A Melanin-Based Natural Antioxidant Defense Nanosystem for Theranostic Application in Acute Kidney Injury

Abstract

Acute kidney injury (AKI) is frequently associated with oxidative stress and causes high mortality annually in clinics. Nanotechnology-mediated antioxidative therapy is emerging as a novel strategy for the treatment of AKI. Herein, a novel biomedical use of the endogenous biopolymer melanin as a theranostic natural antioxidant defense nanoplatform for AKI is reported. In this study, ultrasmall Mn²⁺-chelated melanin (MMP) nanoparticles are easily prepared via a simple coordination and self-assembly strategy, and further incorporated with polyethylene glycol (MMPP). In vitro experiments reveal the ability of MMPP nanoparticles to scavenge multiple toxic reactive oxygen species (ROS) and suppress ROS-induced oxidative stress. Additionally, in vivo results from a murine AKI model demonstrate preferential renal uptake of MMPP nanoparticles and a subsequent robust antioxidative response with negligible side effects according to positron emission tomography/magnetic resonance (PET/MR) bimodal imaging and treatment assessment. These results indicate that the effectiveness of MMPP nanoparticles for treating AKI suggests the potential efficacy of melanin as a natural theranostic antioxidant nanoplatform for AKI, as well as other ROS-related diseases.

Graphical Abstract

Mn²⁺-chelated melanin (MMPP) nanoparticles with ultra-small hydrodynamic size are facilely prepared via a simple coordination and self-assembly strategy, and further PEGylation. MMPP nanoparticles exhibit excellent in vivo circulation and preferential renal uptake in a murine AKI model. Importantly, MMPP nanoparticles can act as efficient ROS scavengers to alleviate AKI via antioxidative protection.

1. Introduction

Acute kidney injury (AKI) is an increasingly common and devastating complication in hospitalized patients, particularly critically ill patients, leading to unacceptably high patient morbidity and mortality.^[1] AKI is commonly defined as an abrupt deterioration in kidney function along with decreased glomerular filtration and increased accumulation of nitrogenous wastes in the blood, with clinical treatment mainly reliant on supportive therapies.^[2] During AKI, toxic reactive oxygen species (ROS) are aberrantly generated in excess and react with biomolecules to trigger oxidative stress, thereby causing abrupt kidney injury and renal dysfunction.^[3] These findings suggest that efficient ROS scavenging in kidneys might alter the renal microenvironment and alleviate pathological progression of AKI mediated by oxidative stress. Recent studies have demonstrated that antioxidative therapy with nanomedicine is an effective strategy to prevent AKI due to its renal targeting and high bioavailability.^[4] However, most nanomedicine is rapidly and nonspecifically taken up from circulation by the mononuclear phagocyte system and not excreted by the kidneys due to the large hydrodynamic size exceeding the kidney filtration threshold (~6 nm), resulting in low renal uptake and potential organ toxicity.^[5] Therefore, it remains challenging to design antioxidative nanomaterials with high renal targeting ability and low systemic toxicity for the treatment of AKI.

As an endogenous biopolymer in most living organisms, including humans, melanin exhibits excellent biocompatibility and biodegradability for biomedical applications accompanied by negligible side effects.^[6] Moreover, the fascinating characteristics of melanin, such as near-infrared absorbance and strong chelating capability, enable innovative design of melanin-based smart nanotheranostics for molecular imaging and photothermal therapy for cancer.^{[6a, 6c, 7]} Notably, melanin contains abundant antioxidant groups and can act as an effective antioxidant to treat a series of ROS-related diseases, such as ischemic stroke, periodontal disease, acute peritonitis, and acute lung injury.^[8] However, recent treatment strategies primarily rely on in situ administration of melanin-based nanoantioxidants, thereby hindering its applicability to other types of ROS-related diseases, including AKI. To date, the potential of melanin in antioxidative therapy for AKI remains poorly explored.

