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Melanin nanoparticles enhance the neuroprotection of mesenchymal stem cells against hypoxic‐ischemic injury by inhibiting apoptosis and upregulating antioxidant defense

Abstract Polydopamine nanoparticles are artificial melanin nanoparticles (MNPs) that show strong antioxidant activity. The effects of MNPs on the neuroprotection of mesenchymal stem cells (MSCs) against hypoxic‐ischemic injury and the underlying mechanism have not yet been revealed. In this study, an oxygen–glucose deprivation (OGD)‐injured neuron model was used to mimic neuronal hypoxic‐ischemic injury in vitro. MSCs pretreated with MNPs and then cocultured with OGD‐injured neurons were used to investigate the potential effects of MNPs on the neuroprotection of MSCs and to elucidate the underlying mechanism. After coculturing with MNPs‐pretreated MSCs, MSCs, and MNPs in a transwell coculture system, the OGD‐injured neurons were rescued by 91.24%, 79.32%, and 59.97%, respectively. Further data demonstrated that MNPs enhanced the neuroprotection against hypoxic‐ischemic injury of MSCs by scavenging reactive oxygen species and superoxide and attenuating neuronal apoptosis by deactivating caspase‐3, downregulating the expression of proapoptotic Bax proteins, and upregulating the expression of antiapoptotic Bcl‐2 proteins. These findings suggest that MNPs enhance the neuroprotective effect of MSCs against hypoxic‐ischemic injury by inhibiting apoptosis and upregulating antioxidant defense, which could provide some evidence for the potential application of combined MNPs and MSCs in the therapy for ischemic stroke.


| INTRODUCTION
Ischemic stroke is one of the leading causes of long-term disability and mortality worldwide and is in urgent need of new therapeutic strategies. Current therapeutic interventions for ischemic stroke are limited to intravenous administration of tissue plasminogen activator and endovascular treatment, which aim to recanalize blood flow before major brain damage occurs (Goyal et al., 2015;Muchada et al., 2014).
However, these therapies have strict time window requirements, thus only a small number of people can benefit from them. Mesenchymal stem cell (MSC) infusion is a potential treatment for ischemic stroke for patients with no other options. Both bench experiments and clinical trials have shown that MSCs are safe, feasible, and potentially efficacious in the treatment of stroke (Moniche et al., 2012;Prasad et al., 2012;Savitz et al., 2011). MSC transplantation could also promote the improvement of neurological function and reduce infarct volume after cerebral ischemia in a stroke model (Bao et al., 2011;Lee et al., 2016).
However, a large number of transplanted MSCs undergo cell death, which affects the therapeutic effect of transplanted MSCs due to the adverse microenvironment induced by hypoxia-ischemia (N. Liu et al., 2011;Zhu et al., 2006). The low survival rate of transplanted MSCs may be related to many factors, such as intense inflammation and oxidative stress (OS), a large number of proapoptotic factors and chemokines, and the lethal effect of ischemia-reperfusion injury on transplanted cells. Therefore, it is urgent to explore innovative methods to promote the survival of transplanted stem cells under hypoxicischemic conditions to enhance the efficacy of stem cells in the treatment of ischemic stroke.
With the development of nanomedicine, a wide range of nanoparticles with antioxidation effects have been formulated for the treatment of ischemic stroke. Among these, natural and artificial melanin nanoparticles (MNPs), including polydopamine nanoparticles (PDA NPs), have the potential to be translated to the clinic due to their potent antioxidation and good biocompatibility. PDA NPs remarkably reduced intracellular reactive oxygen species (ROS) levels in murine macrophages challenged with either H 2 O 2 or lipopolysaccharide. In murine models of both acute peritonitis and acute lung injury (ALI), PDA NPs diminished ROS generation, reduced proinflammatory cytokines, attenuated neutrophil infiltration, and alleviated lung tissue damage (Zhao et al., 2018). Ultrasmall Mn2+ -chelated melanin (MMP) nanoparticles incorporated with polyethylene glycol (MMPP) could scavenge multiple ROS and suppress ROS-induced OS in vitro (Sun et al., 2019). Recently, researchers revealed that melanin-like nanoparticles derived from the self-polymerization of 1,8-dihydroxynaphthalene (PDH nanoparticles) could not only act as radical scavengers to alleviate OS but could also chelate calcium overload to suppress the endoplasmic reticulum stress response and showed a significant anti-inflammation therapeutic effect on ALI mice (Lou et al., 2021). To date, the potential of MNPs in antioxidative therapy for ischemic stroke remains poorly explored. A study demonstrated that MNPs reduced cell death by reducing ROS levels, preventing Bax upregulation and Bcl-2 downregulation in Neuro2A cells treated with CoCl 2 and significantly decreasing the infarct area in an ischemic stroke model . Since MNPs have potential anti-inflammatory and antioxidant effects, MNPs may enhance the survival of transplanted stem cells in an ischemic and hypoxic environment, thereby enhancing the neuroprotective effects of stem cells. Moreover, MNPs can be directly internalized by MSCs without any noticeable influence on cell viability (Cai et al., 2020). However, whether MNPs internalized by MSCs can enhance the neuroprotective effect of MSCs remains unclear. In this study, we aimed to establish the potential effects of MNPs on the neuroprotection of MSCs and investigate the underlying mechanism.

