Neurodegenerative diseases, such as Alzheimer’s (AD) and Parkinson’s disease (PD), are chronic pathologies with a long clinical progression. They are characterised by the loss of specific neuron populations in selective areas of the brain, which leads to a gradual worsening of cognitive and motor functions. According to recent estimates, 50 million people and 6 million people worldwide are currently affected by AD and PD, respectively. However, the prevalence of these conditions and their social and economic burden is expected to increase by 2050, due to the increase in life expectancy. For this reason, the development of disease-modifying therapies is of utmost importance. To date, altering or blocking the progression of these pathologies is not possible, and the available treatments are only aimed at alleviating the symptoms. Moreover, the blood-brain barrier constitutes a physical obstacle to the diffusion of compounds from the bloodstream to the brain, further reducing the range of potential drugs which can be approved for human clinical use. Therefore, alternative ways to deliver molecules to the central nervous system have been explored. Intranasal (IN) delivery is one of the most promising non-invasive approaches, as it takes advantage of the high permeability of the nasal epithelium to increase the bioavailability of a drug at the brain level. The exact mechanisms are still not well understood. However, the trigeminal and olfactory nerves seem to play a key role, since they constitute a direct anatomic connection between the nose and the cerebral environments. This enables a rapid delivery of molecules to the brain, bypassing the blood-brain barrier. The defence mechanisms of the nasal cavity do not allow a prolonged availability of the drugs at the mucosal site. Therefore, a technological solution is the use of delivery systems which allow a controlled and sustained release of the bioactive molecules and delay the physiological clearance. In this regard, hydrogels seem ideal candidates for this application, due to their biocompatibility, their versatility, their tunable mechanic properties and the possibility of achieving gelation in situ. The present work fits into this context, and was aimed at developing an in vitro model of nasal epithelium and to study the nose-to-brain transport of a novel drug delivery system based on collagen derived hydrogels and a molecular chaperone protein to treat neurodegenerative diseases, with a special focus on AD and PD. The main objectives of the work have been: 1. The development and characterisation of a nasal epithelium model, based on RPMI 2650 cells, the only immortalised cell line of human origin. 2. The development of a controlled release delivery system. Natural or semi-synthetic hydrogel matrices were considered, in particular they were either made of bovine type I collagen (COLL) and low molecular weight hyaluronic acid or of COLL and poly(ethylene glycol) with two different molecular weights (2000 Da and 3350 Da). The molecular chaperone protein Heat shock protein 70 kDa (Hsp70), conjugated to the Trans-activator of transcription (Tat) peptide, was selected as the candidate drug because of its neuroprotective properties already demonstrated in the literature, also by our group. 3. The development of a model of controlled delivery across the nasal epithelium. To reproduce the in vivo conditions, a “3D-like” model was obtained by layering the epithelial cells with hydrogels. Different thickness values were considered for each matrix. 4. The development of a complete model of IN delivery, in order to simulate as best as possible the transport of drugs from the nasal cavity to the brain. For this reason, cell models recapitulating the key features of AD and PD should be integrated in the previously optimised model. Results showed that the RPMI 2650 cell line is an adequate model of the nasal barrier, due to its ability to develop tight junctions and to its bioelectric and paracellular permeability properties, which are similar to the physiological condition. In particular, the air-to-liquid interface culture has emerged as having a better performance with respect to the traditional liquid-covered culture setup. For what concerns the materials considered in the present study, we observed that the scaling-down of the hydrogel matrices, which is necessary in view of an application in the nasal cavity, influenced the COLL fibrillogenesis process. In particular, a decrease in the gel thickness led to a reduction of the COLL fibrils diameter. Tat-Hsp70 release studies demonstrated that the selected matrices allow a gradual and sustained protein release up to 168 hours, due to a combination of two processes, i.e. passive diffusion of the drug and polymer network degradation. Studies on the 3D-like model allowed to assess the cytocompatibility of the hydrogels. 1.5 mm thick matrices were selected as optimal for the application in the nasal cavity. Given the properties of the epithelial cell line, the release profile of the hydrogel matrices and the features of the 3D-like model, this system is particularly useful for applications, not exceeding 7-10 days. In conclusion, the 3D-like model developed in the present work represents an interesting tool to evaluate the transport of drugs across the epithelial barrier. Moreover, the tested hydrogel matrices can be considered ideal candidates for the controlled and sustained delivery of bioactive molecules.
