L-Arginine-Loaded Gold Nanocages Mitigates Myocardial Ischemia/Reperfusion Injury by Promoting NO Production and Maintaining Mitochondrial Functions

doi:10.21203/rs.3.rs-1889805/v1

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L-Arginine-Loaded Gold Nanocages Mitigates Myocardial Ischemia/Reperfusion Injury by Promoting NO Production and Maintaining Mitochondrial Functions

https://doi.org/10.21203/rs.3.rs-1889805/v1

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Nitric oxide (NO) has the potential to ameliorate myocardial ischemia/reperfusion injury (MI/RI), and has emerging as key molecular for drug design. However, NO can be oxidized to more toxic peroxynitrite in the presence of reactive oxygen species (ROS), and cause secondary damage to cardiomyocytes. Herein, L-Arginine-loaded selenium-coated gold nanocages (AASP) were designed and synthesized. AASP after PCM modification achieved cardiomyocytes-targeted recognition, enhanced cellular uptake and photoacoustic imaging in vitro and in vivo, which significantly inhibited oxygen glucose deprivation/re-oxygenation (OGD/R)-induced H9C2 cells cytotoxicity and apoptosis. Mechanism investigation revealed that Nano-selenium firstly exhausted ROS, which prevented NO oxidation. L-arginine (Arg) loaded in gold nanocore was catalyzed by nitric oxide synthase (eNOS) to produce NO, which in turn blocked ROS release by regulating mitochondrial permeability transition pore (mPTP) closing, which maintained mitochondria functions, inhibited apoptosis. AASP administration in vivo effectively improved myocardial functions and vascular remodeling, inhibited myocardial apoptosis and fibrosis, and eventually attenuated MI/RI of rats. Transcriptomic analysis revealed that AASP relieved MI/RI of rats mainly by maintaining mitochondrial functions and regulating NO signal. Moreover, AASP showed good safety and biocompatibility in vivo. Taken together, our findings validated the rational design that L-Arginine-loaded selenium-gold nanocages ameliorated MI/RI of rats by promoting nitric oxide production and maintaining mitochondrial functions, which could be a more efficient way to treat MI/RI.

myocardial ischemia/reperfusion injury

L-Arginine

nitric oxide

reactive oxygen species

mitochondrial functions

myocardial functions

Cardiovascular disease is the leading cause of death worldwide, and ischemic heart disease accounts for approximately 50% of all deaths¹. Coronary artery occlusion leads to the development of myocardial ischemia, and results in myocardial cell death and fibrotic scarring^2,3. Clinical strategies include vasodilation and reperfusion therapy. Although reperfusion of ischemic myocardium is critical for survival, it also triggers myocardial ischemia/reperfusion injury (MI/RI), including oxidative damage, inflammatory response, cell death, and dilated cardiomyopathy, which are lack of effective clinical solution^4–7.

Mesenchymal stem cells and pluripotent stem cells used for cardiac regenerative therapy have attracted great interest^8–11. Numerous studies have found that it is very difficult to differentiate adult stem cells into cardiomyocytes sufficient for treatment¹². These cells promote cardiac regeneration, inhibit fibrosis and inflammation mainly through multiple factors secreted by paracrine action. However, cell therapy has its limitations, such as low viability of ischemic cardiomyocytes and slow efficacy. Biotherapeutics, including cell therapy, small molecule drugs and growth factors, are not ideal for restoring cardiac function and preventing adverse remodeling of the heart muscle^13–15. Therefore, there is great needs for new therapeutic approaches that can simultaneously reduce apoptosis of damaged cells, attenuate inflammatory responses and prevent adverse remodeling.

Nanotechnology holds great promise for the development of MI/RI therapeutic strategies. Recently, nano-drugs development that target the heart after ischemic injury has received increasing attention^16–18. Gold nanocages are a new type of metal nanoparticles with hollow, surface mesoporous structure and plasmonic properties. Gold nanocages can be effectively used for the encapsulation, delivery and release of drug molecules under specific conditions. Such nanoparticles have good light scattering and absorption capabilities in the near-infrared region and are widely used in photoacoustic imaging and photothermal therapy research^19–23. PCM is a cardiomyocytes-targeted peptide screened by phage display technology, which can specifically recognize tenascin X (TNX) on the surface of cardiomyocytes and has been widely used in modifying drugs and drug carriers^24,25.

Nitric oxide (NO), a multifunctional signaling molecule, plays an important role in the regulation of cardiovascular homeostasis. L-arginine (L-Arg) was catalyzed by nitric oxide synthase in the presence of O₂ and NADPH. NO can reduce MI/RI by relaxing vascular tone, inhibiting platelet aggregation, and regulating inflammatory responses^26–29. Zhu et al. found that NO inhibited the activity of complex I in the mitochondrial electron transport chain, thereby limited the generation of mitochondrial ROS, further prevented the opening of the mitochondrial permeability transition pore (mPTP), reduced the release of cytochrome c and cardiomyocyte apoptosis³⁰. However, NO reacts rapidly with overproduced reactive oxygen species (ROS) in MI/RI myocardial tissue to generate peroxynitrite, which further damages the tissue^{31, 32}. Therefore, cascading ROS inhibition and NO generation will be the key factors for this therapeutic strategy. Selenium displays novel antioxidant properties and also acts as a component of glutathione peroxidase. Selenium deficiency can lead to a significant decrease in glutathione peroxidase activity, and result in increased oxidative stress^33–35. Nano-selenium (Se NPs) has been widely studied for its higher antioxidant activity, lower toxicity and easier modification. Li et al. confirmed that 11-mercapto-1-undecanol-modified selenium nanoparticles had free radical scavenging activity and antagonized cisplatin-induced nephrotoxicity³⁶. However, little information about the protective effect of Se NPs on MI/RI is available. Hence, design of novel selenium nanocomposites for therapy of MI/RI is of great significance.

