The final phase of cellular respiration, located in the mitochondrial membrane, is the process by which nutrients are broken down into simpler molecules, releasing energy in the form of ATP through proton transport. The enzymatic chain responsible for this process consists of five enzymatic complexes, among which complex III plays a fundamental role. The malfunction of this complex is responsible for the development of serious diseases, such as myopathies, encephalomyopathies, cerebellar ataxias, and various mental disorders, often with fatal outcomes for affected patients. Recent studies conducted by the research group led by Prof. Szabò have demonstrated that molecules with a phenazine structure, known as redox cyclers, such as pyocyanin produced by the bacterium Pseudomonas aeruginosa, can restore the proper functioning of the mitochondrial respiratory chain by acting as electron carriers, replacing complex III. Pyocyanin’s poor water solubility (<0.5 mg/mL) and small size lead to its rapid elimination from the body following administration, limiting its clinical development. The aim of this thesis project was therefore to design and evaluate different formulations of pyocyanin to efficiently deliver it without the use of organic solvents and to increase its half-life. The first part of the study focused on the formulation of an inclusion complex of pyocyanin in cyclodextrins to improve its water solubility. To this end, Hydroxypropyl-β-Cyclodextrins (HPB-CD) were used at increasing concentrations to determine the optimal concentration and the maximum solubilizing capacity, found to be 50 mg/mL. Subsequently, a hydrogel for subcutaneous administration based on hyaluronic acid and alginic acid was developed. The obtained gel was rheologically characterized and loaded with the inclusion complex of pyocyanin in HPB-CD. Release studies showed a rapid initial release within 48 hours, followed by a slower release up to 72 hours, as desired. However, preliminary in vivo studies conducted by collaborators showed a too-rapid release after administration. The second part of the thesis project therefore focused on the development of a particulate system to deliver pyocyanin, which was then inserted into the hydrogel in order to slow the release of the molecule and prolong its plasma half-life. Three nanoparticulate systems were evaluated: polylactic-co-glycolic acid (PLGA) particles, liposomes, and solid lipid nanoparticles (SLNs). Regarding the PLGA particles, several tests were performed by varying parameters such as the preparation technique, the organic solvent, and the polymer composition. In all cases, RP-HPLC analysis of the particles, after purification, showed the system's inability to retain the loaded pyocyanin. Similar results were obtained with the second carrier investigated, the liposomal one. The final encapsulation attempt was carried out by preparing SLNs using microfluidics, employing Milli-Q water as the aqueous phase and a solution of cholesterol, HSPC, and pyocyanin in acetone as the organic phase. In this case, after purification through dialysis, it was possible to detect, via spectrophotometric analysis, the presence of pyocyanin loaded into the lipid particles, which were obtained with a size of 106.1 nm and a PDI of 0.1538. Future studies will focus on optimizing the loading of pyocyanin into SLNs by varying the lipid composition and incorporating the particles into the hydrogel to then investigate their behavior in vitro through release studies, and eventually in vivo.
La fase finale della respirazione cellulare, con sede nella membrana mitocondriale, è il processo tramite il quale i nutrienti vengono demoliti in molecole più semplici, ottenendo energia sotto forma di ATP grazie al trasporto di protoni. La catena enzimatica responsabile di tale processo è formata da cinque complessi enzimatici, tra i quali il complesso III che svolge un ruolo fondamentale. Il malfunzionamento di tale complesso è responsabile dello sviluppo di gravi patologie quali miopatie, encefalomiopatie, atassie cerebellari e vari disturbi mentali, con esito infausto per i pazienti che ne sono affetti. Studi recenti condotti dal gruppo di ricerca della Prof.ssa Szabò hanno dimostrato come molecole con struttura fenazinica, chiamate ciclatori redox, come la piocianina prodotta dal batterio Pseudomonas aeruginosa, siano in grado di ripristinare il corretto funzionamento della catena respiratoria mitocondriale, agendo come trasportatori di elettroni, sostituendosi al complesso III. La scarsa solubilità in acqua (<0,5 mg/mL) e le ridotte dimensioni della piocianina causano una rapida eliminazione dall’organismo in seguito a somministrazione, ponendo un limite allo sviluppo clinico. Lo scopo del presente progetto di tesi è stato quindi quello di progettare e valutare diverse formulazioni di piocianina per veicolarla efficientemente senza l’utilizzo di solventi organici e per aumentarne il tempo di emivita. La prima parte dello studio si è concentrata sulla formulazione di un complesso di inclusione di piocianina in ciclodestrine per migliorarne la solubilità in acqua. A tal fine sono state utilizzate Hydroxypropyl-β-Ciclodestrine (HPB-CD) a concentrazioni crescenti, per determinare la concentrazione ottimale e la massima capacità solubilizzante, risultata essere di 50 mg/mL. Successivamente è stato sviluppato un idrogele per somministrazione sottocutanea a base di acido ialuronico e acido alginico. Il gel ottenuto è stato caratterizzato reologicamente e caricato con il complesso di inclusione di piocianina in