Controlled Release of Hydrogen‐Carrying Perfluorocarbons for Ischemia Myocardium‐Targeting ¹⁹F MRI‐Guided Reperfusion Injury Therapy

Abstract

Hydrogen gas is recently proven to have anti‐oxidative and anti‐inflammation effects on ischemia‐reperfusion injury. However, the efficacy of hydrogen therapy is limited by the efficiency of hydrogen storage, targeted delivery, and controlled release. In this study, H₂‐PFOB nanoemulsions (NEs) is developed with high hydrogen loading capacity for targeted ischemic myocardium precision therapy. The hydrogen‐carrying capacity of H₂‐PFOB NEs is determined by gas chromatography and microelectrode methods. Positive uptake of H₂‐PFOB NEs in ischemia‐reperfusion myocardium and the influence of hydrogen on ¹⁹F‐MR signal are quantitatively visualized using a 9.4T MR imaging system. The biological therapeutic effects of H₂‐PFOB NEs are examined on a myocardial ischemia‐reperfusion injury mouse model. The results illustrated that the developed H₂‐PFOB NEs can efficaciously achieve specific infiltration into ischemic myocardium and exhibit excellent antioxidant and anti‐inflammatory properties on myocardial ischemia‐reperfusion injury, which can be dynamically visualized by ¹⁹F‐MR imaging system. Moreover, hydrogen burst release induced by low‐intensity focused ultrasound (LIFU) irradiation further promotes the therapeutic effect of H₂‐PFOB NEs with a favorable biosafety profile. In this study, the potential therapeutic effects of H₂‐PFOB NEs is fully unfolded, which may hold great potential for future hydrogen‐based precision therapeutic applications tailored to ischemia‐reperfusion injury.

Schematic illustration of hydrogen therapy strategy and mechanisms of enhanced therapeutic effect of H₂‐PFOB NEs combined with LIFU. The synthesized H₂‐PFOB NEs with high biosafety and excellent hydrogen carrying capacity, exhibited stronger anti‐oxidant and anti‐pyroptosis effects and exerted excellent therapeutic effect on I/R injury. In addition, H₂‐PFOB NEs can passively infiltrate into ischemic myocardial tissue, exert further enhanced therapeutic effects when combined with LIFU, realizing the effective release and utilization of hydrogen, thus facilitating the purpose of precise treatment of I/R injury.

1. Introduction

Cardiovascular disease (CVD) is the leading cause of mortality and morbidity globally, and ischemic heart disease (IHD) accounts for approximately half of CVD deaths.^{[ 1 ]} Revascularization therapy is the most commonly used treatment method for IHD, however, ischemia‐reperfusion (I/R) injury caused by revascularization therapy significantly affects the prognosis of IHD.^{[ 2 ]} The underlying mechanisms of I/R injury are complex and primarily involve calcium overload, mitochondrial oxidative stress damage, and the release of a large number of inflammatory factors.^{[ 3 ]} Thus, new therapeutic strategies are needed to mitigate above influential factors in myocardial I/R injury tissue to improve the clinical outcomes of IHD patients.

Hydrogen therapy is a promising emerging treatment based on molecular hydrogen (H₂) as a new type of safe and effective therapeutic agent.^{[ 4 ]} In 2007, Ohta et al. found that hydrogen can selectively reduce toxic hydroxyl free radicals to alleviate cerebral I/R injury.^{[ 5 ]} In the past decade, hydrogen molecules have been found to have highly effective therapeutic effects on a series of oxidative stress and inflammatory diseases, such as arteriosclerosis, cancer, arthritis and diabetes.^{[ 6 ]} These studies add to a growing body of evidence showing that hydrogen has important clinical implications and high translational potential.^{[ 7 ]} However, the efficacy of hydrogen‐based treatment of myocardial infarction is limited by the efficiency of its targeted delivery and controlled release. Direct inhalation of hydrogen gas is convenient but generally accompanied by multiple drawbacks, such as low H₂ concentration in the target area and the potential risk of explosion. In addition, the therapeutic efficacy of hydrogen in water or saline solution is limited by the low solubility and high diffusivity of hydrogen gas. The emergence of nanomaterial‐based platforms provides a promising alternative strategy for hydrogen delivery, and a variety of nanomaterials with hydrogen storage and hydrogen‐producing ability have already been developed for the treatment of various diseases.^{[ 8 ]} However, current hydrogen‐carrying nanomaterials present important limitations for clinical transformation, particularly the biosafety profiles of some inorganic or hybrid materials, which challenges their further development for clinical applications.^{[ 9 ]} Thus, the development of hydrogen carriers with outstanding clinical translational potential would be a significant step forward in the treatment of myocardial I/R injury.

Perfluorinated compounds (PFCs) are inert organic compounds that improve tissue oxygenation due to their high affinity for oxygen and perfect biocompatibility.^{[ 10 ]} These compounds are widely used for several clinical applications, such as artificial blood substitution, organ preservation, ultrasound imaging, and ¹⁹F magnetic resonance imaging.^{[ 11 ]} In addition, perfluorooctylbromide nanoemulsions (PFOB NEs) have large solubilization capacity for oxygen via van der Waals interactions,^{[ 12 ]} and the release of gas contents in PFOB NEs could be facilitated by low‐intensity focused ultrasound (LIFU) at targeted site with excellent biosafety profile due to its low intensity and low frequency ultrasonic characteristics.^{[ 13 ]} However, it remains unclear whether PFOB NEs may also be used to dissolve hydrogen gas and exert biological effects.

In this study, we report a novel therapeutic approach with high clinical translational potential for the treatment of myocardial I/R injury therapy based on the multifunctional advantages of a perfluorocarbon nanoplatform (Figure 1).

Figure 1.

Schematic illustration of hydrogen therapy strategy and mechanisms of enhanced therapeutic effect of H₂‐PFOB NEs combined with LIFU. We synthesized H₂‐PFOB NEs with high biosafety, and the excellent hydrogen carrying capacity of PFOB NEs was confirmed by gas chromatograph and microelectrode for hydrogen concentration determination. We for the first time revealed its excellent therapeutic effect on I/R injury and found that H₂‐PFOB NEs has stronger anti‐oxidant and anti‐pyroptosis effects than H₂‐water. Remarkably, H₂‐PFOB NEs can passively infiltrate into ischemic myocardial tissue, this may be an important reason to enhance the therapeutic effect in addition to the high hydrogen loading capacity of H₂‐PFOB NEs, and the combination with LIFU further enhances its therapeutic effect, realizing the effective release and utilization of hydrogen, thus facilitating the purpose of precise treatment of I/R injury. PFOB NEs, Perfluorooctylbromide nanoemulsions; LIFU, low‐intensity focused ultrasound; NLRP3, Nucleotide‐binding oligomerization domain 3; GSDMD, Gasdermin D.

