ex Vivo Study of Liposomes Loaded With Everolimus in Chronic Lung Allograft Disfunction

Overview

Patients subjected to lung transplantation develop acute rejection or chronic rejection with an incidence of 45% after the first year. The major clinical phenotype of chronic rejection is bronchiolitis obliterans syndrome (BOS), which cause an establishment of chronic lung inflammation status with an aberrant proliferation of myofibroblasts leading to fibrotic obstruction of small airways. The therapeutic regimen available for BOS patients is still scarce and is not able to revert the disease. This project aims to design a new targeted therapy based on nanoparticles that can deliver drug directly inside lungs by aerosol administration to revert BOS. By this project will be exploit a liposomes preparation synthesized modifying surface with hyaluronic acid (HA), the physiologic ligand of CD44, a glycoprotein overexpressed by myofibroblasts forming fibrotic lesions. These targeted liposomes are loaded with everolimus (LIP(ev)-HA400kDa), a mTOR inhibitors already used for BOS patients but with significant side effects leading to a discontinuation of therapy. Loading everolimus inside liposomes allows the administration of drug directly to the lungs and decreases its side effects. LIP(ev)-HA400kDa will be tested on different experimental settings: in vitro, ex vivo, in vivo. This approach will allow us to have a complete observation regarding the effects and the distribution of liposomes preparation, from the modulation of their specific targeting (myofibroblasts) by in vitro experiments, the analysis of LIP(ev)-HA400kDa behavior on human lung tissues and, finally, their ability to revert BOS in animal model. Results obtained with this project will open to a new therapeutic option for BOS affected patients, the first therapy that can revert the disease prolonging the long-term survival of patients.

LFs will be isolated from BAL of BOS-affected or subjected to TX. After isolation and culture, LFs will be incubated with LIP(ev)-HA400kDa, LIP(ev) and everolimus alone, comparing the effect to control cells. At the end of the incubation, we will collect supernatants and proteins of lysed cells. Proteins extracted will be quantified and western blot will be performed to assess the expression of collagen I, fibronectin and laminin. Regarding supernatants we will evaluate cytokines concentration, in particular: IL6, IL8, TGF-beta and IL10. LIP-HA400kDa fluorescently labelled will be incubated onto PCLSs derived from BOS explanted lungs comparing their distribution with LIP-PEG, using confocal microscopy. Moreover, using LIP(ev)-HA400kDa, LIP(ev) and everolimus alone we will assess general toxicity on PCLSs using LDH assay and cell proliferation with Ki67 expression at different time points. We will also assess the release of cytokines involved in pro-inflammation and pro-fibrotic processes.