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Biological and Medical Applications of Materials and Interfaces

A Conductive Hydrogen Sulfide-releasing Hydrogel Encapsulating ADSCs for Myocardial Infarction Treatment Wei Liang, Jingrui Chen, Lingyan Li, Min Li, Xiaojuan Wei, Baoyu Tan, Yingying Shang, Guanwei Fan, Wei Wang, and Wenguang Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01886 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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ACS Applied Materials & Interfaces

A Conductive Hydrogen Sulfide-releasing Hydrogel Encapsulating ADSCs for Myocardial Infarction Treatment Wei Liang 1, Jingrui Chen 2,3, Lingyan Li 2,3, Min Li 2,3, Xiaojuan Wei 4, Baoyu Tan 1, Yingying Shang 1, Guanwei Fan 2,3,*, Wei Wang 1,*, Wenguang Liu 1,* 1 School of Materials Science and Engineering, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin University, Tianjin, 300072, China 2 First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China 3 Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China 4 Institute of Microsurgery on Extremities, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiaotong University, Shanghai, 200233, China *Corresponding author (Guanwei Fan, Email: [email protected]); Wei Wang, Email: [email protected]; Wenguang Liu, Email: [email protected])

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ABSTRACT Hydrogen sulfide (H2S) exhibits extensive protective actions in cardiovascular system, such as anti-inflammatory and stimulating angiogenesis, but the therapeutic potential is severely discounted by the short half-life and the poorly controlled releasing behavior. Herein, we developed a macromolecular H2S prodrug by grafting 2aminopyridine-5-thiocarboxamide (APTC, a small molecule H2S donor) on partially oxidized alginate (ALG-CHO) to mimic the slow and continuous release of endogenous H2S. In addition, tetraaniline (TA, a conductive oligomer) and adipose-derived stem cells (ADSCs) were introduced to form stem cells-loaded conductive H2S-releasing hydrogel through Schiff base reaction between ALG-CHO and gelatin. The hydrogel exhibited adhesive property to ensure a stable anchoring to the wet and beating hearts. After myocardial injection, longer ADSCs retention period and elevated sulfide concentration in rat myocardium were demonstrated, accompanied by upregulation of cardiac related mRNA (Cx43, α-SMA and cTnT) and angiogenic factors (VEGFA and Ang-1), down-regulation of inflammatory factors (TNF-α). Echocardiography and histological analysis strongly demonstrated an increase in ejection fraction (EF) value and smaller infarction size, suggesting a remarkable improvement of the cardiac functions of SD rats. The ADSCs-loaded conductive hydrogen sulfide-releasing hydrogel dramatically promoted the therapeutic effects, offering a promising therapeutic strategy for treating MI.

2


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KEYWORDS: myocardial infarction, injectable hydrogel, adipose-derived stem cells, conductivity, hydrogen sulfide INTRODUCTION Myocardial infarction (MI) remains the major cause of death and continues to be a difficult problem to overcome in clinic.

1-2

However, the development of effective

therapies is still facing enormous challenge. 3 As a gasotransmitter, hydrogen sulfide (H2S) can speedily diffuse across cell membranes for intercellular signaling without the mechanism of reuptake or degradation. 4-5 It has been proven that the administration of both endogenous and exogenous H2S can produce extensive protective actions such as anti-inflammatory effects, mitochondrial function preservation, vasodilation and protection against oxidative stress in cardiovascular systems.

5-8

Sodha et al. reported

that H2S-treated Yorkshire pigs achieved a remarkable reduction in myocardial infarcted size, an amelioration in fractional shortening (FS), and an enhancement in microvascular reactivity. 9 Moreover, the levels of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha (TNF-α) in cardiac tissue were decreased. 5, 10-11 Thiocarboxamide, one of the most used H2S donors, has been widely explored in clinical trials. 12 Among them, 2-aminopyridine-5-thiocarboxamide (APTC) is an organic thiols-dependent H2Sreleasing compound with a fairly conservative release mechanism.

13

However, the

therapeutic potential is limited by the inherent limitations of small molecule drugs including poor water solubility, high clearance rates and toxicity.

14-15

To circumvent

these limitations, a macromolecular H2S prodrug was developed by grafting APTC on 3



partially oxidized alginate (ALG-CHO) with the aim to reduce toxic side effects of APTC without impairing its H2S-releasing ability in this study. Another challenge for MI therapies is that the conduction of electrical impulse signals between myocardial cells is interdicted after MI. 16-19 Conductive hydrogels may reestablish synchronous contraction and relaxation of the heart by facilitating the conduction of electrical impulse in the infarcted area, which shows benefits to the recovery of cardiac functions.

