Injectable Fullerenol/Alginate Hydrogel for Suppression of Oxidative Stress Damage in Brown Adipose-Derived Stem Cells and Cardiac Repair Tong Hao,† Junjie Li,*,† Fanglian Yao,‡ Dianyu Dong,†,‡ Yan Wang,† Boguang Yang,†,‡ and Changyong Wang*,† †
Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Sciences, No. 27, Taiping Road, Beijing 100850, China ‡ Department of Polymer Science and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China S Supporting Information *
ABSTRACT: Stem cell implantation strategy has exhibited potential to treat the myocardial infarction (MI), however, the low retention and survival limit their applications due to the reactive oxygen species (ROS) microenvironment after MI. In this study, the fullerenol nanoparticles are introduced into alginate hydrogel to create an injectable cell delivery vehicle with antioxidant activity. Results suggest that the prepared hydrogels exhibit excellent injectable and mechanical strength. In addition, the fullerenol/alginate hydrogel can effectively scavenge the superoxide anion and hydroxyl radicals. Based on these results, the biological behaviors of brown adipose-derived stem cells (BADSCs) seeded in fullerenol/alginate hydrogel were investigated in the presence of H2O2. Results suggest that the fullerenol/alginate hydrogels have no cytotoxicity effects on BADSCs. Moreover, they can suppress the oxidative stress damage of BADSCs and improve their survival capacity under ROS microenvironment via activating the ERK and p38 pathways while inhibiting JNK pathway. Further, the addition of fullerenol can improve the cardiomyogenic differentiation of BADSCs even under ROS microenvironment. To assess its therapeutic effects in vivo, the fullerenol/alginate hydrogel loaded with BADSCs were implanted in the MI area in rats. Results suggest that the fullerenol/alginate hydrogel can effectively decrease ROS level in MI zone, improve the retention and survival of implanted BADSCs, and induce angiogenesis, which in turn promote cardiac functional recovery. Therefore, the fullerenol/alginate hydrogel can act as injectable cell delivery vehicles for cardiac repair. KEYWORDS: fullerenol, alginate, hydrogel, signal pathway, cardiac tissue engineering
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efficiency and low retention of stem cells in the MI area, which lost to compressive forces and the pulmonary circulation, remain significant challenges in cell-based therapies for treating the injured myocardium.7 In addition, the reactive oxygen species (ROS) microenvironment after MI can induce the oxidative stress damage of retained stem cells and hinder their adhesion to the extracellular matrices (ECM) of the heart tissue by disrupting focal contacts,8 further decreasing the survival of
yocardial infarction (MI), which is widely known as heart attack, is the most prevalent cause of mortality in the world. The current strategies for treatment (e.g., drug therapy and interventional treatment) are ineffective due to the limited regenerative capacity of adult myocardium.1 Recently, many studies2−4 demonstrated that stem cell implantation strategy exhibited potential to treat the MI via secreting paracrine factors. Especially, the discovery of the cardiac stem cells in brown adipose tissue (brown adiposederived stem cells, BADSCs) opened a new method for myocardial regeneration due to their high cardiomyogenic differentiation potential, minimal invasiveness, and large production in cell harvesting.5,6 However, the low delivery © 2017 American Chemical Society
Received: January 11, 2017 Accepted: June 7, 2017 Published: June 7, 2017 5474
DOI: 10.1021/acsnano.7b00221 ACS Nano 2017, 11, 5474−5488
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Scheme 1. Schematic Illustrations of the Effects of Fullerenol/Alginate Hydrogel on the Biological Behaviors of BADSCs in Vitro and Cardiac Repair in Vivo
