subsequently, the size of liposomes developed by NanoAssemblr, an instrument based on the microfluidics tecnhnique, was examined using Dynamic Light Scattering (DLS) and then were evaluated the differences between liposomes that were subjected to electroporation in comparison with those were not. The results obtained have demonstrated that there are no significant differences between liposomes treated and untreated through electroporation. Liposomes with a molar ratio of 67.5:27.5:5, on average, showed larger sizes compared to those with a ratio of 47.5:47.5:5 (Tables 3, 4, and 5), but even the latter remained in the order of a few hundred nanometers. Liposomes were loaded with an antibiotic belonging to the class of aminoglycosides, Gentamicin sulfate, and analyzed their encapsulation efficiency (EE%) using ninhydrin colorimetric assay. The highest values of EE% were found in liposomes with a lipid composition of 67.5:27.5:5, showing a significant difference compared to liposomes of 47.5:47.5:5. Specifically, for liposomes with a composition of 67.5:27.5:5, the encapsulation efficiency was calculated as 29.20% ± 3.11 (cationic liposomes) and 53.06% ± 8.53 (anionic liposomes), while for those with a composition of 47.5:47.5:5, it was 5.65% ± 3.28 (cationic liposomes) and 2.43% ± 3.88 (anionic liposomes), as reported in Table 6. Were also studied drug release by employing a dialysis membrane and placing them in an oven at 37°C, comparing these conditions with liposomes that underwent electroporation. Under static conditions, DSPC:Chol:DOTAP and DSPC:Chol:DOPS liposomes showed 100% release of GS within 24 hours and 72 hours, respectively. A release of 42.20% was observed after only 30 minutes (batch #12) and 40.03% after 2 hours (batch #10), as visible in Figure 17. Releases of 45.78% (batch #12) and 33.38% (batch #10) were achieved immediately, however, subjecting the same batches of liposomes to electroporation, specifically at 200 V (Figure 19).
L'obiettivo del mio progetto di tesi è quello di combinare l'elettroporazione con la nanotecnologia come trattamento innovativo per la cura del cancro. L'elettroporazione è una tecnica ben consolidata per il trattamento di vari tipi di tumori cutanei e delle mucose, che coinvolge l'applicazione di impulsi elettrici a voltaggi specifici per creare pori transitori nella membrana cellulare, agevolando il passaggio di numerose molecole come farmaci (es: Bleomicina), enzimi e peptidi. L’innovazione qui proposta prevede l’uso di questo approccio terapeutico in concomitanza con la nanotecnologia, tramite la quale vengono creati liposomi per veicolare il chemioterapico di interesse, con l'obiettivo finale di colpire selettivamente le cellule tumorali con il farmaco antitumorale. Come proof of concept, è stato studiato quale fosse la migliore formulazione dei liposomi, in termini di composizione chimica attraverso la scelta di lipidi anionici o cationici (DSPC: Chol: DOPS o DSPC: Chol: DOTAP), considerando il rapporto molare tra di essi (47.5:47.5:5 o 67.5:27.5:5). Successivamente, è stata esaminata la dimensione dei liposomi sviluppati con il NanoAssemblr, uno strumento basato sulla tecnica della microfluidica, utilizzando il Dynamic Light Scattering (DLS), per poi analizzare le differenze tra i liposomi sottoposti al trattamento di elettroporazione rispetto a quelli che non sono stati trattati. I risultati ottenuti hanno dimostrato che non vi sono differenze significative tra i liposomi trattati e non trattati tramite elettroporazione; i liposomi con rapporto molare 67.5:27.5:5 hanno mostrato mediamente dimensioni maggiori rispetto a quelli 47.5:47.5:5 (Tabelle 3,4 e 5), ma anche questi ultimi sono rimasti nell’ordine di qualche centinaia di nanometri. Sono stati creati liposomi contenenti come farmaco modello un antibiotico appartenente alla classe degli aminoglicosidi, la gentamicina solfato, di cui ne è stata analizzata l'efficienza di incapsulazione (EE%) e il drug content (DC%) utilizzando il saggio colorimetrico della ninidrina. I valori più elevati di EE% sono stati riscontrati nei liposomi con composizione lipidica 67.5:27.5:5, mostrando una differenza significativa