2. Results

2.1. Synthesis and Characterization of H2‐PFOB NEs

PFOB NEs were synthesized and then hydrogenated by hydrogen absorption (Figure 2a). H₂‐PFOB NEs are spherical, small sized (DLS ≈150 nm) and uniformly dispersed, as revealed by TEM (Figure 2b,c). The zeta potential of H₂‐PFOB NEs (ζ potential ≈−30 mV) is consistent with that of un‐hydrogenated PFOB NEs (Figure 2d). Moreover, DLS measurements showed that H₂‐PFOB NEs maintain a similar size at different temperatures even after long periods of time, revealing good reproducibility and stability (Figure 2e). The Rhodamine B absorbance peak of H₂‐PFOB NEs and PFOB NEs measured by UV‐vis is ≈575 nm (Figure 2f). Elemental analysis showed that C, N, F, O, P, and Br are evenly distributed in PFOB NEs (Figure 2g). To determine the hydrogen‐carrying capacity of PFOB NEs, chromatography and hydrogen microelectrodes were used to measure hydrogen concentration. As shown in Figure 2h, PFOB NEs have an excellent hydrogen‐carrying capacity of ≈ 6 mM, which is about eight times higher than that of water. Moreover, the release of hydrogen in H₂‐PFOB NEs is slower when compared with H₂‐water, indicating that H₂‐PFOB NEs can produce hydrogen for long periods and are more conducive to hydrogen storage and utilization (Figure 2i).

Figure 2.

Synthesis and characterization of H₂‐PFOB NEs. a) Representative image of the H₂‐PFOB NEs. b) TEM image of H₂‐PFOB NEs. Scale bar 100 nm. c) DLS data of PFOB NEs and H₂‐PFOB NEs NEs. d) Zeta potential of PFOB NEs and H₂‐PFOB NEs. e) DLS of H₂‐PFOB NEs at different temperatures and time (Days). f) UV‐vis spectra of PFOB NEs and H₂‐PFOB NEs. g) H₂‐FPOB TEM mapping. h) Comparison of H₂ concentration of H₂‐Water and H₂‐PFOB NEs. i) Residual H₂ concentration of H₂‐Water and H₂‐PFOB NEs with increasing time. Data are shown by mean ± SD.

2.2. Behavior and Mechanism of H₂‐PFOB NEs Targeted Delivery to Ischemic Myocardium

Since Rhodamine B‐labeled H₂‐PFOB NEs possess fluorescent signal, we first performed ex vivo fluorescence imaging using the IVIS fluorescence imaging system to assess the metabolic distribution of H₂‐PFOB NEs in normal mice models. The results displayed that the majority of H₂‐PFOB NEs were metabolized by the liver, spleen and intestine, and cleared from the body after 72 h, whereas a small part of H₂‐PFOB NEs was metabolized by the respiratory and urinary system (Figure  3a,b). Further, the fluorescent characteristics of I/R group and sham group hearts were also explored by IVIS imaging system, excitingly, the results demonstrated that the excellent targeted accumulation properties of H₂‐PFOB NEs in I/R myocardium, meanwhile, the fluorescence intensity was also maintained for a prolonged period of time compared with the sham group hearts (Figure 3c,d). This targeted delivery behavior to ischemic myocardium indicates the potential therapeutic advantages of H₂‐PFOB NEs in treating I/R injury.

Figure 3.

Macrophage mediated targeting of ischemic myocardium of H₂‐PFOB NEs. a) Representative IVIS images of different times. b) Quantification of IVIS fluorescence intensity, n = 5. c) Representative IVIS images of Sham and I/R mice heart at different time points after injection. d) Quantification of IVIS fluorescence intensity, n = 5. e–g). Immunofluorescence staining of myocardial tissue of I/R mice after injection of H₂‐PFOB NEs (×20, scale bar 50 µm). h) TEM images of myocardial microtissues (×10 k, scale bar 100 nm). Yellow circles represent macrophages and the red triangle points to the lysosome. i) Peripheral blood mononuclear cells (PBMCs) from a control mouse and a mouse subjected to myocardial infarction and treated with FITC‐labeled H₂‐PFOB NEs were analyzed for FITC fluorescence by flow cytometry. j) Histograms display specifically stained leukocytes from control (blue) and I/R(red) mouse, leukocytes from I/R mouse was gated on FITC+ cells. Numbers indicate the percentage of FITC+ cells expressing the specific cell marker. Max, maximum. k–m) Gated on WBLs and specific cell markers, which shows FITC+ fluorescence from I/R mouse injected with FITC‐labeled H₂‐PFOB NEs. n) The percentage of Ly6C positive cells in CD11b⁺ cell population, and the percentage of Ly6C⁺ FITC⁺ in CD11b+Ly6C⁺ cell population. o) Flow cytometry analysis of specifically stained single cell suspension from control (blue) and I/R(red) mouse heart, cells form I/R mouse was gated on FITC⁺ cells. p) Gated on specific cell markers, which shows CD11b+FITC+ fluorescence from I/R mouse injected with FITC‐labeled H₂‐PFOB NEs. q) The percentage of F4/80 positive cells in CD11b⁺ cell population, and the percentage of F4/80⁺ FITC⁺ in CD11b⁺F4/80⁺ cell population. Data are shown by mean ± SD, *P < 0.05 **; P < 0.01; *** P < 0.001.