In the present study, PCM-modified selenium-coated gold nanocages (AASP) were designed and synthesized. AASP after PCM modification achieved cardiomyocytes-targeted recognition, enhanced cellular uptake and photoacoustic imaging in vitro and in vivo. Nano-selenium firstly exhausted ROS, which prevented NO oxidation. L-arginine (Arg) loaded in gold nanocore was catalyzed by nitric oxide synthase (eNOS) to produce NO, which in turn blocked ROS release by regulating mitochondrial permeability transition pore (mPTP) closing (Scheme 1). AASP in vitro significantly blocked oxygen glucose deprivation/re-oxygenation (OGD/R)-induced H9C2 cells apoptosis by inhibiting oxidative damage and mitochondrial dysfunction. AASP administration in vivo effectively improved myocardial function and vascular remodeling, inhibited myocardial apoptosis and fibrosis, and eventually attenuated MI/RI of rats by inhibiting oxidative damage and regulating NO signal. Our findings validated the rational design that PCM-modified selenium-coated gold nanocages to cascade ROS inhibition and NO generation could be a highly efficient way to achieve targeted heart repair after MI/RI.

Synthesis and characterization of nanosystems

A graphical abstract for elucidating the synthetic route and the underlying mechanism was firstly presented (Fig. 1A), and our findings validated the rational design that L-Arg-loaded selenium-gold nanocages had the potential to ameliorate myocardial ischemia/reperfusion injury by promoting nitric oxide production and maintaining mitochondrial functions. TEM and SEM characterization results showed that hollow gold nanocages with diameters of 100 nm ± 12 nm and wall thicknesses of 7 ± 1.4 nm were prepared through the electro-displacement reaction between HAuCl₄ and Ag nanocubes. Layer-by-layer self-assembly of cardiomyocyte-targeted peptides and nano-selenium on the surface of gold nanocages prepared AASP with a diameter of 150 ± 13 nm (Fig. 1B-F and S1A). Elemental analysis confirmed that Au and Se elements were uniformly distributed in AASP (Fig. 1G). Similar to the TEM and SEM results, the success of each surface modification of AASP was accompanied by an increase in size and an alternation of positive and negative Zeta potential (Figure S1C and S1D). The chemical structure of AASP was verified by UV-vis-NIR and FTIR spectroscopy techniques. As shown in Fig. 1H, the characteristic absorption peaks at 900 nm and 275 nm in the AASP spectrum originate from the red-shift of the Au nanocages absorption peaks and the intersection of PCM and nano-selenium absorption peaks, respectively. As shown by the FTIR spectrum (Fig. 1I) of AASP, the N-H bending vibration absorption peak at 1560 cm^− 1 and the C = O stretching vibration absorption peak at 1750 cm^− 1 are derived from L-arginine and PCM. The crystal structure of the AASP was further determined by XRD (Fig. 1J), showing the same diffraction peaks as those of the trigonal Se (JCPDS No. 73–0465), the cubic Au (JCPDS No. 04-0784) and the oblique phase crystals Ag3AuSe2 (JCPDS No. 72–0392). The XPS analysis of Se3d in AASP showed binding energy peaks of 54.63, 54.93, 55.38 and 55.88 eV, attributed to Se²⁻3d_5/2, Se⁰3d_5/2, Se²⁻3d_3/2 and Se⁰3d_3/2, respectively. Au4f with its binding energy peaks of 83.78, 84.13, 87.48 and 87.88 eV were attributed to Au⁰4f_7/2, Au¹⁺4f_7/2, Au⁰4f_5/2 and Au¹⁺4f_5/2, respectively (Fig. 1K and 1L). The modified amounts of L-arginine and PCM in AASP were calculated by the regression equation established by UV spectrophotometry to be 119.2 µg/mg and 21.5 µg/mg, respectively (Figure S1E and S1F). The crystal violet spectrophotometry in Fig. 1M confirmed that AASP concentration-dependently removed the ROS generated by the Fenton reaction, exhibiting a loss of surface selenium nanoparticles and a decrease in particle size (Figure S1B). Subsequently, L-arginine in AASP was released continuously and reached equilibrium within 24 h (Figure S1G). The results of dimensional stability and kinetic stability studies of AASP showed that the nanoparticles in pure water, PBS or DMEM medium containing 10% fetal bovine serum (FBS) still had good dispersibility within 72 h (Figure S1H). In this study, gold nanocages can not only serve as drug release carriers but also as contrast agents to achieve targeted photoacoustic imaging of the heart (Fig. 1N). These results indicated that PCM-modified L-Arg-loaded selenium-gold nanocages was successfully designed and synthesized.

