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Nature Communications volume 15, Article number: 8720 (2024 ) Cite this article non ionic wetting agent
The optimization of the enzyme-like catalytic selectivity of nanozymes for specific reactive oxygen species (ROS)-related applications is significant, and meanwhile the real-time monitoring of ROS is really crucial for tracking the therapeutic process. Herein, we present a mild oxidation valence-engineering strategy to modulate the valence states of Mo in Pluronic F127-coated MoO3-x nanozymes (denoted as MF-x, x: oxidation time) in a controlled manner aiming to improve their specificity of H2O2-associated catalytic reactions for specific therapy and monitoring of ROS-related diseases. Experimentally, MF-0 (Mo average valence 4.64) and MF-10 (Mo average valence 5.68) exhibit exclusively optimal catalase (CAT)- or peroxidase (POD)-like activity, respectively. Density functional theory (DFT) calculations verify the most favorable reaction path for both MF-0- and MF-10-catalyzed reaction processes based on free energy diagram and electronic structure analysis, disclosing the mechanism of the H2O2 activation pathway on the Mo-based nanozymes. Furthermore, MF-0 poses a strong potential in acute kidney injury (AKI) treatment, achieving excellent therapeutic outcomes in vitro and in vivo. Notably, the ROS-responsive photoacoustic imaging (PAI) signal of MF-0 during treatment guarantees real-time monitoring of the therapeutic effect and post-cure assessment in vivo, providing a highly desirable non-invasive diagnostic approach for ROS-related diseases.
Nanozymes can mimic the activity of natural enzymes, and have attracted considerable attention due to their advantages of low cost, high stability, mass production and durability1,2. However, the main defect of nanozymes is poor reaction selectivity because of the lack of enzyme-like molecular recognition units with the spatial and componential cooperation observed in natural enzymes3. Oxidoreductases such as catalase (CAT)4, superoxide dismutase (SOD)5, peroxidase (POD)6, and oxidase (OXD)7 are in the spotlight for their ability of scavenging or generating reactive oxygen species (ROS) which are associated with the occurrence and development of various diseases8,9,10. Therefore, it is of great significance to manipulate the selectivity of such nanozymes. To this end, plenty of efforts have been made on the intrinsic engineering of active centers in nanozymes, including atomic doping11,12, coordination environment adjustment13 and crystal facet regulation14,15,16. However, the modulation of selectivity of multiple enzymatic reaction pathways involving the same substrate has rarely been explored mechanistically and remains a challenge for use in specifically catalytic therapy of diseases. In particular, nanozymes with both CAT- and POD-like activities require H2O2 as substrate but produce O2 and hydroxyl radicals (·OH), respectively, which will inevitably compete for H2O2 to a certain extent, affecting H2O2 utilization and performance in target applications14. Recently, we have proved that the valence engineering is a fascinating and powerful strategy in modulating the performance (activity) of nanozymes17. Specifically, the valence states of active sites have been adjusted by varying the calcination conditions for Co/TiO2 single-atom nanozymes17, doping metal into spinel oxide ZnMn2O418 and investigating the size effects on ruthenium nanoparticles (RuNPs)19. Correspondingly, with the increasing content of Co2+, Mn4+ or Ru4+, single or multiple enzyme-like activities remarkably improved, facilitating the treatment of ROS-related models such as tumor, inflammatory bowel disease and liver injury. To date, however, few guidelines exist for the design and regulation of reaction selectivity (pathway) of nanozymes with multi-activities through valence-engineering approach.
Acute kidney injury (AKI) has been considered as one of the most common clinical complications with high morbidity and mortality, which is asymptomatic and has no characteristic clinical manifestations until extremely loss of renal function characterized by decreased renal excretion function and increased nitrogen metabolism accumulation30,31. Therefore, effective treatment, precise diagnosis and post-cure assessment of AKI are urgently needed and of great clinical significance. Oxidative stress is the most dominant pathophysiological mechanism in the occurrence and development of AKI, which may lead to the abnormality of renal oxidative metabolism, thereby ROS produced in excess by renal-infiltrating triggers the damaging of kidneys and causes AKI. Thus ROS is definitely a key target for the prevention and treatment of AKI32,33,34. Currently, the antioxidant N-acetyl cysteine (NAC) is mainly used in the clinical therapy of AKI35, but it obviously suffers from high dose and low bioavailability. As such, nanozymes with promising ROS scavenging ability was proven to be excellent alternatives for the treatment of acute colitis36, acute gout37, acute kidney injury38, androgenetic alopecia39 and other ROS-related diseases40,41.