To investigate the I/R myocardium targeting mechanisms of H₂‐PFOB NEs, I/R mice, underwent 60 min of myocardial ischemia, were sampled after 1 h injection of H₂‐PFOB NEs to explore the cellular uptake properties of H₂‐PFOB NEs at the early stage of myocardial reperfusion. Corresponding immunofluorescence results indicated that H₂‐PFOB NEs highly co‐localized with the myocardial necrosis area (Figure 3e). To further examine the cell population containing the H₂‐PFOB NEs, I/R myocardium tissue sections were incubated with FITC‐labelled anti‐CD11b antibody. As shown in Figure 3f‐g, the fluorescent signal of Rhodamine B‐labelled H₂‐PFOB NEs overlapped to a large extent with macrophages in the ischemic area and border zone, but not in the non‐ischemic myocardium. TEM was used to observe the detailed distribution of H₂‐PFOB NEs in the myocardium. A significantly higher number of macrophages was detected in the ischemia area of the myocardium, and a majority of H₂‐PFOB NEs were engulfed by macrophages via phagocytosis (Figure 3h). After tail vein injection of FITC labeled H₂‐PFOB NEs and subsequent collection of blood samples, we analyzed different cell populations containing the H₂‐PFOB NEs. As shown in Figure 3i‐q, 1 h after injection of FITC‐labeled H₂‐PFOB NEs, cells positive (+) for FITC were detected. The large majority of the labeled cells exhibited the monocyte/macrophage marker CD11b, and only a marginal amount of lymphocytes (CD3) positive for FITC. Furthermore, we found that more than 90% of CD11b positive cells expressing Ly6c (inflammatory monocytes), and which account for 86.6% FITC⁺ CD11b⁺ cells. The above flow cytometric results revealed that H₂‐PFOB NEs are avidly taken up by inflammatory monocytes in circulating blood. In addition, the flow cytometry analysis of myocardial tissue further delineated that H₂‐PFOB NEs were mainly obtained by CD11b⁺ F4/80⁺ macrophages. We also notice that CD11b⁺ cells were significantly enhanced in I/R mice, and about half of them positive for FITC, of which more than 90% cells expressed F4/80 macrophages. Most importantly, macrophages account for 97% of FITC⁺ CD11b⁺ cells. Above results fully illustrated that the inflammatory monocytes/macrophages in circulating blood or I/R myocardial tissue mainly contributed to H₂‐PFOB NEs uptake and distribution. The significant increase in H₂‐PFOB NEs uptake was also observed in macrophages and cardiomyocytes subjected to LPS‐induced inflammation and hypoxia/reoxygenation (H/R), respectively (Figure S1 and S2, Supporting Information).

2.3. Hydrogen Controlled Release Profiles and ¹⁹F‐MR Detection Ability

To investigate the hydrogen‐controlled release profiles of H₂‐PFOB NEs, low intensity focused ultrasound (LIFU) was utilized to excite the nanoparticles. As expected, gas chromatography of H₂‐PFOB NEs + LIFU or H₂‐PFOB NEs showed hydrogen burst release only in the H₂‐PFOB NEs + LIFU group (Figure 4a). Next, the targeting ability of H₂‐PFOB NEs to the I/R myocardium was determined by measuring cardiac hydrogen levels in I/R injury models or normal mice with microelectrodes. Hydrogen concentration in I/R mice hearts peaked at higher values and was sustained for longer than in normal mice hearts (Figure 4b). To further investigate the controlled burst release of hydrogen from H₂‐PFOB NEs in the I/R myocardium, hydrogen concentration in I/R myocardial tissue combined with LIFU was measured immediately following the injection of H₂‐PFOB NEs. LIFU rapidly increased hydrogen concentration in the I/R myocardium, exceeding the peak cardiac H₂ concentration after intravenous H₂‐PFOB NEs (Figure 4c). These results suggest that combining H₂‐PFOB NEs with LIFU is an optimal strategy to achieve the controlled blast release of hydrogen molecules.

Figure 4.

LIFU responsiveness and visualization of hydrogen release of H₂‐FPOB NEs. a) The concentrations of H₂ released from H₂‐Water and H₂‐PFOB NEs were determined by gas chromatography. b) H₂ concentration in normal mice and I/R mice were measured by H₂ microelectrodes at different time after inject of H₂‐FPOB NEs. c) Hydrogen concentrations were measured in the myocardium of I/R mice with and without LIFU within 20 min after injection of H₂‐FPOB NEs using H₂ microelectrodes. d) ¹⁹F‐MRI of H₂‐PFOB NEs in normal mice and I/R mice heart (MRI examination at the beginning of reperfusion). e) Representative ¹⁹F‐MRI phantom images of PFOB NEs, H₂‐PFOB NEs and H₂‐PFOB NEs + LIFU at different time points. f) Corresponding ¹⁹F‐MRI signal intensity calculation of above groups, n = 5. ching cardiac images of I/R mice at different time points after injection (MRI examination at the beginning of reperfusion). h) The ¹⁹F signal‐to‐noise ratio (SNR) was calculated over time in the myocardium of above groups, n = 5. Data are shown by mean ± SD, *P < 0.05 **; P < 0.01; *** P < 0.001.

To further validate the I/R myocardium specific targeting capability of H₂‐PFOB NEs at the in vivo level, cardiac ¹⁹F‐MR imaging and ¹⁹F magnetic resonance spectra were performed after the mice were intravenous injected with H₂‐PFOB NEs. The schematic diagram of cardiac ¹⁹F‐MRI imaging is shown in Figure S3 (Supporting Information). Corresponding ¹⁹F magnetic resonance spectra of H₂‐PFOB NEs was shown in Figure S4 (Supporting Information). Most importantly, as can be seen from the ¹⁹F‐MR signal in Figure 4d, compared to normal myocardium, H₂‐PFOB NEs was highly infiltrated into the left ventricular free wall of ischemia myocardial tissue following ischemia‐reperfusion, in addition, which was highly consistence with the TTC&Evan Blue staining results, while almost no specific ¹⁹F‐MR signal was observed in the normal myocardium. Similarly, H₂‐PFOB NEs produce an enhanced ultrasound signal in the left ventricular (LV) cavity after injection (Figure S5, Supporting Information). To determine whether H₂ affects the fluorine signal of PFOB NEs, phantoms of hydrogenated PFOB NEs with or without LIFU were immediately scanned and ¹⁹F‐MR images were dynamically acquired at different time points. Intriguingly, hydrogen could lead to quenching of ≈50% of the PFOB NEs ¹⁹F‐MR signal (Figure 4e). As the hydrogen was released, the signal was recovered over time. As expected, a more rapid restoration was observed in the H₂‐PFOB NEs + LIFU group (Figure 4f). Next, we assessed whether ¹⁹F‐MR imaging can detect H₂‐PFOB NEs hydrogen‐controlled release in a I/R injury mice model in vivo (Figure 4g). The ¹⁹F‐MR signal in the I/R injury + H₂‐PFOB NEs group was influenced by hydrogen at every time point, but especially during the first 30 min after reperfusion, which can be attributed to the fluorine signal quenching effect of H₂‐PFOB NEs. The blasting release of hydrogen from H₂‐PFOB NEs controlled by LIFU reached a peak value within 20 min, while a ¹⁹F‐MR signal fluctuation in the I/R + H₂‐PFOB NEs group occurred ≈30 min. Compared with the I/R + PFOB NEs group, the decrease of fluorine signal was more pronounced in I/R + H₂‐PFOB NEs + LIFU group at 50 min, 70 min and 90 min after reperfusion (P<0.01) (Figure 4h), showing that H₂‐PFOB NEs have therapeutic potential to regulate inflammatory response in the I/R myocardium. Moreover, these data also indicate that LIFU can significantly accelerate hydrogen release from H₂‐PFOB NEs.