Fullerenol belongs to a group of antioxidant nanoparticles named fullerene derivatives. Not like other carbon nanomaterials (e.g., carbon nanotube, graphene, etc.), fullerenol exhibits excellent water solubility, which is beneficial to prepare a homogeneous hydrogel.19 Fullerenol nanoparticles have much lower cytotoxicity compared with graphite and single-walled carbon nanotube.20,21 Moreover, ROS can easily adhere to the electron-deficient position on the surface of fullerenol nanoparticles, further the ROS on the adjacent electron-deficient position may induce the destruction of ROS via transferring electrons to the fullerenol cage, resulting in the fullerene derivatives effectively suppressing the oxidant stress damage in cell caused by ROS, which is similar to that of the quenching of superoxide dismutase.22,23 In addition, fullerenol nanoparticles can penetrate the cell membrane, diffuse into cell nucleus, and translocate into organelles due to their small size (∼0.7 nm in diameter),24 further modulating the expression of MAPK signaling proteins (ERK, p38 and JNK) related to stem cell survival, proliferation, apoptosis, and cardiomyogenesis.19,25 However, few studies focus on the effects of fullerenol-based hydrogel on the survival, proliferation, and cardiac differentiation of stem cells under ROS microenvironment in vitro, modulation effects on MI microenvironment and repair effects for damaged myocardium in vivo. The molecular mechanism underlying these effects is controversial. The aim of this study is to develop an injectable hydrogel with excellent antioxidant activity as stem cell delivery vehicles for cardiac repair. A series of fullerenol/alginate hydrogels were prepared via ionic cross-linking. We hypothesized that the fullerenol/alginate hydrogel may suppress the oxidant stress damage in cells and improve the cardiac repair when they were delivered to the heart tissue after MI (cf. Scheme 1). To test this hypothesis, we first investigated whether BADSCs embedded in fullerenol/alginate hydrogels decreased the deterioration in survival, proliferation, and cardiomyogenic differentiation caused by ROS in vitro. To understand the molecular mechanism underlying these effects, the expression of MAPK signal pathways (including ERK, p38 and JNK) and
implanted stem cells. Moreover, the uneven distribution of implanted stem cells in MI area can exacerbate the already disrupted alignment of the interrupting cell−cell communication and further limit the therapeutic efficacy for cardiac repair. The use of an injectable hydrogel as the cell vehicle to deliver stem cells into myocardium can effectively improve the stem cells retention within the MI area.9 Furthermore, the hydrogel can provide the structural support for the left ventricle.10 It is expected that the hydrogel can be the antioxidant agent to remove the ROS of MI microenvironment and further suppress the oxidative stress damage. The ionotropic alginate hydrogel has been widely used as a vehicle for cell delivery to induce the regeneration and function restoration of various tissues,11 owing to its mild gelatin process and the structural resemblance to the ECM. The alginate-based hydrogel can improve the stem cells delivery to MI area and the cell retention (50−62%) from 0 to 24 h is higher compared to directly injected cells.12,13 Previous studies suggested that the injection of alginate hydrogel cannot induce any material deposition or myocardial damage in healthy hearts, moreover, it can temporarily replace the damaged ECM, reverse left ventricular remodeling after MI, and improve the cardiac repair.14,15 However, the adhesion and proliferation of cells in alginate hydrogel is poor due to its high hydrophilicity, for example, Granja et al.16 found that the cell viability of human umbilical vein endothelial cells (HUVECs) seeded in injectable alginate hydrogel rapidly decreased throughout time. It is not conducive to the biological function of play of transplanted cells. In addition, the transplanted cells may suffer to the oxidant stress damage under ROS microenvironment after MI due to the low antioxidant activity. Hydrogel/nanoparticles-based stem cell delivery system can be used to develop advanced hydrogels with antioxidant activity for cardiac repair. Recently, carbon nanotube and graphene have been incorporated into injectable hydrogel to improve the therapeutic efficacies in MI.17,18 Nevertheless, these hydrogel/ nanoparticles-based stem cell delivery systems are lacking consideration of the ROS microenvironments after MI. 5475
DOI: 10.1021/acsnano.7b00221 ACS Nano 2017, 11, 5474−5488
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Figure 1. Preparation and properties of fullerenol/alginate hydrogel. (A) Formation and structure of fullerenol/alginate hydrogel. Scavenging effect (%) of fullerenol/alginate hydrogel on (B) hydroxyl radical and (C) DPPH radicals.