HPB-CD. Studi di rilascio effettuati hanno evidenziato un rapido rilascio iniziale entro le 48 ore, seguito da un rilascio lento fino alle 72 ore, come desiderato. Tuttavia, studi preliminari in vivo effettuati dai collaboratori hanno mostrato un rilascio troppo rapido dopo la somministrazione. La seconda parte del progetto di tesi si è pertanto focalizzata sullo sviluppo di un sistema particellare per veicolare la piocianina, da inserire poi all’interno dell’idrogele, in modo da rallentare il rilascio della molecola e prolungarne l’emivita plasmatica. Sono stati valutati tre sistemi nanoparticellari: particelle di acido polilattico-co-glicolico (PLGA), liposomi e particelle lipidiche solide (SLN). Per quanto riguarda le particelle di PLGA, sono state effettuate diverse prove variando parametri quali la tecnica preparativa, il solvente organico e la composizione polimerica. In tutti i casi, le analisi RP-HPLC delle particelle, dopo purificazione, hanno mostrato l’incapacità del sistema di trattenere la piocianina caricata. Risultati simili sono stati ottenuti con il secondo carrier investigato, quello liposomiale. L’ultimo tentativo di incapsulamento è stato effettuato preparando SLN tramite microfluidica, utilizzando come fase acquosa acqua Milli-Q e come fase organica una soluzione di colesterolo, HSPC e piocianina in acetone. In questocaso, dopo purificazione mediante dialisi, è stato possibile rilevare, tramite analisi spettrofotometrica, la presenza di piocianina caricata nelle particelle, che sono state ottenute con una dimensione di 106,1 nm e un PDI di 0,1538. Studi futuri si concentreranno sull’ottimizzazione del caricamento di piocianina in SLN, variando la composizione lipidica, e sull’inclusione delle particelle nell’idrogele, per indagare poi il comportamento in vitro mediante studi di rilascio, ed eventualmente in vivo.
Formulazione di piocianina in idrogeli per il trattamento di patologie mitocondriali
ZANGANI, ENRICO
2023/2024
Abstract
The final phase of cellular respiration, located in the mitochondrial membrane, is the process by which nutrients are broken down into simpler molecules, releasing energy in the form of ATP through proton transport. The enzymatic chain responsible for this process consists of five enzymatic complexes, among which complex III plays a fundamental role. The malfunction of this complex is responsible for the development of serious diseases, such as myopathies, encephalomyopathies, cerebellar ataxias, and various mental disorders, often with fatal outcomes for affected patients. Recent studies conducted by the research group led by Prof. Szabò have demonstrated that molecules with a phenazine structure, known as redox cyclers, such as pyocyanin produced by the bacterium Pseudomonas aeruginosa, can restore the proper functioning of the mitochondrial respiratory chain by acting as electron carriers, replacing complex III. Pyocyanin’s poor water solubility (<0.5 mg/mL) and small size lead to its rapid elimination from the body following administration, limiting its clinical development. The aim of this thesis project was therefore to design and evaluate different formulations of pyocyanin to efficiently deliver it without the use of organic solvents and to increase its half-life. The first part of the study focused on the formulation of an inclusion complex of pyocyanin in cyclodextrins to improve its water solubility. To this end, Hydroxypropyl-β-Cyclodextrins (HPB-CD) were used at increasing concentrations to determine the optimal concentration and the maximum solubilizing capacity, found to be 50 mg/mL. Subsequently, a hydrogel for subcutaneous administration based on hyaluronic acid and alginic acid was developed. The obtained gel was rheologically characterized and loaded with the inclusion complex of pyocyanin in HPB-CD. Release studies showed a rapid initial release within 48 hours, followed by a slower release up to 72 hours, as desired. However, preliminary in vivo studies conducted by collaborators showed a too-rapid release after administration. The second part of the thesis project therefore focused on the development of a particulate system to deliver pyocyanin, which was then inserted into the hydrogel in order to slow the release of the molecule and prolong its plasma half-life. Three nanoparticulate systems were evaluated: polylactic-co-glycolic acid (PLGA) particles, liposomes, and solid lipid nanoparticles (SLNs). Regarding the PLGA particles, several tests were performed by varying parameters such as the preparation technique, the organic solvent, and the polymer composition. In all cases, RP-HPLC analysis of the particles, after purification, showed the system's inability to retain the loaded pyocyanin. Similar results were obtained with the second carrier investigated, the liposomal one. The final encapsulation attempt was carried out by preparing SLNs using microfluidics, employing Milli-Q water as the aqueous phase and a solution of cholesterol, HSPC, and pyocyanin in acetone as the organic phase. In this case, after purification through dialysis, it was possible to detect, via spectrophotometric analysis, the presence of pyocyanin loaded into the lipid particles, which were obtained with a size of 106.1 nm and a PDI of 0.1538. Future studies will focus on optimizing the loading of pyocyanin into SLNs by varying the lipid composition and incorporating the particles into the hydrogel to then investigate their behavior in vitro through release studies, and eventually in vivo.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/80643