2.4. In Vivo H₂‐PFOB NEs Myocardial Ischemia‐Reperfusion Injury Therapeutic Effect

Given the hydrogen‐carrying capacity, passive targeting and LIFU‐controlled hydrogen releasing ability of H₂‐PFOB NEs, we tested the hypothesis that H₂‐PFOB NEs may exert favorable therapeutic effects on myocardial I/R injury on mice models (Figure  5a). TTC and Evans blue staining showed no significant differences in the left ventricle area at risk (AAR/LV) between any of the groups (Figure 5b,c). However, infarct area ratio (IFN/AAR) results showed that myocardial injury was significantly reduced in the H₂‐PFOB group when compared to the H₂‐water group (Figure 5d). When further combined with LIFU (H₂‐PFOB + LIFU), myocardial death in risk areas was almost completely inhibited when compared with H₂‐PFOB (P<0.05). Cardiac biomarkers were then analyzed by ELISA in the myocardial I/R injury model to evaluate the degree of myocardial damage. While a reduction in cardiac troponin I (c‐TnI) and lactate dehydrogenase (LDH) levels was detected in the saturated H₂‐Water group (P<0.05), in the H₂‐PFOB and H₂‐PFOB + LIFU groups the myocardial injury index was nearly completely reversed (Figure 5e,f).

Figure 5.

Effects of H₂‐PFOB NEs on infarct size and cardiac function in I/R mice. a) Schematic illustration of LIFU combined H₂‐PFOB NEs treatment strategies. b) Representative TTC&Evans blue staining images; c) AAR/LV (%), n = 7. d) INF/AAR (%), n = 7. e) Serum c‐TnI concentrations of each group, n = 7. f Serum LDH concentrations of each group, n = 7. g) Representative echocardiography image of mouse hearts; h) EF (%), n = 15. i) FS (%), n = 15. j) %ID/g, n = 5. k) Representative ¹⁸F‐FDG PET image of mice's heart. Data are shown by mean ± SD, *P < 0.05 **; P < 0.01; *** P < 0.001.

To further explore the effects of H₂‐PFOB NEs on cardiac function, we conducted cardiac ultrasound on myocardial I/R injury mice (Figure 5g–i). Cardiac contractile function measured by ejection fraction (EF) and fractional shortening (FS) indexes decreased significantly in the H₂‐PFOB NEs group (EF≈39%, FS≈54%) and to a lesser extent in the H₂‐water group (EF≈21%, FS≈28%). Notably, when H₂‐PFOB NEs were combined with LIFU, cardiac contractile function was restored to a greater extent than in the other groups (EF≈60%, FS≈76%). ¹⁸F‐FDG PET imaging were performed to evaluate the therapeutic efficacy of each treatment groups on I/R myocardium, the results of %ID/g further demonstrated that H₂‐PFOB NEs have a stronger therapeutic effect in alleviating myocardial damage and improving cardiac function than H₂‐water, especially when combined with LIFU (Figure 5j,k).

2.5. H₂‐PFOB NEs Myocardial Ischemia‐Reperfusion Injury Therapy Mechanisms

Next, we explored the mechanisms underlying the H₂‐PFOB NEs therapeutic effects described above. TEM reduced numbers of mitochondria and altered mitochondrial morphology, including increased size and disorganized cristae, suggesting that myocardial I/R injury causes mitochondrial oxidative stress (Figure  6a). Importantly, mitochondrial damage in myocardial ischemia was significantly attenuated in the H₂‐PFOB and H₂‐PFOB + LIFU groups. Moreover, the levels of the oxidative stress markers 8‐OHdG and malondialdehyde (MDA) decreased significantly the H₂‐PFOB group, particularly in combination with LIFU (Figure 6b,c). Similarly, measurements of hydroxyl radical (OH) concentrations reveled that H₂‐PFOB NEs, and H₂‐PFOB NEs + LIFU have better antioxidant properties than H₂‐water (Figure 6d). Finally, changes in the mitochondrial damage indicator ATP and oxidative stress markers superoxide dismutase (SOD) and glutathione (GSH), further revealed that mitochondrial oxidative stress in myocardial I/R injury is significantly reduced by H₂‐PFOB NEs in combination with LIFU (Figure 6e–g).

Figure 6.

Effects of H₂‐PFOB NEs on mitochondrial oxidative stress and NLRP3‐mediated pyroptosis in I/R mice. a) TEM images illustrating the cardiomyocytes (×10k, scale bar 100 nm). b) 8‐OHdG concentrations of each group, n = 7. c) Heart MDA concentrations of each group, n = 7. d) Relative ·OH concentrations of each group, n = 7 e) Relative ATP production of each group, n = 7. f Heart SOD activity of each group, n = 7. g) Heart GSH concentrations of each group, n = 7. h) Relative NLRP3 protein level, n = 5. i) Relative ASC protein level, n = 5. j) Relative IL‐1β protein level, n = 5. k) Relative GSDMD protein level, n = 5. l) Relative Caspase‐1 protein level, n = 5 Data are shown by mean ± SD, *P < 0.05 **; P < 0.01; *** P < 0.001.

To further examine the therapeutic effects of H₂‐PFOB NEs, we analyzed multiple markers of NLRP3‐mediated pyroptosis in myocardial I/R injury models. NLRP3 immunostainings showed that H₂‐PFOB NEs inhibit the expression of NLRP3 protein in myocardial tissue, and LIFU further enhanced this effect (Figure S6, Supporting Information). Moreover, western blot assays revealed that H₂‐PFOB NEs + LIFU significantly reduces the expression of the pyroptosis‐related proteins ASC, Caspase‐1, Gasdermin D and IL‐1β in myocardial I/R injury mice models (Figure 6h–l), suggesting that H₂‐PFOB NEs have a strong anti‐inflammatory effect on the NLRP3 inflammasome. Together these results strongly suggest that H₂‐PFOB NEs have important therapeutic effects on myocardial I/R injury by reducing oxidative stress and decreasing NLRP3 inflammasome activation‐mediated pyroptosis.

