Gold Nanoparticle-Functionalized Reverse Thermal Gel for Tissue Engineering Applications

Abstract

Utilizing polymers in cardiac tissue engineering holds promise for restoring function to the heart following myocardial infarction, which is associated with grave morbidity and mortality. To properly mimic native cardiac tissue, materials must not only support cardiac cell growth but also have inherent conductive properties. Here, we present an injectable reverse thermal gel (RTG)-based cardiac cell scaffold system that is both biocompatible and conductive. Following the synthesis of a highly functionalizable, biomimetic RTG backbone, gold nanoparticles (AuNPs) were chemically conjugated to the backbone to enhance the system's conductivity. The resulting RTG-AuNP hydrogel supported targeted survival of neonatal rat ventricular myocytes (NRVMs) for up to 21 days when cocultured with cardiac fibroblasts, leading to an increase in connexin 43 (Cx43) relative to control cultures (NRVMs cultured on traditional gelatin-coated dishes and RTG hydrogel without AuNPs). This biomimetic and conductive RTG-AuNP hydrogel holds promise for future cardiac tissue engineering applications.

Keywords: cardiac tissue engineering; gold nanoparticles; injectable polymer; reverse thermal gel; tissue engineering.

Figure 1.

AuNP characterization. (A) AuNPs-COOH chemical structure. (B) UV/vis reading showed an absorbance peak around 528 nm. (C) AuNPs-COOH present a rounded morphology with a diameter of ~32 nm, as shown via TEM analysis.

Figure 2.

Characterization of the RTG-lysine and RTG−AuNP hydrogels. (A) A single weight loss monitored by TGA analysis demonstrates the chemical conjugation of the AuNPs to the RTG-lysine. (B) Resistance measurements demonstrating that the RTG−AuNP system is more conductive than the RTG-lysine system. p value: ****<0.0001. Data are presented as mean ± S.D. (C) The RTG−AuNP system presents significantly higher mechanical properties to those of the RTG-lysine system. Data are presented as mean ± S.D. (D) The viscosities of both the RTG−AuNP and RTG-lysine systems at 3% (w/w) concentration are similar to that of the NRVM cell culture media (N/S: non-significant). Data presented as mean ± S.D.

Figure 3.

High-resolution XPS spectra relevant to Au 4f regions of RTG−AuNP and RTG-lysine hydrogels, respectively. (A) Characteristic peaks of Au4f were observed at 87 and 84.5 eV, which confirm the presence of AuNPs in the RTG−AuNPs. (B) Elemental analysis of both hydrogels further indicates the AuNPs within the RTG−AuNPs.

Figure 4.

(A) Morphological characterization of the RTG−AuNPs was analyzed in vertical and horizontal cuts. (B) Vertical cuts of the hydrogel demonstrate a laminar sheet-like configuration. (C) Horizontal cuts of the hydrogel showed a highly interconnected porosity.

Figure 5.

Immunocytochemistry labeling of NRVMs and CFs cultured in 2D and 3D systems for 21 days. (A) Antibody staining against α-actinin (green) and vimentin (pink) label NRVMs and CFs, respectively, with nuclei labeled using DAPI (blue). (B) Quantification of immunocytochemistry staining against α-actinin indicates the percentage of cells likely to be NRVMs, showing both 3D systems to contain a greater percentage of NRVMs than the 2D gelatin control. Scale bar 40 μm. p values: *<0.023, **<0.0017, and ****<0.0001. Data are presented as mean ± S.D.

Figure 6.

Immunocytochemistry labeling of gap junctions in NRVMs cultured in 2D and 3D systems for 21 days. (A) Antibody staining against connexin 43 (Cx43) (red) and α-actinin (green), with nuclei labeled using DAPI (blue). (B) Quantification of immunocytochemistry staining against Cx43 to indicate the surface area of NRVMs positive for this gap junction protein, showing the RTG-AuNP system to contain the largest Cx43-positive area. Scale bar: 40 μm. p values: **<0.0021 and *** <0.0002. Data are presented as mean ± S.D.

Scheme 1.

Representation of the 3D Cell Culture Method Using the RTG Hydrogels

Scheme 2.

Representation of the RTG-AuNP Synthesis