摘要
Open AccessCCS ChemistryCOMMUNICATION1 May 2022Nanomedicine-Leveraged Intratumoral Coordination and Redox Reactions of Dopamine for Tumor-Specific Chemotherapy Bowen Yang, Yuedong Guo, Yuemei Wang, Jiacai Yang, Heliang Yao and Jianlin Shi Bowen Yang State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050 Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049 , Yuedong Guo State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050 Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049 , Yuemei Wang State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050 Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049 , Jiacai Yang State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050 Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049 , Heliang Yao State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050 and Jianlin Shi *Corresponding author: E-mail Address: [email protected] State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050 https://doi.org/10.31635/ccschem.021.202100930 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Great efforts have been made in investigating the neurotoxicity of dopamine (DA) in the presence of manganous ions. In contrast, here, we probe the possibility of DA-based cancer chemotherapy by leveraging intratumoral redox reactions of DA for producing cytotoxic species in situ. For this purpose, we have constructed a Mn-engineered, DA-loaded nanomedicine. Based on the unique size effect of the nanocarrier, this nanomedicine will not enter the central nervous system but can effectively accumulate in the tumor region, after which the nanocarrier can degrade to release Mn2+ and DA in response to the mild acidic intracelluar microenvironment of cancer cells. DA can chelate Mn2+ to form a binary coordination complex, where the strong metal–ligand interaction significantly promotes electron delocalization and elevates the reducibility of Mn center, favoring two sequential one-electron oxygen reduction reactions forming H2O2, which can be further converted into highly oxidizing •OH under the cocatalysis by Mn2+ and intracellular Fe2+. Additionally, as a two-electron oxidation product of DA ligand, DA-o-quinone is potent in exhausting cellular sulfhydryl and depleting reduced glutathione, inhibiting the intrinsic antioxidative mechanism of cancer cells, finally triggering severe oxidative damages in a synergistic manner. It is expected that such a strategy of nanotechnology-mediated metal–ligand coordination and subsequent nontoxicity-to-toxicity transition of DA in tumor may provide a promising prospect for future chemotherapy design. Download figure Download PowerPoint Introduction Dopamine (DA, 3-hydroxytyramine) is a naturally occurring catecholamine biosynthesized in the dopaminergic neuron terminals of a mammalian brain and is one of the most critical neurotransmitters in the central nervous system (CNS).1,2 DA contributes significantly to the neurophysiological control of attention, arousal, perception, motivation, emotion, and movement,3 while abnormal levels of cerebral DA can be indicative of, or lead to, a number of neurological disorders, such as Parkinson’s disease,4 schizophrenia,5 and Tourette’s syndrome.6 Manganism is a major cause of dopaminergic system dysfunction, as the manganous ions (Mn2+) can be chelated by DA to catalyze the two-electron oxidation of this pyrocatechol accompanied by two sequential oxygen reduction reactions (ORRs), after which stoichiometric hydrogen peroxide (H2O2) and DA-o-quinone (DQ) are generated, both of them can elevate cerebral oxidative stress and trigger toxicity to normal neurocytes in substantia nigra.7 On the other hand, cancer is a major disease initiated by malignant proliferation of cancer cells. Triggering toxicity specifically in cancer cells for inhibiting tumor growth without harmful side effect to normal tissues is supposed to be an advanced cancer therapeutic modality. Therefore, we expect that such a nontoxicity-to-toxicity transition of DA in the presence of Mn catalyst will be instructive to the design of anticancer therapy. To achieve this goal, two major scientific issues should be addressed: (1) How to mitigate the side effects of DA and Mn2+ after administration, either alone or in combination, to normal organs especially to the nervous system? (2) How to deliver the two agents efficiently to the tumor region and trigger the in situ coordination reaction between them as well as subsequent redox reactions for cancer therapy? Nanomedicines with diverse physicochemical properties have