2.6. In Vivo H₂‐PFOB NEs Myocardial Ischemia‐Reperfusion Injury Prognostic Effect

Patients with ischemic reperfusion injury have a high probability of long‐term cardiac remodeling and heart failure, which seriously affect the quality of life and survival of patients. Therefore, based on the favorable therapeutic effects of H₂‐PFOB NEs during the acute phase of I/R injury, we further evaluated the long‐term outcomes of I/R (1 h of ischemia and 28 days of reperfusion) mouse treated with H₂‐PFOB NEs. The study schema is shown in Figure  7a, corresponding results were powered to show a reduction in mortality of I/R mouse treated with H₂‐PFOB NEs (Figure 7b). Compared with H₂‐water group, H₂‐PFOB and H₂‐PFOB + LIFU groups could further reduce the concentration of myocardial injury index BNP in serum (P<0.05) (Figure 7c). Transthoracic echocardiography was used to assess cardiac structure and function of mice in different treatment groups. Improvement in left ventricular ejection fraction (EF) occurs in H₂‐PFOB + LIFU group when compared with other groups (57.4% versus 54.7% versus 48.8%). In addition, the left ventricular fractional shortening (FS) was measured and the results demonstrated that H₂‐PFOB and H₂‐PFOB + LIFU groups are better able to improve systolic function in infarcted mouse hearts than H₂‐Water group. The measurement results of left ventricular end‐diastolic internal diameter (LVIDd) and left ventricular end‐systolic internal diameter (LVIDs) were illustrated that H₂‐PFOB and H₂‐PFOB + LIFU groups could better prevent the onset of the subsequent deterioration of long‐term cardiac function and cardiac remodeling in I/R mice (Figure 7d–h). Furthermore, to assess the cardiac remodeling and myocardial fibrosis, paraffin‐embedded heart sections were stained with Masson trichrome staining. As expected, H₂‐PFOB NEs significantly reduced myocardial fibrosis, especially in the H₂‐PFOB + LIFU group (Figure 7i,k). WGA and CD31 immunohistochemical staining results showed that H₂‐PFOB NEs could moderate hypertrophy of surviving cardiomyocytes and promote the development of neovascularization, which further contributes to the prevention of the long‐term heart failure and cardiac remodeling caused by I/R injury (Figure 7j–n).

Figure 7.

Effects of H₂‐PFOB NEs on long term cardiac structure and myocardial fibrosis in I/R mice. a) Schematic illustration of LIFU combined H₂‐PFOB NEs treatment strategies. b) Survival curve of rats of each experimental group. c) The Serum BNP concentration of each group, n = 5; d) Representative echocardiography image of mice's heart. e–h) EF (%); FS (%); LVIDd and LVIDs, n = 7; i) The representative Masson images of mice heart; j) The representative immunohistochemical staining images of wheat germ agglutinin (WGA) expression in each group (×20, scale bar 50 µm). k) Collagen volume fraction (CVF) of left ventricle, n = 5; l) Quantitative analysis of left ventricular tissue sections stained with WGA, n = 5; m,n) The representative immunohistochemical staining images of CD31 and quantitative analysis of CD31 positive rate, n = 5 (×20, scale bar 50 µm). Data are shown by mean ± SD, *P < 0.05 **; P < 0.01; *** P < 0.001.

2.7. Cytotoxicity and Biosafety of H₂‐PFOB NEs

H₂‐PFOB NEs cytotoxicity was assessed in primary cardiomyocytes using standard CCK‐8 cell viability assays. H₂‐PFOB NEs exhibited negligible cytotoxicity in primary cardiomyocytes even at high concentrations (Figure S7, Supporting Information). Moreover, no obvious body weight loss was observed in balb/c mice treated with H₂‐PFOB NEs (Figure S8, Supporting Information). In vivo toxicity of H₂‐PFOB NEs was assessed by histological analyses of major organs (brain, heart, liver, spleen, lung, and kidneys) harvested 24 h after H₂‐PFOB NEs NEs injection. As shown in Figure S9 (Supporting Information), H₂‐PFOB NEs dosed at 10 µL g⁻¹ 30% (v/v) induced no obvious pathological changes, including on cytoplasm loss, cell atrophy or inflammation, further suggesting that these nanoemulsions have excellent histocompatibility.

In addition, standard biochemical analyses revealed no changes in blood urea (UREA) or creatinine (CREA) levels in the H₂‐PFOB NEs group, indicating normal kidney function. Alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST) levels also remained normal in the H₂‐PFOB group at days 1 and 7 after injection, demonstrating healthy liver function. Finally, no significant changes were detected in red blood cells (RBCs), mean corpuscular hemoglobin (MCH), white blood cells (WBCs), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT), hemoglobin (HGB), mean corpuscular volume (MCV), hematocrit (HCT), mean platelet volume (MPV), or platelet distribution width (PDW), showing that mice injected with H₂‐PFOB NEs have no apparent infections or defects in the physiological regulation of their immune system (Figure S10, Supporting Information).

3. Discussion

Myocardial ischemia‐reperfusion (I/R) injury has an important negative impact on the therapeutic effects of revascularization therapy, thereby challenging the development of effective therapeutic drugs.^{[ 14 ]} The deleterious role of reactive oxidative stress (ROS) on cardiac muscle has been comprehensively studied in I/R injuries, and it is well established that a burst of mitochondrial‐derived ROS contributes to muscle damage.^{[ 15 ]} Moreover, the involvement of ROS in oxidative stress‐induced myocardial dysfunction is particularly relevant to the development of targeted therapies.^{[ 16 ]} Hydrogen has a protective ability against oxidative stress and inflammation, and extensive research has demonstrated the preclinical and clinical efficacy of hydrogen in the treatment of a variety of inflammatory‐associated diseases, including diabetes, atherosclerosis and cancer.^{[ 4} , ^{17 ]} In a previous study, our research has shown that hydrogen can reduce myocardial I/R injury by inhibiting ROS‐mediated pyroptosis. Moreover, hydrogen also plays a major role in reducing the no‐reflow phenomenon and in alleviating cardiac remodeling and myocardial fibrosis after myocardial infarction.^{[ 18 ]} However, current hydrogen delivery strategies release low amounts of uncharged hydrogen molecules that can quickly enter the cytoplasm, mitochondria and even the nucleus,^{[ 19 ]} which affects hydrogen dosage and therefore reduces therapeutic efficacy.^{[ 8 ]} Thus, the development of more advanced methods for targeted delivery and controlled release of hydrogen is critical to improve hydrogen‐based therapy.