In addition, current clinical diagnostic and post-cure evaluation for AKI mainly depends on the measurement of renal function indicators such as blood urea nitrogen (BUN) and serum creatinine (CRE) which are insensitive to early-stage kidney dysfunction. Although imaging techniques like magnetic resonance imaging (MRI), positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have been employed to evaluate different stages of renal dysfunction, however, they suffer from low sensitivity, radiation risk or high cost. Non-invasive optical imaging in the near-infrared (NIR) region with deep penetration, high resolution and sensitivity, fast feedback and non-ionizing radiation enables the direct visualization and monitoring of deep tissues such as kidney in vivo42,43,44,45. As such, it is highly desirable to develop renal activatable NIR optical sensors that specifically respond to the kidney related biomarkers (e.g. ROS-responsive). In especial, the theranostic probes for real-time imaging and amelioration of AKI are still lacking and challenging.
The x refers to the oxidation treatment time (hrs) for MF-x.
Interestingly, MF-0 posed a strong potential in AKI treatment, showing excellent therapeutic efficacy in vitro and in vivo, as well as desirable stability and biocompatibility. More intriguingly, MF-0 was able to induce the photoacoustic (PA) signal (driven by NIR light) variations with the consumption of ROS during treatment, guaranteeing the real-time and in situ monitoring of ROS in deep tissues. In AKI mice, the diminished PA signal (“off”) caused by the excess ROS, was turned on as ROS in the kidneys were scavenged in AKI-cured mice (treatment with MF-0) (Fig. 1). The ROS-responsive switchable PAI circumvented false-positive signals from nonspecific retention and successfully realized real-time monitoring of therapeutic process and post-cure assessment for AKI in vivo. This work provides a promising example on tuning the H2O2 activation pathway on Mo-based nanozymes for highly efficient catalytic therapy and post-cure assessment via non-invasive PAI of ROS-related diseases.
Transmission electron microscope (TEM) image indicated that the hydrophobic MoO3-x nanoparticles (NPs) were obtained with a uniform morphology and TEM observed size of ~8 nm (Fig. 2a). As shown in Fig. 2b, high-angle annular dark-field scanning transmission election microscope (HAADF-STEM) image further presented regular lattice fringes with lattice spacing of 0.247 and 0.351 nm, corresponding to the (–2 1 1) and (–1 1 1) crystal planes of tugarinovite MoO2, respectively. Meanwhile, Mo and O were observed uniformly distributed in the particles according to the elemental mapping. In addition, XRD pattern of MoO3-x NPs (Supplementary Fig. 1) matched that of MoO2 (JCPDS 32-0671), further proving the successful preparation of molybdenum oxide NPs.
TEM image (a) and HAADF-STEM image and elemental mapping (scale bar: 10 nm) (b) of MoO3-x. The experiments were independently repeated three times with similar results. c Mo 3d XPS spectra of MF-0, MF-2, MF-3.5, MF-5 and MF-10. d Percentage contents of Mo6+, Mo5+ and Mo4+ and the average valence of Mo in the corresponding samples. e UV-vis-NIR absorption spectra of each sample (inset: photographs of each aqueous solution). Source data are provided as a Source Data file.
