Design of nanoplatforms for ocular delivery of SIRT1 agonists in diabetic retinopathy [Progettazione di nanopiattaforme per la somministrazione oculare di agonisti di SIRT1 nella retinopatia diabetica]

Degenerative eye diseases such as diabetic retinopathy (DR) pose significant therapeutic challenges due to the involvement of oxidative stress and inflammation. Sirtuin 1 (SIRT1), a histone deacetylase protein, acting as crucial regulator of cellular stress responses, plays a key role in protecting ocular tissues such as optic nerve and retina. Downregulation of SIRT1 becomes a key factor of DR condition leading to a degeneration in posterior segment of the eye in terms of vascular and neuronal degeneration. Literature studies show that polyphenolic compounds such as resveratrol (RSV), curcumin (CUR), and melatonin (MEL) act as agonists or activators of the SIRT1 pathway, offering potential therapeutic strategies for degenerative eye diseases by enhancing antioxidant and anti-inflammatory effects. However, the therapeutic application of these compounds is hindered by low bioavailability, poor solubility in water, limited permeability through ocular barriers, and rapid degradation in physiological conditions. The objective of this thesis was to overcome these limitations by encapsulating SIRT1-activating molecules in novel nanotechnological systems compatible with ocular administration. Several systems were explored, including nanostructured lipid carriers (NLC), self-nanoemulsifying drug delivery systems (SNEDDS) and hydrogel scaffold incorporating liposomes, to improve stability, drug delivery efficiency, and patient compliance. These nanocarriers were designed to target the posterior eye segment non-invasively, with the overarching goal of enhancing therapeutic outcomes for ocular degenerative diseases as DR. Thus, the thesis focused on the design of different platforms following two types of approach: formulation intended for topical ocular delivery and biodegradable implant with the aim to deliver SIRT1 agonist into minimal invasive formulation. All the systems intended for topical delivery were designed following the QbD procedure. Specifically, the Design of Experiment (DoE) software was used to optimize lipid systems to obtain formulation with suitable parameters intended for topical ocular administration. 1.Nanostructured lipid carriers The first systems investigated were Nanostructured lipid carriers (NLC), a type of lipid nanoparticles constituted of a matrix composed of solid and liquid lipids for drug entrapping. The encapsulation of diosmin, a SIRT1 agonist with anti-inflammatory and antioxidant properties, in NLC allows to increase Diosmin apparent solubility in water and stability. Response surface methodology (RSM) was performed to optimize NLC formulation for Diosmin ocular delivery. The resulting NLC demonstrated optimal size (<100 nm), polydispersity index (PDI <0.3), and negative surface charge according to materials used that could be compatible with topical ocular delivery administration. Moreover, pH and osmolarity were close to physiological values. Diosmin-loaded NLC exhibited enhanced anti-inflammatory effects in retinal pigment epithelium (ARPE-19) cells exposed to TNF-α, indicating their potential for treating DR. NLC formulations can effectively enhance retinal drug delivery and improve patient compliance by providing a topical administration route as investigated from our research group in a subsequent work focused on development of Axitinib-loaded NLC for DR treatment. 2. Self-nanoemulsifying drug delivery systems The next formulation developed was based on Self-nanoemulsifying drug delivery systems (SNEDDS), a formulation typically used for oral delivery of lipophilic drugs but investigated in this thesis for ocular application. This is a novelty in the ocular field since very few works investigated this type of nanosystems. The first work about SNEDDS aimed to develop the systems, using two SIRT1 agonist as model molecules. Using RSM, SNEDDS were optimized to achieve narrow size (less than 100 nm), rapid emulsification, and high transmittance %, in order to be very compatible with administration, avoiding blurred vision and irritation. Two SIRT1 agonist as model molecules were loaded resulting in RSV-loaded SNEDDS and MEL-loaded SNEDDS suitable for ocular delivery in terms of drug encapsulation efficiency, no drug precipitation after reconstitution and stability during storage and under ocular conditions. The systems resulted non cytotoxic in corneal cells. After that, subsequent work was focused on delivery of CUR according to optimized formulation of previous work. CUR was entrapped very