20-23

In our previous work, the hydrogel crosslinked by

tetraaniline (TA) nanoparticles (NPs) showed the similar conductivity with that of native myocardium and resulted in an obvious upregulation of the expression of connexin 43 (Cx43) in infarcted myocardium. 24 Based on this fact, TA was employed to modify the above mentioned macromolecular H2S prodrugs to form a copolymer (ALG-TA-APTC). The copolymer self-assembled into NPs with TA and APTC as a core through hydrophobic and π-π interactions, which could protect APTC from the attack of thiols to obtain a controllable H2S releasing behavior. Over the past two decades, more studies have identified that stem cells show a significant potential in cardiovascular diseases as regenerative therapy. 25 However, the low cell retention rate in the target area is a significant barrier for the improvement of therapeutic effects because over 90% of the cells are ‘washed out’ (regardless of delivery route and cell type) within a few hours.

26-31

Here, an adhesive injectable

hydrogel was developed with the ability to adapt to the wet and beating conditions. The hydrogel was expected to integrate with myocardial tissue and stay in the target area 4


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for a long time, thus increasing the retention rate of adipose-derived stem cells (ADSCs) encapsulated in the hydrogel. In this study, an ADSCs-loaded hydrogel with properties of conductivity and controllable H2S releasing behavior has been constructed to target the complex symptoms of MI. TA and APTC were grafted on ALG-CHO to endow the system with the ability of conductivity and H2S-releasing. The conductivity of the hydrogel will bridge the electrical pulse conduction between myocardial cells while H2S has the effects of promoting neovascularization and anti-inflammatory. These two factors can effectively improve the unfavorable microenvironment in the MI area. Moreover, the as-formed hydrogel developed by the reversible Schiff base reaction between aldehyde and amine groups has the bio-adhesive ability. It can be tightly integrated with the myocardial tissue, providing good mechanical strengths to adapt to the dynamic cardiovascular condition thus enhancing the ADSCs retention in the MI zone. RESULTS AND DISCUSSION Synthesis and Characterization of ALG-TA-APTC. The increased degradation efficiency of alginate by slight oxidation through sodium iodate (NaIO4) inspires potential applications of alginate hydrogel as a vehicle for drug and cell delivery.

32

Moreover, the uronate groups provide open sites to react with other polymer chains and thus endow the hydrogel with the desired functionalities.

32-33

Here, alginate was

oxidized by NaIO4 to produce ALG-CHO and moderately oxidized ALG-CHO (DO = 42.8%, Mw = 4.2×105 Da, Table S1) was obtained. Then, TA (conductive elements， 5



Figure S1) and APTC (H2S donor) were employed to fabricate a multifunctional copolymer ALG-TA-APTC (Figure S2). The successful synthesis of ALG-TA-APTC was confirmed by 1H NMR (7.00-7.20 ppm, m, H in benzene ring; 8.25 ppm, s, H adjacent to N on the pyridine ring) and FT-IR (1507 cm-1, s, νC=C in benzenoid unit) (Figure S3-S4). To further confirm the chemical structure of ALG-TA-APTC, EDX was used to characterize the elemental composition (Table S2). Based on the content of N and S, the mass fractions of TA and APTC are calculated as 12.2 wt% and 2.6 wt% respectively. It can be seen from the TEM photograph (Figure S5) that the ALG-TAAPTC self-assembles into a NP with TA and APTC as a hydrophobic core and alginate as a hydrophilic shell. The particle size is 320 ± 18 nm according to the DLS measurement (Figure S6). Preparation

and

Characterization

of

ALG-CHO/ALG-TA-APTC/Geln

Multifunctional Hydrogel. Hydrogel was fabricated via covalent cross-linking of ALG-CHO and gelatin. ALG-TA-APTC was embedded to confer electroactivity and H2S-release functions (Figure 1). For in vivo application of an injectable hydrogel, a proper gelation time is important. 24 The gelation time of hydrogels with different ALGTA-APTC concentrations (0 wt%, 2.5 wt%, 5 wt%, and 7.5 wt%) were examined. As observed in Figure 2A and Figure S7, the ALG-CHO/Geln hydrogel can be formed within 41 seconds. The gelation time becomes longer as the concentration of ALG-TAAPTC increases, and the highest concentration (7.5 wt%) leads to the longest (2.5 minutes) gelation time. This feature provides great convenience for the injection in vivo because the hydrogels can maintain sticky and injectable characteristics during the sol6