PFCs, a group of chemically inert synthetic molecules with excellent biocompatibility, have been widely studied and used in the clinic for various purposes, including artificial blood substitution, organ preservation, ultrasound imaging, and fluorine magnetic imaging.^{[ 11 ]} The major advantage of PFCs stems from their unique ability to dissolve significant amounts of oxygen, owing to van der Waals forces.^{[ 20 ]} To our knowledge, this is the first study to explore the hydrogen‐carrying capacity of PFOB NEs. We have successfully developed PFOB NEs with high hydrogen‐carrying capacity, LIFU‐controlled release, and optimal biocompatibility. PFOB NEs offer multiple advantages, such as ischemic myocardium‐specific targeting and ¹⁹F‐MR‐based dynamic visualizing of hydrogen release. In contrast to other hydrogen carriers,^{[ 21 ]} these properties make H₂‐PFOB NEs a promising candidate in precision medicine for the treatment of various diseases. Indeed, given their excellent biosafety profile, H₂‐PFOB NEs have significantly better clinical translational potential than current hydrogen carriers.^{[ 22 ]}

Since myocardial I/R injury mediated by oxidative stress and inflammation is more evident in the early phase of the injury process,^{[ 23 ]} we administrated H₂‐PFOB NEs at the beginning of the reperfusion period. H₂‐PFOB NEs can rapidly infiltrate into the targeted site and release hydrogen efficiently, particularly when used in combination with LIFU. Hydrogen concentration in ischemic area reaches a peak at the early stage of reperfusion, thereby effectively alleviating oxidative stress damage and inflammasome activation of I/R injury, which in turn reduces infarct size and restores cardiac function. Thus, H₂‐PFOB NEs used in combination with LIFU maximize protection against myocardial I/R injury.

Previously developed hydrogen‐loaded nanocarriers have therapeutic applications mostly for the treatment of cancer and osteoarthritis.^{[ 24 ]} In this study, we report a novel hydrogen carrier, H₂‐PFOB NEs, and describe its therapeutic properties and molecular mechanisms of myocardial I/R injury recovery in preclinical models. Thus, we have uncovered a new ¹⁹F‐MR imaging‐guided strategy for the diagnosis and precise treatment of myocardial I/R injury and expanded the scope of clinical applications of hydrogen carriers. With exciting translational prospects ahead, future large animal in vivo investigations are necessary to further assess the role of H₂‐PFOB NEs in I/R myocardium precision therapy, moreover, the potential therapeutic benefits of H₂‐PFOB NEs in other diseases certainly deserves further investigation.

4. Conclusion

In this study, we developed an advanced precise treatment strategy for myocardial I/R injury with potential clinical applications in other diseases. H₂‐PFOB NEs have high hydrogen load capability and can passively infiltrate into ischemic myocardial tissue via a macrophage‐mediated process. In combination with LIFU, H₂‐PFOB NEs exert remarkable therapeutic effects on I/R injury due to the LIFU‐mediated controlled release of hydrogen in targeted injury areas. Moreover, to our knowledge H₂‐PFOB NEs is the first ¹⁹F‐MRI molecular imaging probe that allows the dynamic visualization of hydrogen release in vivo. Finally, our data suggest that H₂‐PFOB NEs have excellent antioxidant stress and anti‐inflammatory properties which further contribute to the reversal of I/R injury. Thus, H₂‐PFOB NEs in combination with LIFU is a novel hydrogen delivery therapeutic agent with great potential for clinical translation.

5. Experimental Section

Animals

Balb/c mice weighing 22–25 g were obtained from Charles River Labs (Beijing, China). The animals were bred and housed under standard conditions (12 h light‐dark cycle, 24 °C), with free access to water and standard laboratory chow. The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of Harbin Medical University (Approval No. 2021WZYSLLSC‐20).

Chemicals and Preparation of Perfluorocarbon Nanoemulsions (NEs)

Unless otherwise listed, all solvents and reagents were purchased from Aldrich Chemical Co. and used as received. Phospholipids were purchased from Avanti Polar Lipids, Inc. (Alabama, USA). Perfluorooctylbromide (PFOB, C₈BrF₁₇) was purchased from Exfluor Research Corporation (Round Rock, TX) and used as acquired.

The fluorescently tagged perfluorocarbon nanoemulsions (NEs) were synthesized using microfluidization similar to prior reports.^{[ 25 ]} Briefly, the nanoparticulate emulsions were comprised of 30% (v/v) PFOB, 3% (w/v) of a surfactant commixture. The surfactant included 80 mol% dipalmitoyl phosphatidylcholine (DPPC), 0.5 mol% 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphoethanolamine (DPPE), 0.2 mol% dipalmotoylphosphatidylglycerol (DPPG), 19.3 mol% cholesterol, 0.02 mol% lissaminerhodamine B sulfonyl (16:0 LissRhod PE). The lipids were dissolved in a mixture of methanol and chloroform, filtered through a small bed of cotton, evaporated under reduced pressure using a rotary evaporator at 45 °C to form a thin film, and then further dried in a vacuum oven (45 °C) for 24 h. The resuspended surfactant was combined with PFOB and water, and then emulsified in a M110P Microfluidics emulsifier (Microfluidics, Newton, MA) at 20,000 psi for 4 min. The completed emulsions were placed in crimp‐sealed vials, blanketed with argon, and stored at 4 °C until use.

Physical and Chemical Characterization of the H₂‐PFOB NEs

Hydrodynamic diameter distribution, polydispersity, and zeta potential (ζ) of the PFC nanoparticles were determined by dynamic light scattering (DLS) with a Malvern Nano ZS Zetasizer (Malvern Instruments Ltd, Malvern, UK). All determinations were made in multiples of three consecutive measurements. The absorbance of Rhodamine B was measured by using a multiplate reader using the option UV Absorbance Spectrophotometer (BioTek, Winooski, VT).

The morphology and microstructure of the PFOB NEs were determined by transmission electron microscopy (TEM) on a Hitachi‐7700 microscope at an accelerating voltage of 220 kV. Briefly, a drop of the sample was deposited on a carbon grid coated with copper and the excess sample was drawn off with filter paper 1 min later and was then left for 5 min to be dried. Digital TEM photographs of the PFOB NEs were obtained.

Measurement of Hydrogen Release Behavior of the H₂‐PFOB NEs

The amount of dissolved hydrogen (H₂) and the release rate of H₂ in pure water and perfluorocarbon (30 % v/v) solution were measured with a gas chromatograph and a hydrogen microelectrode. Specifically, saturated hydrogen water was diluted with pure water in different proportions to prepare a standard curve. Next, hydrogen concentrations in H₂‐PFOB NEs, and hydrogen concentrations in the heart after H₂‐PFOB NEs injection were measured.