AASP attenuated OGD/R-induced H9C2 cytotoxicity

Cellular uptake of AASP nanoparticles in H9C2 cells was initially assessed. As shown in Fig. 2A, H9C2 cells exposed to 30 µg/mL AASP for 8 h showed bright green fluorescence in cytoplasm, indicating that AASP can effectively across the cell membrane and accumulate in H9C2 cells. The real-time fluorescence quantitation further confirmed the time-dependent intracellular uptake of AASP (Fig. 2B). The intracellular fluorescence of SSAP reached the peak at 6 h, and showed better effect than that of AAS, revealing that PCM modification achieved cardiomyocytes-targeted recognition and enhanced the intracellular uptake of AASP. Figure 2C showed that free PCM competitively combined with tenascin X (PCM receptor), and dose-dependently blocked AASP accumulation in H9C2 cells, which directly confirmed the cardiomyocytes-targeted accumulation of AASP. The fluorescence quantitative analysis further confirmed this conclusion (Fig. 2D). Inhibitory effect of AASP against OGD/R-induced H9C2 cytotoxicity was subsequently evaluated. As shown in Fig. 2E, OGD/R treatment significantly decreased the H9C2 cell viability to 43.5%, suggesting that OGD/R caused obvious cytotoxicity with a dose-dependent manner. Co-treatment of AASP significantly inhibited OGD/R-induced H9C2 cytotoxicity and showed better protective effect than that of AS, ascorbic acid and AAS. The single-cell raman spectroscopy showed that AASP significantly improved the ability of cells to metabolize heavy water, which indirectly reflected the improved cells viability (Figure S2). The cell morphological changes further confirmed this protective effects (Figure S3). The Live/Dead cells staining clearly demonstrated that AASP significantly inhibited OGD/R-induced H9C2 cells death, as indicated by the decreased red fluorescence (Figure S4A and S4B). The pre-, co- and post-treatment of AASP all significantly attenuated OGD/R-induced H9C2 cytotoxicity (Fig. 2F), and the real-time monitoring also suggested that co-treatment of AASP and OGD/R was the optimal design. Moreover, flow cytometry results further classified that AASP co-treatment significantly inhibited OGD/R-induced H9C2 cells apoptosis, and the statistical analysis of apoptotic cells further confirmed this conclusion (Figure S4C and S4D). Taken together, these results suggested that PCM modification achieved cardiomyocytes-targeted recognition and enhanced the intracellular uptake of AASP, which eventually attenuated OGD/R-induced H9C2 cytotoxicity and apoptosis.

AASP promoted NO production

A time point of experiment design in vitro was firstly presented (Fig. 3A). L-arginine as a substrate can be catalyzed by TNOS (total nitric oxide synthase) to produce NO. Increasing studies have confirmed that NO as an important messenger molecule plays key role in regulating cardiovascular remodeling by participating in mitochondrial function and energy metabolism of myocardium^37,38. Herein, we firstly examined TNOS activity by a TNOS kit. As shown in Fig. 3B, H9C2 cells after OGD/R treatment showed blue colour and decreased absorbance value (Figure S6A), indicating the decreased NOS activity. However, AASP co-treatment significantly recovered NOS activity, as showed by the red colour of H9C2 cells. Secondly, NO production was detected by DAF-FMDA probe. As shown in Fig. 3C, OGD/R treatment significantly decreased NO production, as convinced by the vanished green fluorescence. However, AASP co-treatment significantly promoted NO production of H9C2 cells, as demonstrated by the enhanced green fluorescence. These results clearly revealed that AASP co-treatment restored NOS activity and promoted NO generation, which showed better effects than that of AAS and Arg + AS. To further confirm the significance of NO, a NO inhibitor (cPTIO) was employed, and the results showed that inhibition of NO by cPTIO signigficantly attenuated AASP-mediated protective effects in NO production and H9C2 cells viability (Fig. 3C, D).

Activation of Complex I in mitochondria was positively correlated with ROS generation. To investigate the causal relationship between NO generation and ROS inhibition, Complex I activity was examined by a NO inhibitor (cPTIO). As show in Fig. 3E, OGD/R treatment resulted in obvious Complex I activation, which indirectly reflected the ROS generation. AASP co-treatment significantly inhibited Complex I activation in OGD/R-treated H9C2 cells. cPTIO addition significantly blocked AASP-induced inhibition against Complex I activation. These results revealed that AASP promoted NO production, which in turn blocked ROS generation.

The opening of mitochondrial permeability transition pore (mPTP) can lead to ROS release. To classify the underlying mechanism by which NO blocked ROS generation, the mPTP opening was evaluated by Calcein-AM and CoCl₂. Calcein-AM can cross normal cells membrane and mitochondrial membrane and shows green fluorescence. CoCl₂ can cross normal cells membrane and quench the green fluorescence of Calcein-AM in cytoplasm, but CoCl₂ could not cross normal mitochondrial membrane to quench the green fluorescence of Calcein-AM in mitochondria. As shown in Fig. 3F, H9C2 cells after OGD/R treatment showed notable green fluorescence, and CoCl₂ co-treatment significantly quenched the green fluorescence. However, H9C2 cells after co-treatment of AASP and OGD/R still showed significant green fluorescence, which revealed that AASP co-treatment promoted NO generation, and NO generation in turn regulated mPTP closing, and blocked ROS release from mitochondria. Addition of NO inhibitor (cPTIO) further confirmed this conclusion (Fig. 3F). Taken together, these results above suggested that AASP promoted NO production, which blocked ROS release from mitochondria by regulating mPTP closing.

AASP maintained mitochondria functions of H9C2 cells

Mitochondrial membrane potential (MMP) was firstly evaluated in H9C2 cells. Figure 4A showed that AASP co-treatment effectively improved the MMP in OGD/R-treated H9C2 cells, as convinced by the fluorescence shift from green to red. The flow cytometry results and quantitative analysis both further confirmed this conclusion (Fig. 4A, B). ATP synthase in mitochondrias can catalyze and synthesize ATP, and play key role in regulating cellular metabolism. Dysfunction of ATP synthase was accepted as one of the important mechanisms of mitochondrial dysfunctions and myocardial apoptosis^39,40. Herein, ATP synthase activity in H9C2 cells was assayed by co-fluorescent staining of ATP synthase and mitochondrias. As shown in Fig. 4C, OGD/R treatment significantly decreased ATP synthase activity and mitochondrial number, as demonstrated by the weaken green and red fluorescence, respectively. The fluorescent quantitation further confirmed this result (Fig. 4D). Cells ultrastructure by TEM showed that OGD/R treatment caused obvious cell membrane damage, mitochondrial reduction and nucleus destruction. However, AASP co-treatment significantly improved cells ultrastructure, restored ATP synthase activity and mitochondrial number, and complemented ATP content in OGD/R-treated H9C2 cells (Fig. 4E). AASP co-treatment in these results all showed better protective effects than that of AS, ascorbic acid and AAS.