been extensively investigated in various biomedical applications, especially in anticancer therapy.8 Importantly, their unique nanodimension effect benefits their therapeutic applications when they cross several physiological structures of the human body: (1) blood–brain barrier (BBB) is a highly selective interface between CNS and ambient blood capillaries.9 It can prevent nanoparticles from entering CNS and thus protect cerebral normal nervous activities. It is here conceived that, if a nanomedicine coloaded with DA and Mn2+ is constructed for treating extracranial tumors, BBB can prevent the entrance of the two chemicals into CNS. In addition, as the DA receptors are only located in CNS,10 the composite nanomedicine is considered to be unable to influence systemic neural activities. (2) Enhanced permeability and retention (EPR) effect is an intrinsic biophysical mechanism of tumors, which leads to automatic accumulation and retention of nanoparticles in tumors.11 It is expected that nanomedicine encapsulating DA and Mn2+ can target tumor regions passively. This strategy, if applicable, may offer a promising approach to initiate DA-based coordination and redox reactions in tumor regions rather than the brain, thus making anticancer therapy feasible. In addition to nano–bio interactions, a second consideration for leveraging DA chemistry for cancer therapy is to design a proper drug delivery nanosystem enabling the co-releases of DA and Mn2+ specifically in the tumor region in a tumor-responsive manner. Mesoporous silica nanoparticles (MSNs) have been widely explored as drug delivery systems due to their unique mesoporous structure and abundant surface chemistry.12 In addition, the composition of their –Si–O–Si– framework is tunable and can be doped with metallic elements to form a –Si–O–M– hybrid structure (M = Fe, Mn, Cu, Mg, and Ca), conferring the nanocarrier with additional physicochemical performances favoring cancer therapy, such as triggered drug release and degradation behaviors.13 In this work, we construct a composite nanomedicine for cancer chemotherapy by loading DA in a Mn-doped, polyethylene glycol (PEG)-modified hollow MSN (Mn-HMSN-PEG-DA, denoted MHPD) (Scheme 1), which can respond to the intracellular mild acidic environment of cancer cells and subsequently degrade to release Mn2+ and DA. DA can chelate Mn2+ to form a binary coordination complex, where the strong metal–ligand interaction significantly promotes electron delocalization and elevates the reducibility of Mn center, favoring ORRs and catalytic DA ligand oxidation to generate H2O2 and DQ. As the intracellular Fe2+ level of cancer cells is higher than that of normal ones,14–16 Mn2+ can cooperate with labile Fe2+ in cancer cells to induce a distinct cocatalytic effect promoting the conversion of H2O2 to highly oxidizing hydroxyl radical (•OH), significantly upregulating oxidative stress.17,18 More importantly, as a two-electron oxidation product of the DA ligand, DQ is electron-deficient and can covalently bind to cellular nucleophiles especially sulfhydryl groups of reduced glutathione (GSH),19 inactivating this antioxidant and thus inhibiting the endogenous self-detoxifying system of cancer cells, finally potentiating oxidative damages to cancer cells in synergy with catalytic •OH generation. Both in vitro and in vivo results have demonstrated the distinct anticancer effect of MHPD with mitigated side effect, suggesting that such a strategy of leveraging DA chemistry for cancer chemotherapy may be achievable by rational nanomedicine design. Scheme 1 | Chemical mechanism of MHPD nanomedicine-triggered coordination and redox reactions of DA enabling enhanced cancer chemotherapy with mitigated side effect. Download figure Download PowerPoint Results and Discussion Synthesis and characterizations of MHPD The synthesis of Mn-engineered hollow MSN (Mn-HMSN) is based on a hydrothermal reaction process in which pristine MSN serves as a hard template (Figure 1a).20 Monodispersed MSNs were first prepared based on a typical sol–gel approach using tetraethyl orthosilicate (TEOS) as a silica precursor, cetyltrimethylammonium chloride (CTAC) surfactant as a structure-directing agent, and triethanolamine (TEA) as an alkaline catalyst. According to transmission electron microscopy (TEM) imaging and selected area electron diffraction (SAED) patterning ( Supporting Information Figure S1), the as-prepared MSNs are amorphous and around 80 nm in diameter. To prepare Mn-HMSN, these nanoparticles were redispersed in aqueous solution with the addition