Development of a topical ocular drug delivery system using polymeric nanoparticles

Common eye diseases such as dry eye syndrome affect 15% of the population. Although eye drops are the most common treatment for these diseases, over 95% of the drugs applied through eye drops are quickly cleared away due to blinking and tear turnover. Consequently, patients struggle with the multiple daily applications required and the resulting side effects. Nanoparticle (NP) drug carriers have gained significant traction recently because of several advantages they provide over conventional eye drop delivery methods. NP surfaces can be tuned to achieve specific properties such as binding affinity towards the ocular surface. NPs can also carry a large amount of drugs and release them in a sustained manner over a long period. Due to their small size, NPs do not cause abrasive sensations on the eye upon patient application. With these unique advantages, NP drug carriers may drastically improve patient compliance while reducing side effects. The thesis focuses on developing an ocular drug delivery platform using NPs to improve retention of ocular therapeutics on the precorneal surface. We developed a method to synthesize an amphiphilic block copolymer composed of poly(D,L-lactide) (PLA) and dextran (Dex) that can self-assemble into NP drug carriers. The size of the NPs can be tuned between 15 and 70 nm by adjusting the molecular weights of PLA and/or Dex. The PLA-b-Dex NPs form the foundation of the ocular drug delivery platform developed in this thesis. A targeted delivery system is crucial for ocular drug delivery due to the rapid clearance by tear drainage on the corneal surface. The PLA-b-Dex NPs were surface modified with phenylboronic acid (PBA) molecules, which can undergo covalent binding with the mucous membrane to circumvent the rapid clearance. Due to the abundance of functional groups on the dextran, we were able to tune the density of PBA on the NP surface to optimize the binding affinity between the NPs and the mucin. While maximizing the PBA density on the NP surface improved the covalent interaction between the NPs and the mucin, it also compromised the NP colloidal stability. The PBA modified NPs demonstrated encapsulation of Cyclosporine A (CsA), a dry eye treatment drug, and sustained release for up to 5 days in vitro, showing their potential as a long-term eye drop delivery platform. We then performed biocompatibility and efficacy studies on these NPs using animal models. Biocompatibility is of the utmost importance in developing new drug delivery formulations. During the 12 weeks of study, no physical signs of irritation or discomfort were detected nor was any inflammatory response or ocular tissue damage observed in the eyes administered with NPs. Topical administration of CsA-loaded NPs on dry eye induced mice using once a week dosing demonstrated complete elimination of the inflammatory response as well as full recovery of the integrity of the ocular tissues. In the same study, the commercial eye drop form of CsA, Restasis®, administered thrice daily only eliminated the inflammatory infiltrates without recovering ocular goblet cells. By delivering CsA through PLA-b-Dex-g-PBA NPs, we can significantly reduce the dose and the frequency of eye drop administration without compromising the treatment efficacy. In vitro mucoadhesion as a result of PBA’s on NP surfaces was demonstrated. We proceeded to further demonstrate this mucoadhesion using in vivo models. Indocyanine green (ICG), a near-infrared fluorescent dye, was encapsulated in the NPs and administered to rabbit eyes to track its ocular retention. ICG delivered via PBA modified NPs showed ocular retention beyond 24 hours on rabbit eyes, whereas free ICG or ICG delivered via unmodified NPs were mostly cleared within the first 3 hours. When the weekly dosing of CsA loaded PLA-b-Dex-g-PBA NPs was repeated for 4 weeks on dry eye induced mice, we observed the same elimination of inflammatory infiltrates but also the damaged ocular tissue structures. When the concentration of the CsA in the weekly dosing of NPs was further reduced 5 times, the treatment effect was much more pronounced, showing both the elimination of the inflammation and the full recovery of the ocular surface tissues. Overall, by using mucoadhesive nanoparticle drug carriers, we reduced the dosage of CsA at least 50-fold compared with the commercial product, Restasis®, without compromising the dry eye treatment efficacy. Finally, we developed a scalable method to synthesize PLA-b-Dex-g-PBA block copolymers using a semi-solid state reaction chemistry. The previous method of conjugating PBA to the Dex required long reaction hours with multiple reaction and purification steps. In contrast, the new method combines the quickness of a semi-solid state reaction with the simplicity of a Williamson ether chemistry to graft PBA to Dex. The results showed that the new method achieved a similar range of tunability of PBA density onto Dex using reaction times as short as 10 minutes. This thesis demonstrates the development process of a polymeric NP as a topical ocular drug delivery system. The PLA-b-Dex-g-PBA NPs demonstrated delivery of a clinically relevant dosage of dry eye therapeutics, controlled release of therapeutics over prolonged period of time, and mucoadhesive properties resulting in prolonged ocular surface retention of drugs. These mucoadhesive NPs show remarkable promise as a long-term topical ocular drug delivery system that significantly reduces the dose and the administration frequency of the eye drops while minimizing side effects