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gel transition. Movie S1 proves that the ALG-CHO/ALG-TA-APTC/Geln hydrogel can be conveniently injected in PBS solution through a 28-gauge needle. Alginate is slightly oxidized in order to increase its degradation efficiency under physiological condition. Gelatin is selected since it is the main component of the extracellular matrix (ECM) and it can be completely degraded in vivo. 34 The hydrogel exhibits a fast degradation rate during a week in PBS (Figure S8). After 4 weeks in vivo, the hydrogel was fully degraded since no hydrogel was observed in the myocardium from Masson's-trichrome staining sections. XRD and Raman were carried out to analyze the functional structure of the hydrogels. As shown in Figure 2B, the diffraction peak (2θ = 21.5o) of TA can be clearly observed in the XRD pattern of the ALG-CHO/ALG-TA-APTC/Geln hydrogel. Typically, the peak at 1607 cm-1 in the Raman spectra is ascribed to the C=C structure of benzene and pyridine (Figure 2C). The gelation kinetic was assessed through dynamic time sweep tests (Figure 2D). As the content of ALG-TA-APTC increases, the G’ value (storage modulus) increases from 538.7 ± 12.2 Pa to 1085.4 ± 63.6 Pa and G’’ value (loss modulus) increases from 16.1 ± 2.3 Pa to 102.1 ± 9.4 Pa, indicating that the additional ALG-TA-APTC can strengthen the hydrogels. The accurate G’-G’’ crossover can hardly be observed because of the rapid sol-gel transition and the unavoidable time required for loading gel samples. Figure 2E-F show that the as-formed ALG-CHO/ALG-TA-APTC/Geln hydrogels exhibit a soft nature and are very stable under a frequency range from 0.1 to 50 Hz. The microstructure of ALG-CHO/ALG-TA-APTC/Geln hydrogel was further confirmed by SEM. Compared with ALG-CHO/Geln (Figure 2G), there are numerous 7



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spherical NPs on the pore walls of the ALG-CHO/ALG-TA-APTC/Geln hydrogel (Figure 2H-I), indicating the uniform distribution of ALG-TA-APTC NPs. H2S Releasing Profile. APTC is an organic thiols-dependent H2S-releasing compound that exhibits suitable H2S releasing profiles. cytotoxicity because of poor biocompatibility.

14-15

35

However, it may result in

In this study, we developed a

macromolecular H2S prodrug (ALG-TA-APTC) with the aim to reduce toxic side effects of APTC without impairing its H2S-releasing ability.

36-37

ALG-TA-APTC

presents an opportunity to overcome the above-mentioned challenges, but its progress toward clinical applications has been hindered due to an inability to release H2S at a desired location with a controllable rate. Therefore, we incorporate ALG-TA-APTC into an injectable hydrogel to form H2S-releasing hydrogels. H2S-loaded hydrogel allows for site-specific delivery of H2S, which would sharply minimize the required dosage of H2S donor when compared with the systemic H2S administration. The hydrogel was expected to release H2S controllably at an appropriate concentration for cardiac tissue repair. 38-39 The H2S-releasing rate of different samples was determined by Methylene Blue assay as shown in Figure 3A. Two parameters, Cmax (maximal concentration of H2S) and t1/2 (time required to reach 1/2 of the Cmax) were calculated from the H2S-releasing curves (Table S3) in order to describe the release kinetics accurately. Free APTC shows a fast and significant release of H2S when triggered by L-cysteine. In contrast, sustained and controllable release of H2S is achieved in the ALG-CHO/ALG-TA-APTC/Geln 8


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hydrogel group. By encapsulating ALG-TA-APTC into the hydrogel, t1/2 is extended from 3 min to 270 min. More importantly, the hydrogel can release H2S controllably over 24 h (Figure S9). Free APTC in the hydrogel is set as a control, and the H2Sreleasing rate is between that of ALG-TA-ATPC and ALG-CHO/ALG-TA-APTC/Geln hydrogel. These results indicate that double-protection for APTC is achieved by encapsulating the ALG-TA-APTC NPs into the hydrogel. Due to the extremely low concentration and the rapid clearance of H2S, it is difficult to quantify the release of H2S in vivo. In this study, methylene blue method was used to detect the total sulfide concentration to indirectly illustrate the H2S concentration in the myocardium. As exhibited in Figure 3B, the concentration of sulfide in normal myocardium is detected as 0.16 μmol/g. After MI, the sulfide keeps decreasing to a relatively low level of 0.12 μmol/g. In order to verify the ability to release H2S in vivo, H2S-releasing hydrogel was injected into the myocardium. As can be seen from the figure, the hearts injected with ALG-CHO/Geln share the same trend with MI while ALG-CHO/ALG-TA-APTC/Geln

and

ALG-CHO/ALG-TA-APTC/Geln/ADSCs

hydrogel show an increase in sulfide during the first week post MI. After implantation, the H2S-loaded hydrogels can release H2S over a longer period of time. We consider that the different environment (in PBS and in myocardium) causes the different H2Sreleasing behavior. As we all know, the physiological environment is very complicated, and PBS cannot exactly mimic the myocardial environment. Therefore, different H2Sreleasing profile between PBS and myocardium is reasonable. The in vitro release profile of H2S was measured in the presence of excess cysteine. Whereas, the limited 9



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access of the H2S donor to the thiol trigger led to slower and more sustained H2Sreleasing behavior after implantation. Electroactivity of the Hydrogel. After MI, the process of converting electrical signals into contractile coupling in scar tissue is interrupted.