of alkaline precursor disodium maleate and Mn precursor manganese(II) sulfate monohydrate. The hydrolysis of disodium maleate generates carboxylate and a mild alkaline environment ( Supporting Information Scheme S1), the latter enables gradual MSN dissolution into Si-containing oligomers such as orthosilicic acid (Si(OH)4) during hydrothermal process (step A in Figure 1a). Active sites are generated subsequently on nanoparticle surfaces to adsorb Mn2+, which can decrease the activation energy for carboxylate decomposition and CO2 bubble generation favoring reactant exchange (step B in Figure 1a). The continuous reaction between Mn2+ and Si(OH)4 results in the deposition of a Mn-doped silica layer, and further alkali etching of inner MSN template leads to the formation of hollow cavity (step C in Figure 1a). These reactions enable the evolution of the –Si–O–Si– framework of MSN template to a –Si–O–Mn– hybrid framework (Figures 1b and 1c), conferring additional chemical characteristics to the nanocarier. Figure 1 | Synthesis and characterizations of Mn-HMSN. (a) Chemical mechanism for the synthesis of Mn-HMSN. (b) Chemical structure for the –Si–O–Si– framework of pristine MSN. (c) Chemical structure for the –Si–O–Mn– hybrid framework of Mn-HMSN. (d) SEM image of as-prepared Mn-HMSNs showing a rough surface topography. Scale bar, 50 nm. (e) TEM image of Mn-HMSNs showing a porous structure. Scale bar, 50 nm. (f) High-angle annular dark-field (HAADF) image and element mappings of Mn-HMSNs. Scale bar, 50 nm. (g) XRD patterns of MSN and Mn-HMSN. (h) Mn 2p spectrum of XPS spectra of Mn-HMSN. (i) 29Si solid-state MAS NMR spectra of MSN and Mn-HMSN. (j) Accumulated release profiles of Mn element from Mn-HMSN in SBF with different pH values. Download figure Download PowerPoint Scanning electron microscopy (SEM) images of Mn-HMSNs show that these nanoparticles are around 80 nm in diameter with a rough surface topography (Figure 1d). TEM imaging reveals their hollow structure and abundant porosity (Figure 1e and Supporting Information Figure S2). N2 adsorption–desorption isotherm and pore-size distribution analysis further evidence the mesoporous structure of Mn-HMSNs ( Supporting Information Figure S3). SAED patterning manifests the weak crystallinity of Mn-HMSNs ( Supporting Information Figure S4), in agreement with the result of X-ray diffraction (XRD, Figure 1g), which favors their degradation in a biological environment. Elemental mappings of Mn-HMSN sample demonstrate a homogeneous Mn distribution on the hollow nanoparticles (Figure 1f), while the energy-dispersive spectroscopy (EDS) profile indicates a high Mn-doping concentration ( Supporting Information Figure S5). According to X-ray photoelectron spectroscopy (XPS) spectra (Figure 1h), the main peak in Mn 2p spectrum can be divided into three characteristic peaks at binding energies of 641, 642, and 644 eV, corresponding to Mn2+, Mn3+, and Mn4+ species. The results of 29Si solid-state magic angle spinning nuclear magnetic resonance (MAS NMR) show that the distinctive peaks at chemical shifts of −100 ppm (Q3, Si(OSi)3(OH)) and −110 ppm (Q4, Si(OSi)4) of pristine MSN sample disappear after Mn doping (Figure 1i), as a result of a new phase formation with abundant –Mn–O– bonding. As the bonding energy of –Mn–O– bond is much lower than that of –Si–O– bond,21 the –Si–O–Mn– hybrid framework of Mn-HMSN is unstable in an acidic environment and Mn ions will be released upon H+ attack (Figure 1j). It is noted that among the released Mn ions, Mn3+ and Mn4+, are highly active and can react with H2O spontaneously generating Mn2+: 4 Mn 3 + + 2 H 2 O → 4 Mn 2 + + O 2 + 4 H + (1) 2 Mn 4 + + 2 H 2 O → 2 Mn 2 + + O 2 + 4 H + (2)The extraction of Mn from the hybrid framework leads to silanol group (Si-OH) generation and defect formation,22 which further promotes nanoparticle degradation ( Supporting Information Figures S6 and S7). TEM images show that Mn-HMSNs undergo morphological deformation after immersion in mild acidic simulated body fluid (SBF) (pH 6.8) for 6 h, and completely degrades in 60 h of treatment ( Supporting Information Figure S8). Comparatively, in neutral SBF (pH 7.4), Mn-HMSNs still keep their morphological integrity in 6 h and only degrade slightly even after 60 h of treatment. These results further evidence that Mn-HMSNs are sensitive to acidic environment and their degradation rate is pH-dependent. Additionally, negligible degradation of Mn-HMSN has been observed in fetal bovine serum (FBS) in 60 h of dispersion ( Supporting Information Figure S9), further