Le malattie neurodegenerative, come la malattia di Alzheimer (AD) ed il morbo di Parkinson (PD), sono patologie croniche a decorso progressivo caratterizzate dalla perdita neuronale in specifiche aree del cervello e dal graduale deterioramento delle funzioni cognitive e motorie. Attualmente, si stima che i pazienti affetti da AD e PD a livello mondiale siano, rispettivamente, circa 50 e 6 milioni. Tuttavia, con l’aumento dell’aspettativa di vita, la prevalenza di queste condizioni e l’impatto economico e sociale ad esse legato sono destinati ad aumentare. Per questo, l’esigenza di sviluppare terapie che riescano a fermare il processo di degenerazione neuronale è sempre più forte. Ad oggi, non è possibile modificare definitivamente il decorso di malattie come AD e PD, infatti i trattamenti farmacologici disponibili sono solo volti ad alleviare i sintomi. La presenza della barriera ematoencefalica, inoltre, costituisce un ostacolo fisico alla diffusione di sostanze dal circolo ematico all’ambiente cerebrale, restringendo così il range di farmaci, anche promettenti, che potrebbero trovare un’applicazione clinica nell’uomo. Per questo, la ricerca scientifica si è focalizzata su meccanismi alternativi, possibilmente non invasivi, per veicolare molecole di interesse al sistema nervoso centrale. Tra questi approcci spicca la via di somministrazione intranasale (IN), che trae vantaggio dall’elevata permeabilità della mucosa nasale per aumentare la biodisponibilità dei farmaci a livello cerebrale. I meccanismi alla base di questo fenomeno non sono ancora del tutto chiari, tuttavia i nervi trigemino e olfattivo sembrano avere un ruolo chiave. Infatti, essi stabiliscono una connessione anatomica diretta tra i due distretti, permettendo il trasporto rapido di molecole dalla mucosa nasale al cervello ed aggirando la barriera ematoencefalica. I vari meccanismi di difesa della cavità nasale non permettono un contatto prolungato dei farmaci con la mucosa. Lo sviluppo di sistemi che rilascino molecole bioattive in modo controllato nel tempo e che rallentino i processi fisiologici di eliminazione costituisce quindi uno sviluppo tecnologico di interesse. In particolare, gli idrogeli sembrano essere i candidati ideali per questa applicazione, data la loro generale biocompatibilità, versatilità, la possibilità di modulare le loro caratteristiche meccaniche e di ottenere una gelazione in situ. Il presente lavoro di tesi si inserisce in questo contesto, e ha avuto come scopo lo sviluppo di un modello in vitro per lo studio della somministrazione IN di un nuovo sistema per il rilascio controllato di farmaci (drug delivery systems) basato su idrogeli contenenti collagene e una proteina chaperone per il trattamento delle malattie neurodegenerative, con una particolare attenzione per AD e PD. Gli obiettivi principali sono stati i seguenti: 1. Realizzare e caratterizzare un modello di epitelio nasale, ottenuto a partire dalla linea cellulare RPMI 2650, l’unica derivante da tessuto nasale umano. 2. Sviluppare un sistema a rilascio controllato e prolungato di molecole bioattive. In particolare, sono state prese in considerazione matrici idrogeliche naturali o semi-sintetiche, a base di collagene bovino di tipo I (COLL) e di acido ialuronico a basso peso molecolare e di COLL e polietilenglicole di due diversi pesi molecolari (2000 Da e 3350 Da). La proteina Heat shock protein 70 kDa (HSP70), coniugata al peptide Trans-activator of transcription (Tat), è stata invece selezionata come molecola bioattiva, date le sue proprietà neuroprotettive già dimostrate in letteratura anche dal nostro gruppo di lavoro. 3. Verificare la biocompatibilità del sistema a rilascio controllato con l’epitelio nasale. Al fine di simulare la condizione in vivo, è stato realizzato un modello denominato “simil-3D” ottenuto ricoprendo l’epitelio con uno strato di gel di spessore variabile. 4. Sviluppare un modello completo di somministrazione IN che simuli al meglio il trasporto di sostanze bioattive dalla mucosa nasale all’ambiente cerebrale. A tal fine, è stata prevista l’integrazione del modello simil-3D precedentemente ottimizzato con modelli cellulari che ricapitolino gli aspetti chiave di AD e PD. I risultati ottenuti hanno mostrato come la linea cellulare RPMI 2650 costituisca un adeguato modello di barriera epiteliale, in grado di formare tight junctions e con proprietà bioelettriche e una permeabilità paracellulare comparabili alla condizione in vivo. In particolare, è stato messo in luce come l’utilizzo di un modello air-to-liquid interface sia migliorativo rispetto al tradizionale metodo di coltura. Per quanto riguarda i materiali utilizzati nel presente studio, è emerso che la riduzione dello spessore delle matrici idrogeliche, necessaria dato il volume limitato della cavità nasale, influenza la fibrillogenesi del COLL. In particolare, si è visto come, al diminuire dello spessore della rete polimerica, si riduca anche il diametro delle fibrille di COLL. Gli studi sugli idrogeli caricati con Tat-Hsp70 hanno inoltre dimostrato come le tre matrici testate permettano un rilascio graduale e prolungato della proteina nel tempo fino a 168 ore, con un meccanismo duplice, dovuto sia alla diffusione della molecola attraverso la rete polimerica, sia alla degradazione di quest’ultima. Lo studio del modello simil-3D ha permesso invece di verificare la citocompatibilità di tutti gli idrogeli considerati, ed ha inoltre consentito la scelta delle matrici con spessore di 1,5 mm come sistemi ottimali per l’applicazione nella cavità nasale. Considerati le proprietà del modello cellulare impiegato, il profilo di rilascio degli idrogeli e le caratteristiche del modello simil-3D, questo sistema si dimostra particolarmente utile per applicazioni a breve termine, in particolare per periodi che non superino i 7-10 giorni. In conclusione, il modello simil-3D sviluppato nel presente lavoro di tesi rappresenta uno strumento interessante per valutare il trasporto di farmaci anche innovativi (come le proteine ricombinanti) attraverso l’epitelio nasale, e le matrici idrogeliche testate si sono dimostrate ottimi candidati per la somministrazione controllata e prolungata di molecole bioattive.