In this study, we designed a novel strategy to synthesize ultrasmall Mn²⁺-chelated melanin (MMP) nanoparticles via a simple coordination and self-assembly strategy. The polyethylene glycol-incorporated (PEGylated) MMP (MMPP) nanoparticles exhibited an ultrasmall hydrodynamic size, good physiological stability, high r₁ relaxivity, stable radiolabeling performance, and excellent antioxidative activities toward various toxic ROS. Importantly, T₁-weighted magnetic resonance (MR) and positron emission tomography (PET) imaging revealed excellent in vivo circulation and preferential renal uptake of MMPP nanoparticles in a murine AKI model, with in vivo studies demonstrating that the MMPP nanoparticles can act as efficient ROS scavengers to alleviate AKI via antioxidative protection. Overall, for the first time, the MMPP nanoparticles were applied as a multifunctional nanotheranostic system for PET/MR bimodal imaging-guided AKI therapy, which may inspire further development of nanotechnology and endogenous biopolymers in AKI therapy.

2. Results and Discussion

2.1. Synthesis and characterization of MMPP nanoparticles

As illustrated in Figure 1a, ultrasmall MMP nanoparticles were easily prepared through a simple coordination and self-assembly method and acted as a smart nanoplatform for PET/MR bimodal imaging-guided AKI therapy. In brief, the melanin granular powder, Mn²⁺ and polyvinylpyrrolidone (PVP) were dissolved in dimethyl sulfoxide (DMSO). Because of the poor water-solubility and strong chelating capabilities of melanin and surface modification of PVP,^{[6a, 6c, 9]} a self-assembly process to form water-soluble MMP nanoparticles was initiated by adding the above mixture to deionized water at a 1:10 (v/v) ratio. During self-assembly, the melanin coordinated with Mn²⁺ to form nanoparticles, which underwent PVP modification to improve their stability. The resulting water-soluble MMP nanoparticles showed good dispersity (Figure 1b and d), which was attributed to the PVP modification and a highly negative potential (−21.5 ± 1.9 mV) (Figure S1) that efficiently blocked nanoparticle aggregation through steric and electrostatic repulsion. Compared with MMP nanoparticles, the control sample prepared with melanin and PVP showed an ultrasmall size and a slightly worse monodispersity (Figure S2c). However, no well-shaped nanoparticles were obtained in control experiments without the addition of either melanin or PVP (Figure S2a, b, and d), indicating the critical role of melanin in forming the nanostructure, as well as the important role of PVP to control the ultrasmall size of the obtained nanostructure. Fourier transform infrared (FT-IR) spectra subsequently confirmed the successful PVP modification and preservation of the melanin structure in MMP nanoparticles (Figure S3).

Figure 1.

Synthesis and characterization of MMPP nanoparticles. (a) Schematic illustration of the MMPP nanoparticle synthesis process and their activity as a naturally antioxidative platform for PET/MR bimodal imaging-guided AKI therapy. Transmission electron microscopy (TEM) images of (b) MMP and (c) MMPP nanoparticles. Scale bar: 20 nm. (d) The hydrodynamic size of MMP and MMPP nanoparticles measured by dynamic light scattering (DLS). (e) UV-Vis-NIR spectra of melanin, MMP and MMPP aqueous dispersions. Inset is the corresponding photograph of melanin, MMP and MMPP nanoparticles dispersed in water.

To improve the physiological stability of MMP nanoparticles, thiol-terminated polyethylene glycol (HS-PEG) was incorporated via a Michael addition reaction.^[10] The resulting MMPP nanoparticles exhibited excellent physiological stability in various media (Figure 1c and Figure S4), and compared with the MMP nanoparticles, the hydrodynamic size of MMPP nanoparticles increased to ~4.5 nm (Figure 1d). Moreover, the surface potential of MMPP nanoparticles decreased to −7.8 ± 1.0 mV (Figure S1) due to the introduction of PEG chains, which was confirmed by FT-IR analysis (Figure S3). The characteristic absorption peaks of MMPP nanoparticles at 2880 cm⁻¹ (the alkyl C-H stretching vibration) and 1110 cm⁻¹ (the C-O-C stretching vibration) were attributed to the PEG molecules.^[11] As shown in Figure 1e, melanin, MMP and MMPP aqueous dispersions showed similar absorption spectra, indicating that the melanin molecules were well preserved in the MMPP nanoparticles. Additionally, the absorbance of the melanin aqueous dispersion at 808 nm exhibited a good linear relationship with melanin concentrations (Figure S5), providing the basis for quantification of melanin content in MMPP nanoparticles. Evaluation of Mn²⁺ content in the MMPP nanoparticles using inductively coupled plasma atomic emission spectroscopy (ICP-AES) revealed a mass ratio of melanin to Mn²⁺ of ~112.7, providing a basis for further MR imaging experiments.