Hydrophobic MoO3-x was then transferred to hydrophilic NPs through coating of amphiphilic Pluronic F127, which were then oxidized for different times (0, 2, 3.5, 5 and 10 h) under the mild oxidation condition to form MF-x, namely, MF-0, MF-2, MF-3.5, MF-5 and MF-10, respectively, showing various enzyme-like activities. TEM images indicated that MF-x samples were well dispersed (Supplementary Fig. 2) with an average dynamic light scattering (DLS) size from ~21 to ~24 nm (Supplementary Fig. 3a), and zeta potential from –10.7 to –14.6 mV (Supplementary Fig. 3b). These slight variations in size and zeta potential would have negligible effects on the enzyme-like activity of nanozymes46,47,48. Meanwhile, HAADF-STEM image and the elemental mapping of MF-0 (Supplementary Fig. 4) presented consistent microstructure with that of hydrophobic MoO3-x NPs. FT-IR spectra (Supplementary Fig. 5) and thermogravimetric (TG) analysis (Supplementary Fig. 6) further confirmed the connectivity of organic components for the as-prepared nanozyme, that is, the presence of oleylamine and oleic acid as the surface ligands of hydrophobic MoO3-x NPs, as well as the F127 polymer coating on hydrophilic MF-0. Furthermore, XPS fitting spectra for Mo 3d (3d3/2 and 3d5/2) revealed the mixed-valence states of Mo6+, Mo5+ and Mo4+ in MF-x, with significant differences in the content of each valence state among the samples (Fig. 2c). As illustrated in Fig. 2d, the percentage content of Mo4+ decreased from 56.3% for MF-0 to 6.5% for MF-10, and the percentage of Mo6+ increased from 20.5% to 74.8%. Accordingly, the average valence of Mo increased from 4.64 for MF-0 to 5.68 for MF-10. As expected, the elevation of high valence (Mo6+) content and the average valence of Mo were positively correlated with the oxidation time for valence tuning of Mo, thus performing the regulation of enzyme-like activity. UV-vis-NIR absorption spectra in Fig. 2e exhibited noticeable decrease of absorption from MF-0 to MF-10, with obviously different colors (MF-0 and MF-2: dark grey, MF-3.5: navy blue, MF-5 and MF-10: yellow), due to the variation of valence state of Mo.
a Oxygen production ability of MF-0, MF-2, MF-3.5, MF-5 and MF-10 treated with H2O2 under pH 7.4, respectively. b Absorbance intensity difference (at 650 nm) between different time points and 0 min of TMB aqueous solution treated with the corresponding samples in the presence of H2O2 (100 μM) under pH 6.5 (means ± SD, n = 3 independent experiments). c ESR spectra of ·OH trapped by DMPO in different samples treated with H2O2 under pH 6.5. d ESR spectra of MF-0 and MF-10 (powder), performed in a quartz tube at room temperature. e Free energy diagram of the reaction process responsible for the CAT- and POD-like activities of MF-0 and MF-10 (inset: the surface configuration of MF-0 and MF-10 at different stages). f The calculated Mo 4d PDOS of MF-0 and MF-10. g Charge density difference of the 2*OH or *O2 on MF-0 and MF-10, respectively. Isosurface value: 0.003 e/Bohr3. h Schematic illustration of the relationship between the average valence of Mo and catalysis selectivity. Small spheres of different colors in (e) and (g) stand for various kinds of atoms. Green: Mo in MF-0, purple: Mo in MF-10, grey: O in MF-0 or MF-10, red: O in oxygen species, blue: H. Source data are provided as a Source Data file.
The POD-like activity enhanced with the increment of the average valence of Mo (from 4.64 of MF-0 to 5.68 of MF-10), while it was uncertain whether the summit has been reached (Fig. 3b), thus the oxidation time was prolonged to prepare MF-20 and MF-30 with higher proportion of Mo6+ for further elucidating the evolution of the POD-like activity. As expected, the percentage of Mo6+ in MF-20 and MF-30 increased to 79.4% and 89.5%, correspondingly the average valence of Mo attained to 5.73 and 5.89, respectively (Supplementary Fig. 10). UV-vis-NIR absorption spectra of MF-20 and MF-30 were analogous to that of MF-10 (Fig. 2e), likewise the DLS size and zeta potential (Supplementary Fig. 11). TMB oxidation assay in Supplementary Fig. 12 demonstrated that the POD-like activity of MF-20 was obviously higher than MF-30 but weaker as compared to MF-10 at pH 6.5 (Fig. 3b). Correspondingly, the production of ROS was not observed at pH 7.4, which is pH-dependent. Altogether, within a certain range, the POD-like activity of MF-x enhanced with the increase of the average Mo valance, and then declined.
