Recuperación funcional de la superficie ocular en el ojo seco experimentaldesarrollo de un plásmido de muc5ac

Résumé

OBJECTIVE The general objective of this thesis work was to develop and characterize new mucoadhesive NP systems made of materials capable of efficiently interacting with the ocular mucosa to deliver plasmids coding for MUC5AC. METHODOLOGY -The first step was the design of a plasmid capable of inducing the expression of MUC5AC, (pMUC5AC) in ocular surface epithelial cells. -Then, we performed the in vitro and in vivo evaluation of the potentially suitable nanoparticle systems for the administration of pMUC5AC plasmid. Specifically, hyaluronic acid and chitosan-based nanoparticles (HA-CS NPs) were evaluated. -In line with the nanoparticles studies, and taking into account that ocular inflammation was the cornerstone of the thesis project, we aimed to study the in vitro effect of inflammation in the corneal epithelial barrier, because the disruption of this barrier has been described in many inflammatory pathologies of the ocular surface. -The physicochemical characteristics of HA-CS NPs, along with the large size of the pMUC5AC plasmid made it really difficult to use the HA-CS NPs as a plasmid vehicle. For this reason, new cationized gelatin-based nanosystems were developed and tested in vitro.. -The last step of the projects was the in vivo study of cationized gelatin-based nanoparticles loaded with the pMUC5AC plasmid, evaluating the tolerance and the therapeutic effect in an animal model of ocular surface inflammatory disease. RESULTS -The newly designed plasmid pMUC5AC induced the expression of the modified MUC5AC protein in ocular surface epithelial cells in vitro and in vivo, without altering cell viability. -HA-CS NPs were internalized by corneal and conjunctival cell lines by an active transport mechanism mediated by HA-receptors through a caveolin-dependent endocytic pathway. Utilizing this pathway, plasmid-loaded HA-CS NPs achieved significant transfection efficiency in both cell lines without altering cell viability. -HA-CS NPS were internalized in vivo by corneal and conjunctival epithelial cells and had an excellent tolerance. -In vitro inflammatory conditions lead to an alteration of corneal barrier function by modulating tight junctions. To a lesser extent, the adherent junctions were also altered. Pro-inflammatory molecules TNF¿, TGFß, IL-13, IL-17, and IL-6 induced a structural and functional disruption of the epithelial barrier. In contrast, IL-10 enhanced barrier protective effects. -CG-based NPs induced the expression of the modified MUC5AC mRNA and protein in the ocular surface epithelial cells, both in vivo and in vitro. pMUC5AC-NPs were well tolerated and induced the expression of the modified MUC5AC in healthy murine conjunctiva. They also induced the expression of modified MUC5AC in EDE animals. Moreover, expression of the novel MUC5AC plasmid in EDE mice correlated with significant reduction of inflammation and, consequently, with remarkable improvement of the clinical parameters associated with this condition. These included improved integrity of the corneal epithelial barrier, increased tear production and goblet cell numbers, and reduced CD4+ T cell infiltrations in the conjunctiva. CONCLUSIONS -The plasmid designed and constructed to encode the MUC5AC protein induces the expression of that protein, both in vitro and in vivo, without altering cell viability. -GC-based nanoparticle systems loaded with pMUC5AC plasmid are able to restore MUC5AC levels in the ocular surface of mice with EDE. -The normalization of MUC5AC levels leads to reduction of the underlying inflammation, and, consequently, to improvement of the clinical parameters associated with this condition. -pMUC5AC-NPs induce an improvement in the integrity of the corneal epithelial barrier in murine EDE. -pMUC5AC-NPs are proposed as a new potential therapeutic modality for the treatment of dry eyes.