2.2. Multi-antioxidative activities of MMPP nanoparticles

Based on the naturally antioxidative properties of melanin, we systematically evaluated the potential of MMPP nanoparticles to act as robust ROS scavengers in vitro. Two physiologically relevant ROS, including superoxide anion radical (O₂•⁻) and hydroxyl radical (•OH), were selected to test the ROS-scavenging capacity of MMPP nanoparticles. As shown in Figure 2a, MMPP nanoparticles were highly sensitive to O₂•⁻, showing successful scavenging of 79.4 ± 4.7% of O₂•⁻ at a low melanin concentration (25 μg/mL). Moreover, as MMPP concentration increased, we observed a concentration-dependent scavenging behavior toward O₂•⁻. As an extremely reactive radical, •OH can cause DNA damage, lipid peroxidation, and protein fragmentation, making it among the most potent type of ROS.^[12] As shown in Figure 2b, MMPP nanoparticles scavenged ~68.4 ± 2.5% of •OH at a melanin concentration of 100 μg/mL. Furthermore, we assessed MMPP-mediated free radical scavenging using a 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, which verified the concentration-dependent scavenging of free radicals according to elimination of ~85.2 ± 2.3% of ABTS^+• at a melanin concentration of 100 μg/mL (Figure 2c). These results confirmed admirable multi-antioxidative activities of MMPP nanoparticles toward various ROS in vitro.

Figure 2.

ROS-scavenging activity of MMPP nanoparticles in vitro. The ability of MMPP nanoparticles at different concentrations to scavenge (a) O₂•⁻, (b) •OH, and (c) free radicals (n = 4; ***p < 0.001 vs. the blank group). (d) Representative ROS staining of HEK293 cells under different treatments. Scale bar: 100 μm. (e) Relative ROS levels (n = 4) and (f) cell viabilities (n = 3) following stimulation with H₂O₂ (250 μM) and different concentrations of MMPP nanoparticles. ns, nonsignificant; *p < 0.05, **p < 0.01, and ***p < 0.001 vs. H₂O₂ control group. MMPP concentrations were established according to their melanin concentrations. All data are presented as the mean ± s.d.

2.3. MMPP-mediated protection of H₂O₂-stimulated cells

The kidney receives about one-fourth of the total blood supply, rendering it vulnerable to oxidative stress during a rhabdomyolysis attack and subsequent development of AKI.^[13] Based on the demonstrated effectiveness of MMPP nanoparticles as robust ROS scavengers, we investigated their protective effects in human embryonic kidney 293 (HEK293) cells against ROS-induced oxidative stress. MTT assay results revealed that MMPP nanoparticles were biocompatible, with no obvious cytotoxicity observed under the experimental conditions (Figure S6). We then initiated oxidative stress via ROS accumulation by the administration of H₂O₂ (250 μM) (Figure 2d and e). Compared with the blank control group, we confirmed that no excess ROS was induced in HEK293 cells following introduction of MMPP nanoparticles. Moreover, cells pretreated with MMPP nanoparticles at various concentrations exhibited significantly decreased levels of intracellular ROS in a dose-dependent manner following H₂O₂ treatment relative to untreated cells (Figure 2e), indicating the high-efficiency ROS scavenging activity of MMPP nanoparticles. Furthermore, H₂O₂ stimulation initiated ROS-induced cell death in untreated cells, whereas MMPP-pretreated cells displayed reduced levels of cell death (Figure 2f).

