Nanomedicine is defined as the application of nanotechnology to medicine for developing new nanomaterials and nanosystems able to overcome the limits of the current medicine in diagnostics and treatment of human diseases. In particular, throughout these last decades, the attention has been posed on theranostic nanomedicine, which links the fields of diagnostic and therapy, with the development of systems suitable for both drug delivery and imaging. Liposomes are one of the most versatile and promising carriers to transport drugs and contrast agents (CAs), thanks to their structure that allows them to encapsulate both hydrophilic and hydrophobic molecules and to their surface that can be easily functionalized. The goal of this experimental thesis was the development of a protocol for the preparation of bimodal theranostic liposomes suitable for 19F-Magnetic Resonance Imaging (19F-MRI) and fluorescence microscopy analyses. In 2014 the laboratory of Nanostructured Fluorinated Materials (NFMLab) developed PERFECTA [tetra(perfluorotertbutyl)pentaerythritol], a new superfluorinated molecular probe possessing excellent properties for 19F-MRI. Gold nanoclusters (AuNCs) functionalized with PERFECTA have been loaded into liposomes via the film hydration method as already reported in the literature for the loading of gold nanoparticles (AuNPs) and magnetic nanoparticles in unilamellar liposomes. Despite the promising results (loading efficiency, polydispersity and stability), the relaxations times of nanoclusters (NCs)-loaded liposomes were not suitable for 19F-MRI. Therefore, the developed formulation, although successful in the loading of fluorinated gold nanoclusters (F-AuNCs) into stable liposomes, did not retain the same relaxivities of the fluorinated probes (NCs) thus not offering the possibility of using it as 19F-MRI CAs. Therefore, another approach to obtain bimodal theranostic fluorinated liposomes was attempted by loading PERFECTA directly into the lipid bilayer of the liposomes without linking it to gold NCs. PERFECTA-loaded liposomes were first prepared through heating-sonication cycles, but this method led to highly polydisperse liposomes. Thus, PERFECTA-loaded liposomes were prepared through the method developed for the encapsulation of fluorinated nanoclusters into liposomes, leading to promising loading efficiency, polydispersity and stability. Relaxation times of PERFECTA-loaded liposomes still have to be investigated in order to assess their suitability for Magnetic Resonance Imaging (MRI) applications and, in case of satisfying results, they should be functionalized with fluorescent lipids and loaded with drugs to obtain bimodal theranostic liposomes. Qualitative and quantitative characterization of liposomes has been performed by Dynamic Light Scattering (DLS), Inductively Coupled Plasma emission (ICP-AES), UltraViolet-Visible (UV-Vis) spectroscopy, Fourier-Transform InfraRed (FT-IR) spectroscopy, Nuclear Magnetic Resonance (NMR), and Transmission Electron Microscopy (TEM).
La nanomedicina è definita come l’applicazione della nanotecnologia alla medicina, con lo scopo di sviluppare nuovi nanomateriali e nanosistemi in grado di superare i limiti della medicina odierna nella diagnostica e nella terapia delle più diffuse patologie umane. In particolare, negli ultimi anni l’attenzione si è concentrata sulla nanomedicina teranostica, che unisce diagnostica e terapia sviluppando sistemi che possono essere utilizzati sia per il drug delivery che per l’imaging. In quest’ottica, i liposomi sono la soluzione più versatile e promettente per il trasporto di farmaci e agenti di contrasto, grazie alla loro struttura che permette loro di incapsulare molecole idrofile e idrofobe e alla loro superficie che può essere facilmente funzionalizzata. Lo scopo di questa tesi sperimentale è lo sviluppo di un protocollo per la preparazione di liposomi bimodali teranostici idonei per analisi 19F-MRI e microscopia a fluorescenza. Nel 2014, NFMLab ha sviluppato PERFECTA, un nuovo probe molecolare superfluorurato con eccellenti proprietà per applicazioni nel campo della 19F-MRI. Liposomi caricati con AuNCs funzionalizzati con PERFECTA sono stati preparati mediante il film hydration method integrato con cicli di freeze and thaw ed estrusione, come riportato in letteratura nel caso di liposomi caricati con AuNPs e nanoparticelle magnetiche. Nonostante i risultati promettenti in termini di stabilità, efficienza di carico e polidispersità, l’analisi dei tempi di rilassamento di questi liposomi ha messo in evidenza che questi non sono adatti per applicazioni nel campo dell’imaging. Quindi, nonostante il protocollo sviluppato sia valido per il caricamento di nanoparticelle (NPs) all’interno di liposomi stabili, ha portato a una modifica dei tempi di rilassamento dei probes fluorurati (NCs), rendendoli non idonei come CAs per 19F-MRI. Di conseguenza, si è cercato di preparare liposomi fluorurati bimodali teranostici caricando PEFECTA direttamente nel doppio strato lipidico dei liposomi senza legarla a AuNCs. Questi liposomi sono stati inizialmente preparati attraverso cicli di sonicazione-riscaldamento, come descritto nella sintesi dell’emulsione di lecitina e PERFECTA sviluppata dal NFMLab, ma questo metodo ha portato alla formazione di liposomi altamente polidispersi. Quindi, si è provato a sintetizzare liposomi caricati con PERFECTA attraverso il metodo precedentemente sviluppato per i liposomi caricati con i NCs, e i risultati ottenuti sono stati positivi in termini di stabilità, efficienza di carico e polidispersità. I tempi di rilassamento di questi liposomi andranno analizzati per verificare la loro idoneità per applicazioni nel campo dell’imaging e, in caso di risultati positivi, potranno essere funzionalizzati con lipidi fluorescenti e caricati con farmaci in modo da ottenere i liposomi teranostici inizialmente voluti. La caratterizzazione qualitativa e quantitativa effettuata nell’ambito di questa tesi è stata condotta tramite Dynamic Light Scattering (DLS), Inductively Coupled Plasma emission (ICP-AES), spettroscopia ultravioletta-visible (UV-Vis), spettroscopia infrarossa (FT-IR) e risonanza magnetica nucleare (NMR), microscopio elettrico a trasmissione (TEM).
Study and characterization of engineered liposomes for 19F-MRI applications
LIOSI, KORINNE
2015/2016
Abstract
Nanomedicine is defined as the application of nanotechnology to medicine for developing new nanomaterials and nanosystems able to overcome the limits of the current medicine in diagnostics and treatment of human diseases. In particular, throughout these last decades, the attention has been posed on theranostic nanomedicine, which links the fields of diagnostic and therapy, with the development of systems suitable for both drug delivery and imaging. Liposomes are one of the most versatile and promising carriers to transport drugs and contrast agents (CAs), thanks to their structure that allows them to encapsulate both hydrophilic and hydrophobic molecules and to their surface that can be easily functionalized. The goal of this experimental thesis was the development of a protocol for the preparation of bimodal theranostic liposomes suitable for 19F-Magnetic Resonance Imaging (19F-MRI) and fluorescence microscopy analyses. In 2014 the laboratory of Nanostructured Fluorinated Materials (NFMLab) developed PERFECTA [tetra(perfluorotertbutyl)pentaerythritol], a new superfluorinated molecular probe possessing excellent properties for 19F-MRI. Gold nanoclusters (AuNCs) functionalized with PERFECTA have been loaded into liposomes via the film hydration method as already reported in the literature for the loading of gold nanoparticles (AuNPs) and magnetic nanoparticles in unilamellar liposomes. Despite the promising results (loading efficiency, polydispersity and stability), the relaxations times of nanoclusters (NCs)-loaded liposomes were not suitable for 19F-MRI. Therefore, the developed formulation, although successful in the loading of fluorinated gold nanoclusters (F-AuNCs) into stable liposomes, did not retain the same relaxivities of the fluorinated probes (NCs) thus not offering the possibility of using it as 19F-MRI CAs. Therefore, another approach to obtain bimodal theranostic fluorinated liposomes was attempted by loading PERFECTA directly into the lipid bilayer of the liposomes without linking it to gold NCs. PERFECTA-loaded liposomes were first prepared through heating-sonication cycles, but this method led to highly polydisperse liposomes. Thus, PERFECTA-loaded liposomes were prepared through the method developed for the encapsulation of fluorinated nanoclusters into liposomes, leading to promising loading efficiency, polydispersity and stability. Relaxation times of PERFECTA-loaded liposomes still have to be investigated in order to assess their suitability for Magnetic Resonance Imaging (MRI) applications and, in case of satisfying results, they should be functionalized with fluorescent lipids and loaded with drugs to obtain bimodal theranostic liposomes. Qualitative and quantitative characterization of liposomes has been performed by Dynamic Light Scattering (DLS), Inductively Coupled Plasma emission (ICP-AES), UltraViolet-Visible (UV-Vis) spectroscopy, Fourier-Transform InfraRed (FT-IR) spectroscopy, Nuclear Magnetic Resonance (NMR), and Transmission Electron Microscopy (TEM).| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/121404