20

It is reported that

conductive biomaterials can be used to synchronize contractions by connecting isolated cardiac cells. 40 Thus, we designed a conductive injectable hydrogel with TA acting as the conductive element for treating acute MI. Firstly, the electroactivity of ALG-TAAPTC is detected by UV spectroscopy (Figure S10). TA (the blue solid line) shows a single peak at 325 nm, which is attributed to the π-π* transition of the benzene ring. Upon doping with HCl, another peak appears at 430 nm, which is the polaron absorption peak (the blue dotted line). This is because the protonation band of the TA backbone increases the conjugation length.41 In the case of ALG-TA-APTC, a new peak appears at 422 nm when doped with HCl, indicating that the electroactivity of the copolymer originates from TA. The good electroactivity of the hydrogel was confirmed by both Four-probe and Cyclic Voltammogram. As shown in Table 1, the enhanced conductivity is attributed to the loading of ALG-TA-APTC. The ALG-CHO/ALG-TAAPTC (7.5%)/Geln hydrogel has the highest conductivity (G = 3.7 ± 0.3×10-4 S/cm) which is consistent with the conductivity of native myocardium (from 5×10-5 to 1.6×103

S/cm). The hydrogel with such conductivity value is appropriate for cardiac repair. 42

When aniline oligomers are treated by oxidizing and reducing agents as well as different voltages, they will experience three different oxidation states.

43

To confirm the

conductivity of the hydrogels, Cyclic Voltammogram was employed. With the changes 10


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of the voltage, the TA segments in the hydrogel undergo two oxidation transitions (Figure S11A), suggesting a good conductivity of the hydrogel, which is similar to that of TA (Figure S11B). Based on these results, the ALG-CHO/ALG-TA-APTC (7.5%)/Geln hydrogel will be employed for the further study. Adhesive Strength of the Hydrogels. Hydrogels with tissue adhesiveness can be tightly integrated with the tissue, providing good mechanical strength to adapt to the moist and dynamic conditions.

43-44

The aldehyde groups in ALG-CHO/ALG-TA-

APTC/Geln hydrogels can react with the nucleophilic groups such as amino groups in the tissue, resulting in an outstanding tissue adhesion.

44-47

Here, a universal test

machine (UTM) was used to assess the adhesive strength of the hydrogels through lap shear tests. Figure 4A-B reveals that the adhesive strength of the ALG-CHO/ALG-TAAPTC (7.5%) /Geln hydrogel is 5.3 kPa, which is slightly higher than that of fibrin glue adhesive (Greenplast®) (about 5 kPa). 48 The binding ability of the hydrogels to tissues was further evaluated by cutting the chicken heart in half to expose the injected hydrogel. As shown in Movie S2, the hydrogel can endure violent water flushing and adhere firmly to the myocardium without detachment. Moreover, the hydrogel can adhere to the rubber gloves and endure a large and fast stretching (Figure 4C, Movie S3). We also compared the calcium cross-linked alginate hydrogels (ALG/Ca2+) with the ALG-CHO/ALG-TA-APTC/Geln hydrogel in adhesion difference. The former slides down the substrate surface quickly while the latter remains adherent to the surface (Figure 4D, Movie S4). Further, the excellent binding strength and the ability to bear a considerable mechanical load of our hydrogel were examined by dynamic tensile tests. 11



The rate was 1 HZ which is the heartbeat frequency of adult human. 100 μL of ALG/Ca2+ (blue) and ALG-CHO/ALG-TA-APTC/Geln hydrogel (black) were injected into the chicken myocardium respectively. Immediately after the start of the stretching, the ALG/Ca2+ is extruded from the pinhole (Figure 4E), while ALG-CHO/ALG-TAAPTC/Geln hydrogel remains intact and fixes to the injection site after 1×104 cycles of stretching (Figure 4F, Movie S5). All the results indicate that the covalently crosslinked hydrogel is competent in maintaining their structural integrity and achieving retention in the dynamic environment of the heart. In Vivo Cell Retention. To verify if an adhesive injectable hydrogel can increase cell retention, the fluorescence intensity (FI) was measured by In Vivo Imaging System (IVIS) 15 min, 1 day and 3 day after the operation. As shown in Figure 5, 15 min after ADSCs are injected into the MI heart, the FI values of the two adhesive hydrogel groups (297.6 ± 8.3 × 106 and 306.2 ± 10.8 × 106 respectively) are stronger than those of the PBS (202.7 ± 8.3 × 106) and ALG/Ca2+ (226.1 ± 10.5 × 106) group. The high initial retention is achieved because the hydrogels can be tightly bind to the tissue during the sol-gel transition so as to retain structural stability for a long time. The fluorescence gradually diminishes over time. Actually, CM-DiI signal was still detected 7 days after injection though the intensity was extremely weak (only 0.13% for ALG-CHO/Geln and 0.22% for ALG-CHO/ALG-TA-APTC/Geln remained). This means very few injected cells still persist in the recipient hearts and the adhesive hydrogel can increase the retention rate of ADSCs.