An in vitro model for hydrogel-based intranasal drug delivery targeting neurodegenerative disorders
Caddeo, Veronica
2019/2020
Abstract
Neurodegenerative diseases, such as Alzheimer’s (AD) and Parkinson’s disease (PD), are chronic pathologies with a long clinical progression. They are characterised by the loss of specific neuron populations in selective areas of the brain, which leads to a gradual worsening of cognitive and motor functions. According to recent estimates, 50 million people and 6 million people worldwide are currently affected by AD and PD, respectively. However, the prevalence of these conditions and their social and economic burden is expected to increase by 2050, due to the increase in life expectancy. For this reason, the development of disease-modifying therapies is of utmost importance. To date, altering or blocking the progression of these pathologies is not possible, and the available treatments are only aimed at alleviating the symptoms. Moreover, the blood-brain barrier constitutes a physical obstacle to the diffusion of compounds from the bloodstream to the brain, further reducing the range of potential drugs which can be approved for human clinical use. Therefore, alternative ways to deliver molecules to the central nervous system have been explored. Intranasal (IN) delivery is one of the most promising non-invasive approaches, as it takes advantage of the high permeability of the nasal epithelium to increase the bioavailability of a drug at the brain level. The exact mechanisms are still not well understood. However, the trigeminal and olfactory nerves seem to play a key role, since they constitute a direct anatomic connection between the nose and the cerebral environments. This enables a rapid delivery of molecules to the brain, bypassing the blood-brain barrier. The defence mechanisms of the nasal cavity do not allow a prolonged availability of the drugs at the mucosal site. Therefore, a technological solution is the use of delivery systems which allow a controlled and sustained release of the bioactive molecules and delay the physiological clearance. In this regard, hydrogels seem ideal candidates for this application, due to their biocompatibility, their versatility, their tunable mechanic properties and the possibility of achieving gelation in situ. The present work fits into this context, and was aimed at developing an in vitro model of nasal epithelium and to study the nose-to-brain transport of a novel drug delivery system based on collagen derived hydrogels and a molecular chaperone protein to treat neurodegenerative diseases, with a special focus on AD and PD. The main objectives of the work have been: 1. The development and characterisation of a nasal epithelium model, based on RPMI 2650 cells, the only immortalised cell line of human origin. 2. The development of a controlled release delivery system. Natural or semi-synthetic hydrogel matrices were considered, in particular they were either made of bovine type I collagen (COLL) and low molecular weight hyaluronic acid or of COLL and poly(ethylene glycol) with two different molecular weights (2000 Da and 3350 Da). The molecular chaperone protein Heat shock protein 70 kDa (Hsp70), conjugated to the Trans-activator of transcription (Tat) peptide, was selected as the candidate drug because of its neuroprotective properties already demonstrated in the literature, also by our group. 3. The development of a model of controlled delivery across the nasal epithelium. To reproduce the in vivo conditions, a “3D-like” model was obtained by layering the epithelial cells with hydrogels. Different thickness values were considered for each matrix. 4. The development of a complete model of IN delivery, in order to simulate as best as possible the transport of drugs from the nasal cavity to the brain. For this reason, cell models recapitulating the key features of AD and PD should be integrated in the previously optimised model. Results showed that the RPMI 2650 cell line is an adequate model of the nasal barrier, due to its ability to develop tight junctions and to its bioelectric and paracellular permeability properties, which are similar to the physiological condition. In particular, the air-to-liquid interface culture has emerged as having a better performance with respect to the traditional liquid-covered culture setup. For what concerns the materials considered in the present study, we observed that the scaling-down of the hydrogel matrices, which is necessary in view of an application in the nasal cavity, influenced the COLL fibrillogenesis process. In particular, a decrease in the gel thickness led to a reduction of the COLL fibrils diameter. Tat-Hsp70 release studies demonstrated that the selected matrices allow a gradual and sustained protein release up to 168 hours, due to a combination of two processes, i.e. passive diffusion of the drug and polymer network degradation. Studies on the 3D-like model allowed to assess the cytocompatibility of the hydrogels. 1.5 mm thick matrices were selected as optimal for the application in the nasal cavity. Given the properties of the epithelial cell line, the release profile of the hydrogel matrices and the features of the 3D-like model, this system is particularly useful for applications, not exceeding 7-10 days. In conclusion, the 3D-like model developed in the present work represents an interesting tool to evaluate the transport of drugs across the epithelial barrier. Moreover, the tested hydrogel matrices can be considered ideal candidates for the controlled and sustained delivery of bioactive molecules.File | Dimensione | Formato | |
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https://hdl.handle.net/10589/164616