Mitochondrial dysfunction may lead to the mPTP opening and cause the ROS release, which could tirgger oxidative damage of H9C2 cells. Accumulated studies have indicated that OGD/R could induce ROS overproduction, and cause oxidative damage to cardiomyocytes⁴¹. Herein, DCFH-DA was employed to investigate the oxidative status of H9C2 cells. As shown in Figure S5A, OGD/R treatment caused significant ROS generation with a time-dependent manner, and reached a peak at 6 h. However, AASP co-treatment completely eliminated OGD/R-induced ROS generation with a time-dependent manner, and AASP showed better protective effects than that of AS, ascorbic acid and AAS. The weaken green fluorescence (Figure S5A), and fluorescent quantitation (Figure S5B), and flow cytometry results (Figure S5D) all confirmed this conclusion. Overproduced ROS can oxidate lipids in biological membranes to form lipid peroxidation products (MDA), and result in changes in cell structure and function. Hence, the activity of SOD and lutathione peroxidase (GSH-Px), and MDA content were all detected. As shown in Figure S5E-G, AASP co-treatment significantly increased the activity of SOD and GSH-Px, and decreased the MDA content in OGD/R-treated H9C2 cells. We speculated that selenium was the active sites of many selenium-containing enzymes, and selenium supplement by AASP effectively inhibited ROS accumulation. Taken together, AASP displayed enhanced protection against OGD/R-induced oxidative damage by maintaining mitochondria functions of H9C2 cells.

AASP improved myocardial function in MI/RI rats

A time point of experiment design in vivo was firstly presented (Fig. 5A). Advanced nanoprobes with enhanced photoacoustic properties were previously reported for noninvasive in vivo visual imaging of vulnerable atherosclerosis⁴². MI/RI rats was injected with AASP by caudal vein, and cardiomyocyte-targeted property of AASP in vivo was real-timely monitored. Figure 5B showed that photoacoustic (PA) signal was visibly detected in the anterior wall of the left ventricle at 4 h, and the PA signal from AASP was 3 times stronger than that of AAS (Figure S6B). PA signal intensity showed significant decline, indicating that AASP can be quickly metabolized in vivo. These results indicated that AASP after PCM modification achieved cardiomyocyte-targeted photoacoustic imaging in vivo.

Myocardial reperfusion therapy as the most effective way to restore blood and oxygen supply to the ischemic heart may in turn cause lethal tissue damage to heart⁴³. Searching new strategies to mitigate MI/RI remains great challenge in clinic. Therefore, the myocardial function in MI/RI rats after AASP administration was evaluated. Lactate dehydrogenase (LDH) and creatine kinase-isokinase MB (CK-MB) as important indicators of MI/RI were firstly examined⁴⁴. As shown in Figure S6C and 6D, MI/RI rats showed significant increase of LDH and CK-MB level, indicating the successful establishment of MI/RI model. Echocardiographic assessment revealed that MI/RI resulted in significant the left ventricular systolic dysfunction (Fig. 5C). The quantitative analysis indicators (Fig. 5D-I), such as left ventricular ejection fraction (LV-EF), left ventricular fraction shortening (LV-FS), left ventricular internal diameter at end-diastole (LVIDD), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV), all further confirmed the myocardial dysfunction in MI/RI rats. However, AASP co-treatment significantly decreased the LDH and CK-MB level, and normalized the LV-EF, LV-FS, LVIDD, LVEDV and LVEVS. The protective effects of AASP is much better than that of AS and AAS. Taken together, these results indicated that AASP achieved cardiomyocyte-targeted photoacoustic imaging and improved myocardial function in MI/RI rats.

AASP inhibited oxidative damage and myocardial apoptosis in MI/RI rats

AASP-mediated protection mechanism in MI/RI rats was further explored by histological analysis. As shown in Figure S7A, H&E staining results showed obvious inflammatory cell infiltration in ventriculus sinister of MI/RI rats. Active-caspase-3 staining (Figure S7B and S7D) and TUNEL staining (Figure S7C and S7E) showed significant myocardial apoptosis in ventriculus sinister of MI/RI rats. Masson's staining indicated that MI/RI rats showed significant myocardial fibrosis, as convinced by the increased collagen fibers (Figure S7F and G). Moreover, induction of oxidative damage was also found in MI/RI rats, as demonstrated by the decreased activity of antioxidant enzymes SOD (Figure S7H) and GSH-Px (Figure S7I), and increased MDA content (Figure S7J). However, AASP administration in vivo dramatically inhibited inflammatory cell infiltration, myocardial apoptosis, myocardial fibrosis and oxidative damage in MI/RI rats.

Reconstruction of the vascular system is beneficial for myocardial regeneration and repair after MI/RI. eNOS staining showed that AASP administration in vivo significantly promoted eNOS expression (Fig. 6E and 6F), which could induce NO production and promote angiogenesis. CD34 staining indicated that AASP administration in vivo improved the angiogenesis in MI/RI rats (Fig. 6A and 6B). Ki-67 staining revealed that AASP administration in vivo inhibited the abnormal proliferation of cardiomyocytes in MI/RI rats (Fig. 6C and 6D). Increased cross-sectional area of cardiomyocytes is typical of dilated cardiomyopathy and ventricular systolic dysfunction. WGA staining clearly indicated that AASP administration in vivo effectively blocked the expansion of cardiomyocytes (Fig. 6G and 6H). TEM observation showed that AASP administration in vivo effectively attenuated myocardial fiber rupture, mitochondrial damage and increased mitochondrial number (Fig. 6I). AASP showed better effects than that of AS and AAS in all these results. Taken together, these results suggested that AASP administration in vivo effectively improved cardiomyocyte proliferation, angiogenesis and inhibited expansion of cardiomyocytesin in MI/RI rats.

