The Department of Chemical, Biological, Pharmaceutical and Environmental Sciences of the University of Messina is involved in the Liver Smar Drug project in several activities: Development of in vitro models, in vitro biological characterization of LSD systems and selection of the most performing systems.
Following the chemical-physical characterization of the drug-delivery systems by the Institute of Biomedicine and Molecular Immunology "Alberto Monroy", National Research Council (CNR) of Palermo (Italy), in vitro studies on hepato-carcinoma cell cultures have been developed, in order to evaluate the biological effects of LSD systems and to select the most performing systems. For this purpose, at the Department of Chemical, Biological, Pharmaceutical and Environmental Sciences (CHIBIOFARAM) of the University of Messina, a human cell culture of hepato-carcinoma (HepG2) has been set up; the cells were treated with at least 4 types of LSD systems and 2 different drugs compared to the already patented LSD system.
After treatment with LSD systems, with and without medication, cell viability tests (MTS) were performed to evaluate the cytotoxic effects of next-generation LSD systems. Development of in vivo biological models and clinical protocols: After evaluating the efficacy of LSD systems in vitro, at the CHIBIOFARAM Department, a mouse model of hepato-carcinoma was developed using the HepG2 cell line. Specifically, an in vivo xenograft model of hepatocarcinoma was used where 5x106 hepatocarcinoma cells were inoculated subcutaneously in immunodeficient naked mice of the NU(NCr)-Foxn1nu strain. As soon as the tumor mass (about 5 mm) was formed, the animals were divided into experimental groups and treated with LSD systems, both through intraperitoneal and oral administration. Following treatments with LSD systems, the animals were monitored at different time points to evaluate the biological efficacy of individual treatments.
For this purpose, the presence and growth of tumor masses were evaluated, through both palpation of the implant area and measurement with a digital micrometric caliber. Ex-vivo biological characterization of selected LSD systems: After completing the in vivo studies, the ex vivo phase continued, during which the tumor masses were explanted at different time points and prepared for histological and molecular characterization, to verify the effectiveness of LSD systems.
This activity was mainly focused on the morphological and immunohistochemical study of tumor masses of hepatocarcinoma explanted from animals. Specifically, the tumor masses were fixed in formalin and chemically processed until they were included in paraffin. Subsequently, the fixed tissues were dissected to the microtome and colored with standard methods (hematoxylin and eosin) and with histochemical methods (PAS; Alcian-PAS; staining for elastic fibers and lattice). In addition, for each sample, additional sections were obtained for immunohistochemical studies with immunoperoxidase methods. Fresh tissues have been frozen and stored for molecular biology studies such as Western Blot and ELISA kits.
Molecular biology studies have also been conducted using different tumor markers, including those specific to hepatocarcinoma, as well as angiogenic, inflammatory and apoptotic markers, to better define tumor vascularization. The morphological study has helped to highlight the characteristics of neoplasms, emphasizing the degree, architecture, invasiveness and response to therapy in terms of absence or presence of tumor necrosis, inflammation (acute, chronic, granulomatous), reactive fibrosis and apoptosis. Subsequently, to verify the biodistribution of LSD systems, various organs (spleen, liver and kidneys) will be taken, to extract and quantify the systems present, to better understand the biodistribution of the drug and the systems used; these analyzes will be conducted at the CNR of Palermo.