Liposomes Loaded with Everolimus and Coated with Hyaluronic Acid: A Promising Approach for Lung Fibrosis.

Laura Pandolfi,Sara Bozzini,Elias Fattal,Monica Morosini,Vanessa Frangipane,Nicolas Tsapis,Valentina Vertui,Alessandro Marengo,Kamila Bohne Japiassu,Sara Lettieri,Silvia Arpicco,Davide Piloni,Valeria Bincoletto,Veronica Codullo,Federica Meloni

doi:10.3390/ijms22147743

Abstract

Chronic lung allograft dysfunction (CLAD) and interstitial lung disease associated with collagen tissue diseases (CTD-ILD) are two end-stage lung disorders in which different chronic triggers induce activation of myo-/fibroblasts (LFs). Everolimus, an mTOR inhibitor, can be adopted as a potential strategy for CLAD and CTD-ILD, however it exerts important side effects. This study aims to exploit nanomedicine to reduce everolimus side effects encapsulating it inside liposomes targeted against LFs, expressing a high rate of CD44. PEGylated liposomes were modified with high molecular weight hyaluronic acid and loaded with everolimus (PEG-LIP(ev)-HA400kDa). Liposomes were tested by in vitro experiments using LFs derived from broncholveolar lavage (BAL) of patients affected by CLAD and CTD-ILD, and on alveolar macrophages (AM) and lymphocytes isolated, respectively, from BAL and peripheral blood. PEG-LIP-HA400kDa demonstrated to be specific for LFs, but not for CD44-negative cells, and after loading everolimus, PEG-LIP(ev)-HA400kDa were able to arrest cell cycle arrest and to decrease phospho-mTOR level. PEG-LIP(ev)-HA400kDa showed anti-inflammatory effect on immune cells. This study opens the possibility to use everolimus in lung fibrotic diseases, demonstrating that our lipids-based vehicles can vehicle everolimus inside cells exerting the same drug molecular effect, not only in LFs, but also in immune cells.

Highlights

Lung transplantation is the most difficult challenge among solid organ transplantations
After assessing that poly(ethylene glycol) (PEG)-LIP-HA400kDa are efficiently internalized by CD44-positive cells, we evaluated the effect of everolimus loaded liposomes on LFs assessing cell proliferation using CFSE dye since the primary effect of everolimus is blocking cell proliferation
To understand in which phase of cell cycle LFs were accumulated after drug treatment, we analyzed the cell cycle of chronic lung allograft dysfunction (CLAD) and CTD-ILD LFs. By this assay we did not find any difference between PEG-LIP(ev), PEG-LIP(ev)-HA400kDa, and everolimus alone, After assessing that PEG-LIP-HA400kDa are efficiently internalized by CD44-positive cells, we evaluated the effect of everolimus loaded liposomes on LFs assessing cell proliferation using CFSE dye since the primary effect of everolimus is blocking cell proliferation

Summary

Introduction

Lung transplantation is the most difficult challenge among solid organ transplantations. The long-term outcome of lung transplantation is mainly limited by the occurrence of chronic lung allograft dysfunction (CLAD) which represents a chronic fibrotic reaction of the graft involving the small airways or the interstitial sub-pleural spaces caused by chronic allospecific and aspecific inflammatory injuries [1,2,3,4]. Another clinical setting in which repeated chronic auto-immune inflammatory injuries cause a diffuse fibrotic reaction of lung tissue, is the interstitial lung disease associated with collagen tissue diseases (CTD-ILD). These end-stage lung disorders share the pathogenic process in which different chronic auto- or allo-specific triggers (acute cellular rejection episodes, insults induced by tissue-specific antibodies, aspecific inflammation due to bacterial or viral agents, gastroesophageal reflux, or pollutants) induce migration, differentiation, activation, and proliferation of myo-/fibroblasts (LFs) in lung tissue

Objectives

Methods

Results