Peptídeo derivado da lactoferricina bovina: síntese, caracterização, avaliação da atividade frente a linhagens celulares de câncer de mama e desenvolvimento preliminar de formulação lipossomal

Abstract

The purpose of this work was to synthesize and characterize two peptides derived from bovine lactoferricin (26[F] LfcinB (20 – 30)2 and 26[1-NAL] LfcinB (20 – 30)2), to evaluate the in vitro activity against breast cancer cell lines (MCF-7 and MDA-MD231) and subsequently develop a liposomal formulation for the encapsulation of the peptide considered most promising. The peptides were obtained by solid phase synthesis (SPPS) using the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology, purified by SPE chromatography and the collected fractions were analyzed by HPLC-FR, ESI-Q-TOF and MALDI-TOF. The purity was greater than 80% for both peptides. The HPLC-FR results showed that the retention times obtained were similar to those reported in the literature. The ESI-MS mass spectra showed [M+H]+ signals very close to the expected m/z ratios, which confirmed the chemical identity of the peptides. Furthermore, molecular identification was confirmed by MALDI-TOF/MS. The peptides were also evaluated for their influence on the viability of tumor cells (MCF-7 and MDA-MB-231) and normal cells (CCD1059 SK) using the tetrazolium salt reduction method. In this case, the peptide 26[F] LfcinB (20 – 30)2 showed greater selectivity against tumor cells, especially for the MDAMB-231 cell line, considered highly invasive and with high metastatic potential. Therefore, it was chosen to continue the study. When it was evaluated the antimigratory effect (scracth assay) against tumor cell lines, this peptide showed a dosedependent effect with more pronounced inhibitory activity, also for the MDA-MB-231 cell line. Finally, conventional liposomes (Lip) were developed using a nanoprecipitation technique for peptide encapsulation (Lip-Pep), which were characterized by means of hydrodynamic diameter (DHm), polydispersity index, Zeta potential (PZ), encapsulation efficiency (EE) and in vitro release profile. The developed vesicles showed a monodisperse characteristic (PdI < 0.3). The DHm of Lip-Pep (102.6 ± 9.91 nm) was higher than that observed for “empty” Lips (without the addition of the peptide; Lip-B = 74.99 ± 1.32 nm), which constituted the first indication of the occurrence of encapsulation. The EE of the peptide in the liposomes was approximately 100%. The PZ of Lip-Pep (-22.3 ± 3.54 mV) was less negative than that obtained for Lip-B (-32.0 ± 1.82 mV), probably due to electrostatic interactions between the cationic peptide and the anionic phospholipid used to prepare liposomes. This hypothesis may explain the result obtained in the in vitro release assay, which demonstrated that there was no release of the peptide during the 24 hours of the experiment. This behavior made further evaluation of the formulation unfeasible. Thus, although we found promising activity of the peptide against the MDA-MB-231 cell line, in order to continue the study it will be necessary to develop a new liposomal formulation, possibly with a non-inonic phospholipid.