To elucidate the difference of the reaction selectivity between MF-0 and MF-10, ESR spectra of the two samples was first conducted. As shown in Fig. 3d, MF-0 and MF-10 presented resonance signals at g = 2.003 with analogous intensity, indicating that there was little difference in the oxygen vacancy (OV) concentration of the two samples. Thus, the contribution of surface OV to the selectivity of H2O2-associated enzymatic reactions of MF-x samples was considered to be negligible. Furthermore, the rapid cycles of redox process were essential for the activity of nanozymes containing redox-active centers (Mo with different valence states herein)17, thus the valence state of Mo in MF-x was reasonably considered as the key factor to determine the catalysis selectivity. Additionally, density functional theory (DFT) calculations were carried out to elucidate the catalytic origin and underlying mechanism for the catalysis selectivity of MF-0 and MF-10. Firstly, it is noted that the optimization of the surface models (Supplementary Fig. 13 and Supplementary Fig. 14) was described in the section of experimental methods (calculation details). Next, as shown in Supplementary Fig. 15, the charge density difference for MF-0 and MF-10 intuitively displayed that Mo lost more electrons in MF-10 than in MF-0, indicating the higher valence state of Mo in MF-10, which was agree with the XPS results (Fig. 2d), and also verified the reliability of the computational modeling. To obtain the intrinsic rate-determining step (RDS) of the catalytic reaction pathways for the production of ·OH (POD-like activity) and O2 (CAT-like activity) of both MF-0 and MF-10, the corresponding free energy diagrams were further investigated (Fig. 3e, Supplementary Fig. 16 and Supplementary Fig. 17). As for MF-0, the RDS of the ·OH formation pathway was *OH + *OH → *OH + ·OH (·OH desorption), and the RDS of the O2 generation pathway was *O2 → MF-0 + O2 (O2 desorption), with free energy barriers of 2.24 eV and 1.21 eV, respectively. The lower barrier for the RDS of the O2 generation pathway indicated that MF-0 was more likely to catalyze H2O2 to generate O2 (CAT-like activity) rather than ·OH (POD-like activity), which was thermodynamically favorable. Correspondingly for MF-10, the RDS of the ·OH formation pathway was *OH + *OH → *OH + ·OH (·OH desorption), and the RDS of the O2 generation pathway was *O + *H2O2 → *OH + *OOH (proton transfer), with free energy barriers of 0.89 eV and 1.10 eV, respectively, suggesting the remarkable selectivity toward POD-like activity.
Overall, the selectivity of H2O2-associated enzymatic reactions of MF-x could be tuned by a feasible mild oxidation valence-engineering strategy, as summarized in the schematic diagram (Fig. 3h). MF-0 (Mo average valence 4.64) and MF-10 (Mo average valence 5.68) exhibited exclusively efficient CAT- or POD-like activity, respectively.
a UV-vis-NIR absorption spectra of MF-0 (160 μg of Mo per mL) after incubation with H2O2 at different concentrations for 2 h (inset: corresponding photographs). H2O2 (b), ·OH (c) and ABTS· (d) scavenging activity of MF-0 at different Mo concentrations (n = 3 independent experiments). e Viability of MREpiC cells treated with MF-0 at different concentrations (n = 6 independent experiments). f Viability of MREpiC cells under different treatment conditions (n = 6 independent experiments; *P < 0.1, **P < 0.01, ***P < 0.001, P values: 2.1 × 10–5, 5.7 × 10–2, 2.5 × 10–3 and 1.0 × 10–4). g Fluorescence images of MREpiC cells stained by DCFH-DA (ROS probe) for various treatment groups. Group I: cells treated without H2O2 and MF-0; group II: cells treated with H2O2; group III: cells treated with H2O2 and MF-0 (20 μg/mL); group IV: cells treated with H2O2 and MF-0 (50 μg/mL); group V: cells treated with H2O2 and MF-0 (80 μg/mL). h Quantification analysis of DCF fluorescence intensity for groups I ~ V in (g) (n = 20 cells; **P < 0.01, ***P < 0.001, P values: 3.7 × 10–12, 2.3 × 10–3, 9.4 × 10–11 and 1.4 × 10–12). The concentration unit of MF-0 in (f) and (g) is μg/mL; H2O2 concentrations used in (f) and (g) were 250 and 100 μM, respectively. Data in (b–f) and (h) are presented as means ± SD. Significance was calculated by one-sided Student’s t-test. Source data are provided as a Source Data file.