Metabolic Distribution In Vivo

In vivo degradation of the nanoemulsions was monitored with an In Vivo Imaging System (IVIS Lumina XR, USA) after Balb/c mice were intravenously injected with H₂‐PFOB NEs (10 µL g⁻¹, 30% (v/v) PFOB NEs). The mice were sacrificed 1 h, 12 h, 24 h, and 72 h after injection, and all major organs (heart, liver, spleen, lung, kidney, intestine, muscle, and bone) were harvested and imaged ex vivo using an IVIS Spectrum. The average total fluorescence intensity of each organ was semi‐quantified using the IVIS imaging software.

Animal 9.4 T Magnetic Resonance Imaging (MRI) Experiments

All magnetic resonance imaging (MRI) experiments were performed on a Bruker Biospin 9.4T scanner (Bruker BioSpin 94/20 USR system, Germany) with a ¹H/¹⁹F double tune volume coil. After acquisition of the morphological ¹H images, the resonator was tuned to ¹⁹F and the corresponding ¹⁹F‐MR images were recorded from the same FOV using a ¹⁹F chemical shift‐selective (CSSI) pulse sequences with multislice rapid acquisition. Mice were anaesthetized by inhalation with an initial dose of 2 % isoflurane (RWD Life Science) and were maintained spontaneously breathing 1.5% isoflurane, supplied via a nose cone. During imaging, respiratory rates were continuously monitored by a small animal monitoring and gating system (SA Instruments), and the body temperature was maintained at 37 °C using a temperature‐controlled heating system (Thermo). Parameters of ¹⁹F_CSSI sequence: RARE factor, 64; matrix, 64×64; slice thickness, 3 mm; averages, 128; acquisition time, 8.32 minutes. For fusion with ¹⁹F images, additional ¹H data sets with a slice thickness of 1.0 mm were recorded.

Low‐Intensity Focused Ultrasound (LIFU)

The parameters and settings of low‐intensity focused ultrasound (LIFU, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences) were as follows: Focus area of 0.4 cm², pulse wave mode, 50 % duty cycle, 1.1 MHz, 55.8 mW cm⁻², and focal length of 2 cm. LIFU acoustic intensity was confirmed by hydrophone. The schematic diagram of experimental procedure of LIFU H₂‐PFOB NEs therapy applied to myocardial ischemia‐reperfusion injury and animal grouping were shown in Figure 6a. Specifically, H₂‐PFOB NEs (10 µL g⁻¹, 30% (v/v)) were administered to mice 1 h after ischemia that exactly at immediate reperfusion the infusion was done and followed by LIFU irradiation for 20 min to facilitate controlled release of the hydrogen at the targeted area.

Mouse Myocardial Ischemia‐Reperfusion Model

Balb/c mice were anesthetized with Ketamine/Xylazine mixture, intubated, and ventilated with a Ventstar mouse ventilator (RWD; stroke volume, 250 µL; respiratory rate, 105 breaths per minute). An ECG device was used for electrocardiogram (ECG) monitoring of mice in real time to ensure the experimental model was suitable. Following left thoracotomy between the fourth and fifth ribs, a 7‐0 nylon suture was placed around the left coronary artery for ligation, with the aid of a dissecting microscope. The ischemic state of heart was confirmed by evidence of immediate visible changes, including sudden pallor and paralysis of the affected part of the left ventricle, accompanied by an ST‐T segment elevation detected in the ECG. After 60 min of ischemia, the ligature was then released, the chest was closed in layers and the mice were allowed to recover^{[ 3a ]}. Next, H₂‐Water, PFOB NEs and H₂‐PFOB NEs (10 µL g⁻¹, 30% (v/v) PFOB) were injected through the caudal vein. The dosage of PFOB injected refers to previous published studies^{[ 26 ]}. In the sham group, the chest was opened and the suture needle passed through left coronary artery, but it was not ligated.

Infarct Size Measurement

After reperfusion, each mouse group was anesthetized with a Ketamine/Xylazine complex and a thoracotomy was performed. Then, the left ventricle (LV) areas at risk (AAR) were assessed for each mice group with 1 ml of 2 % Evan's blue dye (Sigma, E2129, USA) perfusion while ensuring that the left coronary artery (LCA) was re‐occluded at the same position. Next, the hearts stained with Evan's blue were removed rapidly and frozen at −20 °C for 15 min after sacrifice and then sliced horizontally at 1–2 mm thickness under the ligation line, to compare the myocardial tissues of the different groups. The heart slices were immersed in 1 % 2,3,5‐triphenyltetrazolium chloride (TTC) (Sigma, T8877, USA) phosphate buffer saline (PBS) solution for 15 min at 37 °C. The viable myocardial tissue was displayed in red and the nonviable myocardial tissue appeared to be white. The stained slices were imaged with a digital camera, and the total LV area, the AAR, and the infarct area were quantified using an Image‐Pro Plus software. AAR was expressed as a percentage of the LV (AAR/LV), and infarction (INF) per area at risk was expressed as a percent of the AAR (INF/AAR).

Echocardiographic Assessment of Cardiac Function

Cardiac function was evaluated by 2D transthoracic echocardiography on conscious mice using a VisualSonics Vevo2100 imaging system, as described previously^{[ 27 ]}. Fractional shortening (FS) and ejection fraction (EF) were used as indices of cardiac contractile function. FS was calculated according to the following formula: FS = (LVIDd‐LVIDs)/LVIDd × 100%. EF was calculated as EF = [(LVIDd)³‐(LVIDs)³]/(LVIDd)³ × 100%. All measurements were performed by an experienced operator blinded to the study.

Heart ¹⁸F‐FDG Positron Emission Tomography (PET) Imaging

¹⁸F‐FDG was obtained as an aliquot from the daily production for the clinical positron emission tomography (PET) at the Fourth Affiliated Hospital of Harbin Medical University qualified for administration in humans. ≈0.15≈0.2 mCi of ¹⁸F‐FDG per mouse was administered intravenously. Mice micro‐PET imaging was acquired 45 min after ¹⁸F‐FDG injection using a Super Argus PET scanner (Sedecal, Spain). ROI of myocardial ischemia was outlined by Amide software, and FDG uptake of ischemic myocardium was quantitatively analyzed by % ID/g, as previously reported.^{[ 28 ]}

ELISA

Final blood was collected in 1% EDTA, centrifuged at 3000 rpm for 15 min and the plasma collected. The plasma concentrations of lactate dehydrogenase (LDH) (Cusabio Bio, CSB‐E11723m, China), cardiac troponin I (c‐TnI) (Cusabio Bio, CSB‐E08421m, China) and 8‐hydroxy‐2 deoxyguanosine (8‐OHdG) (Cusabio Bio, CSB‐E10527m, China) were measured using ELISA kits, according to the manufacturer's instructions.