12


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Evaluation of Heart Functions. Before in vivo injection, the cytotoxicity of the hydrogel and cell proliferation were evaluated. The ALG-CHO/ALG-TA-APTC/Geln hydrogel exhibits negligible toxicity to ADSCs and L929 cells, and shows the ability to promote cell proliferation (Figure S12-13). After confirming the successful establishment of the multifunctional hydrogel, the therapeutic effects of the hydrogel on acute MI was investigated. 100 rats were randomly assigned to five groups: (I) Sham; (II) MI; (III) ALG-CHO/Geln; (IV) ALGCHO/ALG-TA-APTC/Geln; (V) ALG-CHO/ALG-TA-APTC/Geln/ADSCs. The multifunctional hydrogel does not have toxic side effects on the heart since the survival rate has no significant difference with other groups (Table S4). Global left ventricle (LV) functions were evaluated by echocardiography on 7, 14, 21, 28 days after the surgery (Figure 6A, Table S5). In order to verify if hydrogels, H2S and stem cells can help restrain LV remolding and restore heart functions, the hydrogels of group III, IV and V are compared with MI (A) group. Notably, the decrease of Ejection fraction (EF) (Figure 6B) and Fractional shortening (FS) (Figure 6C) as well as the increase of Enddiastolic volume (EDV) (Figure 6D) and End-systolic volume (ESV) (Figure 6E) of all the hydrogel groups are effectively suppressed compared to MI group. These results mean the injectable hydrogels can reduce LV cavity size, restore LV geometry, and improve LV functions. Then, we compared the difference of ALG-CHO/Geln, ALGCHO/ALG-TA-APTC/Geln

and


in

enhancing EF values. Surprisingly, ALG-CHO/ALG-TA-APTC/Geln hydrogel (EF = 53.9 ± 5.9 %), with both H2S releasing and improved electrical conductivity leads to 13



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higher EF value, but does not reach statistical significance than ALG-CHO/Geln hydrogel group (EF = 45.3 ± 10.4 %). However, ALG-CHO/ALG-TAAPTC/Geln/ADSCs hydrogel (EF = 66.6 ± 11.7 %) performs much better in enhancing EF value than ALG-CHO/Geln hydrogel. A probable reason is that stem cells are primarily responsible for the improvement in cardiac functions. Stem cell therapy has been widely explored as a potential cure which can not only inhibit the trend of heart failure, but also promote the regeneration of myocardium.49 In this study, ADSCs were employed to reconstruct cardiac functions and promote myocardial regeneration. Although the LV functions of all the hydrogel groups cannot be fully recovered, the occurrence of heart failure is effectively hindered especially in the ALG-CHO/ALGTA-APTC/Geln/ADSCs group. All of the above analyses demonstrate that the conductive H2S-releasing hydrogels bearing ADSCs promoted the cardiac functions. Effects of the Multifunctional Hydrogel on LV Remodeling. MI occurs upon occlusion of a coronary artery, with the consequent reduction of the blood flow to the myocardium. The prolonged ischemic anoxia results in a mass death of cardiomyocytes (CMs), accompanied by an inflammatory response and cardiac fibrosis.

50

Then, LV

remodeling occurs with regional myocardial wall thinning and progressive ventricular dilation. Restraining the adverse-remodeling and improving the heart functions are the key to decrease the incidence of heart failure. Masson and Sirius red staining were employed to determine the positive effects of the injectable hydrogels on cardiac fibrosis and morphology. As shown in Figure 7A-B, the blue collagen deposition area is obvious in the MI group after 4 weeks, demonstrating a severe fibrosis of the 14


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myocardium. The fibrotic area of MI hearts treated by our multifunctional hydrogels decreases sharply, and the effect is more obvious in the stem cells group. A ventricular wall thinning is observed in MI group as well as non-functional hydrogel group, but becomes more mitigatory in multifunctional hydrogel group and stem cells group. The Sirius red staining (Figure 7C-D) and the quantitative data (Figure 7E-F) further prove the above analysis. The fibrosis area decreases from 49.7% to 21.8%, and the left ventricular wall thickness increases from 1.1 mm to 2.5 mm compared to MI group. All these results testify that the conductivity, H2S and ADSCs collectively restrain the LV remolding and promote the reconstruction of cardiac functions. Protective Role of the Multifunctional Hydrogel against MI by RT-PCR. H2S has protective actions against myocardial infarction through various physiological functions,

5, 10-11

such as relaxing blood vessels, anti-inflammatory

angiogenesis etc.8,

51

6

and stimulating

In addition, H2S has been reported to generate numerous

cytoprotective effects. It was demonstrated that H2S-releasing materials are able to enhance the proliferation of mesenchymal stem cells (MSCs) and cardiomyocytes (CMs).