Transcriptomic analysis of molecular mechanism

To investigate the underlying mechanism of AASP-mediated protection against MI/RI in rats, transcriptomic analysis of left ventricle from MI/RI rats was conducted to detect the total mRNA abundance differences. As shown in the Venn diagram (Fig. 7A) and the volcano diagram (Fig. 7B), there were 4688 differential genes between the sham group and model group. The Model group and the AASP-treated group also showed 2653 differential genes. However, there were only 40 differential genes between the sham group and AASP-treated group, which implying that AASP administration in vivo significantly normalized the differential genes in MI/RI rats. Gene Ontology (GO) enrichment analysis of biological process (BP), cellular component (CC) and molecular function (MF) found that ATPase activity, ATP metabolic processes, angiogenesis, myocardial contraction, mitochondrial respiratory chain, oxidoreductase activity, and fatty acid oxidation were all enriched after AASP administration in vivo (Figure S8A). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found that VEGF signaling pathway, PI3K-Akt signaling pathway, cAMP signaling pathway, selenium compound metabolism, glutathione metabolism, arginine and proline metabolism pathways were enriched after AASP administration in vivo (Figure S8B). The heating map specifically found that AASP administration in vivo significantly up-regulated Sod2, Atp5f1, Atp5a1, Vegfb, and Akt, and down-regulated Mki67 and Casp3 expression in MI/RI rats. The protein expression level of Thr308-AKT, Ser473-AKT, Ser1177-eNOS and VEGF detected by western blotting further confirmed this conclusion (Fig. 7E-F). Taken together, these results all suggested that AASP administration in vivo significantly attenuated MI/RI of rats with involvement of improved ATP synthesis, angiogenesis and oxidoreductase metabolism.

Evaluation of AASP safety

The distribution and biocompatibility of nano-drugs in vivo are both important factors in drug design and development. Herein, the bio-distribution and biocompatibility of AASP were evaluated in rats. As shown in Figure S9, AASP mainly accumulated in the heart at 4 h, and then accumulated at the other organs. Liver was the main metabolic organs of AASP, and showed a peak at 12 h (Figure S9A and S9B). The contents of Se (Figure S9C) and Au (Figure S9D) in vivo gradually decreased with a time-dependent manner in main organs, and almost no retention can be detected at 21 d, indicating that AASP can be quickly metabolized. Figure S10 showed that AASP administration in vivo caused no significant changes of hemolytic behavior (Figure S10A) and rats body weight (Figure S10B). Blood biochemical analysis indicated that AASP showed no significant effects on alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CRE) (Figure S10C-S10F). Histopathological analysis by H&E staining suggested that AASP administration in vivo caused no significant histopathological changes in heart, liver, spleen, lung, kidney and brain (Figure S11). Taken together, these results indicated that AASP showed good safety and biocompatibility in vivo.

Materials. All chemical reagents were purchased from Sigma-Aldrich (Mainland-China) or Thermo Fisher Scientific (Mainland-China) and used without further purification. PCM (WLSEAGPVVTVRALRGTGSW) was synthesized by Shanghai Bioengineering Co. The purity of the synthesized PCM was determined to be higher than 95% by mass spectrometry and high performance liquid chromatography detection.

Synthesis of Au Nanoparticles (Au NPs). The Au NPs was synthesized by the substitution reaction between Ag NPs and chloroauric acid (HAuCl₄)⁴⁵. Briefly, 650 mg of Polyvinylpyrrolidone K30 (PVP-K30), 340 mg of AgNO₃ and 10 mg of NaCl were dissolved in 7 mL of ethylene glycol, and then 2 mL of glycerol was added, and reacted at 150°C for 2 h to obtain Ag NPs colloids. 200 µL of Ag NPs colloidal solution was dispersed in 20 mL of deionized water, heated and boiled for 10 min, 5 mL of HAuCl₄ (1 mM) was slowly added, and continue to reflux for 20 min under ultrasonication. The AgCl generated by the displacement reaction can be dissolved and removed with a saturated NaCl solution, and the Au NPs can be obtained by centrifugation (8000 g, 15 min).

Synthesis of L-Arg@Au@Se@PCM nanoparticles (AASP NPs). Eight milliliters of Au NPs (0.1 mg/mL) solution and 2 mL of L-Arg (20 mg/mL) solution were mixed and stirred for 2 h, and centrifuged (8000 g, 15 min) to obtain L-arginine-modified gold nanocages. 500 µL of cetyltrimethylammonium bromide (CTAB, 10 mg/mL) solution and 300 µL of Na₂SeO₃ (17.3 mg/mL) solution were mixed with the re-dispersed L-arginine modified gold nanocage solution (3 mL), and then 1.2 mL of ascorbic acid solution (17.6 mg/mL) was added dropwise. After stirring for 30 min, the solution turned into a stable tan. PCM with a final concentration of 5 mg/mL was added to the reaction system, incubated overnight at 4°C, centrifuged (8000 g, 15 min) and washed to remove unreacted substances.