Figure 2. Cytocompatibility of fullerenol/alginate hydrogel. (A) Acridine orange/propidium iodide (AO/PI) staining images of BADSCs seeded in fullerenol/alginate hydrogel at day 3, 7, and 14. (B) Proliferation behaviors of BADSCs seeded in fullerenol/alginate hydrogel within 14 days (*p < 0.05, vs at day 3, #p < 0.05, vs in pure hydrogel at day 7). (C) 3D live cell images in fullerenol/alginate hydrogel after cultured 14 days. 5476
DOI: 10.1021/acsnano.7b00221 ACS Nano 2017, 11, 5474−5488
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Figure 3. Effects of fullerenol/alginate hydrogel on intracellular ROS of BADSCs in the presence of H2O2. (A) Representative images of intracellular superoxide anion radical activity (DHE) and total intracellular ROS (DCFH-DA) at day 3, 7, and 14. Quantification analysis of (B) DHE and (C) DCFH-DA staining. (*p < 0.05 and **p < 0.01 vs pure alginate groups; #p < 0.05, vs10 μg/mL group).
(Figure 1A) in this study. In the fullerenol/alginate aqueous solution, the guluronic acid (G unite) blocks interact with divalent cations (Ca2+) to form ionic bridges among different alginate chains, resulting in the formation of alginate hydrogel. Fullerenol can homogeneously disperse in alginate network due to their excellent water solubility. The storage modulus (G′) are obviously higher than loss modulus (G″) for all hydrogels (Figure S2), confirming their gel nature. The fullerenol/alginate hydrogels exhibit good injectability, their gelation time is 5−10 min. In addition, the introduction of fullerenol does not influence the mechanical strength, their G′ values range from 600 to 1000 Pa, which is similar to other hydrogel used in cardiac tissue engineering. For example, Seliktar et al.27 suggested that both tetronic-fibrinogen (TF) and PEG-
cardiac specific proteins and genes (including cardiac troponin (cTnT), α-sarcomeric actinin (α-actinin), and connexin-43 (Cx-43)) of BADSCs seeded in alginate/fullerenol hydrogel under ROS microenvironment were also analyzed. Then, we investigated whether the fullerenol/alginate hydrogel loaded with BADSCs can modulate MI microenvironment and improve the therapeutic efficacy in vivo.
RESULTS AND DISCUSSION Formation and Characterization of the Fullerenol/ Alginate Hydrogel. Fullerenol is successfully synthesized (cf. Figure S1A,B) according to previous study,26 which shows excellent water solubility (Figure S1C). Calcium gluconate was used as the cross-linker to create fullerenol/alginate hydrogel 5477
DOI: 10.1021/acsnano.7b00221 ACS Nano 2017, 11, 5474−5488
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Figure 4. Effects of fullerenol/alginate hydrogel on the MAPK pathway of BADSCs in the presence of H2O2. (A) Representative Western blot assay for detecting the levels of ERK, p-ERK, p38, p-p38, JNK, and p-JNK of BADSCs. Quantitative analysis of (B) p-ERK expression, (C) pp38 expression, and (D) p-JNK expression. (*p < 0.05 and **p < 0.01 vs pure alginate hydrogel group, ##p < 0.01 vs 10 μg/mL group).
fibrinogen hydrogels with G′ (400−1800 Pa) exhibited the good rescue of heart function. Nikkhah et al.,28 found that the poly(N-isopropylacrylamide)−gelatin-based injectable hydrogel exhibited viscoelastic behavior (G′: 1260 Pa) to properly accommodate the cardiac cells. Moreover, the introduction of fullerenol can significantly improve the antioxidant activity of hydrogel and the antioxidant activity promotes with the increase of fullerenol concentration. As shown in Figure 1B,C, the pure alginate hydrogel shows a certain scavenging capacity against hydroxyl radicals (·OH, 63.8 ± 1.4%) because their hydroxyl groups can act as electron donors to bind ·OH.29 However, their scavenging capacity against 1′-diphenyl-2-picrylhydrazyl radicals (DPPH) is very low (