52-53

In order to confirm the positive effects of H2S-releasing hydrogel on

treating MI, the expression of inflammatory factors (TNF-α) and proangiogenic growth factors (VEGFA and Ang-1 mRNA) in the myocardium were determined by RT-PCR. As shown in Figure 8A, ALG-CHO/ALG-TA-APTC/Geln/ADSCs hydrogel can dramatically reduce the expression of TNF-α and increase the expression of VEGFA and Ang-1 compared with MI group and other hydrogel groups (Figure 8B-C). That’s probably because H2S can effectively reverse the unfavorable microenvironment and 15



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increase cell survival in the MI area. Based on the above analysis, it can be concluded that H2S-releasing hydrogels can inhibit inflammatory response and promote angiogenesis thus contributing to the restoration of cardiac functions.54 The Cx43 is the main connexin that contributes to the intercellular connection of myocardial cells.55 To determine the specific effects of electroactivity during MI, the expression of Cx43 was detected. As convincingly revealed in Figure 8D, the level of Cx43 related mRNA is significantly up-regulated by the multifunctional hydrogels. For the evaluation of the heart functions after different treatments, α-SMA and cTnT are selected as cardiac related factors (Figure 8E-F). The expression of α-SMA and cTnT can hardly be found in the MI rats, while the expression of these two mRNA is enhanced after hydrogel injections

especially

in

the


group,

suggesting a significantly improvement of the heart functions. CONCLUSION In order to reverse the adverse-remodeling and improve heart functions, ADSCsloaded conductive H2S-releasing hydrogel system was elaborately designed to address the tough challenge of MI therapy. The conductivity and hydrogen sulfide-releasing properties effectively improved the harsh microenvironment in the myocardial infarction zone. The adhesive property derived from aldehyde groups ensured the ability to maintain structural stability of the hydrogel in the beating heart thus enhancing the retention of loaded stem cells. The multi-functional hydrogels system shows a promising therapeutic strategy for treating MI. 16


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EXPERIMENTAL SECTION Materials. Gelatin (medium gel strength, 170-195 g Bloom), sodium alginate (low viscosity, 4-12 cP), N-phenyl-p-phenylenediamine (AR, 98%), L-cysteine (L-cys, AR, 97%), sodium periodate (NaIO4, AR, 99.8%) and 2-aminopyridine-5-thiocarboxamide (APTC, AR, 97%) were purchased from Sigma-Aldrich. Ethylene glycol (AR, 98%), calcium carbonate (CaCO3, AR, 99%), glucono-delta-lactone (GDL, AR, 98%) and chloral hydrate were purchased from Heowns Biochem Technologies LLC. Iron chloride (FeCl3, AR, 98%,) was supplied by Aladdin Industrial Inc. Dimethyl sulfoxide (DMSO, AR, ≥99.5%), N, N-dimethylformamide (DMF, AR, ≥99.5%) and absolute ethanol (AR, ≥99.5%) were purchased from Tianjin Yuanli Chemical Co., Ltd. 3HIndolium,

5-[[[4-(chloromethyl)benzoyl]amino]methyl]-2-[3-(1,3-dihydro-3,3-

dimethyl-1-octadecyl-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-1-octadecyl-, chloride (CellTrackerTM CM-DiI-special packaging) was purchased from Molecular Probes. Hydrogen sulfide detection kit was provided by Nanjing Jiancheng Bioengineering Institute. Synthesis and Characterization of ALG-TA-APTC. The synthesis route of ALGCHO and TA followed the previous reports.

56-57

Successful synthesis of TA is

confirmed by 1H NMR spectrum. The molecular weight (Mw) of sodium alginate is negatively correlated with the degree of oxidation (DO) and the DO is determined by hydroxylamine hydrochloride-potentiometric titration. The ALG-TA-APTC was synthesized by the Schiff base reaction between aldehyde and amino under mild 17



conditions. Briefly, 0.5 g (2.12 mmol aldehyde) ALG-CHO was dissolved in 5 mL deionized water, and then 0.46 g (1.27 mmol) TA in 3 mL DMF was added dropwise and stirred for 4 h in a nitrogen atmosphere. Afterwards, 0.049 g (0.32 mmol) APTC in 4 mL DMSO was added dropwise and stirred for another 2 h at 50 oC in a nitrogen atmosphere. The reaction mixture solution was precipitated with cold ethanol four times to yield a dark blue solid of ALG-TA-APTC. The chemical structure of ALG-TAAPTC was determined by UV-Vis spectrum (200-800 nm), 1H NMR (500 MHz, D2O, VARIAN INOVA), Fourier transform infrared (FTIR, Nicolet 6700), and Energy dispersive X-ray spectroscopy (EDX). The self-assembly of ALG-TA-APTC was detected by Transmission electron microscope (TEM, JEM-2100F) and the particle size distribution of the self-assembled ALG-TA-APTC was analyzed by dynamic light scattering (DLS, Nano ZS). Preparation and Characterization of the Multifunctional Hydrogel. Typically, the ALG-CHO/ALG-TA-APTC (7.5%)/Geln hydrogel was prepared by the following procedure: 50 µL of ALG-CHO (12 wt%), 50 µL of ALG-TA-APTC (30 wt%), and 100 µL of gelatin (6 wt%) were uniformly mixed to form a well-proportioned solution and it would gel within 2.5 min. Other hydrogels were prepared following the same process with different feed ratios. The gelation time of the hydrogels was measured by observing the stop moving of a spinning magnetic bar (n = 5). 24 The crystal structure of TA and APTC in the ALG-CHO/ALG-TA-APTC/Geln hydrogel was detected by Xray diffraction spectroscopy (XRD, 5 - 40o, 4o/min). The chemical structure of the hydrogel was characterized by Raman spectrometer (Raman, 633 nm, DXR) in the 18