| Synthesis of MNPs
MNPs were synthesized according to the reported method (Ju et al., 2011), with only minor modifications. In brief, an aqueous solution containing dopamine (DA) hydrochloride was prepared by dissolving 119.7 mg of DA hydrochloride (Aladdin Biochemical Technology Co., Ltd.) in 90 ml of deionized water and stirring vigorously. The solution was heated from room temperature to 70°C, and then 760 μl of 1N NaOH (Chemical Co., Ltd.) was added and heated at 70°C for 5 h.
After the addition of NaOH, the solution first turned light brown and then gradually turned dark brown. To obtain MNPs, the resulting product was cooled to room temperature at the end of the reaction, washed three times with deionized water, and centrifuged at 16,000 rpm. Then, MNPs were stored in a refrigerator at 4°C for subsequent use.

| Characterizations of MNPs
The morphology and size of MNPs were observed by scanning electron microscopy (SEM; Hitachi S-4800) at an accelerating voltage of 5 kV. The hydrated particle size and zeta potential of the MNPs

| Culture and identification of MSCs
Bone marrow MSCs were isolated as described previously with some modification (Kong et al., 2017). Briefly, bone marrow was harvested by flushing isolated from the tibia and femur of newborn Sprague-Dawley rats. The obtained cell suspension was centrifuged, and then the mononuclear cells were suspended in minimum essential medium-α (Gibco, Invitrogen) with 15% fetal bovine serum (Gibco), counted at 1 × 10 6 /ml and placed in a culture flask. The cells were cultured in a 5% CO 2 incubator at 37°C, and the medium was re-

| Assay for antioxidant activity of MNPs
The free radical-scavenging capacity of MNPs was detected by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging method (Brand-Williams et al., 1995 2.8 | Establishment of in vitro ischemic models of cortical neuron and MSC-neuron coculture systems The coculture system was established in Transwell ® 6-well or 12-well plates with 0.4 μm polycarbonate membranes (Corning Life Sciences).
TANG ET AL.

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| 935 The primary neurons were isolated and cultured as described previously (Kong et al., 2017) and identified as at least 95% MAP2positive cells with immunofluorescence. Primary cortical neurons were cultured in the lower chamber of transwell plates at a density of 2 × 10 6 cells per well (six-well plates) or 1 × 10 6 cells per well (12-well plates). Oxygen-glucose deprivation (OGD) was carried out in cultures after 7 days. The primary cortical neurons were washed with deoxygenated, glucose-free PBS buffer, and then the cultures were transferred to an anaerobic incubator containing 5% CO 2 and 95% N 2 at 37°C for 120 min. The non-OGD control neurons were cultured with PBS containing glucose and incubated in normal culture conditions for 120 min. Immediately after OGD, both OGD and non-OGD

| Intracellular ROS/superoxide radical assay
Total levels of ROS and superoxide were detected by using the intracellular ROS/superoxide detection assay kit (ab139476; Abcam).
Neurons cultivated in coverslips at a density of 1 × 10 6 cells per well

| Statistical analysis
Data are presented as the mean ± SEM. One-way analysis of variance was applied for multiple comparisons followed by Tukey's post-hoc test. Statistical analysis was conducted using SPSS software version 23.0 (SPSS Inc.). Statistical significance was defined as p < .05.

