Lung disorders are becoming a heavy burden for global health both in terms of mortality, morbidity and for the burden for caregivers and increased patient management costs. Among lung pathologies, pulmonary fibroses play a main role on their total incidence. It is interesting to underline that a broad range of clinical disorder, such as tumour, toxicant agent exposure, cardiac impairment, autoimmune diseases, infections, share a common downstream process associated with progressive fibrosis. This common pattern is extremely important in the field of pharmacology because a strategy aimed at delivering anti-inflammatory and/or anti-fibrotic drugs in lungs could strongly reduce the clinical worsening occurring in many different disorders. The clinical progression of fibroses induces parenchymal injury, inflammation and collagen accumulation. Fibrogenesis is a dynamic process characterized by extracellular matrix production, activation of myofibroblasts, and epithelial mesenchymal transition (EMT) induced by inflammatory cytokines. During EMT, the protective layer facilitating oxygen and carbon dioxide exchange gradually diminishes, resulting in respiratory impairment that often leads to patient mortality. Diminishing the signalling associated with cytokine release emerges as one of the primary approaches to impede or at least slow pulmonary dysfunction. One potential target in this regard is modulating the functional expression of activated macrophages, which play a pivotal role in fibrosis by acting as tissue sentinels and producing numerous pro-fibrotic factors. Unfortunately, nearly all therapies commonly employed in clinical practice, especially anti-inflammatory and immune-modulatory agents, have failed in clinical trials due to their low specificity and side effects associated with the spread of these drugs to off-target organs. To overcome this impasse, in my thesis project, I first developed and fully characterized a reliable model of bleomycin-induced acute fibrosis in mice. I systematically compared different fibrogenesis protocols, selecting the one that most closely resembled human pathology in terms of clinical and pathological features. I also examined macrophage responses during the progressive stages of fibrosis. After establishing a reliable model replicating the human disorder, I compared the effects of steroid treatment when administered freely or incorporated into biodegradable and biocompatible nanocarriers. I also evaluated their kinetics depending on the route of administration (systemic or via inhalation). Parameters such as lung tropism, distribution in other organs, penetration into macrophages, and drug release were carefully investigated. The results demonstrated that the use of nanocarriers via intranasal administration was the optimal approach to reach the target. Finally, I applied this approach to test an innovative method for delivering angiotensin-converting enzyme 2 (ACE2) as a decoy system for preventing SARS-CoV-2 infection. Before conducting in vivo studies, we established the in vitro ability of the nanodecoy to reduce viral infectivity by 80-90% within minutes. In vivo studies confirmed the safety and affinity of these decoys for lung parenchyma and macrophages. Overall, this work represent a starting point for the treatment of lung disorders and pave the way for the next steps: 1) the chronic treatment with nanoassemblies functionalized with corticosteroids for the evaluation of therapeutic efficacy and amelioration of the clinical symptoms in the mouse model and 2) the and determination of in vivo efficacy in the prevention of SARS-CoV-2 infection in infected mice. The advancement of nanotechnology-based strategies will provide innovative solutions in the management of major lung diseases that affect millions of people worldwide every year.

I disturbi polmonari rappresentano un carico gravoso per la salute globale, sia in termini di mortalità che di morbilità, nonché per l'incremento dei costi di gestione dei pazienti e l'impatto sull'assistenza. Tra le patologie polmonari, le fibrosi giocano un ruolo predominante nella loro incidenza complessiva. È interessante sottolineare che una vasta gamma di disturbi clinici, come tumori, l'esposizione ad agenti tossici, patologie cardiache, malattie autoimmuni e infezioni, condividono un processo comune associato a una fibrosi progressiva. Tale caratteristica è importante nel campo della farmacologia, poiché una strategia mirata alla somministrazione di farmaci antinfiammatori e/o anti-fibrotici nei polmoni potrebbe ridurre in modo significativo il peggioramento clinico in molteplici patologie. La progressione delle fibrosi induce lesioni del parenchima e infiammazione. La fibrogenesi è un processo dinamico caratterizzato dall'attivazione dei miofibroblasti, dalla produzione di matrice extracellulare e dalla transizione epiteliale mesenchimale (EMT) indotta da citochine pro-infiammatorie. Durante l'EMT, lo strato protettivo che facilita lo scambio di ossigeno e anidride carbonica diminuisce gradualmente, causando un'insufficienza respiratoria e quindi la morte del paziente. La riduzione del rilascio di citochine emerge come possibile approccio per impedire o almeno rallentare la disfunzione polmonare. Un obiettivo potenziale è la regolazione della funzionalità dei macrofagi attivati, i quali rivestono un ruolo fondamentale nella fibrosi agendo come sentinelle e producendo fattori profibrotici. Sfortunatamente, molte delle terapie utilizzate, in particolare gli agenti antinfiammatori e immunomodulanti, hanno fallito negli studi clinici a causa della loro scarsa specificità e degli effetti collaterali legati alla diffusione di tali farmaci in altri organi. Per superare questo ostacolo, nel mio progetto di tesi ho sviluppato e caratterizzato un modello murino di fibrosi acuta indotta da bleomicina. Ho comparato diversi protocolli di fibrogenesi, selezionando quello più vicino alla patologia umana in termini di caratteristiche cliniche e patologiche. Ho inoltre esaminato le risposte dei macrofagi durante le fasi progressive della fibrosi. Dopo aver stabilito un modello affidabile, ho confrontato gli effetti del trattamento con steroidi somministrati liberamente o legati a nanocarrier biodegradabili e biocompatibili. Ho valutato la loro cinetica a seconda della via di somministrazione (sistemica o tramite inalazione). Sono stati studiati parametri come il tropismo polmonare, la distribuzione in altri organi, la penetrazione nei macrofagi e il rilascio del farmaco. I risultati ottenuti hanno dimostrato che l'uso di nanocarrier tramite somministrazione intranasale è l'approccio ottimale per raggiungere il bersaglio. Infine, ho applicato tale approccio per testare un metodo innovativo per l’utilizzo dell'enzima di conversione dell'angiotensina 2 (ACE2) come sistema “esca” per la prevenzione dell'infezione da SARS-CoV-2. È stata inizialmente valutata in vitro l'abilità del sistema di ridurre l'infettività virale dell'80-90% entro pochi minuti. Gli studi in vivo hanno confermato la sicurezza e l'affinità di questi nanocomposti per il parenchima polmonare e i macrofagi. Nel complesso, questo lavoro rappresenta un punto di partenza promettente per il trattamento dei disturbi polmonari e apre la strada per le fasi successive: 1) il trattamento cronico con nanoassemblati funzionalizzati con corticosteroidi per valutare l'efficacia terapeutica e il miglioramento dei sintomi clinici nel modello murino, e 2) la determinazione dell'efficacia in vivo nella prevenzione dell'infezione da SARS-CoV-2 in topi infetti. L'avanzamento delle strategie basate sulla nanotecnologia permette di fornire soluzioni innovative per la gestione delle malattie polmonari che colpiscono milioni di persone ogni anno.

