Formulazione di liposomi ad alta stabilità: caratterizzazione, funzionalizzazione con coniugati dell’acido ialuronico ed effetti biologici in vitro

This project focused the attention on the development of high stability liposomes as drug carriers. Many studies demonstrated that liposomes are one of the best systems to vehiculate drugs at the interest site for a large number of desease, increasing the specific interaction with the target cells using lower amount of dosage, avoiding unwanted drug side effect. Following this way, we investigated the formulation for the lipid film (i.e. the reagents and the proportion among them). We also studied the differences between two solution buffers for the hydration phase of the lipid film: the solfate and citrate ones. The drug was encapsulated with the pH gradient method and the liposomes were characterized under the dimension profile, polydispersity index and Z potential. To assess the stability of these micelles, the release vs. time tests were done at 37°C, 4°C and in serum. We tested furthermore the differences among blank liposomes and liposomes decorated with a conjugate of hyaluronan acid with different molecular weight (14800Da and 44700Da), in order to evaluate the specific interaction HA-CD44 receptor. About this, we tested the two formulation on cell lines CD44high (A549), CD44mid (Calu-3), CD44neg (16HBE) and lung fibroblastic cells come from patients affected by BOS (Bronchiolitic Obliterant Syndrome) after lung transplantation (Tx) and CTD-ILD (Collagen Tissue Disease–associated Interstitial Lung Fibrosis). In details, the in vitro studies assessed the cytotoxicity of the blank liposomes to evaluate the differences among citrate and solfate buffer and the uptake by the cells previously described in order to demonstrate that HA-decorated liposomes are better uptaken compared to blank liposomes. The formulation, based on previously studies, led to the obtainment of high thermic (4°C-37°C) and in time (3 weeks) stability liposomes. As buffer, the citrate one demonstrated to be a good hydration buffer: the drug concentration, entrapment efficiency and dimensions in time gave us better data compared to the solfate buffer. However, in serum stability tests we found a drug concentration 50% less than in HEPES buffer at loading time. It could be due to the different pH between serum and buffer or to the proteins in the serum. About the cytotoxicity, the liposomes hydrated with citrate buffer showed a higher effect on all the cells tested. From the in vitro studies, we found that there is a better uptake for HA-decorated liposomes than blank ones, especially in A549, as aspected. Furthermore, a better uptake was seen for HA-decorated liposomes with a higher molecular weight (44700Da), underlining a receptor-mediated internalization of the liposomes.