2.4. PET imaging of ⁸⁹Zr-MMPP nanoparticles in healthy and AKI mice

Given the intrinsic chelating function of melanin, ⁸⁹Zr (half-life: 78.4 h) was readily and stably chelated by MMPP nanoparticles via a chelator-free labeling method (Figures S7 and S8), with a high ⁸⁹Zr-labeling yield (~87.9 ± 1.2%) achieved by MMPP nanoparticles within 15 min of incubation at 37°C (Figure S8a). Prolonging the incubation time increased the yield to 89.7 ± 0.5% at 1 h post-incubation (Figure S8a), which was similar to the ~86.5% yield measured using a PD-10 purification method (Figure S7). Thin-layer chromatography indicated that >85% of ⁸⁹Zr-MMPP nanoparticles remained intact at 48 h post-incubation (Figure S8b), indicating their high stability in phosphate-buffered saline (PBS) and fetal bovine serum (FBS).

We then assessed differences in MMPP biodistribution between healthy and AKI-model mice by PET imaging. We established the murine model of AKI by water deprivation and subsequent intramuscular injection of 50% glycerol, after which ⁸⁹Zr-MMPP nanoparticles were intravenously injected, and longitudinal PET imaging was performed to monitor the behavior of the ⁸⁹Zr-MMPP nanoparticles in vivo. Healthy mice intravenously injected with ⁸⁹Zr-MMPP nanoparticles were used as control. We observed enhanced accumulation of ⁸⁹Zr-MMPP nanoparticles in the kidneys of AKI mice as compared with that in healthy mice (Figure 3a and d). Interestingly, the PET signal from the heart was still visible at 24 h post-injection, indicating excellent in vivo circulation of the ⁸⁹Zr-MMPP nanoparticles. We observed strong PET signals in the kidneys and bladder during the first 3 h post-injection, suggesting partial secretion of the ⁸⁹Zr-MMPP nanoparticles accompanied by low accumulation in the liver and spleen due to the hydrodynamic size of MMPP nanoparticles (~4.5 nm) below the kidney filtration threshold (~6 nm), which precluded liver accumulation due to the efficient excretion from kidneys via glomerular filtration.^{[5, 14]} Prolonged post-injection time resulted in the increased nonspecific uptake of ⁸⁹Zr-MMPP nanoparticles by the liver, possibly resulting from serum protein adsorption and subsequent uptake by macrophages.^[15]

Figure 3.

PET imaging and biodistribution of ⁸⁹Zr-MMPP in healthy and AKI mice. Representative maximum intensity projection (MIP) PET imaging of ⁸⁹Zr-MMPP in (a) healthy and (b) AKI mice. Images are representative of triplicate experiments. It represents H, heart; K, kidney; L, liver; B, bladder. (c) Blood time-activity curves of ⁸⁹Zr-MMPP in healthy and AKI mice. Quantification of ⁸⁹Zr-MMPP uptake in the blood, liver, spleen, kidney and muscle of (d) healthy and (e) AKI mice at different post-injection time points. (f) Biodistribution of ⁸⁹Zr-MMPP in healthy and AKI mice at 24 h post-injection. Data represent the mean ± s.d. of three independent replicates. ***p < 0.001.

Region-of-interest (ROI) analysis of the PET images quantitatively revealed an extended and similar blood half-life of ⁸⁹Zr-MMPP nanoparticles in healthy and AKI mice (~6.03 h and ~6.25 h, respectively) (Figure 3c), indicating excellent in vivo circulation of MMPP nanoparticles, which is highly desirable for cancer nanotheranostics. Moreover, the healthy and AKI mice showed similar uptake of ⁸⁹Zr-MMPP nanoparticles in the liver and spleen at different post-injection time points (Figure 3d and e). However, the uptake in muscle of AKI mice was higher than that in healthy mice due to the rhabdomyolysis-related damage to both hind limbs in AKI mice. Notably, renal accumulation of the ⁸⁹Zr-MMPP nanoparticles in AKI mice also was higher than that in healthy mice at different post-injection time points (Figure 3d and e). We found that the renal uptake of ⁸⁹Zr-MMPP nanoparticles in AKI mice was 13.5 ± 0.8 %ID/g at 1 h post-injection and 11.0 ± 1.1 %ID/g at 24 h post-injection relative to 9.4 ± 0.8 %ID/g and 4.6 ± 0.4 %ID/g, respectively, in healthy control mice. Importantly, the higher renal uptake in AKI mice also enhanced the opportunity for MMPP nanoparticles to perform their antioxidative functions to address the AKI. As expected, ex vivo biodistribution studies confirmed the higher renal uptake of the ⁸⁹Zr-MMPP nanoparticles (11.3 ± 2.3 %ID/g) in AKI mice at 24 h post-injection as compared with that in healthy mice (5.5 ± 0.4 %ID/g) (Figure 3f), which agreed with quantitative analysis by PET imaging.