Physicochemical Characterization. High-definition transmission electron microscopy (TEM, HT7700, Hitachi, Japan), scanning electron microscopy (SEM, S-4800, Hitachi, Japan) and energy dispersive X-ray spectroscopy (EDS, X-Max-N150, Oxford, UK) were used for morphological observation and elemental analysis of nanoparticles. Nanoparticle size and surface zeta potential were measured by dynamic light scattering (DLS) using a Nano-ZS instrument (Malvern Instruments Limited, UK). Ultraviolet-near infrared (UV-vis-NIR) absorption spectrum were detected by UV-vis-NIR spectrophotometer (UV-2550, Shimadzu, Japan). Fourier transform infrared (FTIR) spectroscopy on a FTIR spectrometer (Nicoletteis50; Thermo Fisher Scientific, USA) in the wavelength range between 4000 − 500 cm^− 1. X-ray photoelectron spectroscopy (XPS, EscaLab Xi⁺, Thermo Fisher Scientific, USA) was used to analyze the elemental composition and valence of nanoparticles. X-ray difraction (XRD, D/max-2400, Tokyo, Japan) was used to analyze the crystal structure of nanospheres. In vivo photoacoustic (PA) imaging was conducted on a PA system (Endra Nexus 128, USA).

Cell culture. H9C2 cells (rat cardiomyocytes) were purchased from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Cells were cultured in high glucose dulbecco's modified eagle medium (DMEM) containing 10% FBS, 100 IU/mL penicillin and streptomycin at carbon dioxide incubator (37 ℃ and 5% CO₂). Oxygen glucose deprivation/re-oxygenation (OGD/R) model was established with glucose-free DMEM and incubated in 95% N₂ and 5% CO₂) for 6 h, then cells were cultured with normal condition for 24 h for re-oxygenation.

Real time cell analysis. The real-time cell electronic sensor system (xCELLigence RTCA-S16, ACEA Bioscience, USA) was used to detect cell proliferation. Briefly, H9C2 cells were cultured in e-plate at a density of 5×10³ cells per well for 24 h at 37℃. Then, AASP NPs (30 µg/mL) were added to the e-plate and treated by OGD/R. Real-time and dynamic cell proliferation was detected within 72 h. Similarly, AASP NPs were added to cells 6 h before OGD/R treatment, or 6 h after OGD/R treatment, and real-time and dynamic cell proliferation was detected within 72 h.

Cellular Uptake of L-Arg@Au@Se NPs and AASP NPs. Fluorescein isothiocyanate (FITC)-labeled nanoparticles were prepared following the previously reported construction method of fluorescent probes^34,46. The H9C2 cells (1×10⁶ cells per well) were implanted in a 6-well plate and cultured for 24 h at 37 ℃. 30 µg/mL FITC-labeled nanoparticles were added to a 6-well plate to observe the changes in cell fluorescence intensity within 8 h. Similarly, the cells were pre-blocked with 0–3 mg/mL Free-PCM for 24 h, and then 30 µg/mL FITC-labeled nanoparticles were added to the 6-well plates to observe the intracellular fluorescence intensity at the 6th hour. A multifunctional microplate reader (Tecan Infinite 200Pro, Austria, Switzerland) was used to measure the fluorescence intensity of FITC (excitation and emission wavelengths were 495 and 519 nm, respectively), and the cell uptake was expressed as the percentage of adsorbed nanoparticles. Distribution of nanoparticles in the cell was determined by co-localizing the cytoskeleton (rhodamine phalloidin, excitation wavelength and emission wavelength were 540 and 570 nm, respectively) and cell nucleus (4',6-diamidino-2-phenylindole, DAPI, excitation wavelength and emission wavelength were 358 and 461 nm, respectively), and recorded by confocal laser microscope (TCS SP8 X, Leica, Germany).

Cell viability assay. The H9C2 cells (5×10³ cells per well) were implanted in a 96-well plate and cultured for 24 h at 37 ℃. Nanoparticle (0–60 µg/mL) was added to 96-well plates and given OGD/R treatment. Cell Counting Kit-8 (ab228554, Abcam) was used to determine cell viability. Briefly, H9C2 cells (1×10⁶ cells/well) were implanted into 6-well plates and incubated for 24 h at 37 ℃. Nanoparticles (30 µg/mL) were added to the 6-well plates while the cells were subjected to OGD/R treatment. Cells were stained for 30 min according to the LIVE/DEAD kit (L3224, Thermo Fisher Scientific). Cells were fluorescently imaged by fluorescence microscopy.

Single-cell Raman spectra detects general metabolic activity of cells. The H9C2 cells (5×10³ cells per well) were implanted in a 96-well plate and cultured for 24 h at 37°C. Add 30 µg/mL nanoparticle to 96-well plates while the cells were subjected to OGD/R treatment (30% Heavy water (v/v)-containing medium). About 20 single cells from each group were randomly sampled for Raman spectroscopy measurements after OGD/R treatment. The C-D ratio, an index to quantify the substitution of D atom in C–H bond and the metabolism degree of single cells, was calculated by dividing the integrated area intensity of C-D band (2040–2300 cm^− 1) by the sum of C-D band and C–H band (2800–3100 cm^− 1) via Ramanome Explorer (RamEX, Qingdao Single cell Biotech, China).

Flow cytometry analysis. The ratio of cell mitochondrial membrane potential drop and cell apoptosis after nanoparticle treatment was analyzed by flow cytometry. H9C2 cells (1×10⁶ cells/well) were implanted in a 6-well plate and cultured for 24 h at 37 ℃. The nanoparticles (30 µg/mL) were added to a 6-well plate and given OGD/R treatment. After treatment, all cells were collected, and the cells were stained according to the instructions of apoptosis detection kit (331200, Thermo Fisher Scientific) and MMP detection kit (V35116, Thermo Fisher Scientific). Then, the stained cells were analyzed by flow cytometry (Cytoflex, Beckman, CA, USA).

Measurement of ROS generation. To confirm the production of intracellular ROS, H9C2 cells were implanted in a 6-well plate at a density of 1×10⁶ cells per well and cultured for 24 h. The nanoparticles (30 µg/mL) were added to a 6-well plate and cells were subjected to OGD/R treatment. After treatment, the medium was removed, and the cells were gently washed three times with PBS. 10 µM of 2', 7'-Dichlorofluorescent yellow diacetate (DCFH-DA) was added to each dish and incubated for 45 min at 37 ℃. The fluorescence intensity in the cells was tested to verify the ROS generation within 0-180 min.