| Characterizations of MNPs
The dimensions of the MNPs were characterized by SEM (Hitachi S-4800) at an accelerating voltage of 5 kV. A small amount of dried powder was dipped on the conductive adhesive, and then the unstuck powder was blown off by using an airbag. After being sprayed with gold, the sample was fixed on the sample holder for SEM observation. As shown by SEM in Figure 1a, the MNPs were uniform nanospheres, with an average diameter of 115.13 ± 8.02 nm.
The size of hydrated particles is the size of nanoparticles in the solution state, also known as the hydrodynamic diameter, including the core of the nanoparticle and the micelle and adsorbed water on the surface. After the nanoparticles were diluted to an appropriate concentration, ultrasonic dispersion was conducted for approximately 1 min, and then the hydration particle size was measured directly. The pH of the diluted sample was adjusted to 7 and then tested for zeta potential. As shown in Figure 1b, the average hydrated particle size of MNPs is 106.5 nm (polymer dispersity index: 0.340 ± 0.010). Zeta potential analysis revealed that the surface of the MNPs was negatively charged (−21.3 mV; Figure 1c).
The FTIR spectra of DA and MNPs are shown in Figure 1d is that DA was oxidized into dopachrome and dopaindole, and the subsequent self-polymerization process led to an obvious absorption from visible light to NIR wavelengths (Liu et al., 2013).
The Raman spectra of DA and MNPs are presented in Figure 1f.
Characteristic peaks at 1594 and 1360 cm −1 can be attributed to the stretching and deformation of the aromatic ring on MNPs (Li et al., 2013).
Under our experimental conditions, the free radical scavenging rate of MNPs increased from 2.04% to 38.58% as the concentration of MNPs increased (Figure 3a). The free radical scavenging activity of MNPs increased in a dose-dependent manner. The

| MNPs enhance the protection of MSCs against OGD-injured neurons
To verify whether MNPs enhance the protection of MSCs against OGD-injured neurons, PI/Hoechst double staining was performed.

| MNPs-MSCs suppressed OGD-induced free radicals in neurons
To determine the antioxidative effect of MNPs-MSCs, an ROS/superoxide detection assay was used to detect the intracellular ROS and F I G U R E 5 (See caption on next page) TANG ET AL.

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| 941 superoxide generation in neurons after OGD exposure and the corresponding treatment. As shown in Figure 5a,