2.5. MR imaging of MMPP nanoparticles in AKI mice

To evaluate the capacity of MMPP nanoparticles for MR imaging, we measured T₁ relaxivity. Compared with commercially available gadobenate dimeglumine (Gd-BOPTA; a gadolinium-based contrast agent), MMPP nanoparticles exhibited excellent concentration-dependent T₁-weighted contrast enhancement (Figure 4a and Figure S9). Notably, the T₁ relaxation rate (r₁) of MMPP nanoparticles was as high as 46.5 mM⁻¹ s⁻¹ at 4.7 T, which was ~7.5-fold higher than that of Gd-BOPTA (6.1 mM⁻¹ s⁻¹) and surpassed that of most reported nanosized Mn-based MR contrast agents.^{[11, 16]} The excellent r₁ relaxivity of MMPP nanoparticles might be attributed to the geometrically confined conformation and the efficient chemical exchange between Mn²⁺ and surrounding protons.^{[16b, 17]} Moreover, Mn as an essential trace element is necessary for general biological processes. Encouraged by the safety of MMPP nanoparticles and their prominent r₁ relaxivity, we performed in vivo MR imaging in AKI mice. As shown in Figure 4b, the T₁-weighted images showed increased MR signals in the kidneys of AKI mice following intravenous injection of MMPP nanoparticles, with this confirmed by quantitative analysis (Figure 4c and d). The enhanced MR signals in the kidneys of AKI mice revealed the renal accumulation of MMPP nanoparticles in vivo, which was consistent with PET analysis.

Figure 4.

MR performance of MMPP nanoparticles and their in vivo MR imaging in AKI model mice. (a) T₁ relaxation rates of different concentrations of MMPP and Gd-BOPTA at 4.7 T. (b) T₁-weighted MR imaging of MMPP nanoparticles in AKI mice at pre-injection (0 h) and different post-injection time points. Kidneys are circled with a yellow dashed line. Quantification of the MR signal-intensity ratios in kidneys and muscle from (c) longitudinal and (d) cross-sectioned MR images (n = 3; mean ± s.d.).

2.6. Antioxidant defense of MMPP nanoparticles in a murine AKI model

As illustrated in Figure 5a, accumulated rhabdomyolysis-related damage was created in mice through water deprivation and subsequent intramuscular injection of 50% glycerol, which triggered injury related to oxidative stress in kidneys and renal dysfunction in this form of AKI.^{[4a, 4b, 18]} Considering the excellent in vitro antioxidant activities and good renal accumulation, MMPP nanoparticles are highly desirable to treat AKI via the antioxidant defense. Subsequently, AKI treatment was performed via intravenous injection of MMPP nanoparticles, and AKI mice intravenously injected with PBS were used as controls. At 24 h post-treatment, severe body weight loss was observed in the PBS-treated AKI mice, whereas this was not observed in AKI mice administered with MMPP nanoparticles (500 μg melanin) (Figure S10). To evaluate the therapeutic effect of MMPP nanoparticles, we analyzed blood urea nitrogen (BUN) and serum creatinine levels as clinical indices of kidney excretory function (Figure 5b and c). We found that PBS-treated AKI mice exhibited increased BUN and serum creatinine levels, which are hallmarks of renal failure. Notably, treatment with the MMPP nanoparticles significantly decreased the BUN and serum creatinine levels in AKI mice, and the renal function of AKI mice was restored at a higher MMPP nanoparticle injection dose. Furthermore, hematoxylin and eosin (H&E) staining of kidney tissues from each group revealed severe renal tubular injury and cast formation (a marker of kidney disease) in PBS-treated AKI mice (Figure 5d), whereas treatment with MMPP nanoparticles alleviated kidney damage in AKI mice according to MMPP-concentration-dependent decreases in the number of damaged kidney structures. Moreover, no renal toxicity was found in healthy mice treated with a higher dose of MMPP nanoparticles. In addition, confocal imaging of dihydroethidium (DHE)-stained kidney tissues was performed to assess renal superoxide production. As shown in Figure 5e, renal ROS generation of AKI mice was obviously inhibited by the treatment of MMPP nanoparticles, which confirmed the robust antioxidant defense capacity of MMPP nanoparticles in AKI therapy.

