Improved Reversion of Calcifications in Porcine Aortic Heart Valves Using Elastin-Targeted Nanoparticles

Figures

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  Figure 1. Induced calcification of porcine aortic valve leaflets. Leaflets were incubated in standard medium or osteogenic medium for indicated days. Leaflets incubated in standard medium did not calcify, whereas leaflets incubated in osteogenic medium presented an increase in calcification. (A). Calcium quantification, mean ± SD; n ≥ 3; ((B), upper two panels). Representative images of von Kossa staining of aortic valve leaflets visualizing calcified areas (brown). The ventricularis is presented on the right side of the images. As control, a native valve leaflet without culturing is shown (the middle panel represents a magnification of the upper panel, scale bars represent 100 µm; n ≥ 3 valve leaflets, n ≥ 3 sections per leaflet analyzed). ((B), lower panel). Leaflets were viable under standard culture conditions for the whole incubation period, while viability in valves cultured in osteogenic medium decreased. As control, normal viability of a native valve leaflet without culturing is shown (MTT (Methylthiazolyldiphenyl-tetrazolium bromide) staining, scale bars represent 100 µm; n ≥ 3 valve leaflets, n ≥ 3 sections per leaflet analyzed). ** p ≤ 0.01; *** p ≤ 0.001.

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  Figure 2. Representative Raman mapping of calcified aortic valves cultured in osteogenic medium for 10 days. (A). Complementary von Kossa image of this section displaying calcified deposits with selected ROI (black square) (upper left panel, scale bar: 200 µm). (B). Representative overview image of this section as unstained, methanol-fixed, and air-dried aortic valve with selected ROI (green square) (upper middle panel). Representative Raman spectrum of the indicated ROI displaying the characteristic peak for BCP at 960 cm−1 (lower panel, Y-axis shows the intensity of the scattered light (arbitrary units)). Representative heat map of BCP calcification after normalization and integration at 960 cm−1, showing the distribution of BCP crystals in the tissue (as determined by the integrated area under the curve at 945–975 cm−1, right panel). n = 2 valve leaflets, 4 sections per valve, 3–5 analyzed locations each.

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  Figure 3. Visualization of targeting of PromoFluor 633P-labeled NPs at porcine aortic valve leaflets. Aortic valve leaflets were pre-cultured for five days under standard or osteogenic conditions prior to four hours of incubation with NP. (A). NP conjugated with anti-elastin antibody (HSA-NP-(PF633)-AntiElastinPig) connected to the calcified aortic valve leaflets (osteogenic medium) and were able to infiltrate the valve, while noncalcified valves (standard medium) were not targeted. Control NPs conjugated with IgG (HSA-NP-(PF633)-IgG) could not be detected at the calcified aortic valve leaflets (n ≥ 3 valve leaflets, n ≥ 3 sections per leaflet analyzed). (B). NPs conjugated with anti-elastin antibody (HSA-NP-(PF633)-AntiElastinPig) targeted to elastin-rich areas in the aortic valve. Nanoparticles: pink, autofluorescence: green, elastin: red. Scale bar: 50 µm (n ≥ 3 valve leaflets, n ≥ 3 sections per leaflet analyzed).

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  Figure 4. Reversion of induced calcification in porcine aortic valve leaflets using NPs. Leaflets were pre-incubated in osteogenic medium for five days to induce calcification prior to administration of 1 mg/mL DTPA, the equivalent of DTPA bound to DTPA(HSA-NP)AntiElastinPig or DTPA(HSA-NP)IgG as control for two days. (A). Quantification of calcium showing reduced calcium content in leaflets incubated with pure DTPA, DTPA(HSA-NP)AntiElastinPig, and DTPA(HSA-NP)IgG compared to control only cultured in osteogenic medium. Mean ± SD; n ≥ 3; ((B), upper two panels). Representative images of von Kossa staining of aortic valve leaflets visualizing calcified areas (brown) in leaflets incubated in osteogenic medium, while no calcification was found in leaflets treated with pure DTPA, DTPA(HSA-NP)AntiElastinPig, and DTPA(HSA-NP)IgG (middle panel represents magnification of upper panel, scale bars represent 100 µm; n ≥ 3 valve leaflets, n ≥ 3 sections per leaflet analyzed). ((B), lower panel). Approximately half of the cells were viable in leaflets cultured under osteogenic culture conditions. No viability was detectable in leaflets incubated with pure DTPA, whereas leaflets administered with…