successfully in the SNEDDS, with little globule size and spherical shape. The systems increased the dissolution and release of the drug in simulated ocular conditions. In vitro studies showed that SNEDDS formulations enhanced the cellular uptake of CUR in retinal cells, probably leading to greater activation of the SIRT1 protein (intracellular localized) and improved antioxidant response. The encapsulated CUR also demonstrated enhanced stability against light, heat, and pH variations compared to free CUR, which degraded rapidly under similar conditions. These findings indicate that SNEDDS formulations can significantly improve the bioavailability and therapeutic potential of poorly soluble SIRT1 agonists in ocular tissues. Results not already published showed that SNEDDS are biocompatible in a HET-CAM analysis both blank and loaded with RSV. Moreover, the goal of achieving retinal target after topical administration was reached. Indeed, RSV-SNEDDS reached retina after 2 hours of topical administration in rats, in a 3-times higher than the free RSV. 3. Biodegradable Hydrogel Implants via 3D Printing Another approach explored in this thesis was the development of biodegradable hydrogel implants for intravitreal administration. This work was conducted in foreign lab during the period abroad in Queen’s University of Belfast (UK). Using 3D printing and microfluidic technologies, a hyaluronic acid/chitosan -based scaffold system was optimized to deliver both CUR and RSV in the same system. The system utilized a polyelectrolyte complex formed of chitosan and hyaluronic acid, with the aim of good biocompatibility and adhesion within the vitreous humor. Coaxial printing enabled the separation of the two molecules, allowing for distinct release profiles and extended drug delivery times. CUR-liposomes were prepared by microfluidics. The formulation resulted stable and suitable for improving CUR retinal delivery in terms of size, pegylation and charge. Liposomes were incorporated in a hydroxyethyl cellulose hydrogel and then printed by coaxial 3D printing as core filament. RSV was freely incorporated in hyaluronic acid/chitosan hydrogel and printed as external matrix. Thanks to coaxial nozzle was possible to deliver two drugs in the same scaffold and release the drug in a different time with rapid and sustained release. This implantable system, loaded with liposomal CUR, was designed to degrade naturally over time, eliminating the need for surgical removal. The hydrogel scaffold exhibited excellent compatibility with retinal cells, retaining its memory shape once implanted and providing a customizable platform for personalized ocular treatments. The works presented in this thesis successfully addressed the challenges of using polyphenolic SIRT1 agonists, such as RSV, CUR, and diosmin, for treating degenerative eye diseases as DR. By encapsulating these molecules in NLC, SNEDDS, liposomes, and biodegradable hydrogel systems, their stability, bioavailability, and ocular delivery will significantly be enhanced. The development of topical formulations, particularly through NLC and SNEDDS, provides a non-invasive alternative to intravitreal injections, improving patient compliance and reducing the risk of complications. Biodegradable hydrogel scaffold could be an alternative less invasive than common non-biodegradable implants on the market. The findings presented in this thesis underscore the potential of nanotechnology in ocular pharmacotherapy, offering new avenues for treating degenerative eye diseases through innovative drug delivery systems that target the posterior eye segment.

Le malattie degenerative dell'occhio, come la retinopatia diabetica (DR), rappresentano una significativa sfida terapeutica a causa del coinvolgimento dello stress ossidativo e dell'infiammazione. La sirtuina 1 (SIRT1), una proteina istone deacetilasi, agisce come un regolatore cruciale delle risposte cellulari allo stress e svolge un ruolo fondamentale nella protezione dei tessuti oculari, come il nervo ottico e la retina. La ridotta espressione di SIRT1 rappresenta un fattore chiave nella DR, portando a una degenerazione del segmento posteriore dell'occhio sia a livello vascolare che neuronale. Studi presenti in letteratura dimostrano che composti polifenolici, come il resveratrolo (RSV), la curcumina (CUR) e la melatonina (MEL), agiscono come agonisti o attivatori della via di SIRT1, offrendo potenziali strategie terapeutiche per le malattie oculari degenerative grazie ai loro effetti antiossidanti e antinfiammatori. Tuttavia, l'applicazione terapeutica di questi composti è ostacolata dalla bassa biodisponibilità, dalla scarsa solubilità in acqua, dalla limitata