Síntese, análise conformacional, avaliação do potencial de liberação em implante de PLGA e ensaios de atividade antiangiogênica e de citotoxicidade do peptídeo PSAIWF-NH2

Abstract

This work reports the synthesis of the peptide PSAIWF-NH2 using the 9fluorenylmethoxycarbonyl (Fmoc) solid phase approach. In a previous investigation performed by our research group, this peptide showed considerable antiangiogenic activity in vivo. Characterizations by high-performance liquid chromatography with ultraviolet detection (HPLC-UV) and mass spectrometry MALDI-ToF confirmed the peptide obtention at higher yield and purity. The conformational preferences of the peptide were investigated by circular dichroism spectroscopic. No conformational preference is observed neither in aqueous environments nor in 2,2,2,-trifluoroethanol (TFE) aqueous solutions. The absence of conformational preference also takes place in the presence of n-Docecylphosphocholine (DPC). On the other hand the spectra of the peptide in the presence of POPC:POPG (3:1) large unilamellar vesicles (LUVs) are consistent with peptide β-conformations, which indicate either affinity of the peptide to anionic membrane or that the anionic liposomes somehow induce this conformational preference. The in vivo assay using chorioallantoic membrane of chicken embryos (CAM) revealed the best profile of angiogenic inhibition at an intermediate concentration, indicating non-linear association between dose and response, corroborating the antiangiogenic potential of the peptide. Furthermore, in vitro assay at pigment epithelium retinal cells (ARPE-19) did not show peptide toxicity at tested concentrations. Finally, the peptide was incorporated into poly(lactic acid-co-glycolic acid) - PLGA implants. As for the hexapeptide release, these implants seemed very efficient in providing controlled and sustained release of the peptide for 39 days. The profile was marked by an initial burst of release and a subsequently decay to concentration levels compatible to that of the angiogenic inhibition observed in the investigated CAM model.