Physisorption of GHHPH Tetra Repeat of HPRG on hydroxyapatite nanocrystals as tunable angiogenic nano platform.

Histidine-Proline Rich Glycoprotein (HPRG) is a known modulator of angiogenesis, that is a major but transient event during the formation and repair of wound tissues ,2. In a tissue-engineered scaffold the angiogenesis process can be triggered by the loading and/or the surface tailoring with growth factors, such as VEGF, FGF, BMP and/or genetically modified cells. Nevertheless, inorganic angiogenic regulators, such as copper ions, are of great interest, due to their low cost, higher stability, and potentially superior safety compared with recombinant proteins or genetic engineering approaches.3 This study tackles the fabrication and physicochemical characterization of hybrid bio-composite scaffolds based on nano-hydroxyapatite (nHAp), functionalized with both copper and the GHHPH peptide, that is the known tetra-repeat sequence in HPRG.4 Specifically, the nHAp nanocrystals were modified by physical adsorption processes of: i) Cu(II) ions, ii) fluorescein amidite-labeled tetra-repeat (GHHPH-FAM) and iii) GHHPH-FAM/Cu(II) complexes. The functionalized composite scaffolds (nHAp/GHHPH-FAM, nHAp/Cu and nHap/GHHPH-FAM/Cu) were characterized by AFM, UV-Vis, fluorescence and FT-IR spectroscopies. Preliminary cell assays with neuroblastoma cells were carried out by confocal microscopy. The dynamic processes of cellular internalization were scrutinized in live imaging experiments by tracking the peptide fluorescent tag and copper chemosensor, respectively.