Prior to assessment of feasibility of MF-0 in cellular level, methyl thiazolyl tetrazolium (MTT) assay was first implemented to check the cytotoxicity of MF-0. Although the viability of MREpiC cells descended slightly with the increase of MF-0 concentration after incubation for 24 h, nearly 90% of the cells survived even at a high concentration of 400 μg/mL (Fig. 4e). Besides, the absorbance at 600 nm and morphology, as well as zeta potential and average DLS size of MF-0 changed little during 14 days of storage (Supplementary Fig. 23). These results showed that MF-0 owned desired stability and biocompatibility for further biomedical applications. Afterwards, as depicted in Fig. 4f, compared with the negative control group (no addition of H2O2 and MF-0), cell viability of the positive control group (only H2O2) significantly declined to 66.5% due to H2O2-induced excessive oxidative stress and thereby cell death. With the following addition of MF-0, the cell viability improved steadily and reached ~90% at the dose of 80 μg/mL (MF-0). Correspondingly, as shown in Fig. 4g, h, the fluorescence imaging of intracellular ROS by using DCFH-DA probe demonstrated that the group II (positive control) emerged the strongest green fluorescence of DCF (from oxidation of DCFH-DA by ROS), indicating the most severe oxidative stress occurred. While in MF-0 treated groups (III, IV, V), intracellular oxidative stress (ROS) was distinctly inhibited, and recovered to a comparative level with the negative control group (I) at a dose of 80 μg/mL. These results were in good agreement with those in above cell survival experiments (Fig. 4f), which confirmed that the introduction of MF-0 could scavenge intracellular ROS effectively and protect cells from oxidative stress-induced cell damages.
Encouraged by the desirable in vitro ROS clearance capability of MF-0, its therapeutic effects on ROS-related acute kidney injury (AKI) in mice were further explored. First, MF-0 was labeled with the fluorescent dye IR780 (IR780@MF-0) to inspect its circulation, targetability, and biodistribution in AKI mice. The in vivo fluorescence imaging showed that IR780@MF-0 could obviously accumulate to the kidney within 10 min, and reached the maximum accumulation in 30 min and lasted for an appropriate period (with a slight attenuation in 60 min) (Supplementary Fig. 24). At the same time, the results of ICP-MS detection of Mo content (Supplementary Fig. 25) indicated that the retention of MF-0 in the kidneys of AKI mice attained the maximum (2.8% ID per gram of kidney) at 30 min of post-injection, and then decreased to around 2.1% at 120 min, which was consistent with the metabolic trend observed in in vivo fluorescence imaging. Furthermore, the high-resolution bio-TEM-EDX elemental mapping images of Mo for renal cortex sections of AKI mice treated with PBS and MF-0 were compared, which demonstrated the obvious presence of MF-0 in glomerular basement membrane (GBM) of AKI mice, although it has not been clearly observed in renal tubules at this stage (30 min of post-injection) (Supplementary Fig. 26). In the meantime, the content of Mo accumulated in the urine of AKI mice treated with MF-0 was significantly higher than that in the control group (AKI mice treated with PBS), and the amount of Mo in urine gradually increased with metabolic time (Supplementary Fig. 27), indicating that MF-0 could be further excreted into urine after passing through kidneys. Besides, the ICP-MS quantification data on the biodistribution of MF-0 in major organs (heart, liver, spleen, lung, kidney and intestine) of AKI mice at different time points of post-injection (0, 0.5, 2, 6, 12, 24, 72, 168 and 336 h) showed that MF-0 was mainly accumulated in the liver, spleen and lung of AKI mice, followed by the kidney, and reached its maximum accumulation in different organs at 2 h (liver, spleen and lung) of post-injection, and then was gradually metabolized out of the body within 14 days (Supplementary Fig. 28). These suggested that the nanoprobes were still retained at most in the reticuloendothelial system (RES) organs including liver, spleen and lung, a certain dose of MF-0 could be targeted to the kidneys of AKI mice via renal metabolic pathways, providing a favorable prerequisite for subsequent treatment design.