rispetto ai liposomi 47.5:47.5:5; per i liposomi 67.5:27.5:5, infatti, si è calcolata un’efficienza di incapsulazione del 29.20% ± 3.11 (liposomi cationici) e del 53.06% ± 8.53 (liposomi anionici), mentre per quelli 47.5:47.5:5 di 5.65% ± 3.28 (liposomi cationici) e di 2.43% ± 3.88 (liposomi anionici), come riportato in Tabella 6. Inoltre, è stato valutato il rilascio del farmaco utilizzando una membrana da dialisi e ponendo i campioni in stufa a 37°C, confrontando queste condizioni con i liposomi che sono stati sottoposti ad elettroporazione. In condizioni statiche, i liposomi DSPC:Chol:DOTAP e DSPC:Chol:DOPS hanno mostrato il 100% del rilascio di GS nell’arco delle 24 e delle 72 ore, rispettivamente. È stato osservato un rilascio del 42.20% dopo soli 30 min (batch #12) e del 40.03% dopo 2 ore (batch #10), come visibile nella Figura 17. Rilasci del 45.78% (batch #12) e del 33.38% (batch #10) si sono ottenuti nell’immediato, tuttavia, sottoponendo gli stessi lotti di liposomi ad elettroporazione e, nello specifico, a 200 V (Figura 19).
Studio formulativo di liposomi contenenti gentamicina e destinati a elettroporazione
BATTAGLIA, MARIAGRAZIA
2022/2023
Abstract
The aim of my thesis project is to combine electroporation with nanotecnology as an innovative treatment for cancers treatment. Electroporation is a well-established technique for the treatment of various tumors, as it involves the application of electric pulses at specific voltages to create transient pores in the cell membrane, facilitating the passage of numerous molecules such as drugs (Bleomycin), enzymes and peptides. The innovation lies in the use of this therapeutic approach in conjunction with nanotechnology, where liposomes are created to deliver the chemotherapy agent of interest with the final goal to selectively target cancer cells with the anti-cancer drug. As proof of concept, was studied what the best formulation of liposomes would be, in terms of chemical composition with the selection of anionic or cationic lipids (DSPC: Chol: DOPS or DSPC: chol: DOTAP), considering the molar ratio between them (47.5:47.5:5 or 67.5:27.5:5). Subsequently, the size of liposomes developed by NanoAssemblr, an instrument based on the microfluidics tecnhnique, was examined using Dynamic Light Scattering (DLS) and then were evaluated the differences between liposomes that were subjected to electroporation in comparison with those were not. The results obtained have demonstrated that there are no significant differences between liposomes treated and untreated through electroporation. Liposomes with a molar ratio of 67.5:27.5:5, on average, showed larger sizes compared to those with a ratio of 47.5:47.5:5 (Tables 3, 4, and 5), but even the latter remained in the order of a few hundred nanometers. Liposomes were loaded with an antibiotic belonging to the class of aminoglycosides, Gentamicin sulfate, and analyzed their encapsulation efficiency (EE%) using ninhydrin colorimetric assay. The highest values of EE% were found in liposomes with a lipid composition of 67.5:27.5:5, showing a significant difference compared to liposomes of 47.5:47.5:5. Specifically, for liposomes with a composition of 67.5:27.5:5, the encapsulation efficiency was calculated as 29.20% ± 3.11 (cationic liposomes) and 53.06% ± 8.53 (anionic liposomes), while for those with a composition of 47.5:47.5:5, it was 5.65% ± 3.28 (cationic liposomes) and 2.43% ± 3.88 (anionic liposomes), as reported in Table 6. Were also studied drug release by employing a dialysis membrane and placing them in an oven at 37°C, comparing these conditions with liposomes that underwent electroporation. Under static conditions, DSPC:Chol:DOTAP and DSPC:Chol:DOPS liposomes showed 100% release of GS within 24 hours and 72 hours, respectively. A release of 42.20% was observed after only 30 minutes (batch #12) and 40.03% after 2 hours (batch #10), as visible in Figure 17. Releases of 45.78% (batch #12) and 33.38% (batch #10) were achieved immediately, however, subjecting the same batches of liposomes to electroporation, specifically at 200 V (Figure 19).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/20.500.14239/16898