Fig 2: Down-regulation of Runx2 expression by C60-PEI/shRNA-Runx2 plasmids polyplexes reduces the expression of osteoblast differentiation markers ALP (A), OSP (B), BSP (C) and BMP-4 (D) in VIC exposed to HGOM. Results, normalized to ß-actin, represent the means ± S.D. of three independent experiments performed in duplicate (n = 6) and represent fold change relative to HGOM condition (considered as 1). Representative blots are shown above the graphs. * p < 0.05, ** p < 0.01, *** p < 0.001 versus HGOM.

Fig 3: Downregulation of Runx2 expression in osteoblast-differentiated VIC. (A) mRNA and (B) protein Runx2 expression in HGOM-activated VIC for 5 days and transfected for 48 h with different type of nanocarriers. NM (lane 1); HGOM (lane 2); V-LPP/shRunx2 (lanes 3, 4); Scr-LPP/shRunx2 (lane 5); C60-PEI/shRunx2 (lane 6); V-LPP/shCTR (lanes 7, 8); Scr-LPP/shCTR (lane 9). (C) mRNA Runx2 level in HGOM-activated VIC subjected to double transfection in the 5th and 12th days, and measured 48 h after the second transfection (14th day). The results were normalized to ß-actin and represent the mean ± S.D. of two independent experiments made in duplicate (n = 4) and represent fold change relative to HGOM condition (considered as 1). The samples considered statistically different were marked with * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to HGOM; # p < 0.05, ## p < 0.01, ### p < 0.001 when compared to NM; & p <0.05 when compared to V-LPP/shRunx2.

Fig 4: Schematic representation of the successive steps in the synthesis of VCAM-1 targeted lipopolyplexes encapsulating the shRNA plasmid lipopolyplexes (V-LPP/shRNA). First, the core-shell structures consisting of fullerene (C60) core and branched low molecular weight polyethyleneimine (PEI) (2kDa) were complexed with the plasmid containing shRNA sequences specific for Runx2 (shRunx2) or scrambled sequences (shCTR). Second, the anionic phospholipid DOPG dissolved in chloroform/methanol was added to the positively charged C60-PEI/shRNA polyplexes to form reverse micelles entrapping the polyplexes and, third, the organic phase was removed by reverse-phase evaporation under reduced pressure in the presence of coating phospholipids (POPC, Metoxi-PEG2000-DSPE, and Mal-PEG2000-DSPE). The resulting lipopolyplexes, namely PEG-stabilized lipid-coated particles containing the C60-PEI/shRNA polyplexes inside were subsequently extruded through polycarbonate membranes to achieve a narrow and unimodal size distribution of the lipopolyplexes suspension. Next, the VCAM-1 recognizing peptide having an amino acid sequence terminating in cysteine was coupled via a reaction between thiol and maleimide-derivatized PEGylated phospholipid (Mal-PEG2000-DSPE) from the liposome’s membranes.

Fig 5: Down-regulation of Runx2 mRNA (A) and protein (B) expression in HGOM-treated VIC and transfected with C60-PEI/shRNA-Runx2 plasmids polyplexes containing different shRNA sequences specific for Runx2 (sh_1, sh_2 and sh_3) and then investigated at 48 h after transfection. Results, normalized to ß-actin, were expressed as mean ± S.D. of three experiments made in duplicate (n = 6) and represent fold change relative to HGOM condition (considered as 1). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with HGOM.