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range of 500 - 3000 cm-1. Scanning electron microscope (SEM, JSM-7800F) was employed to detect the microscopic morphology of the hydrogels after lyophilization. Mechanical Properties. Dynamic rheological measurements were performed on a rheometer (TA, DHR-2, USA). For time-sweep tests, the frequency was set as 1 Hz and the strain was set as 5% to ensure a linear oscillatory deformation. The tests were carried out at a temperature of 37 oC over a time range from 0 to 10 min. Dynamic sweep tests in a frequency mode (temperature: 37 oC, strain: 5%, frequency range: 0.1 to 50 Hz) were performed to determine the dynamic behaviors of the hydrogels. In Vitro Degradation. A mass loss measurement was used to examine the degradation rate of the hydrogel. Hydrogels with initial known dry masses (10 mg) were soaked into 10 mL of PBS solution at 37 oC. At prescribed time points, the hydrogels (n = 5) were dried and weighted on a microbalance, and then the relative mass loss was calculated. H2S Releasing Profile. The release rate of H2S was determined by Methylene Blue assay. Typically, H2S released from APTC was measured as follows: 5 mg of APTC was dispersed in 50 ml of PBS (pH = 7.4) (0.65 mM) to form a uniform suspension, and the solution was purged vigorously with nitrogen gas for 15 min. Then, 100 µL of L-cys was added into the closed flask to ensure a final concentration of 6.5 mM and the reaction was triggered immediately. At scheduled time points, 250 µL of the solution was withdrawn for Methylene Blue tests. The same volume of PBS was added into the flask immediately to avoid the accumulation of free H2S gas. The other three samples 19



were measured by the same method as described above. The total concentration of sulfide in the myocardial tissue was measured to determine the release of H2S in vivo. Briefly, on 1, 7, 14, 28 days after MI, rats were anesthetized and sacrificed. Saline solution was used to clean the fresh rat hearts to remove residual blood. The infarction areas were cut and weighted with a microbalance, and then homogenized for the Methylene Blue tests. Electroactivity Test. The conductivities of the hydrogels with different ratios were measured via four-point probe method (ST2253, China). Cyclic voltammetry tests were used to detect the electroactivity of the ALG-CHO/ALG-TA-APTC/Geln hydrogels. Electrochemical workstation (CHI660D, China) with a three-electrode system was used for the measurement. TA and the ALG-CHO/ALG-TA-APTC (7.5%)/Geln hydrogel were spread on ITO conductive glasses to form conductive films. Then CV detection was performed with the conductive films as a working electrode, Ag/AgCl as a reference electrode and a Pt sheet as a counter electrode respectively, and with 1 M HCl as electrolyte solution. The voltage was in the range of 0-1 V and the scan rate was set at 100 mV/s. Tissue Adhesive Test. The adhesive ability of ALG-CHO/ALG-TA-APTC/Geln hydrogels to the host tissue was measured by lap shear tests through a universal test machine (UTM, LEGENG 2344). Fresh porcine myocardium tissue was cut into rectangular pieces with 3 cm × 1 cm size. The hydrogel (200 μL) was applied as an interlayer, which was sandwiched between the two pieces of the porcine myocardium 20


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tissue. The overlapping area of the hydrogel and the two pieces of tissue was set as 1 cm ×1 cm and the samples were kept at 37 oC for 15 min under a humid condition to allow a complete curing. The tensile rate was set at 5 mm/min and the maximal detachment stress of the hydrogel from the tissue was regarded as the adhesive strength (n = 6). In Vivo Cell Retention. ADSCs (5×105 cells) labeled with CM-DiI were loaded into 100 μL of hydrogel (or 100 μL of PBS) and injected into MI hearts. Four different vehicles (PBS, ALG/Ca2+, ALG-CHO/Geln and ALG-CHO/ALG-TA-ATPC/Geln) were employed to compare their ability to retain stem cells after injection. The rat hearts were harvested 15min, 1 d and 3 d after the operation, and the fluorescence intensity was measured by In Vivo Imaging System (PerkinElmer, IVIS Lumina II). In Vivo Hydrogel Injection. Male Sprague-Dawley rats (SD, 220 ± 20 g) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The adipose-derived stem cells (ADSCs, OriCellTM) were taken from the inguinal fat of SD rats. Flow cytometry identified CD44, CD90, CD29 positive (> 70%), CD34, CD11b, CD45 (< 5%). All of the animal experiments were performed according to the rules and regulations of Tianjin Biomedical Engineering Institute. Animal suffering were minimized during the operation. The LAD (left anterior descending) MI rat model was established according to the previous studies.