Timeline of mouse AKI modeling and treatment was presented in Fig. 5a, the AKI model was established via intramuscular injection of glycerin into both hind legs of dehydrated mice, and the mice subsequently showed symptoms such as oliguria, slow movement and hematuria. After 24 h all the mice were sacrificed, and kidney function test, H&E staining as well as dihydroethidium (DHE) staining were conducted for the therapeutic assessments. As shown in Fig. 5b, c, two crucial renal function indicators of AKI mice, namely, creatinine (CRE) and blood urea nitrogen (BUN) became significantly higher than those of healthy mice, indicating an apparent abnormality in renal function. In contrast, the AKI mice treated with NAC (a commonly used antioxidant) or MF-0 exhibited down-regulation of CRE and BUN, and the MF-0 treated groups recovered to a level comparable to that of healthy mice. Moreover, the survival rate of AKI mice treated with MF-0 was 100% during the treatment, which was obviously better than that of PBS treated group (Fig. 5d), and their body weight also maintained an increasing trend within 10 days (Supplementary Fig. 29). Afterwards, H&E staining of kidneys (Fig. 5e) provided intuitive evidence that in AKI mice, a large number of damaged renal tubules (marked with arrows) and casts (marked with asterisks) formed by precipitation of denatured proteins in the tubules, while in the NAC treatment group, the kidney injury was alleviated to a certain extent as evidenced by the reduction in the number of casts (a marker of more severe tubular damage), but there were still damaged renal tubules. More encouragingly, AKI mice treated with MF-0 recovered as normal, almost no such tissue damages were found, suggesting the superior therapeutic effect of MF-0 on AKI. In addition, DHE staining of renal tissues further evaluated the ROS level (showing red fluorescence) of each treatment group (Fig. 5f). As compared to the PBS and NAC treated groups, ROS level of MF-0 treated AKI mice was effectively inhibited to that of healthy mice, which revealed the desired antioxidant activity and targeting effect of MF-0 for AKI treatment. In other words, MF-0 could effectively eliminate excess ROS in the kidneys of AKI mice, thereby avoiding renal tubule damages and achieving prominent AKI therapeutic efficacy.
a Timeline of AKI modeling and treatment with mice. CRE (b) and BUN (c) levels in the blood serum from each group after indicated treatments (n = 3; Data are presented as means ± SD. *P < 0.1, n.s. no significance. P values in (b): 2.0 × 10–2, 2.1 × 10–2, 1.8 × 10–1 and 2.3 × 10–2, P values in (c): 3.4 × 10–2, 2.9 × 10–2, 2.6 × 10–1 and 5.6 × 10–2). d The survival rate of AKI mice treated with PBS and MF-0, respectively. e H&E staining of renal sections from each treatment group. Arrows indicated damaged tubules and asterisks indicated the formation of casts (a marker of more severe tubular damage). f DAPI (blue fluorescence indicating cell nuclei) and dihydroethidium (red fluorescence indicating ROS level) staining of kidney tissues from each treatment group. NAC used in (b, c, e, f) was a ROS inhibitor. The injection dosage of agents in different treatment groups was 200 μL: NAC (800 μg/mL), MF-0 (800 μg of Mo per mL). Significance was calculated by one-sided Student’s t-test. Source data are provided as a Source Data file.