Detection of SOD, GSH-PX and MDA in H9C2 cells. Superoxide dismutase (SOD) activity was measured by xanthine oxidase assay. Briefly, H9C2 after OGD/R and nanoparticle treatment were collected and fragmented by ultrasonic fragmentation. The supernatant was collected by centrifugation and protein concentration was quantified, followed by determination of absorbance at 450 nm through a multifunctional microplate reader according to the SOD activity assay kit (19160, Sigma-Aldrich) procedure. The activity of GSH-Px was measured by chemical colorimetric method. Cells after treatment as described above, the absorbance was measured at 412 nm according to the GSH-Px kit (CGP1, Sigma-Aldrich) procedure and by a multifunctional microplate reader. Lipid peroxidation activity was measured by thiobarbituric acid assay. Cells after treatment as described above, the absorbance was measured at 532 nm according to the MDA kit (MAK085, Sigma-Aldrich) procedure and by a multifunctional microplate reader.

Production of nitric oxide (NO). H9C2 cells (5×10³ cells/well) were implanted in a 96-well plate and cultured at 37 ℃ for 24 h. Nanoparticles (0–30 µg/mL) was added to a 96-well plate and cells were subjected to OGD/R treatment. Cells were collected and lysed at 0, 6, 12, and 24 h after restore of normal culture conditions. The amount of nitric oxide synthase in 50 µL of cell lysate in each well was measured according to the instructions of the Total Nitric Oxide Synthase (TNOS) Assay Kit (ab211083, Abcam), and then the absorbance was measured at OD_{540 nm} using a multifunctional microplate reader. Briefly, 30 µg/mL nanoparticles were added to a 96-well plate (1 mM nitric oxide scavenger Carboxy-PTIO (cPTIO) was added as a negative control) and cells were subjected to OGD/R treatment. After treatment, 5 µM nitric oxide fluorescent probe DAF-FMDA (excitation wavelength and emission wavelength were 490 nm and 520 nm, respectively) was incubated with the cells for 20 min, and the fluorescence intensity was detected by a inverted fluorescence microscope (Nikon, Ti-E) and a multifunctional microplate reader.

Evaluation of mitochondrial complex 1 activity and mPTP opening. mPTP opening in H9C2 cells was assessed by Calcein-AM/CoCl₂ quenching technique. Briefly, Nanoparticles (30 µg/mL) were added to 96-well plates containing H9C2 cells (1 mM cPTIO was added as a negative control), and cells then were subjected to OGD/R treatment. After treatment, cells were incubated with 1 mM Calcein-AM and 2 mM CoCl₂ for 30 min. Cells were imaged using a fluorescence microscope and relative fluorescence intensities were calculated. The mitochondrial respiratory chain complex I activity was determined according to the operating manual (BC0515, Solarbio). Treated cells in each group were sonicated and mitochondria were isolated. After mixing with the corresponding substrate solution, the activity of complex I was detected spectrophotometrically.

Fluorescence detection of ATP synthase, mitochondrial and cellular ATP levels. Cells after treatment were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% TritonX-100 for 5 min, and incubated with 1% bovine serum albumin at room temperature for 30 min, respectively. Cells were labeled with 5 µg/mL of ATP Synthase beta Monoclonal Antibody for 1 h, 100 nM of MitoLite Red for 30 min, and 0.5 µg/mL of DAPI for 5 min, respectively. The stained cells were observed and recorded by laser confocal microscopy. Cellular ATP levels were detected according to the Luminescent ATP Detection Assay kit (ab113849, Abcam).

TEM observation of ultra-thin sections. H9C2 cells were treated with OGD/R according to the above method. After treatment, cells were collected by centrifugation (2000 g, 5 min) and fixed overnight with 2.5% glutaraldehyde solution, followed by osmium tetroxide (1%) for 1 h. The samples were dehydrated with different concentrations (30%, 50%, 70%, 80%, 90%, 100%) of ethanol gradients, followed by resin embedding, sectioning, and stained with uranyl acetate solution (1%) and lead citrate solution (1%) for 5–10 min. The samples were fixed on copper grids, and the microstructures of the cells before and after treatment were observed by transmission electron microscopy.

Myocardial ischemia-reperfusion injury (MI/RI) model in rats. The animal procedures were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals of China, and the protocols used were approved by the Laboratory Animal Ethics Committee of Weifang Medical University. Fifty male SD rats (300 g ± 20 g, 8–10 weeks old) were randomly divided into five groups: (1) sham-operated group (Sham), (2) myocardial ischemia-reperfusion injury group (MI/RI), (3) AS treatment group, (4) AAS treatment group, (5) AASP treatment group. The rat myocardial ischemia/reperfusion model was referred to the previously reported method with slight modifications⁴⁷. Briefly, SD rats were continuously anesthetized during surgery using isoflurane. Under aseptic conditions, the heart was exposed by left open thoracotomy, acute myocardial ischemia was achieved by temporal ligation of the left anterior descending (LAD) coronary artery for 30 min, and reperfusion injury was achieved by loosening the ligature 30 min later. Rats were injected intravenously with saline (MI/RI group), AS, AAS and AASP (dose: 3 mg/kg) for treatment. One dose was injected immediately after surgery and one dose was injected every two days after 24 h of reperfusion for two weeks of treatment. Therefore, a total of 8 injections were given over a 2-week period. Blood samples were taken before and after surgery, and plasma lactate dehydrogenase and creatine kinase isoenzyme levels were measured.