58

100 rats were randomly assigned to

five groups: (I) Sham; (II) MI; (III) ALG-CHO/Geln; (IV) ALG-CHO/ALG-TAAPTC/Geln; (V) ALG-CHO/ALG-TA-APTC/Geln/ADSCs. The cell suspension was 21



uniformly mixed with the precursor solution and injected into the rat heart to form a hydrogel in situ. Another 60 rats were suffered to detect the retention of ADSCs in MI hearts. During the surgery, 16 rats died after MI, and 10 rats died the day after the experiments. Thus, 134 rats remained for the final analysis. Hydrogel injection procedure was described as follows: after ligation, 100 µL of hydrogel or PBS was immediately injected into three areas adjacent to the MI area with a 28-gauge needle, and the rat was subjected to a chest seal immediately and placed on a hot plate. Echocardiographic Assessment of Left Ventricular Function. Echocardiography was obtained using an echocardiograph (Vevo 2100 Imaging System, Visual Sonics, Canada). The left ventricular functions of the rats were examined by echocardiography on 7, 14, 21 and 28 days after MI referring to the specific procedure reported previously. 24

The LV end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction

(EF), fractional shortening (FS) were measured as indicators of LV function and remodeling. All indicators were calculated as the average of five consecutive cardiac cycles. Histological Evaluation. At postoperative 4 weeks, the rats were anesthetized and sacrificed and the hearts were harvested. The heart samples were fixed with 4% paraformaldehyde at 4 oC for 48 h. Then, all the hearts were dehydrated, embedded and cut into 5-µm thick sections for routine Masson's-trichrome staining and Sirius Red staining. Myocardial fibrosis and morphology were assessed and the proportion of infarction and LV wall thickness were measured by Image J software. 22


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Quantitative Real-time RT-PCR. The rats were anesthetized and sacrificed 1, 7, 14, 28 days after MI. The hearts were harvested and frozen in liquid nitrogen. The total RNA was extracted in strict accordance with HiPure Plant RNA Mini Kit (R4151-03, MAGEN). UV spectrophotometer was used to determine the RNA concentration. The total RNA (1 µg) was reversely transcribed to cDNA according to protocol. After the reaction was completed, the cDNA was diluted 5 times with sterile deionized water and stored at -20 °C. Expressions of TNF-α, VEGFA, Ang-1, Cx43, α-SMA and cTnT were detected (primer sequences are shown in Table S6). Statistical Analysis. The data in the experiment were shown as mean ± SD (at least n = 3). Statistical analysis was accomplished by Student's t-test. The difference was considered to be statistically significant for p < 0.05. ASSOCIATED CONTENT Supporting Information NMR, FT-IR, TEM, DLS, EDX and UV-Vis spectra of the copolymer, oxidation degree of sodium alginate, the gelation process, degradation curves, CV curves and cytotoxicity of hydrogels, ADSCs proliferation, statistical data on cardiac function, primers sequences used for real-time PCR. AUTHOR INFORMATION Corresponding Authors *Email: [email protected] (G.-W. F.). 23



*Email: [email protected] (W.W.). *Email: [email protected] (W.-G. L.). ORCID Wen-Guang Liu: 0000-0002-7776-9622 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS The authors gratefully acknowledge the support for this work from the Excellent Young Scientists Fund by National Natural Science Foundation of China (No. 31822020), National Natural Science Foundation of China (Grant No. 51473117, 31771030, 31870965), National Key Research and Development Program (Grant No. 2016YFC1101301, 2018YFC1105604), and Tianjin Outstanding Youth Science Foundation (17JCJQJC46200).

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Figure Captions Figure 1. Schematic illustration of the ADSCs-loaded conductive H2S-releasing hydrogel for cardiac repair. Figure 2. Characterization of ALG-CHO/ALG-TA-APTC/Geln hydrogels. A) Gelation time of different hydrogel samples; B) XRD spectra of TA powder, APTC powder, and ALG-CHO/ALG-TA-APTC/Geln hydrogel; C) Raman spectra of pure TA powder, APTC powder, and ALG-CHO/ALG-TA-APTC/Geln hydrogel; D) Time sweep analysis of hydrogels at 37 oC; E) Frequency-sweep measurements of the hydrogels at 37 oC over a frequency range from 0.1 to 50 Hz; F) Elastic modulus G’ of different samples; G) SEM images of ALG-CHO/Geln hydrogel, scale bar: 10 µm; H, I) SEM images of ALG-CHO/ALG-TA-APTC/Geln hydrogels, scale bar: 30 µm, 10µm and 1µm (inset). Figure 3. A) H2S releasing profile in vitro; B) Sulfide concentration of the cardiac tissue in MI area (* indicates a significant difference between the experimental group and PBS group, *p

A Conductive Hydrogen Sulfide-releasing ... - ACS Publications

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