demonstrating its high stability in neutral biological environment. Therefore, Mn-HMSN can be a competent candidate for constructing DA-loaded nanomedicine that enables the co-releases of Mn2+ and DA specifically within acidic tumor region. We then fabricated the composite nanomedicine MHPD by PEGylation of Mn-HMSN and subsequent DA loading ( Supporting Information Figure S10). Fourier transform infrared (FTIR) spectra indicated that the characteristic absorption peak at 2887 cm−1 attributable to the stretching vibration of –CH2– of PEG silane appears in the spectrum of modified Mn-HMSN (Figure 2a), demonstrating successful PEG modification. It is also noted that the absorption peak at 1110 cm−1 attributable to the stretching vibration of –Si–O– bond in the spectrum of pristine MSN has slightly shifted to the low wavelength region (1034 cm−1) in the spectrum of Mn-HMSN, as a consequence of Mn doping that makes the composite present physicochemical characteristics partially analogous to metallic oxide. DA can be easily loaded in Mn-HMSNs due to the large hollow cavity and unique mesoporous structure of these nanoparticles. The characteristic peak of DA in UV–vis absorption spectra at 279.5 nm can be observed in the spectrum of MHPD (Figure 2b), indicating that DA has been loaded in the nanocarrier. In this work, we maintain the stoichiometric compositions of Mn/DA in MHPD nanomedicine at 2:1 to enable complete catalytic oxidation of DA. The direct investigation of the release kinetics of DA from MHPD is difficult as the released DA will undergo quick oxidation under catalysis by free Mn2+. The detailed considerations on this issue, as well as the related experimental investigations, will be presented in the following section. Figure 2 | Preparation of MHPD nanomedicine. (a) FTIR spectra of MSN, Mn-HMSN, PEG silane, and Mn-HMSN-PEG. (b) UV–vis absorption spectra of DA, Mn-HMSN-PEG, and MHPD. Download figure Download PowerPoint Redox-regulating mechanism investigation The composite nanomedicine MHPD coloaded with Mn2+ and DA may enable catalytic DA oxidation and subsequent reactive oxygen species (ROS) generation; therefore, the reaction mechanism between DA and Mn2+ has been explored before applying this nanomedicine in biomedical applications. Based on previous studies on metal-catalyzed pyrocatechol oxidation,23,24 Florence and Stauber25 and Lloyd26 have proposed the reaction mechanism of Mn-catalyzed DA oxidation and ORRs forming H2O2 (Scheme 2a). As the reduction of O2 by either Mn2+ or DA is difficult ( Supporting Information Table S1 and Discussion S1), it has been inferred that a manganese-catechol binary coordination complex (i.e., DA-Mn chelate) is formed to trigger ORRs (equation 1 in Scheme 2a), as the redox capability of Mn2+/Mn3+ couples can be significantly regulated by the nature of complexing organic ligand and the strength of interaction between them.27 The Mn-catalyzed DA oxidation can be considered as two successive one-electron reaction processes. The chelated Mn2+ in the DO2-Mn(II) complex is highly reductive due to the formation of a Mn (d-electron)-ligand (p-electron) covalent bond favoring electron delocalization,27 and the metal center can donate one electron to oxygen, leading to the formation of DO2-Mn(III)+ and O2•− (equation 2 in Scheme 2a). Scheme 2 | Chemical mechanism for Mn2+-triggered catalytic reactions. (a) Mn2+-catalyzed DA oxidation forming DQ and H2O2.25 DA is denoted as D(OH)2 for better explanation of the dynamic change of phenolic hydroxyl group during reactions. (b) Cocatalysis between Mn2+ and Fe2+ promoting •OH generation. Download figure Download PowerPoint The Mn3+ is electron-deficient (electronic configuration type: [Ar]3d4) and much harder to donate an additional electron compared with Mn2+ (electronic configuration type: [Ar]3d5, d orbital is half-filled) but has a greater tendency to obtain an electron. According to the chemical structure of DA ligand, the electrons in the big π bond of the aromatic ring are highly delocalized, while the p orbital of phenolic hydroxyl oxygen is conjugated with a π bond (i.e., p-π conjugation), making the aromatic ring further activated to donate one electron to Mn3+ via p orbital of phenolic hydroxyl oxygen (equation 3 in Scheme 2a) ( Supporting Information Discussion S1). Consequently Mn2+ is regenerated, which further transfers this electron to O2•−, forming H2O2 and •DO2-Mn(III)2+ semiquinone radical ions (equation 4 in Scheme 2a).28 This electron-deficient semiquinone is unstable and a second intramolecular electron