Measurement of Malondialdehyde and Hydroxyl Radical Concentrations

Malondialdehyde (MDA) concentration was measured with a commercial kit (Jiancheng BioEngineering Institute, A003‐2‐1, China) to monitor oxidant‐mediated lipid peroxidation. The concentration of hydroxyl radical (OH), a type of toxic ROS, was measured with commercial kit (Jiancheng BioEngineering Institute, A018‐1‐1, China).

Measurement of ATP, Superoxide Dismutase (SOD) and Glutathione (GSH) Level

ATP concentration was measured with a commercial kit (Beyotime, S0026, China) to monitor mitochondrial function. Oxidative stress balance index GSH (Solarbio Life Science, BC1175, China) and SOD concentration (Solarbio Life Science, BC0170, China) were measured by using a commercial kit.

Flow Cytometry

Flow cytometry were conducted to further explore the underlying mechanism of the selective accumulation of H₂‐PFOB NEs in the injured heart. I/R mice were injected with 10 µL g⁻¹ FITC labeled probe, respectively, at 1 h post injury. 30 min after the injection, the blood of the mice was stained with specific antibodies, APC anti‐mouse CD45 (#103111, Biolegend, USA), PE/Cyanine7 anti‐mouse CD3 (#100219, Biolegend, USA), APC/Cyanine7 anti‐mouse CD11b (#101225, Biolegend, USA), PE anti‐mouse Ly‐6C (#128007, Biolegend, USA) and PE anti‐mouse F4/80 (#123109, Biolegend, USA). After centrifugation at 1000 rpm for 5 min, the cells were resuspended in Staining Buffer, and fluorescence signals were detected by BD FACS Aria III. None‐cardiomyocytes single‐cell suspension of tissues in the marginal area of the injury was obtained by using Multi Tissue Dissociation Kit 2 (MiltenyiBiotec, USA) according to the instructions. Briefly, after cut into small pieces (1–2 mm³), harvested heart tissue was digested in Enzyme Mix at 37 °C for 15 min, followed by mechanical agitation through the Program Multi_G of the gentle MACS Dissociator (MiltenyiBiotec, USA), and then repeated once. After filtered through MACS SmartStrainer (70 um), the resulting sample was centrifuged at 600 g for 5 min to separate singe cells. The cell pellet of one sample was resuspended in 10 µL g⁻¹ staining buffer. Follow‐up staining and detection process were the same as described previously.

Western Blotting Analysis

Protein samples were extracted using lysis buffer containing 1 % protease inhibitor (Roche, Switzerland). Protein concentrations were measured using a BCA protein kit (Beyotime Institute of Biotechnology, Shanghai, China). The proteins were separated by 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS) and transferred onto polyvinylidene difluoride membranes (PALL, USA). The membranes were incubated with primary antibodies against NLRP3 (NBP2‐12446, Novus, USA; 1:200), GSDMD (sc‐393581, Santa Cruz Biotechnology, USA; 1:200), IL‐1β (ab9722, Abcam, USA; 1:1000), caspase‐1 (22915–1‐AP, Santa Cruz Biotechnology, China; 1:200), ASC (sc‐514414, Santa Cruz Biotechnology, USA1:200) and β‐actin (1:1000; #bs‐0061R, BIOSS, Boston, MA, USA) at 4 °C overnight. After washing with T‐BST, the membranes were inoculated at room temperature with horseradish peroxidase‐labelled secondary antibodies (SA00001‐1 and 2, Proteintech, China; 1:2000) for one hour. Next, ECL reagent was employed to develop the bands and the protein bands were detected by autoradiography and analyzed with ImageJ software. β‐actin was used as a normalized reference.

Masson and Wheat Germ Agglutinin (WGA) Stain

Hearts were harvested, fixed with 4% paraformaldehyde, and then paraffin‐embedded. After that, 5 mm sections were made. Masson staining were employed to assess cardiac fibrosis along with collagen deposition by a Masson stain kit. The whole heart fibrosis (fibrosis area/whole heart area) was computed as the ratio of fibrosis area to the overall or left ventricular assessed area quantified with the Image J software (V.1.50, Bethesda, USA). The sections of hearts were prepared and stained with WGA following the instruction of manufacture protocol. The green fluorescence indicates the cell membrane, and the surface area of cardiomyocyte was measured by Image J software.

Statistical Analysis

All data were expressed as mean ± SD. Data normality was determined by Shapiro‐Wilk test. Student's t‐test was used for comparisons between two groups. One‐way ANOVA followed by Bonferroni post hoc analysis was used when comparing > 2 groups. Two‐way ANOVA followed by Bonferroni post hoc analysis was used when more than 2 groups and variables were compared. P<0.05 was considered statistically significant. Prism 9.0 (GraphPad Inc, San Diego) and IBM SPSS Statistics 22.0 (IBM Co., Armonk, New York) were used for the statistical analyses.

Conflict of Interest

The authors declare no conflict of Interest.

Author Contributions

C.N., R.A, and J.W. contributed equally to this work. C.N. and X.S. designed the experiment and drafted the manuscript. X.S. supervised whole study and provided invaluable advice and assistance. W.Y. and X.S. developed the research idea. L.W provides PFOB NEs. C.N. and R.A. performed the statistical analyses, as well as interpreted the results and drafted the manuscript. J.W completed the cardiac ¹⁸F‐FDG PET imaging of mice. S.P., R.Z., B.W., S.X., X.H., M.Z., H.W., M.Y. helped with the experiments. The authors read and approved the final manuscript.

Supporting information

Supporting Information

Nie C., A R., Wang J., Pan S., Zou R., Wang B., Xi S., Hong X., Zhou M., Wang H., Yu M., Wu L., Sun X., Yang W., Controlled Release of Hydrogen‐Carrying Perfluorocarbons for Ischemia Myocardium‐Targeting ¹⁹F MRI‐Guided Reperfusion Injury Therapy. Adv. Sci. 2023, 10, 2304178. 10.1002/advs.202304178

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.