Photoacoustic imaging in vivo. Gold nanocages are not only used as a nano-drug delivery system but also endow nanomaterials with near-infrared PA imaging properties due to the surface plasmon resonance effect of gold nanocages⁴⁸. MI/RI rats were injected with 3 mg/kg of AAS and AASP in the tail vein, and PA imaging was performed by PA imaging platform (Excitation wavelength: 808 nm) on rat heart cross-sections at the 0th, 2nd, 8th and 24th hours to observe and calculate the PA signal intensity in the anterior wall region of the left ventricle, respectively.

Echocardiographic assessment of myocardial function. All rats underwent echocardiography using an echocardiography system (VINNO 6VET) equipped with an X6-16L transducer (6.5–18 MHz) after MI/RI and within 2 weeks after treatment. M-mode imaging was performed in the long-axis view of the left ventricular. LV-EF, LV-FS, LVIDD, LVEDV and LVESV were determined separately. All assays were performed randomly and the experimenters knew nothing about the treatment group.

Transcriptome analysis. Total RNA was extracted from left ventricular damaged tissue in sham, model and AASP treatment groups by TRIzol instructions, and a cDNA library was created. After the quality inspection, the Illumina Novaseq6000 platform was used for PE150 sequencing. Differential expression genes (DEGs) analysis was performed by using the edgeR package. False Discovery Rate (FDR)-adjusted P-value ≤ 0.05 as the threshold. The GO term and KEGG pathways that meet this condition were defined as the GO term and the KEGG pathway that were significantly enriched in differential expressed genes, respectively.

Western blotting. Left ventricular injury tissues of rats in sham-operated group, model group and treatment group were lysed and homogenized with RIPA buffer containing 1 mM PMSF. The protein concentration was determined using the BCA protein detection kit, and samples containing equal amounts of protein (40 µg/lane) were loaded into SDS polyacrylamide gels. After electrophoresis, the samples were transferred to PVDF membranes. Blocking, incubation with primary and secondary antibodies, and visualize target protein bands on the membrane using ECL Western Blot Detection Reagent. The proteins expression was quantified by Quantity-One Software, and the expression rate was labeled under the band.

Histology and Immunohistochemistry. After 2 weeks of injection treatment, all rats were executed and heart tissue was collected. Hearts were perfused with PBS and embedded with opti-mum cutting temperature compound (OCT), immediately snap frozen with liquid nitrogen. Ten consecutive frozen sections of 10 µm thickness were cut with a freezing microtome (HM525 NX, Thermo Fisher Scientific) from the left ventricular ligation site to the apex of the left ventricle. All sections were stored at -20 ℃ pending staining. To assess the size of the heart injury, tissue sections were fixed with 4% paraformaldehyde for 2 h and stained with Masson's trichrome method. Apoptosis in the myocardial tissue region was detected using the DeadEnd™ Fluorescent TUNEL System (G3250, Promega). Immunohistochemistry and immunofluorescence were conducted to examine the expression of active caspase-3, CD34, Ki67 and eNOS. All images were taken with a Nikon Ti-E inverted fluorescence microscope.

Metabolism and safety assessment. In quantitative nanomaterial biodistribution, SD rats were injected intravenously with 3 mg/kg of AASP nanoparticles. Rats were executed at 4 h, 8 h, 12 h, 24 h, 7 d, 14 d and 21 d after administration, respectively. The major organs such as heart, liver, spleen, lung and kidney were excised and homogenized by digestion, and the content of Se ions and Au ions in the samples were analyzed by ICP-MS (inductively coupled plasma mass spectrometry, 7500, Agilent).

The erythrocytes in 5 mL of fresh human blood were collected by centrifugation at 1000 g for 10 min, and 5% of erythrocyte stock solution (80 µL) and different concentrations of AASP solution (20 µL) were incubated for 1 h at 37 ℃. Hemolysis was measured at OD_{405 nm} using a multifunctional microplate reader. 0.1 M PBS buffer and acetic acid (HAc) were used as negative and positive controls, respectively.

The in vivo toxicity of AS, AAS and AASP in SD rats was evaluated. Briefly, AS (3 mg/kg), AAS (3 mg/kg) and AASP (3/9 mg/kg) were injected once daily at a fixed time through the tail vein, and an equal amount of saline was injected as a control. After 21 days of continuous administration, blood was collected from the orbit and measured by a fully automated hematology analyzer (LH750, Beckman Coulter) for biochemical parameters. The heart, liver, spleen, lung, kidney and brain of the rats were collected and stained with H&E for histopathological observation.

Statistical analysis. All experiments were performed at least three times, and the results are expressed as mean ± S.D. One-way ANOVA with Dunnett’s post-hoc test was used to calculate the significant difference between different groups. *P < 0.05 and **P < 0.01, suggesting significant differences and very significant differences, respectively.

Funding

This work was supported by National Natural Science Foundation of China (21401002, 81501106，91639108，81770272), Key projects of youth talent fund in Anhui Provincial Universities (gxyqZD2020006), Key R&D Projects in Anhui Province (202104f06020028) and Shandong Introduction and Education Program of Young Innovative Talents (RCJF007).

Competing interests

The authors have declared that no competing interest exists.

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L-Arginine-Loaded Gold Nanocages Mitigates Myocardial Ischemia/Reperfusion Injury by Promoting NO Production and Maintaining Mitochondrial Functions

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Abstract

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Introduction

Results And Discussion

Synthesis and characterization of nanosystems

AASP attenuated OGD/R-induced H9C2 cytotoxicity

AASP promoted NO production

AASP maintained mitochondria functions of H9C2 cells

AASP improved myocardial function in MI/RI rats

AASP inhibited oxidative damage and myocardial apoptosis in MI/RI rats

Transcriptomic analysis of molecular mechanism

Evaluation of AASP safety

Materials And Methods

Declarations

References

Additional Declarations

Supplementary Files

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