transfer takes place spontaneously between the aromatic ring of DA ligand and Mn3+,29 finally the complex decomposes into DQ and Mn2+ (equation 5 in Scheme 2a). For the whole redox reaction process, the valence of Mn does not change but the DA ligand loses two electrons enabling the redox cycling between Mn2+ and Mn3+ during catalyzing the ORRs. Therefore, Mn serves as a bridge favoring electron transfer between DA and oxygen. In this work, the codelivery of Mn specie and DA by MHPD is considered to enable such a metal–ligand coordination promoting catalytic H2O2 generation via ORRs. For anticancer applications, the generated H2O2 should be converted into highly oxidizing •OH to trigger oxidative damage to cancer cells. However, Mn2+ alone is not able to trigger distinct Fenton-like reactions. In conventional Fenton reactions ( Supporting Information Scheme S2), Fe2+ reacts with H2O2 to generate Fe3+ and •OH (equation iii in Supporting Information Scheme S2), after which Fe3+ is reduced into Fe2+ via reaction with H2O2, forming hydroperoxy radical (HOO•) (equation iv in Supporting Information Scheme S2, rate-limiting step). The HOO• can further reduce Fe3+ into Fe2+ (equation v in Supporting Information Scheme S2).30 Comparatively, although Mn2+ can also react with H2O2 to generate Mn3+ and •OH (equation vi in Supporting Information Scheme S2); however, as the redox potential of Mn3+/Mn2+ couple is much higher than others in the order of E0(Mn3+/Mn2+) > E0(O2•−, 2H+/H2O2) > E0(Fe3+/Fe2+) ( Supporting Information Table S1), Mn3+ can oxidize H2O2 into superoxide anion (O2•−) rather than HOO• (equation vii in Supporting Information Scheme S2), which will counteract •OH generated in equation vi (equation viii in Supporting Information Scheme S2), resulting in much compromised •OH-generating efficiency, allowing the whole catalytic process to present the characteristic of H2O2 decomposition into O2. In cancer cells, the overproduction of O2•− due to mitochondrial metabolic dysregulation enables this radical ion to attack the [4Fe-4S] cluster of iron regulatory protein, after which Fe2+ is released (equation 7 in Scheme 2b).31 Therefore, the intracellular level of free Fe2+ in cancer cells is higher than that in normal cells.14,15 It is noted that during the reaction between Mn2+ and H2O2, the addition of Fe2+ can favor the reduction of generated Mn3+ efficiently, forming Mn2+ and Fe3+ (equation 8 in Scheme 2b). Mn2+ returns to equation 6 and continues to enable •OH production while Fe3+ can be further reduced to Fe2+ by H2O2 and HOO• (equations 9 and 10 in Scheme 2b, identical to equations iv and v in Supporting Information Scheme S2). Such a cooperation between Mn2+ and Fe2+ leads to a distinct cocatalytic effect ( Supporting Information Scheme S3), resulting in significantly elevated •OH-generating efficiency than that triggered by Mn2+ or Fe2+ alone. In cancer cells, Mn2+ released from MHPD nanomedicine is considered to cooperate with cellular excessive labile Fe2+ for synergistic enhancement of oxidative damage. Combining the two catalytic processes, that is, DA oxidation and H2O2 decomposition into •OH, Mn2+ released from MHPD nanomedicine catalyzes overall ORRs from O2 to O2•− and H2O2, and finally to •OH. The direct experimental evaluation on the catalytic performance of MHPD is rather difficult as the releases of Mn2+ and DA from MHPD are both time-dependent during which the concentrations of Mn2+ and DA keep changing, making the reaction process complicated for quantitative investigation. However, inspired from differential calculus developed in advanced mathematics, if we could differentiate the whole catalytic reaction process with respect to time, the initial concentrations of Mn2+ and DA will be meaningful at a particular moment. Therefore, in this work, we used the acidic buffer solution (pH 6.8) containing Mn2+ and DA of certain initial concentrations ([Mn2+]0 = 10 mM, [DA]0 = 5 mM) to mimic a differentiated reaction for precisely figuring out the catalytic mechanism and reaction trend in a much-simplified manner. It was observed that the colorless mixed buffer solution turned black in 10 min, and the base line of UV–vis spectra of the solution kept increasing (Figure 3a), indicating a black substance generation. According to equation 5 in Scheme 2a, DQ will be generated during catalytic DA oxidation. However, this chemical is rather reactive and can automatically cyclize to form 5,6-dihydroxyindole (DHI), by desquamating two protons and two electrons and subsequent internal rearrangement (equation ix in Supporting Information Scheme S4).32 DHI can aggregate spontaneously to form polydopamine (melanin) when there is no other reactive reactant in the reaction system (equation x in Supporting Information Scheme S4).33 Therefore, these phenomena provide indirect evidences for DA oxidation and DQ formation in the presence of Mn catalyst. As the acidity-triggered releases of Mn2+ and DA from MHPD will lead to the coordination reaction between the two chemicals, finally DQ will be produced to form melanin; therefore, this experimental phenomenon can be used to confirm DA release from the nanomedicine. Distinct melanin generation was monitored in acidic buffer solution containing MHPD after 12 h of dispersion ( Supporting Information Figure S11), while in neutral buffer solution containing equivalent concentration of MHPD negligible amount of melanin was generated, demonstrating that DA can be released from the nanomedicine specifically in acidic environment. Additionally, minimal amount of melanin was generated in FBS in 12 h of MHPD dispersion ( Supporting Information Figure S12), evidencing that DA can be hardly oxidized in neutral biological environment. Figure 3 | Redox-regulating mechanism investigation. (a) UV–vis absorption spectra of 4-morpholineethanesulfonic acid hydrate (MES) buffer solution containing DA and Mn2+ at different time points of reaction, indicating melanin generation. The insets are digital photos of the solutions before (1) and after (2) reaction for 10 min. (b) CV curves investigating the electrochemical behavior and redox capability of Mn2+ in acidic buffer solution (pH 6.8) with or without the addition of DA. (c) ESR spectra indicating •OH generation in different reaction systems. (d) RhB decolorization in acidic buffer solution (pH 6.8) of different chemical environments revealing synergistic actions among DA, Mn2+, and Fe2+. (e) UV–vis absorption spectra of RhB in acidic buffer solution (pH 6.8) of different chemical environments for 20 h revealing the cooperation of MHPD and Fe2+ promoting RhB degradation. (f) The reaction system of a buffer solution containing RhB, MHPD, and Fe2+ was further added with SOD or catalase for 20 h to investigate the generation of O2•− and H2O2 intermediates during pro-oxidation reactions. Blank group is the buffer solution containing RhB, MHPD, and Fe2+ without SOD or catalase addition. (g) Chemical mechanism of GSH depletion by DQ via the formation of 5-S-glutathionyl-DA adduct. (h) Time-dependent GSH depletion by DQ or the mixture of DA, Mn2+, and Fe2+. Download figure Download PowerPoint The redox capability of Mn2+/Mn3+ couple in the DA-Mn chelate compound was investigated by measuring the electrochemical cyclic voltammetry (CV) behavior of buffer solution containing Mn2+ before and after DA addition (Figure 3b). The oxidation peak of buffer solution containing single Mn2+ is attributed to the electron transfer from Mn2+ to the outside reaction system showing significant electrical behavior. This oxidation peak almost disappeared after DA addition, resulting from the formation of DO2-Mn(II) complex where the DA ligand significantly increased the reducibility of Mn2+ to enable the electron transfer to O2 for ORRs ( Supporting Information Table S2). Additionally, the reduction peak of the buffer solution has been shifted toward the positive direction after DA addition, demonstrating that the reduction of DO2-Mn(III)+ to DO2-Mn(II) is more difficult than that of Mn3+ to Mn2+. In reverse, the oxidation of DO2-Mn(II) to DO2-Mn(III)+ is therefore easier than that of Mn2+ to Mn3+, in consistence with the previous conclusion that the DO2-Mn(II) chelate presents higher reducibility than Mn2+. Moreover, according to equation 3 of Scheme 2a, the intramolecular electron transfer will occur in DO2-Mn(III)+, forming the semiquinone transition-state •DO2-Mn(II)+ with high reducibility. Therefore, it is much easier for DO2-Mn(III)+ to transfer one electron for enabling ORRs rather than accepting one electron to generate DO2-Mn(II), different from the case that Mn3+ alone can be reduced in aqueous solution into Mn2+ spontaneously. CV results provide evidence of two-electron oxidation of DA-Mn complex, which encourages us to further investigate the •OH-generating efficiency of the reaction system in the presence of Fe2+. Electron spin resonance (ESR) spectroscopic profiles indicate distinct characteristic 1∶2∶2∶1 •OH signal in buffer solution containing DA, Mn2+, and Fe2+ (Figure 3c), while the absence of Fe2+ or Mn2+ will lead to much compromised •O