| DISCUSSION
In this study, we found that MNPs enhance the neuroprotective effect of MSCs against hypoxic-ischemic injury by inhibiting apoptosis and upregulating antioxidant defense.
In this study, the observed size and shape of the prepared MNPs (Figures 1a and 3i) were very similar to those reported (Ju et al., 2011; F I G U R E 5 Treatment with MNPs-MSCs significantly attenuated ROS and superoxide production in neurons after OGD toxicity. Primary cortical neurons were cultured on coverslips in 12-well culture plates at a density of 1 × 10 6 cells per well for 7 days, and the groups were treated as described previously. Neurons cultivated in coverslips after corresponding treatment for 24 h were washed with fresh medium and loaded with the oxidative stress detection reagent (green fluorescence) and superoxide detection reagent (red fluorescence) for 30 min at 37°C. F I G U R E 6 Treatment with MNPs-MSCs provides better protection against OGD-induced neuronal apoptosis than treatment with MNPs or MSCs only. Primary cortical neurons were cultured on cover slides at a density of 1 × 10 6 cells per well in a 12-well culture plate or a density of 2 × 10 6 cells per well in a six-well culture plate for 7 days. The treatment methods for each group were as described above. (a) Representative images of cleaved caspase-3 immunostaining in our experimental settings. Scale bar = 50 μm. (b) Treatment with MNPs-MSCs, MSCs, or MNPs significantly decreased the number of cleaved caspase-3-positive cells compared with OGD vehicle, and the cleaved caspase-3 expression in the OGD-MNPs-MSCs group was significantly lower than that in the OGD-MSCs group or OGD-MNPs group.
(c-f) The protein levels of cleaved caspase-3, Bcl-2, and Bax were detected by western blot analysis and quantified using ImageJ software. Western blot analysis showed that the levels of cleaved caspase-3 protein were significantly decreased with treatment with MNPs-MSCs, MSCs, or MNPs relative to the OGD vehicle group, and the levels of cleaved caspase-3 protein in the OGD-MNPs-MSCs group were significantly lower than those in the OGD-MSCs group or OGD-MNPs group. Treatment with MNPs-MSCs or MSCs prevented Bax upregulation and Bcl-2 downregulation relative to the OGD vehicle. (g) The ratio of Bcl-2/Bax. The Bcl-2/Bax ratio in the OGD-MNPs-MSCs group was significantly higher than that in the OGD-MSCs group or OGD-MNPs group. The data are presented as the mean ± SEM from six separate experiments. # p < .01 relative to the normal control; **p < .01 compared with the OGD-vehicle group or OGD-MNPs-MSCs group, *p < .05 compared with the OGD-MNPs-MSCs group. MNP, melanin nanoparticle; MSC, mesenchymal stem cell; OGD, oxygen-glucose deprivation TANG ET AL.

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| 943 It has been well documented that OS, including ROS and reactive nitrogen species, followed by reperfusion is responsible for most ischemia-reperfusion injuries and thus leads to brain tissue damage (Orellana-Urzua et al., 2020). The brain is highly sensitive to ROSinduced injury because the brain exhibits a high concentration of lipid peroxide, a low level of protective antioxidants, high oxygen consumption, and a high level of iron, which makes OS have a profound effect on the pathogenesis of stroke (Saeed et al., 2007). Therefore, modulating OS by scavenging ROS might be an effective measure to attenuate hypoxic-ischemic and reperfusion injury. According to previous reports (Rageh & El-Gebaly, 2018;Zhong et al., 2019), our data showed that MNPs exhibited the capacity to scavenge ROS and superoxide, which is beneficial for protecting against hypoxicischemic injury. Previous studies have shown that antioxidation is one of the protective mechanisms of MSCs against ischemic brain injury (Calió et al., 2014). We, therefore, wondered whether combined treatment with MSCs and MNPs exhibits a synergistic protective effect against hypoxic-ischemic injury. To our surprise, our study demonstrated that combined treatment with MSCs and MNPs provided better neuroprotection against ischemia than treatment with either MSCs or MNPs alone, and the underlying mechanism was related to their synergistic effect on antioxidants. These data provide favorable evidence for the clinical use of combined treatment with MSCs and MNPs as a therapy for ischemic stroke.
Previous studies have revealed that apoptosis may contribute to a significant proportion of neuronal death, especially in the ischemic penumbra, following acute brain ischemia (Uzdensky, 2019). Therefore, the identification of an efficient neuroprotector capable of rescuing neurons in the ischemic penumbra is the main goal for stroke therapy. Inhibitors of caspases, which play an important role in both the signal conduction and execution stage of the cascade reaction of apoptosis, can block the process of cell death in animal models of ischemic stroke, reduce the volume of cerebral infarction and significantly improve neurological function deficits after ischemic stroke (Shabanzadeh et al., 2015). Previous literature has shown that the expression of proapoptotic genes in the Bcl family, including Bax and Bcl-XS, was significantly increased, while the expression of antiapoptotic genes, including Bcl-2 and Bcl-XL, was significantly decreased after global cerebral ischemia in rats (Gillardon et al., 1996). A recent study revealed that the neuroprotection of MNPs was correlated with the prevention of Bax upregulation and Bcl-2 downregulation in Neuro2A cells treated with CoCl 2 . In line with a previous report , the current study de-

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