CAMPERI Silvia Andrea
congresos y reuniones científicas
Innovative nanotechnological polyelectrolyte complexes loaded with an anti-inflammatory peptide for the treatment of chronic inflammatory vascular diseases.
A. BLACHMAN; F. FUNEZ ; A.C. EIGUREN; S. L. SAAVEDRA; S. A. CAMPERI; G. ROMINA; LAZARO J. M. ; CALABRESE G.
Congreso; IV International Congress in Translational Medicine; 2018
Introduction. In spite of the fact that atherosclerosis, a chronic inflammatory disease, affects the vascular wall, there is still no pharmaceutical treatment that directly targets the blood vessel wall instead of just controlling the risk factors.We have produced polyelectrolyte complexes (PECs) by polyelectrolyte complexation method, between low molecular mass dermatan sulfate (DS) and chitosan (CS); with a hydrodynamic diameter of 729±11 nm.1Moreover, PECs show specific uptake by healthy or injured endothelial cells; when they were cultured alone or in the presence of macrophages. This specific uptake, mediated by DS, could be related with hialuronic acid receptors (CD44; HARE).2 In the context of endothelial cells as targeted destination, the aim of the present work was to study these complexes as a drug delivery platform of an anti-inflammatory egg white ovotransferrin-derived tripeptide Ile-Arg-Trp, (IRW).Methods.The tri-peptide IRW was obtained on solid phase employing Fmoc chemistry in Rink-Amide-MBHA resin. PECs/IRW were produced by the ionotropic gelification method, in the presence of IRW (5, 25 or 50 µM).1 Beside, CS labeled with fluorescein isothiocyanate were employed to obtain FITC-PECs/IRW for confocal microscopy studies.1 The hydrodynamic diameter (Dh), size distribution (PDI) and zeta potential (Z-potential) of PECs/IRW were determined by dynamic light scattering (DLS). High-resolutions-NMR experiments (13C CP-MAS and 1H-MAS) were used to analyze the chemical structures of both DS/CS-PECs and PECs loaded with IRW (PECs/IRW). Murine endothelial cell line (H5V) were incubated with FITC-PECs (10 µg/mL, according to their DS concentration) 3 for 2 hours after bacterial lipopolysaccharide (LPS; 1.5 µg/mL) treatment. Hialuronic acid receptor CD44 and HARE were evaluated by semiquantitative reverse transcriptase-polymerase chain reaction analysis. Zimography studies were carried out in order to analyze the modulator effect on the inflammatory response. Results. PECs/IRW (5, 25 or 50 µM), produced by the ionotropic gelification method, exhibited a main size distribution of ? ± ? nm (? %); an average PDI value of ? ± ? and a Z value ?. (n= ?). The 13C resonance signals confirmed a new nanocomposite structure. 1H-MAS spectrum corresponding to PECs/IRW showed signals corresponding to the polymer structure at 1H = 4.5 ppm together with different resonance signals assigned to the dispersion of the IRW peptide. PECs/IRW (5, 25 or 50 µM) uptake was evaluated on H5V cells after 60 min the addition. Cells presented a homogeneous green dotted signal for all PECs/IRW samples analyzed, accompanied by a significative increase in CD44 receptor expression after LPS treatment. Furthermore, confocal studies confirmed PECs/IRW cell internalization. On the other hand, zimographic analyzes showed that IRW anti-inflammatory properties were maintained in PECs/IRW formulation. Conclusions. (1) DS guarantee PECs/IRW specific uptake by endothelial cells; while CS limits lysosomal degradation and support the gradual release of IRW inside the cell. (2) Tri-peptide included in PECs formulation was able to modulate the endothelial inflammatory response associated with early stages of vascular diseases