In addition to the effective treatment of AKI, MF-0 was also endowed with ROS-responsive photoacoustic imaging (PAI) features for non-invasive and real-time post-cure assessment of AKI. It is well known that the PA signal of a material is highly dependent on its absorption, which was obviously reflected in the color change of MF-0 solution from dark grey to pale yellow after incubation with H2O2 (Fig. 6a). UV-vis-NIR absorption spectra presented a noticeable decreasing absorption of MF-0 solution along with the increment of H2O2 from 0 to 1 mM (Fig. 6b). Meanwhile, the absorption intensity ratio (A/A0) of MF-0 (at 730 nm) incubated with 1 mM of H2O2 declined quickly in 5 min and reached the plateau in 10 min (Fig. 6c). Accordingly, PA intensity of MF-0 solution (at 730 nm) was observed negatively proportional to the concentration of H2O2 (Fig. 6d). Encouraged by the rapid and sensitive H2O2-responsive performance of MF-0, the in vivo PAI was further carried out to intuitively evaluate the therapeutic effect on AKI mice. As illustrated in Fig. 6e, PAI was performed at different post-injection time points for AKI mice and AKI-cured mice. Distinguished from healthy mice, ROS level in kidneys of AKI mice was much higher, causing a diminished PAI signal (“off” state). After treatment with MF-0, the excess ROS in kidneys were scavenged, meanwhile, the absorption of MF-0 was tuned by ROS consumption, thus switching on the PAI signal (turn “on”) in AKI-cured mice. As depicted in Fig. 6f, g, the evolution trend of PAI signal in kidneys among the three groups during the post-injection period of 120 min was analogous. PAI signal appeared in 10 min after injection of MF-0, then reached the maximum in 30 min, and attenuated to the level as the beginning of MF-0 injection in 120 min, indicating the rapid accumulation and metabolism of MF-0 in kidneys. As expected, the PAI intensity difference in AKI-cured mice at 30 min was ~2.1-fold stronger than that in AKI mice, and almost the same level as in healthy mice, successfully achieving ROS-responsive PAI for AKI therapeutic effect and post-cure assessments in vivo.
a Photographs of MF-0 solution before and after incubation with H2O2 (3 mM). b UV-vis-NIR absorption spectra of MF-0 (60 μg of Mo per mL) after incubation with H2O2 at different concentrations. c Normalized absorbance intensity (at 730 nm) evolution of MF-0 solution (60 μg of Mo per mL) incubated with H2O2 (1 mM) within 30 min (n = 3 independent experiments). d PA intensity (at 730 nm) of MF-0 solution (60 μg of Mo per mL) incubated with H2O2 at different concentrations (n = 3 independent experiments, inset: corresponding PA images). e Timeline of photoacoustic imaging for AKI mice and AKI-cured mice. In vivo PA images (f) and the corresponding quantitative analysis (g) of the kidney regions at 730 nm in healthy mice, AKI mice and AKI-cured mice at different post-injection time points (0, 10, 30, 60 and 120 min, n = 3). ΔI indicated PA intensity difference (at 730 nm) between different time points and 0 min. MF-0 was intravenously (i.v.) injected into the mice (800 μg of Mo per mL, dosage: 200 μL). Data in (c), (d) and (g) are presented as means ± SD. Source data are provided as a Source Data file.
Absolutely, the biosafety assessment of MF-0 was also very worthy of attention. The hemolysis assay demonstrated that MF-0 did not cause hemolysis even at a concentration of 400 μg/mL (Supplementary Fig. 30). More importantly, the biological toxicity studies of the mice revealed that no obvious difference was found in liver function indicators and hematological parameters among the groups: healthy mice treated with PBS, AKI mice treated with PBS and AKI mice treated with MF-0. (Supplementary Fig. 31). In addition, it was evidenced by H&E staining (Supplementary Fig. 32) that MF-0 treated AKI mice had no significant damage such as necrosis, congestion, and hemorrhage in major organs (heart, liver, spleen, lung and intestine), further suggesting the favorable biosafety of MF-0.