Ex-vivo and In-vivo Characterization of Human Accommodation

A completely satisfying approach to restoring accommodation still needs to be developed. Besides, there are considerable discrepancies between objective and subjective trials to evaluate the therapeutic success. A substantial biomechanical understanding of all structures and processes involved in accommodation as well as presbyopia are needed to develop promising new strategies. This contribution focuses on developing advanced imaging techniques to create a basic understanding of accommodation and presbyopia and to evaluate existing concepts for restoring accommodation. Besides, the emphasis is also on replacing stiff presbyopic lenses by a material that imitates the young crystalline lens

rational design of functionalized lipids with antioxidant and scavenging activity as components of innovative artificial tears

openDurante i tre anni di dottorato mi sono occupato del design razionale di lipidi funzionalizzati ad attività antiossidante da utilizzare per la formulazione di lacrime artificiali. Abbiamo scelto i liposomi per veicolare le molecole antiossidanti testate; siamo partiti utilizzando l’Edaravone (EDR), il quale è stato opportunamente funzionalizzato (EDR-C18), senza diminuire le proprietà antiossidanti della molecola. Abbiamo condotto studi sperimentali evidenziando che l’EDR-C18 mantiene elevate capacità antiossidanti, solo a questo punto abbiamo condotto simulazioni di dinamica molecolare utilizzando un sistemi lipidici puri contenti fosfatidilcolina (POPC), e differenti concentrazioni di EDR-C18. I dati di simulazione hanno evidenziato una elevata stabilità dell’EDR-C18 in membrana. Abbiamo quindi determinato la concentrazione ottimale al fine di ottenere una intatta fluidità del sistema e allo stesso tempo una elevata quantità di antiossidante. Partendo da questo modello di simulazione, abbiamo creato altri sistemi di simulazione in cui abbiam investigato l’effetto dei Sali. Abbiamo testato soluzioni saline già comunemente utilizzate nella formulazione di lacrime artificiali, ed abbiamo riscontrato nel CaCl2 il sale maggiormente utile per la nostra strategia. Tali sistemi sono estremamente promettenti per la formulazione di gocce oculari. Un’altra molecola testata è l’epigallocatechin3-Gallato (EGCG). Essa ha la capacità di interagire spontaneamente con sistemi lipidici, perciò non è stata funzionalizzata. Abbiamo condotto delle simulazioni di dinamica molecolare creando sistemi lipidici misti per indagare l’effetto della matrice sulla capacità di inglobamento dell’EGCG. Abbiamo inoltre modulato la concentrazione salina ed abbiamo individuato nel sistema lipidico anionico con una quantità di Magnesio pari a 5:1 in rapporto molare con EGCG, il sistema in cui tutto l’EGCG introdotto viene inglobato, aumentando la biodisponibilità della molecola al massimo possibile.During three years of my PhD course, I studied the rational design of functionalized lipid with antioxidant activity to be used for the formulation of artificial tears. We chosen the liposomes to carrier the antioxidant molecules that we studied; we started using the edaravone (EDR), which has been suitably functionalized (EDR-C18), without decrease the antioxidant properties of the molecule. For this purpose, we made experimental studies showing that the EDR-C18 maintains high antioxidant capacity, only at this point we carried out the molecular dynamics (MD) simulations using pure lipid systems containing POPC and different EDR-C18 concentrations. The simulation data showed a high EDR-C18 stability in the membrane. We determined the optimal concentration to obtain an intact fluidity of the system and at the same time a high amount of antioxidant. On the basis on this simulation model, we created other MD systems in which we have investigated the effects of salts. We tested salt solutions already commonly used in the formulation of artificial tears, and we found that CaCl2 is better salt to use for our strategy. Such systems are extremely promising for the formulation of eye drops. Another antioxidant that we studied is the epigallocatechin3-gallate (EGCG). It has the ability to interact spontaneously with lipid system, for this reason, it was not functionalized. We carried out MD simulations creating mixed lipid systems to investigate the effects of the matrix on the EGCG encapsulation. In addition, we modulated on salt concentration and we found that the best model is the anionic lipid system with an amount of magnesium equal to 5:1 in molar ratio with EGCG the system in which all the EGCG introduced is encapsulated, maximizing the bioavailability of the antioxidant compound, that is able to reach the lipid medium of lacrimal tear and to retain in it.SCIENZE DELLA VITA E DELL'AMBIENTEembargoed_20181001Laudadio, EmilianoLaudadio, Emilian