permeabilità attraverso le barriere oculari e dalla rapida degradazione in condizioni fisiologiche. L'obiettivo di questa tesi è il superamento di tali limitazioni attraverso l'incapsulamento di molecole attivanti SIRT1 in nuovi sistemi nanotecnologici compatibili con la somministrazione oculare. Sono stati esplorati diversi sistemi, tra cui nanostrutture lipidiche (NLC), sistemi auto-emulsionanti (SNEDDS) e impianti idrogel contenenti liposomi, per migliorare la stabilità, l'efficienza nella somministrazione del farmaco e la compliance del paziente. Questi nanocarrier sono stati progettati per il targeting del segmento posteriore dell'occhio in modo non invasivo, con l'obiettivo generale di migliorare gli esiti terapeutici per le malattie degenerative oculari come la DR. La tesi si è dunque focalizzata sulla progettazione di diverse piattaforme seguendo due approcci principali: formulazioni destinate alla somministrazione oculare topica e impianti biodegradabili con lo scopo di veicolare gli agonisti di SIRT1 attraverso formulazioni minimamente invasive. Tutti i sistemi per la somministrazione topica sono stati progettati seguendo la metodologia Quality by Design (QbD). In particolare, è stato utilizzato il software Design of Experiment (DoE) per ottimizzare i sistemi lipidici al fine di ottenere formulazioni con parametri adeguati alla somministrazione oculare. I primi sistemi indagati sono stati i Nanostructured Lipid Carriers (NLC), un tipo di nanoparticelle lipidiche costituite da una matrice di lipidi solidi e liquidi per l'intrappolamento del farmaco. L'incapsulamento di diosmina, un agonista di SIRT1 con proprietà antinfiammatorie e antiossidanti, negli NLC ha permesso di aumentarne la solubilità apparente in acqua e la stabilità. Tramite la metodologia della superficie di risposta (RSM), è stata ottimizzata la formulazione di NLC per la somministrazione oculare di diosmina. La formulazione NLC ha mostrato dimensioni ottimali (<100 nm), indice di polidispersione (PDI <0,3) e carica superficiale negativa, compatibili con la somministrazione oculare. Inoltre, pH e osmolarità erano vicini ai valori fisiologici. NLC caricati con diosmina hanno mostrato migliorati effetti antinfiammatori in cellule dell'epitelio pigmentato retinico (ARPE-19) esposte a TNF-α, evidenziando il loro potenziale per il trattamento della DR. Il successivo sistema sviluppato si basa sui Self-Nanoemulsifying Drug Delivery Systems (SNEDDS), una formulazione comunemente usata per la somministrazione orale di farmaci lipofili, ma investigata in questa tesi per applicazioni oculari. Questo approccio rappresenta una novità nel campo oftalmico. Gli SNEDDS sono stati ottimizzati utilizzando la RSM per ottenere dimensioni ridotte (<100 nm), rapida emulsificazione e alta trasmittanza, migliorando così la compatibilità con l'amministrazione oculare ed evitando visione offuscata o irritazione. Sono stati veicolati RSV e MEL, con risultati promettenti in termini di efficienza di incapsulamento e stabilità. Studi successivi hanno permesso di formulare CUR in SNEDDS, aumentando la dissoluzione del farmaco e la stabilità contro luce, calore e pH. Un ulteriore approccio esplorato è stato lo sviluppo di impianti biodegradabili in idrogel per la somministrazione intravitreale. Questo lavoro è stato svolto durante un periodo di ricerca all’estero presso la Queen’s University di Belfast (Regno Unito). Utilizzando tecnologie di stampa 3D e la microfluidica, è stato ottimizzato un sistema a base di acido ialuronico/chitosano per il rilascio di CUR e RSV. La stampa coassiale ha permesso il rilascio controllato e prolungato dei farmaci nello stesso sistema. L'impianto, progettato per degradarsi naturalmente, ha mostrato eccellente compatibilità con le cellule retiniche. I risultati presentati dimostrano che l'incapsulamento di agonisti polifenolici di SIRT1, come RSV, CUR e diosmina, in NLC, SNEDDS, liposomi e sistemi biodegradabili migliora significativamente la loro stabilità, biodisponibilità e somministrazione oculare. La possibilità di somministrare farmaci per via topica, in particolare attraverso NLC e SNEDDS, offre un'alternativa non invasiva alle iniezioni intravitreali, migliorando l'aderenza del paziente e riducendo i rischi di complicazioni. Gli impianti biodegradabili in idrogel rappresentano un’alternativa meno invasiva rispetto agli impianti non biodegradabili attualmente disponibili sul mercato. Questi risultati sottolineano il potenziale della nanotecnologia nella farmacoterapia oftalmica, aprendo nuove prospettive per il trattamento delle malattie oculari degenerative mediante sistemi innovativi di rilascio di farmaci mirati al segmento posteriore dell'occhio.