Figure 5.

Formation of the AKI model and its treatment with MMPP nanoparticles. (a) Schematic illustration of the establishment of an AKI model in mice and their treatment with MMPP nanoparticles. Blood serum levels of the kidney function indicators (b) blood urea nitrogen (BUN) and (c) creatinine in healthy mice and AKI mice treated with PBS or MMPP nanoparticles (according to melanin concentration: L-MMPP group, 200 μg melanin/mouse; H-MMPP group, 500 μg melanin/mouse; n = 4). Data represent the mean ± s.d. ***p < 0.001 vs. PBS-treated AKI controls. (d) Images of H&E-stained kidney tissues from healthy mice and AKI mice treated with PBS or MMPP nanoparticles. Arrows indicate damaged kidney tubules, and asterisks indicate the cast formation. Scale bar: 50 μm. (e) Confocal images of dihydroethidium (DHE) and DAPI-stained kidney tissues from healthy mice and AKI mice treated with PBS or MMPP nanoparticles. Scale bar: 100 μm.

2.7. In vivo toxicity assessment of MMPP nanoparticles

To confirm the safety of MMPP nanoparticles, mice were intravenously injected with MMPP nanoparticles at a high dose of 1 mg melanin, followed by collection of blood samples and the primary organs (heart, liver, spleen, lung, and kidneys) at 24 h post-injection. We observed no clear damage in kidney tissue from the MMPP-treated group relative to the control group, indicating absence of renal toxicity (Figure 6a). Additionally, H&E staining of other primary organs revealed no adverse side effects from MMPP administration. Moreover, the kidney function (Figure 6b), liver function (Figure 6c), and hematologic indices (Figure 6d) in the MMPP-treated group were all within normal ranges and similar to those observed for the control group. These results demonstrated the excellent in vivo biocompatibility and low toxicity of MMPP nanoparticles, supporting their significant potential for use in clinical applications.

Figure 6.

In vivo toxicity assessment of MMPP nanoparticles. (a) Images of H&E staining of primary organs (kidneys, heart, liver, spleen and lung) from healthy mice treated with PBS (control group) or MMPP nanoparticles (1 mg melanin/mouse). Scale bar: 50 μm. (b) Serum levels of the kidney function indicators blood urea nitrogen (BUN) and creatinine in mice at day 1 post-injection. (c) Serum levels of the liver function indicators aspartate transaminase (AST) and alanine transaminase (ALT) in mice at day 1 post-injection. (d) Hematology analysis of whole-blood parameters in mice treated with PBS or MMPP nanoparticles. Data represent the mean ± s.d. of five independent experiments.

3. Conclusion

In summary, we demonstrated the efficacy of the endogenous biopolymer melanin as a natural antioxidant defense platform for PET/MR bimodal imaging-guided AKI therapy in vivo. We prepared ultrasmall MMP nanoparticles using a simple coordination and self-assembly strategy, followed by PEGylation to create MMPP nanoparticles exhibiting excellent antioxidative activity against various types of toxic ROS. We found that MMPP nanoparticles acted as efficient ROS scavengers to protect HEK293 cells against harmful oxidative stress. Additionally, chelator-free ⁸⁹Zr radiolabeling of MMPP nanoparticles assembled T₁-weighted MR and PET imaging modalities, allowing visualization of highly efficient in vivo circulation and enhanced renal uptake in a murine AKI model. Importantly, the biocompatible MMPP nanoparticles efficiently alleviated a murine AKI model via robust antioxidative protection. These results suggest the efficacy of MMPP nanoparticles for applications as a smart theranostic nanoplatform for AKI and other injuries associated with oxidative stress.