(D-122) Nitroxide-Mediated Enzymatically Polymerized Hydrogel Attenuates Post‐Infarct Cardiac Dysfunction

As the world moves towards reducing its reliance on fossil-fuel energy and addressing environmental concerns, hydrogels have become increasingly important in various industries, particularly the biomedical field. Hydrogels are highly absorbent three-dimensional networks of polymer chains, offering unique properties for applications ranging from drug delivery to tissue engineering. Moreover, enzymes are increasingly attracting interest as a tool for designing and synthesizing functional polymers and hydrogels mildly and efficiently. A well-established radical initiating system consisting of horseradish peroxidase (HRP), β-diketones (e.g., acetylacetone (ACAC) or 1,3-cyclopentanedione), and H₂O₂ can initiate free radical polymerization in aqueous media, producing polymers and hydrogels at room temperature.¹ However, this enzymatic reaction may have an irreproducible induction period (lasting 45 − 360 minutes), and the use of β-diketones can harm mammalian health, limiting their potential biomedical applications. Here, we report the development of a fast-gelling gelatin methacryloyl (GelMA) hydrogel loaded with exosomes derived from mesenchymal stem cells (MSC-EXOs) via nitroxide-mediated enzymatic polymerization. As shown in Figure 1, we used a nontoxic N-hydroxyimide compound, N-hydroxy-5-norbornene-2,3-dicarboximide (HONB), to enable HRP-catalyzed nitroxide radical initiation for producing the exosome-laden hydrogels, avoiding the use of the conventional toxic ACAC. We evaluated the efficacy of the hydrogels prepared using either HONB- or ACAC-mediated enzymatic polymerization in preserving and vascularizing the hearts in a rat model of myocardial ischemia/reperfusion (I/R) injury. Our findings demonstrate the potential of HONB as a safe and efficient alternative to β-diketones as an enzymatic substrate in the development of biofunctional hydrogels, leading to more effective and safer biomedical applications.

We utilized electron paramagnetic resonance (EPR) spectroscopy to detect free radical formation. MSC-EXOs were generated from human bone marrow-derived mesenchymal stem cells using the ExoQuick-TC ULTRA isolation kit (SBI). Subsequently, we synthesized a biodegradable, adhesive, and exosome-laden GelMA hydrogel via HONB-mediated enzymatic polymerization. To create the hydrogel patches, we mixed a sterile GelMA solution (20 wt%) with MSC-EXOs (5 × 10¹¹ exosomes/mL) at 37°C and then added HONB, HRP, and H₂O₂ sequentially to initiate free radical polymerization ([HONB]/[HRP]/[H₂O₂] = 60.4/0.015857/0.44 mM). The mixture was immediately transferred to silicone-gasketed chambers (7 mm × 7 mm × 1 mm per chamber) and incubated at 37°C to produce hydrogel patches (denoted HCG). Control hydrogel patches (denoted ACG) were prepared in the same way, using ACAC instead of HONB (Figure 1). We evaluated the compatibility of the hydrogels with cardiac cells using a CCK8 assay and assessed the angiogenic capacity of cardiac endothelial cells in the presence of different hydrogel leaching solutions using a tube formation assay. All animal procedures were approved by the Institutional Animal Care and Use Committee at Duke University. Sprague-Dawley rats (6-8 weeks old, Charles River Laboratories) underwent myocardial I/R injury and were randomized into three groups to receive either: 1) HCG patch (n = 8 rats), 2) ACG patch (n = 8 rats), or 3) saline placebo (n = 6 rats). Transthoracic echocardiogram was performed to assess cardiac function at baseline (pre-MI) and 4 weeks post-treatment. The hearts were harvested for histological analysis after 4 weeks of treatment.

EPR spectroscopic analysis showed that the HONB-HRP-H₂O₂ ternary system produced an initiating radical in just 5 minutes at room temperature. The spectrum of this species showed a doublet of sextets (hyperfine coupling constants: g = 2.0067, a^N = 13.15 G, a^H_β = 7.05 G, and a^H_g = 2.2 G), indicating the formation of a nitroxide radical (Figure 2). The HONB-HRP-H₂O₂ enzymatic initiator system generated the exosome-laden GelMA hydrogel at 37°C in approx. 5 min, much faster than the conventional ACAC-HRP-H₂O₂ ternary system, which required over 15 min at a comparable substrate concentration. The leaching solution of the HCG hydrogel was compatible with H9c2 cardiomyoblasts and human coronary artery endothelial cells (HCAECs), with high cellular viability ( >90%) and the ability to support the tube formation of HCAECs. In contrast, the leaching solution of the ACG hydrogel significantly impaired the viability of the above cardiac cells (approx. 70%) and the angiogenic capacity of HCAECs. After 4 weeks of treatment, the rat hearts that received the HCG hydrogel treatment showed a preserved left ventricular dimension and a decrease in the end-systolic ventricular volume, leading to a marked recovery of cardiac pump function as measured by left ventricular ejection fraction (LVEF) compared to those treated with ACG and saline placebo, respectively (Figure 3). Immunohistological analysis showed that HCG-recipient hearts had the highest α-SMA-positive blood vessel density in the peri-infarct region after 4 weeks of treatment (Figure 4A). Furthermore, there were fewer TUNEL-positive apoptotic cells in the HCG-treated hearts than those treated with ACG and saline placebo (Figure 4B). Since the discovery of HRP-mediated free radical polymerization of vinyl monomers in the early 1990s, β-diketones have been the primary initiating radical-generating substrates. However, the potential chronic toxicity of β-diketones can harm mammalian health and limit their biomedical application. Therefore, developing an HRP-mediated initiating system without β-diketones has been a challenge. Our findings demonstrate the potential of HONB as a safe and innovative alternative to β-diketones as an enzymatic substrate for biofunctional hydrogel development. This work paves the way for future research into nitroxide-mediated enzymatic polymerization for more effective and safer biomedical applications.

1. Corrigan et al. Reversible-deactivation radical polymerization (Controlled/living radical polymerization): From discovery to materials design and applications. Progress in Polymer Science 2020, 111, 101311.