Balancing functions of antifouling, nitric oxide release and vascular cell selectivity for enhanced endothelialization of assembled multilayers

Abstract

Surface endothelialization is a promising way to improve the hemocompatibility of biomaterials. However, current surface endothelialization strategies have limitations. For example, various surface functions are not well balanced, leading to undesirable results, especially when multiple functional components are introduced. In this work, a multifunctional surface was constructed by balancing the functions of antifouling, nitric oxide (NO) release and endothelial cell promotion via layer-by-layer (LBL) self-assembly. Poly(sodium p-styrenesulfonate-co-oligo(ethylene glycol) methacrylate) (negatively charged) and polyethyleneimine (positively charged) were deposited on silicon substrates to construct multilayers by LBL self-assembly. Then, organic selenium, which has a NO-releasing function, and the cell-adhesive peptide Gly-Arg-Glu-Asp-Val-Tyr, which selectively promotes endothelial cells, were introduced on the assembled multilayers. Poly(oligo(ethylene glycol) methacrylate) is a hydrophilic component for antifouling properties, and poly(sodium p-styrenesulfonate) is a heparin analog that provides negative charges. By modulating the contents of poly(oligo(ethylene glycol) methacrylate) and poly(sodium p-styrenesulfonate) in the copolymers, the NO release rates catalyzed by the modified surfaces were regulated. Moreover, the behaviors of endothelial cells and smooth muscle cells on modified surfaces were well controlled. The optimized surface strongly promoted endothelial cells and inhibited smooth muscle cells to achieve endothelialization effectively.

Keywords: endothelialization; heparin analogs; layer-by-layer self-assembly; nitric oxide release; surface modification.

Graphical abstract

Figure 1.

Chemical structure (A),¹H-NMR spectra using heavy water (D₂O) as the solvent (B) and FTIR spectra (C) of the PSO_n copolymers.

Figure 2.

The water contact angle (A) and FTIR spectra (B) of Si and Si-OH surfaces. The water contact angle (C) and thickness (D) of multilayers during the LBL self-assembly process. FTIR spectra of surfaces with different numbers of bilayers. (E) The outermost layer was PEI. (F) The outermost layer was PSO₃.

Figure 3.

(A) Water contact angle, (B) XPS, (C), (D) FTIR and (E) AFM.

Figure 4.

(A) Absorbance of the PBS solution before and after soaking the PEI/PSO₃-Se-G samples for 7 days. Thicknesses (B) and FTIR spectra (C) of the PEI/PSO₃-Se-G samples before and after 7 days of incubation in PBS solution.

Figure 5.

(A) NO Release rates of the PEI/PSO_n-Se-G samples within a period of 11 days. NO release rate of Si was approximately 0.065 × 10⁻¹⁰mol·min⁻¹·cm⁻². (B) Cumulative release amount of NO catalyzed by different samples.

Figure 6.

Fluorescence images (A), cell density (B) and cell coverage ratio (C) of HUVSMCs on different samples after 24 h of incubation.

Figure 7.

Fluorescence images (A), cell density (B) and cell coverage ratio (C) of HUVSMCs on different samples after 72 h of incubation.

Figure 8.

cGMP concentrations in HUVSMCs cultured on Si and PEI/PSO₃-Se-G samples for 2 h.

Figure 9.

Western blotting analysis of α-SMA in the HUVSMCs on PEI/PSO₃-Se-G samples after 72 h of incubation. (A) GAPDH and (B) α-SMA.

Figure 10.

Fluorescence images (A), cell density (B) and cell coverage ratio (C) of HUVECs on different samples after 24 h of incubation.

Figure 11.

Fluorescence images (A), cell density (B) and cell coverage ratio (C) of HUVECs on different samples after 72 h of incubation.

Figure 12.

Immunofluorescence images of HUVECs on different samples after 24 h (A) and 72 h (B) of incubation.

Figure 13.

Fluorescence images showing the migration of HUVECs cultured on different samples after scratching with a pipette tip. Migration of HUVECs after 24 h of incubation without the donor (A) and with the donor (B).

Figure 14.

Competitive growth behaviors of HUVECs and HUVSMCs on Si and PEI/PSO₃-Se-G samples. Fluorescence images after co-culture for 2 h (A) and 24 h (B). density ratios of HUVECs/HUVSMCs after co-culture for 2 h (C) and 24 h (D).

Scheme 1.

Construction of multifunctional surfaces. (1) and (2) PEI and PSO_n were deposited on piranha-treated Si to construct multilayers by LBL self-assembly. (3) SeCA was grafted on the multilayers. (4) The GREDVY peptide was immobilized on the surface as a top coating by electrostatic force.