Nanoparticle Biofunctionalization of Bioprosthetic Heart Valves to Modulate Mechanisms of Structural Valve Deterioration

Figures

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  Figure 1. Immobilization of PPN on Pericardium (A) PPN coating process (scale bar represents 100 nm). (B) Quantification of percentage area of pericardium surface covered after PPN conjugation, with and without pretreatment with Cys. (Data presented as mean ± SEM compared with 2-way ANOVA with Dunnett's multiple comparisons test, 3 per group). (C) Scanning electron microscopy and photographs of pericardium after coating with PPN (brown) with and without Cys pretreatment (scale bar represents 1 μm). (D) Experimental setup to test PPN retention under flow. (E) Scanning electron microscopy images and photographs of PPN-coated pericardium before washing (prewash) and after (postwash) (scale bar represents 500 nm). (F) Quantification of the surface coverage of postwash pericardium samples as a percentage of prewash samples. Simulated time was calculated as per the methods (3 per group). Statistical significance is indicated by ∗∗∗P < 0.0001. Cys = L-cysteine; PPN = plasma polymerized nanoparticle.

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  Figure 2. Reduction of Residual Aldehyde Groups and Its Effect on Biocompatibility (A) Aldehyde reduction using Cys on pericardium surfaces. (B) Control (untreated), Cys, or Cys + PPN treated pericardium stained with Schiff’s reagent, which stains aldehyde groups purple. (C) Human coronary artery endothelial cell seeding methodology. (D) Human coronary artery endothelial cell attachment after 1 hour (scale bar represents 20 μm). F-actin shown in red. (E) Quantification of human coronary artery endothelial cell number 1 hour after seeding. (F) Quantification of human coronary artery endothelial cell F-actin staining after a 1-hour attachment. (G) Quantification of EC viability after 24 hours. (Data presented as mean ± SEM compared using 1-way ANOVA with Dunnett's multiple comparisons test, 3 per group). Statistical significance is indicated by ∗P < 0.05, ∗∗∗P < 0.001. ECs = endothelial cells; other abbreviations as in Figure 1.

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  Figure 3. Mechanical and Surface Properties of PPN-Coated Pericardium (A) Biaxial testing setup. A refers to axial and T refers to transversal direction in inset. (B) Results for stress test and maximum load (C) for control, Cys treated, and Cys + PPN treated pericardium in both the axial and transversal directions (Data presented as mean ± SEM compared using 2-way ANOVA with Dunnett's multiple comparisons test, 3 per group). (D) Quantification of maximum load degradation over 10 cycles (Data presented as mean ± SEM compared using 2-way ANOVA with Dunnett's multiple comparisons test, 3 per group). Values are given as a percentage of initial maximum load test. (E) Water contact angle testing (scale bar represents 1 mm). (F) Quantification of water contact angle testing across control, Cys treated, and Cys + PPN treated pericardium. (Data presented as mean ± SEM compared using 1-way ANOVA with Dunnett's multiple comparisons test, 3 per group). Statistical significance is indicated by ∗P < 0.05, ∗∗P < 0.01. Abbreviations as in Figure 1.

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  Figure 4. Functionalization of Pericardium With APX to Reduce Thrombosis (A) Molecular cargo dip-coating functionalization step. (B) Scanning electron microscopy images using back-scattered electron detector of gold-tagged APX (bright white dots) functionalized onto PPN-coated pericardium. (C) Whole blood clotting assay (scale bar represents 5 mm). (D) Quantification of blood clot weight. (Data presented as mean ± SEM compared using Student t test, 3 per group). (E) Scanning electron microscopy images of platelet attachment and aggregation on pericardium after 1-hour incubation. Platelets are false colored in orange (scale bar represents 4 μm). (F) PF4 enzyme-linked immunosorbent assay results after whole blood clotting assay. (Data presented as mean ± SEM compared using Student t test, 3 per group). Statistical significance is indicated by ∗P < 0.05, ∗∗P < 0.01. APX = apixaban; PF4 = platelet factor 4; Abbreviations as in Figure 1.

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  Figure 5. Functionalization of Pericardium With MCC950 to Inhibit NLRP3-Mediated Inflammatory Responses (A) Control, stimulated only (Stim), stimulated and PPN coated, and stimulated and PPN-MCC950 coated (PPN-MCC) pericardium with macrophages seeded on the surface. All groups were pretreated with L-cysteine to allow for macrophage adhesion and stimulation. Cells stained with rhodamine phalloidin (red) to visualize F-actin (scale bar represents 50 μm). (B and C) Interleukin-1β (B) and tumor necrosis factor-α (C) enzyme-linked immunosorbent assay quantification from macrophages seeded on differently coated pericardium samples poststimulation. (Data presented as mean ± SEM compared using 1-way ANOVA with Dunnett's multiple comparisons test, 3 per group). (D) Rat subcutaneous implant model. (E) Masson's Trichrome staining with the pericardium implant outlined in black and fibrotic capsule denoted by a star (scale bar represents 30 μm). (F) Quantification of fibrotic capsule thickness surrounding the implant. (Data presented as mean ± SEM compared using 1-way ANOVA with Dunnett's multiple comparisons test, 5 per group). Statistical significance is indicated by ∗P < 0.05, ∗∗∗P < 0.001.…

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  Figure 6. Functionalization of Pericardium With PA to Prevent Calcification (A) In vitro human fibroblast calcification model that underwent treatment with calcification media (scale bar represents 1 mm). (B) Quantification of Alizarin red staining. (Data presented as mean ± SEM compared using 1-way ANOVA with Dunnett's multiple comparisons test, 3 per group). (C) PA functionalization and mechanism of action. Free calcium and phosphate ions 1) traditionally bind together to form hydroxyapatite crystals on pericardium surface. PA functionalized on PPN-coated pericardium preferentially binds calcium ions, inhibiting phosphate ion interactions, 2) and preventing hydroxyapatite crystal formation. 3) (D) Scanning electron microscopy images of differently treated pericardium samples incubated with a calcium phosphate solution showing hydroxyapatite crystal formation false colored in yellow (scale bar represents 10 μm). (E) Quantification of hydroxyapatite crystal coverage across samples. (Data presented as mean ± SEM compared using 1-way ANOVA with Dunnett's multiple comparisons test, 3 per group). (F) Alizarin red staining in the pericardium implant outlined in black (scale bar represents 500…

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  Figure 7. Translatability of PPN Coating on Commercial Transcatheter Aortic Valve Replacement Valve (A) Dip-coating process used to conjugate PPN to an expired Medtronic Evolut R 26-mm transcatheter valve. Scanning electron microscopy images show the surface of the pericardium leaflets before and after PPN coating. (Scale bar represents 500 nm). (B) Pulse duplicator system. (C) Evolut R valve inside pulse duplicator system with and without PPN coating. (D-F) Quantification of RF (D), EOA (E), and mean pressure gradient (F). (Data presented as mean ± SEM compared using Student t test, 1 valve, 10 individual values represent individual pulses). EOA = effective orifice area; FFV = flow forward volume; PPD = peak pressure drop; RF = regurgitation fraction; Abbreviations as in Figure 1.