VCAM-1 Targeted Lipopolyplexes as Vehicles for Efficient Delivery of shRNA-Runx2 to Osteoblast-Differentiated Valvular Interstitial Cells; Implications in Calcific Valve Disease Treatment

Figures

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  Figure 1. 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…

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  Figure 2. Particle size intensity distribution of C60-PEI/shCTR (A) and V-LPP/shCTR (C) as measured by dynamic light scattering and Zeta potential distribution of C60-PEI/shCTR (B) and V-LPP/shCTR (D) measured by electrophoretic light scattering at the initial time and after storage at 4 °C for up to one month.

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  Figure 3. Characterization of VCAM-1 targeted lipopolyplexes encapsulating the shRNA plasmid. (A) Electron micrographs of negatively-stained V-LPP/shCTR lipopolyplexes showing its spherical morphology. (B) Colloidal stability of C60-PEI/shCTR polyplexes and V-LPP/shCTR lipopolyplexes in PBS determined by DLS. (C) Electrolyte-induced flocculation of C60-PEI/shCTR polyplexes and V-LPP/shCTR lipopolyplexes. Note the stability of V-LPP/shCTR lipopolyplexes in comparison with C60-PEI/shCTR polyplexes. Results are reported as mean ± S.D. for three individual measurements.

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  Figure 4. (A) VCAM-1 expression on VIC surface. Percentages of VCAM-1 positive cells as determined by flow cytometry experiments on VIC cells cultured in normal medium (NM) or high glucose osteogenic medium (HGOM) for 1, 2, or 7 days and plotted from a representative experiment performed in duplicate. The bar graph shows data as mean ± S.D. The overlays of representative flow cytometry histograms are presented above the graphs. (B) Fluorescence microscopy exemplifying the uptake of V-LPP/Cy3 labeled plasmid by VIC exposed to NM or HGOM for 5 days before incubation with lipopolyplexes for 24 and 48 h in the absence or the presence of an excess of V-BP (V-LPP/Cy3 (red); scale bar: 100 μm). (C) Quantification of lipopolyplexes uptake expressed as fluorescence intensity of red pixels percentage for each image field (each point represents media of 6 fields) using ImageJ software. The bar graph shows results as mean ± S.D. (D) Flow cytometry data showing the uptake of V-LPP/Cy3 labeled plasmid by VIC exposed to NM or HGOM for 5 days before incubation with lipopolyplexes for 48 h in the absence or the presence of excess V-BP. The results are determined as Mean Fluorescence Intensity (MFI) and…

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  Figure 5. 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 β-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.

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  Figure 6. (A–F). The reduction of mRNA and protein expression of osteogenic molecules, OSP (A,B), BSP (C,D), and BMP-2 (E,F) in VIC exposed to HGOM for 7 days and transfected with a different type of nanocarriers on the fifth day. Western blots: 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). (G–I) The gene expression of OSP, BSP, and BMP-2 in VIC exposed to HGOM for 14 days and subjected to a second transfection on the 12th day. As controls, transfection using V-LPP/shCTR and Scr-LPP/shCTR were employed. Results, normalized to β-actin, were expressed as mean ± S.D. of two independent experiments made in duplicate (n = 4) and represented as 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.

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  Figure 7. Cyto-/hemocompatibility evaluation of lipopolyplexes. (A) Cytotoxicity of V-LPP/shCTR lipopolyplexes measured as release of AK at 48 h after transfection performed on the 5th and 12th days. Data are presented as mean ± S.D. of one representative experiment done in three replicates. (B) Quantification of hemolysis in erythrocytes incubated with V-LPP/shCTR at different plasmid concentrations. The corresponding photographs of erythrocytes after their sedimentation are shown above the graph. (C) Evaluation of erythrocyte aggregation after incubation with lipopolyplexes by light microscopy. PBS [Ctr (−)] and 0.5% Triton X-100 [Ctr (+)]. Scale bar: 200 μm. Note that there are no significant changes in erythrocyte aggregation at any V-LPP/shCTR concentration used.