Propriedades físico-químicas e biológicas de lipossomas termossensíveis de cisplatina funcionalizados com ácido hialurônico.

Abstract

The continuous administration of cisplatin (CDDP), a chemotherapeutic agent used to treat various types of solid tumors, is limited due to adverse reactions, such as nephrotoxicity, hepatotoxicity, neurotoxicity, myelotoxicity, and ototoxicity. In an attempt to circumvent these inconveniences and improve the therapeutic index of CDDP, pharmacotechnical modifications have been proposed, such as the encapsulation of CDDP in drug delivery systems, like liposomes. In this sense, it was proposed to develop a thermosensitive liposome of nonfunctionalized cisplatin (TSL-CDDP), functionalized with hyaluronic acid (HA) by electrostatic interaction (TSL-CDDP-HA-1) and functionalized with HA by covalent bond (TSL-CDDP-SA-HA-2), in order to provide an active targeting for the tumor region. Initially, a detailed study was carried out to characterize the formulations developed, through the association of dynamic light scattering (DLS), microcalorimetry, and low angle X-ray scattering (SAXS) techniques. The CDDP encapsulation rate was evaluated by high performance liquid chromatography using a diethyldithiocarbamate derivatization method. Then, cytotoxicity against MDA-MB-231 tumor cells was evaluated, as well as in vivo toxicity in healthy Swiss mice. The characterization results showed an average diameter less than 200 nm and a polydispersity index (PDI) suggestive of homogeneous formulations (< 0.2), indicating the absence of vesicle aggregation even after functionalization with HA. The efficiency of the HA-coating of liposome was attributed to values of zeta potential close to neutrality. The DLS data showed a significant reduction in the mean diameter and Kcps (Kilo counts per second) of the formulations evaluated at 40oC, and the coating with HA did not alter the phase transition temperature of the formulations. CDDP encapsulation was 9.3 ± 0.7% for TSL-CDDP-SA-HA, 16.1 ± 1.9% for TSL-CDDP-HA and 13.7 ± 0.9% for TSL -CDDP. The profile obtained by SAXS for all the formulations studied was characteristic of a lamellar organization independent of the evaluated temperature and presented dilation of the bilayer, caused by disorganization in the lipid structure, confirming the conformational alteration due to heating. However, the in vitro release profile suggested possible drug adsorption in the phospholipid bilayer causing a diffusion rate of CDDP before reaching Tc (42oC), for TSLCDDP and TSL-CDDP-SA-HA. For TSL-CDDP-HA, the presence of the polymer may be modulating this diffusion, generating a more controlled and slower release profile. The biological results showed that TSL-CDDP have greater cytotoxicity when compared to free CDDP, in addition to leading to an increase in the occurrence of senescence. The use of hyperthermia in the groups treated with LTS-CDDP and LTS-CDDP-HA increased the occurrence of apoptosis compared to groups without hyperthermia. In the in vivo study, greater toxicity of the cationic formulation was observed for healthy cells, with the occurrence of nephrotoxicity in animals treated with LTS-CDDP. While, with the use of the HA ligand, the known adverse effects related to the use of CDDP were eliminated, since the animals treated with this formulation did not show significant changes in hematological and biochemical tests, as well as in histological analyzes. Thus, the proposed formulation of CDDP, thermosensitive and containing HA by electrostatic interaction, has adequate characteristics for cancer treatment intravenously, in association with moderate hyperthermia, and may be able to reduce CDDPrelated toxicity, representing a potential alternative for the delivery of this drug.