(2024). Characterization of the nanocarrier-mediated targeting in a murine model of lung inflammation and fibrosis. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2024).

Characterization of the nanocarrier-mediated targeting in a murine model of lung inflammation and fibrosis

MORELLI, ANNALISA
2024

Abstract

Lung disorders are becoming a heavy burden for global health both in terms of mortality, morbidity and for the burden for caregivers and increased patient management costs. Among lung pathologies, pulmonary fibroses play a main role on their total incidence. It is interesting to underline that a broad range of clinical disorder, such as tumour, toxicant agent exposure, cardiac impairment, autoimmune diseases, infections, share a common downstream process associated with progressive fibrosis. This common pattern is extremely important in the field of pharmacology because a strategy aimed at delivering anti-inflammatory and/or anti-fibrotic drugs in lungs could strongly reduce the clinical worsening occurring in many different disorders. The clinical progression of fibroses induces parenchymal injury, inflammation and collagen accumulation. Fibrogenesis is a dynamic process characterized by extracellular matrix production, activation of myofibroblasts, and epithelial mesenchymal transition (EMT) induced by inflammatory cytokines. During EMT, the protective layer facilitating oxygen and carbon dioxide exchange gradually diminishes, resulting in respiratory impairment that often leads to patient mortality. Diminishing the signalling associated with cytokine release emerges as one of the primary approaches to impede or at least slow pulmonary dysfunction. One potential target in this regard is modulating the functional expression of activated macrophages, which play a pivotal role in fibrosis by acting as tissue sentinels and producing numerous pro-fibrotic factors. Unfortunately, nearly all therapies commonly employed in clinical practice, especially anti-inflammatory and immune-modulatory agents, have failed in clinical trials due to their low specificity and side effects associated with the spread of these drugs to off-target organs. To overcome this impasse, in my thesis project, I first developed and fully characterized a reliable model of bleomycin-induced acute fibrosis in mice. I systematically compared different fibrogenesis protocols, selecting the one that most closely resembled human pathology in terms of clinical and pathological features. I also examined macrophage responses during the progressive stages of fibrosis. After establishing a reliable model replicating the human disorder, I compared the effects of steroid treatment when administered freely or incorporated into biodegradable and biocompatible nanocarriers. I also evaluated their kinetics depending on the route of administration (systemic or via inhalation). Parameters such as lung tropism, distribution in other organs, penetration into macrophages, and drug release were carefully investigated. The results demonstrated that the use of nanocarriers via intranasal administration was the optimal approach to reach the target. Finally, I applied this approach to test an innovative method for delivering angiotensin-converting enzyme 2 (ACE2) as a decoy system for preventing SARS-CoV-2 infection. Before conducting in vivo studies, we established the in vitro ability of the nanodecoy to reduce viral infectivity by 80-90% within minutes. In vivo studies confirmed the safety and affinity of these decoys for lung parenchyma and macrophages. Overall, this work represent a starting point for the treatment of lung disorders and pave the way for the next steps: 1) the chronic treatment with nanoassemblies functionalized with corticosteroids for the evaluation of therapeutic efficacy and amelioration of the clinical symptoms in the mouse model and 2) the and determination of in vivo efficacy in the prevention of SARS-CoV-2 infection in infected mice. The advancement of nanotechnology-based strategies will provide innovative solutions in the management of major lung diseases that affect millions of people worldwide every year.
BIGINI, PAOLO
fibrosi polmonare; infiammazione; corticosteroidi; nanomedicina; modello murino
pulmonary fibrosis; inflammation; corticosteroids; nanomedicine; murine model
BIO/15 - BIOLOGIA FARMACEUTICA
English
7-feb-2024
36
2022/2023
embargoed_20250807
(2024). Characterization of the nanocarrier-mediated targeting in a murine model of lung inflammation and fibrosis. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2024).
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Descrizione: Characterization of the nanocarrier-mediated targeting in a murine model of lung inflammation and fibrosis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/460578
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